研究者データベース

原島 秀吉(ハラシマ ヒデヨシ)
薬学研究院 医療薬学部門 医療薬学分野
教授

基本情報

所属

  • 薬学研究院 医療薬学部門 医療薬学分野

職名

  • 教授

学位

  • 薬学博士(東京大学)

ホームページURL

J-Global ID

研究キーワード

  • マイトポーター   核酸ナノ医薬品   個別化医療   ナノ医療   pH-応答性カチオニック脂質   リポソーム   薬物送達システム   エンドサイトーシス   siRNA   細胞内動態制御   MEND   オクタアルギニン   がん   エンドソーム   細胞内動態   遺伝子診断   遺伝子ベクター   遺伝子デリバリー   遺伝子治療   多機能性エンベロープ型ナノ構造体   KALA   非ウイルスベクター   ミトコンドリア   ワクチン   ターゲティング   薬物速度論   

研究分野

  • ライフサイエンス / 薬系分析、物理化学
  • ライフサイエンス / 医療薬学
  • ライフサイエンス / 生体材料学
  • ライフサイエンス / 生体医工学
  • ライフサイエンス / 薬系化学、創薬科学
  • ライフサイエンス / 薬理学
  • ライフサイエンス / 薬系衛生、生物化学
  • ライフサイエンス / 感染症内科学

職歴

  • 2009年04月 - 現在 北海道大学大学院薬学研究院 未来創剤学研究室 教授(兼務)
  • 2006年04月 - 現在 北海道大学 大学院薬学研究院薬剤分子設計学研究室 教授
  • 1999年07月 - 2006年03月 北海道大学 大学院薬学研究科 薬剤分子設計学分野 教授
  • 1989年07月 - 1999年06月 徳島大学 薬学部 助教授
  • 1987年07月 - 1989年06月 スタンフォード大学 医学部麻酔科 JSPS特別研究員
  • 1985年10月 - 1987年07月 東京大学 薬学部 助手

学歴

  • 1983年04月 - 1985年10月   東京大学   薬学系大学院   博士課程退学
  • 1981年04月 - 1983年03月   東京大学   薬学系大学院   修士課程修了
  • 1977年04月 - 1981年03月   東京大学   薬学部卒業

所属学協会

  • 日本核酸医薬学会   AAPS: American Association of Pharmaceutical Sciences   日本癌学会   Controlled Release Society   日本薬物動態学会   日本DDS学会   公益社団法人日本薬学会   公益社団法人日本薬剤学会   

研究活動情報

論文

  • Fumika Kubota, Satrialdi, Yuta Takano, Masatoshi Maeki, Manabu Tokeshi, Hideyoshi Harashima, Yuma Yamada
    Journal of biophotonics e202200119  2022年09月01日 
    Photodynamic therapy (PDT) is a cancer therapy that uses a photosensitizer (PS) in the presence of oxygen molecules. Since singlet oxygen is highly reactive, it is important to deliver it to the target site. Thus, an efficient drug delivery system (DDS) is essential for enhancing the efficacy of such a treatment and protecting against the side effects of PDT. Here, we report on attempts to increase the therapeutic effect of PDT by using a DDS, a lipid nanoparticle (LNP). We prepared a porphyrin analog, rTPA (PS) that was encapsulated in LNPs using a microfluidic device. The findings indicated that the internal structure of the prepared particles changed depending on the amount of rTPA in LNPs. The photoactivity and cell-killing effect of PS in LNPs also changed when the amount of the cargo increased. These results suggest that the internal structure of LNPs is important factors that affect drug efficacy. This article is protected by copyright. All rights reserved.
  • Alaa M Khalifa, Takashi Nakamura, Yusuke Sato, Takanori Sato, Mamoru Hyodo, Yoshihiro Hayakawa, Hideyoshi Harashima
    International journal of pharmaceutics 624 122034 - 122034 2022年08月25日 
    Programmed cell death 1 (PD-1) blockade combination to other drugs have attracted the interest of scientists for treating tumors resistant to PD-1 blockade. In this study, the impact of the interval, order of administration, and number of cycles of immunotherapeutic combination of stimulator of interferon genes (STING) pathway agonist loaded lipid nanoparticle (STING-LNP) and PD-1 antibody for inducing the optimal combined antitumor activity against a melanoma lung metastasis is reported. One cycle had no effect, but two and three cycles resulted in a combinedantitumor effect. The interval between the administration was found to influence the induction of the combined effect. The second and third doses increased the gene expression of the NK cell activation marker, interferon γ (IFN-γ), PD-1 and a ligand of PD-1 (PD-L1), whereas the first dose failed. NK cells in the lung showed an increase in the expression of the activation markers and PD-1 after the second dose. The combined antitumor effect of this combination therapy against melanoma lung metastasis model could be dependent on the interval as well as the number of doses of STING-LNP.These findings suggest the importance of the protocol setting when combining a nano system loaded with an immune adjuvant and PD-1 antibody.
  • Takashi Nakamura, Yusuke Sato, Yuma Yamada, Mahmoud M Abd Elwakil, Seigo Kimura, Mahmoud A Younis, Hideyoshi Harashima
    Advanced drug delivery reviews 188 114417 - 114417 2022年07月03日 
    A new era of nanomedicines that involve nucleic acids/gene therapy has been opened after two decades in 21st century and new types of more efficient drug delivery systems (DDS) are highly expected and will include extrahepatic delivery. In this review, we summarize the possibility and expectations for the extrahepatic delivery of small interfering RNA/messenger RNA/plasmid DNA/genome editing to the spleen, lung, tumor, lymph nodes as well as the liver based on our studies as well as reported information. Passive targeting and active targeting are discussed in in vivo delivery and the importance of controlled intracellular trafficking for successful therapeutic results are also discussed. In addition, mitochondrial delivery as a novel strategy for nucleic acids/gene therapy is introduced to expand the therapeutic dimension of nucleic acids/gene therapy in the liver as well as the heart, kidney and brain.
  • Kento Okuda, Yusuke Sato, Kazuki Iwakawa, Kosuke Sasaki, Nana Okabe, Masatoshi Maeki, Manabu Tokeshi, Hideyoshi Harashima
    Journal of controlled release : official journal of the Controlled Release Society 348 648 - 659 2022年06月18日 
    The use of lipid nanoparticles (LNPs) for nucleic acid delivery is now becoming a promising strategy with a number of clinical trials as vaccines or as novel therapies against a variety of genetic and infectious diseases. The use of microfluidics for the synthesis of the LNPs has attracted interest because of its considerable advantages over other conventional synthetic methods including scalability, reproducibility, and speed. However, despite the potential usefulness of large particles for nucleic acid delivery to dendritic cells (DCs) as a vaccine, the particle size of the LNPs prepared using microfluidics is typically limited to approximately from 30 to 100 nm. In this study, focusing on Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, the effect of some synthetic parameters, including total flow rate, flow rate ratio, buffer pH, lipid concentration, molar ratio of PEG-lipid as well as salt concentration, on particle size was systematically examined by means of the design of experiment approaches. The findings indicated that the simple addition of salt (e.g. NaCl) to a buffer containing nucleic acids contributed greatly to the synthesis of large LNPs over 200 nm and this effect was concentration-dependent with respect to the salt. The effect of salt on particle size was consistent with a Hofmeister series. The systemic injection of larger mRNA-loaded LNPs resulted in a higher transgene expression in mouse splenic DCs, a higher activation of various splenic immune cells, and had a superior effect as a therapeutic cancer vaccine in a syngeneic mouse model compared to the smaller-sized counterpart with constant lipid composition prepared with lower NaCl concentration. Collectively, size-regulation by the simple addition of salt is a promising strategy for developing potent LNPs.
  • Takafumi Fukui, Hironao Tateno, Takashi Nakamura, Yuma Yamada, Yusuke Sato, Norimasa Iwasaki, Hideyoshi Harashima, Ken Kadoya
    International journal of molecular sciences 23 12 2022年06月15日 
    Despite recent advancements in therapeutic options for disorders of the central nervous system (CNS), the lack of an efficient drug-delivery system (DDS) hampers their clinical application. We hypothesized that liposomes could be optimized for retrograde transport in axons as a DDS from peripheral tissues to the spinal cord and dorsal root ganglia (DRGs). Three types of liposomes consisting of DSPC, DSPC/POPC, or POPC in combination with cholesterol (Chol) and polyethylene glycol (PEG) lipid were administered to sciatic nerves or the tibialis anterior muscle of mature rats. Liposomes in cell bodies were detected with infrared fluorescence of DiD conjugated to liposomes. Three days later, all nerve-administered liposomes were retrogradely transported to the spinal cord and DRGs, whereas only muscle-administered liposomes consisting of DSPC reached the spinal cord and DRGs. Modification with Cholera toxin B subunit improved the transport efficiency of liposomes to the spinal cord and DRGs from 4.5% to 17.3% and from 3.9% to 14.3% via nerve administration, and from 2.6% to 4.8% and from 2.3% to 4.1% via muscle administration, respectively. Modification with octa-arginine (R8) improved the transport efficiency via nerve administration but abolished the transport capability via muscle administration. These findings provide the initial data for the development of a novel DDS targeting the spinal cord and DRGs via peripheral administration.
  • Yuma Yamada, Yusuke Sato, Takashi Nakamura, Hideyoshi Harashima
    Journal of controlled release : official journal of the Controlled Release Society 348 357 - 369 2022年06月09日 
    The recent rapid progress in the area of drug delivery systems (DDS) has opened a new era in medicine with a strong linkage to understanding the molecular mechanisms associated with cancer survival. In this review, we summarize new cancer strategies that have recently been developed based on our DDS technology. Cancer immunotherapy will be improved based on the concept of the cancer immunity cycle, which focuses on dynamic interactions between various types of cancer and immune cells in our body. The new technology of genome editing will also be discussed with reference to how these new DDS technologies can be used to introduce therapeutic cargoes into our body. Lastly, a new organelle, mitochondria will be the focus of creating a new cancer treatment strategy by a MITO-Porter which can deliver macromolecules directly to mitochondria of cancer cells via a membrane fusion approach and the impact of controlled intracellular trafficking will be discussed.
  • Yuma Yamada, Takuya Ishimaru, Hideyoshi Harashima
    Clinical and Translational Discovery 2 2 2022年06月
  • Yuma Yamada, Sen Ishizuka, Manae Arai, Minako Maruyama, Hideyoshi Harashima
    Expert opinion on biological therapy 1 - 11 2022年05月11日 
    INTRODUCTION: After the emergence of lipid nanoparticles (LNP) containing therapeutic mRNA as vaccines for use against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the clinical usefulness of nucleic acid-encapsulated LNPs is now a fact. In addition to the nucleus and cytoplasm, mitochondria, which have their own genome, are a site where nucleic acids function in the cell. Gene therapies targeting mitochondria are expected to pave the way for the next generation of therapies. AREAS COVERED: Methods for delivering nucleic acids to mitochondria are needed in order to realize such innovative therapies. However, only a few reports on delivery systems targeting mitochondria have appeared. In this review, we summarize the current state of research on RNA-based therapeutics targeted to mitochondria, with emphasis on mitochondrial RNA delivery therapies and on therapies that involve the use of mitochondrial genome editing devices. EXPERT OPINION: We hope that this review article will focus our attention to this area of research, stimulate more interest in this field of research, and lead to the development of mitochondria-targeted nucleic acid medicine. It has the potential to become a major weapon against urgent and unknown diseases, including SARS-CoV-2 infections.
  • Takashi Nakamura, Kyoko Kawakami, Momoka Nomura, Yusuke Sato, Mamoru Hyodo, Hiroto Hatakeyama, Yoshihiro Hayakawa, Hideyoshi Harashima
    Journal of controlled release : official journal of the Controlled Release Society 345 200 - 213 2022年05月 
    Since the effect of cancer immunotherapy is largely dependent on the status of the immune system in the tumor microenvironment (TME), choice of therapy and the development of new therapies based on the immune status in the TME would be predicted to be effective. Unfortunately, the development of delivery systems for such therapy has been slow. Here, we defined a parameter of immune status in TME showing antitumor effects and demonstrated the cancer immunotherapy with an adjuvant loaded lipid nanoparticle (LNP), which was taken advantage the parameter. An analysis was carried out to determine the relationship between antitumor effects and gene expression (22 target genes) in tumors (MC38 and E.G7-OVA) that respond to the programmed cell death 1 (PD-1) antibody and non-responding tumors (B16-F10 and 4T1). The immune status showing an effective antitumor effect, which consisted of 10 genes, was then extracted. Treatment with the adjuvant loaded LNP caused a significant antitumor effect against an E.G7-OVA tumor, and the gene expression in the E.G7-OVA tumor was completely within the range of gene expression for showing an effective antitumor effect, as defined by the identified immune status panel (IS-panel-10). Although the treatment with the adjuvant loaded LNP failed to induce a sufficient antitumor effect against the 4T1 tumor, we succeeded in enhancing the antitumor effect by using a combination therapy that was adopted based on the analysis by the IS-panel-10 in the TME. The 10 genes were found to affect the prognosis in a variety of human cancers. Collectively, the findings reported herein demonstrate the potential of immune status analysis in the TME for developing cancer immunotherapies using a delivery system.
  • Seigo Kimura, Hideyoshi Harashima
    Neural regeneration research 17 4 785 - 787 2022年04月 [査読有り][招待有り]
  • Daisuke Sasaki, Jiro Abe, Atsuhito Takeda, Hideyoshi Harashima, Yuma Yamada
    Scientific reports 12 1 4344 - 4344 2022年03月22日 
    Given the potential for myocardial stem cell transplantation as a promising treatment for heart failure, numerous clinical trials have been conducted and its usefulness has been clearly confirmed. However, the low rate of engraftment of transplanted cells has become a clinical problem, and this needs to be improved in the case of transplanting cells to the heart. To address this issue, we report on attempts to prepare mitochondria-activated stem cells (MITO cells) for use in transplantation. MITO cells, which is cardiac progenitor cells (CPCs) activated by the mitochondrial delivery of resveratrol with an anti-oxidant and mitochondrial activation effects were successfully prepared using a mitochondrial targeting nanocarrier (MITO-Porter). The purpose of this study was to validate the therapeutic effect of cell transplantation by the MITO cells using a mouse model of myocardial ischemia-reperfusion. Mouse CPCs were used as transplanted cells. The transplantation of CPCs and MITO cells were conducted after myocardial ischemia-reperfusion, and the therapeutic effect was determined. The MITO cells transplanted group showed increase in postoperative weight gain, improve cardiac function and inhibition of fibrosis compared to the non-transplanted group and the CPC group. The transplantation of MITO cells to the ischemic myocardium showed a stronger transplantation effect compared to conventional CPC transplantation.
  • Nako Maishi, Yu Sakurai, Hiroto Hatakeyama, Yui Umeyama, Takashi Nakamura, Rikito Endo, Mohammad Towfik Alam, Cong Li, Dorcas Akuba-Muhyia Annan, Hiroshi Kikuchi, Hirofumi Morimoto, Masahiro Morimoto, Kosuke Akiyama, Noritaka Ohga, Yasuhiro Hida, Hideyoshi Harashima, Kyoko Hida
    Cancer science 2022年03月09日 
    Tumor blood vessels play important roles in tumor progression and metastasis. Targeting tumor endothelial cells (TECs) is one of the strategies for cancer therapy. We previously reported that biglycan, a small leucine-rich proteoglycan, is highly expressed in TECs. TECs utilize biglycan in an autocrine manner for migration and angiogenesis. Furthermore, TEC-derived biglycan stimulates tumor cell migration in a paracrine manner leading to tumor cell intravasation and metastasis. In this study, we explored the therapeutic effect of biglycan inhibition in the TECs of renal cell carcinoma using an in vivo siRNA delivery system known as a multifunctional envelope-type nanodevice (MEND), which contains a unique pH-sensitive cationic lipid. To specifically deliver MEND into TECs, we incorporated cyclo(Arg-Gly-Asp-D-Phe-Lys) (cRGD) into MEND because αV β3 integrin, a receptor for cRGD, is selective and highly expressed in TECs. We developed RGD-MEND-encapsulating siRNA against biglycan. First, we confirmed that MEND was delivered into OS-RC-2 tumor-derived TECs and induced in vitro RNAi-mediated gene silencing. MEND was then injected intravenously into OS-RC-2 tumor-bearing mice. Flow cytometry analysis demonstrated that MEND was specifically delivered into TECs. Quantitative RT-PCR indicated that biglycan was knocked down by biglycan siRNA-containing MEND. Finally, we analyzed the therapeutic effect of biglycan silencing by MEND in TECs. Tumor growth was inhibited by biglycan siRNA-containing MEND. Tumor microenvironmental factors such as fibrosis were also normalized using biglycan inhibition in TECs. Biglycan in TECs can be a novel target for cancer treatment.
  • Takao Tsujioka, Daisuke Sasaki, Atsuhito Takeda, Hideyoshi Harashima, Yuma Yamada
    International journal of molecular sciences 23 1 2021年12月22日 
    The development of drug delivery systems for use in the treatment of cardiovascular diseases is an area of great interest. We report herein on an evaluation of the therapeutic potential of a myocardial mitochondria-targeting liposome, a multifunctional envelope-type nano device for targeting pancreatic β cells (β-MEND) that was previously developed in our laboratory. Resveratrol (RES), a natural polyphenol compound that has a cardioprotective effect, was encapsulated in the β-MEND (β-MEND (RES)), and its efficacy was evaluated using rat myocardioblasts (H9c2 cells). The β-MEND (RES) was readily taken up by H9c2 cells, as verified by fluorescence-activated cell sorter data, and was observed to be colocalized with intracellular mitochondria by confocal laser scanning microscopy. Myocardial mitochondrial function was evaluated by a Seahorse XF Analyzer and the results showed that the β-MEND (RES) significantly activated cellular maximal respiratory capacity. In addition, the β-MEND (RES) showed no cellular toxicity for H9c2 cells as evidenced by Premix WST-1 assays. This is the first report of the use of a myocardial mitochondria-targeting liposome encapsulating RES for activating mitochondrial function, which was clearly confirmed based on analyses using a Seahorse XF Analyzer.
  • Satrialdi, Yuta Takano, Eri Hirata, Natsumi Ushijima, Hideyoshi Harashima, Yuma Yamada
    NANOSCALE ADVANCES 3 20 5919 - 5927 2021年10月 
    A photochemical reaction mediated by light-activated molecules (photosensitizers) in photodynamic therapy (PDT) causes molecular oxygen to be converted into highly reactive oxygen species (ROS) that are beneficial for cancer therapy. As the active oxygen consumer and the primary regulator of apoptosis, mitochondria are known as an important target for optimizing PDT outcomes. However, most of the clinically used photosensitizers exhibited a poor tumor accumulation profile as well as lack of mitochondria targeting ability. Therefore, by applying a nanocarrier platform, mitochondria-specific delivery of photosensitizers can be materialized. The present research develops an effective mitochondria-targeting liposome-based nanocarrier system (MITO-Porter) encapsulating a pi-extended porphyrin-type photosensitizer (rTPA), which results in a significant in vivo antitumor activity. A single PDT treatment of the rTPA-MITO-Porter resulted in a dramatic tumor inhibition against both human and murine tumors that had been xenografted in a mouse model. Furthermore, depolarization of the mitochondrial membrane was observed, implying the damage of the mitochondrial membrane due to the photochemical reaction that occurred specifically in the mitochondria of tumor cells. The findings presented herein serve to verify the significance of the mitochondria-targeted nanocarrier system for advancing the in vivo PDT effectivity in cancer therapy regardless of tumor type.
  • Takashi Nakamura, Taisei Nakade, Koharu Yamada, Yusuke Sato, Hideyoshi Harashima
    International journal of pharmaceutics 609 121140 - 121140 2021年09月27日 [査読有り]
     
    The use of natural killer (NK) cells in cell therapy is an attractive next generation strategy for cancer immunotherapy. NK-92 cells (a human NK cell line) have been tested in clinical trial stages, making them an off-the-shelf medicine. Controlling gene expression in NK-92 cells by an artificial delivery system is an available for enhancing NK-92 cell therapy. We report here on the development of a siRNA-loaded lipid nanoparticle (LNP) composed of CL1H6 (CL1H6-LNP), an optimized, pH-sensitive cationic lipid, with efficient gene silencing and low cytotoxicity in NK-92 cells. The hydrophilic head group of the lipid molecule used in preparing these particles largely influences the pKa of the final LNP, and lipids with an amino moiety substituted with a methyl group showed a high gene silencing activity. Compared with myristate and palmitate, the hydrophobic tail of oleate had a high gene silencing activity and cell viability. Analyses of intracellular trafficking indicated that the CL1H6-LNP appeared to escape from the endosomes via membrane fusion, without disrupting the membrane. The mechanism of endosomal escape should contribute to our understanding of efficient gene silencing with a low degree of cytotoxicity. These results therefore suggest that a CL1H6-LNP has promise for delivering siRNA to NK-92 cells.
  • Saed Abbasi, Haruki Higashino, Yusuke Sato, Keiko Minami, Makoto Kataoka, Shinji Yamashita, Hideyoshi Harashima
    Molecular pharmaceutics 18 9 3281 - 3289 2021年09月06日 [査読有り]
     
    Lipid-based formulations, such as self-microemulsifying drug-delivery systems (SMEDDSs), are promising tools for the oral delivery of poorly water-soluble drugs. However, failure to maintain adequate aqueous solubility after coming into contact with gastrointestinal fluids is a major drawback. In this study, we examined the use of a novel cinnamic acid-derived oil-like material (CAOM) that binds drugs with a high affinity through π-π stacking and hydrophobic interactions, as an oil core in a SMEDDS for the oral delivery of fenofibrate in rats. The use of the CAOM in the SMEDDS resulted in an unprecedented enhancement in fenofibrate bioavailability, which exceeded the bioavailability values obtained using SMEDDSs based on corn oil, a conventional triglyceride oil, or Labrasol, an enhancer of intestinal permeation. Further characterization revealed that the CAOM SMEDDS does not alter the intestinal permeability and has no inhibitory activity on P-glycoprotein-mediated drug efflux. The results reported herein demonstrate the strong potential of CAOM formulations as new solubilizers for the efficient and safe oral delivery of drugs that have limited water solubility.
  • Yuma Yamada, Takuya Ishimaru, Kohei Ikeda, Hideyoshi Harashima
    Journal of pharmaceutical sciences 111 2 432 - 439 2021年08月31日 [査読有り]
     
    Large amounts of ATP are produced in mitochondria especially in the brain and heart, where energy consumption is high compared with other organs. Thus, a decrease in ATP production in such organs could be a cause of many diseases such as neurodegenerative diseases and heart disease. Based on thus assumption, increasing intracellular ATP production in such organs could be a therapeutic strategy. In this study, we report on the delivery of vitamin B1, a coenzyme that activates the tricarboxylic acid (TCA) cycle, to the inside of mitochondria. Since the TCA cycle is responsible for ATP production, we hypothesized delivering vitamin B1 to mitochondria would enhance ATP production. To accomplish this, we used a mitochondrial targeted liposome a "MITO-Porter" as the carrier. Using SH-SY5Y cells, a model neuroblast, cellular uptake and intracellular localization were analyzed using flow cytometry and confocal laser scanning microscopy. The optimized MITO-Porter containing encapsulated vitamin B1 (MITO-Porter (VB1)) was efficiently accumulated in mitochondria of SH-SY5Y cells. Further studies confirmed that the level of ATP production after the MITO-Porter (VB1) treatment was significantly increased as compared to a control group that was treated with naked vitamin B1. This study provides the potential for an innovative therapeutic strategy in which the TCA cycle is activated, thus enhancing ATP production. Relative ATP ratio (%) = IS/IU × 100, where IS and IU represent the intracellular ATP amounts for the treated and untreated cells with samples, respectively.
  • イメージングと操作で見るミトコンドリア機能 ミトコンドリア機能を操作するDrug Delivery System
    山田 勇磨, 原島 秀吉
    バイオイメージング 30 2 38 - 38 日本バイオイメージング学会 2021年08月
  • Takashi Nakamura, Takanori Sato, Rikito Endo, Shun Sasaki, Naomichi Takahashi, Yusuke Sato, Mamoru Hyodo, Yoshihiro Hayakawa, Hideyoshi Harashima
    Journal for immunotherapy of cancer 9 7 2021年07月 [査読有り]
     
    BACKGROUND: Resistance to an immune checkpoint inhibitor (ICI) is a major obstacle in cancer immunotherapy. The causes of ICI resistance include major histocompatibility complex (MHC)/histocompatibility locus antigen (HLA) class I loss, neoantigen loss, and incomplete antigen presentation. Elimination by natural killer (NK) cells would be expected to be an effective strategy for the treatment of these ICI-resistant tumors. We previously demonstrated that a lipid nanoparticle containing a stimulator of an interferon gene (STING) agonist (STING-LNP) efficiently induced antitumor activity via the activation of NK cells. Thus, we evaluated the potential of reducing ICI resistance by STING-LNPs. METHODS: Lung metastasis of a B16-F10 mouse melanoma was used as an anti-programmed cell death 1 (anti-PD-1)-resistant mouse model. The mice were intravenously injected with the STING-LNP and the mechanism responsible for the improvement of anti-PD-1 resistance by the STING-LNPs was analyzed by RT-qPCR and flow cytometry. The dynamics of STING-LNP were also investigated. RESULTS: Although anti-PD-1 monotherapy failed to induce an antitumor effect, the combination of the STING-LNP and anti-PD-1 exerted a synergistic antitumor effect. Our results indicate that the STING-LNP treatment significantly increased the expression of CD3, CD4, NK1.1, PD-1 and interferon (IFN)-γ in lung metastases. This change appears to be initiated by the type I IFN produced by liver macrophages that contain the internalized STING-LNPs, leading to the systemic activation of NK cells that express PD-1. The activated NK cells appeared to produce IFN-γ, resulting in an increase in the expression of the PD ligand 1 (PD-L1) in cancer cells, thus leading to a synergistic antitumor effect when anti-PD-1 is administered. CONCLUSIONS: We provide a demonstration to show that a STING-LNP treatment can overcome PD-1 resistance in a B16-F10 lung metastasis model. The mechanism responsible for this indicates that NK cells are activated by stimulating the STING pathway which, in turn, induced the expression of PD-L1 on cancer cells. Based on the findings reported herein, the STING-LNP represents a promising candidate for use in combination therapy with anti-PD-1-resistant tumors.
  • 心筋虚血再灌流モデルラットに対するヒトミトコンドリア強化心筋幹細胞移植療法の治療効果の検討
    佐々木 大輔, 山田 勇磨, 後藤 悠太, 白石 真大, 武田 充人, 原島 秀吉
    日本DDS学会学術集会プログラム予稿集 37回 99 - 99 日本DDS学会 2021年06月
  • ミトコンドリア人工共生による細胞機能制御の試み
    山田 勇磨, 日比野 光恵, 伊藤 百, 荒井 愛永, 佐々木 大輔, 真栄城 正寿, 渡慶次 学, 太田 善浩, 原島 秀吉
    日本DDS学会学術集会プログラム予稿集 37回 111 - 111 日本DDS学会 2021年06月
  • Ian Baudi, Masanori Isogawa, Federica Moalli, Masaya Onishi, Keigo Kawashima, Yuji Ishida, Chise Tateno, Yusuke Sato, Hideyoshi Harashima, Hiroyasu Ito, Tetsuya Ishikawa, Takaji Wakita, Matteo Iannacone, Yasuhito Tanaka
    PLoS pathogens 17 5 e1009228  2021年05月 [査読有り]
     
    Virus infection, such as hepatitis B virus (HBV), occasionally causes endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) is counteractive machinery to ER stress, and the failure of UPR to cope with ER stress results in cell death. Mechanisms that regulate the balance between ER stress and UPR are poorly understood. Type 1 and type 2 interferons have been implicated in hepatic flares during chronic HBV infection. Here, we examined the interplay between ER stress, UPR, and IFNs using transgenic mice that express hepatitis B surface antigen (HBsAg) (HBs-Tg mice) and humanized-liver chimeric mice infected with HBV. IFNα causes severe and moderate liver injury in HBs-Tg mice and HBV infected chimeric mice, respectively. The degree of liver injury is directly correlated with HBsAg levels in the liver, and reduction of HBsAg in the transgenic mice alleviates IFNα mediated liver injury. Analyses of total gene expression and UPR biomarkers' protein expression in the liver revealed that UPR is induced in HBs-Tg mice and HBV infected chimeric mice, indicating that HBsAg accumulation causes ER stress. Notably, IFNα administration transiently suppressed UPR biomarkers before liver injury without affecting intrahepatic HBsAg levels. Furthermore, UPR upregulation by glucose-regulated protein 78 (GRP78) suppression or low dose tunicamycin alleviated IFNα mediated liver injury. These results suggest that IFNα induces ER stress-associated cell death by reducing UPR. IFNγ uses the same mechanism to exert cytotoxicity to HBsAg accumulating hepatocytes. Collectively, our data reveal a previously unknown mechanism of IFN-mediated cell death. This study also identifies UPR as a potential target for regulating ER stress-associated cell death.
  • Amisha Sanghani, Konstantinos N Kafetzis, Yusuke Sato, Salsabil Elboraie, Julia Fajardo-Sanchez, Hideyoshi Harashima, Aristides D Tagalakis, Cynthia Yu-Wai-Man
    Pharmaceutics 13 3 2021年03月13日 [査読有り]
     
    The master regulator of the fibrosis cascade is the myocardin-related transcription factor/serum response factor (MRTF/SRF) pathway, making it a key target for anti-fibrotic therapeutics. In the past, inhibitors and small interfering RNAs (siRNAs) targeting the MRTF-B gene have been deployed to counter fibrosis in the eye, with the latter showing promising results. However, the biggest challenge in implementing siRNA therapeutics is the method of delivery. In this study, we utilised the novel, pH-sensitive, cationic lipid CL4H6, which has previously demonstrated potent targeting of hepatocytes and endosomal escape, to safely and efficiently deliver an MRTF-B siRNA into human conjunctival fibroblasts. We prepared two lipid nanoparticle (LNP) formulations, incorporating targeting cleavable peptide cY in one of them, and measured their physicochemical properties and silencing effect in human conjunctival fibroblasts. Both proved to be non-cytotoxic at a concentration of 50 nM and effectively silenced the MRTF-B gene in vitro, with the targeting cleavable peptide not affecting the silencing efficiency [LNP with cY: 62.1% and 81.5% versus LNP without cY: 77.7% and 80.2%, at siRNA concentrations of 50 nM (p = 0.06) and 100 nM (p = 0.09), respectively]. On the other hand, the addition of the targeting cleavable peptide significantly increased the encapsulation efficiency of the LNPs from 92.5% to 99.3% (p = 0.0005). In a 3D fibroblast-populated collagen matrix model, both LNP formulations significantly decreased fibroblast contraction after a single transfection. We conclude that the novel PEGylated CL4H6-MRTF-B siRNA-loaded LNPs represent a promising therapeutic approach to prevent conjunctival fibrosis after glaucoma filtration surgery.
  • Mahmoud A Younis, Ikramy A Khalil, Yaser H A Elewa, Yasuhiro Kon, Hideyoshi Harashima
    Journal of controlled release : official journal of the Controlled Release Society 331 335 - 349 2021年03月10日 [査読有り]
     
    Hepatocellular carcinoma (HCC) is a fatal disease with limited therapeutic choices. The stroma-rich tumor microenvironment hinders the in vivo delivery of most nanomedicines. Ultra-small lipid nanoparticles (usLNPs) were designed for the selective co-delivery of the cytotoxic drug, sorafenib (SOR), and siRNA against the Midkine gene (MK-siRNA) to HCC in mice. The usLNPs composed of a novel pH-sensitive lipid, a diversity of phospholipids and a highly-selective targeting peptide. A microfluidic device, iLiNP, was used and a variety of factors were controlled to tune particle size aiming at maximizing tumor penetration efficiency. Optimizing the composition and physico-chemical properties of the usLNPs resulted in an enhanced tumor accumulation, selectivity and in vivo gene silencing. The optimized usLNPs exerted potent gene silencing in the tumor (median effective dose, ED50~0.1 mg/Kg) with limited effect on the healthy liver. The novel combination synergistically-eradicated HCC in mice (~85%) at a surprisingly-low dose of SOR (2.5 mg/Kg) which could not be achieved via individual monotherapy. Toxicity studies revealed the biosafety of the usLNPs upon either acute or chronic treatment. Furthermore, the SOR-resistant HCC established in mice was eradicated by 70% using this approach. We conclude that our strategy is promising for potential clinical applications in HCC treatment.
  • 福井 隆史, 角家 健, 舘野 寛直, 中村 孝司, 山田 勇磨, 佐藤 悠介, 岩崎 倫政, 原島 秀吉
    日本整形外科学会雑誌 95 2 S123 - S123 (公社)日本整形外科学会 2021年03月
  • ミトコンドリア強化ヒト心筋幹細胞(hMITO Cell)の製造および心虚血再灌流モデルを用いた細胞移植療法の検証
    後藤 悠太, 山田 勇磨, 日比野 光恵, 佐々木 大輔, 武田 充人, 真栄城 正寿, 渡慶次 学, 原島 秀吉
    日本薬学会年会要旨集 141年会 29V09 - pm09S (公社)日本薬学会 2021年03月
  • Yuta Hagino, Ikramy A Khalil, Seigo Kimura, Kenji Kusumoto, Hideyoshi Harashima
    Molecular pharmaceutics 18 3 878 - 888 2021年03月01日 [査読有り]
     
    This study describes the development of lipid nanoparticles (LNPs) for the efficient and selective delivery of plasmid DNA (pDNA) to the lungs. The GALA peptide was used as a ligand to target the lung endothelium and as an endosomal escape device. Transfection activity in the lungs was significantly improved when pDNA was encapsulated in double-coated LNPs. The inner coat was composed of dioleoylphsophoethanolamine and a stearylated octaarginine (STR-R8) peptide, while the outer coat was largely a cationic lipid, di-octadecenyl-trimethylammonium propane, mixed with YSK05, a pH-sensitive lipid, and cholesterol. Optimized amounts of YSK05 and GALA were used to achieve an efficient and lung-selective system. The optimized system produced a high gene expression level in the lungs (>107 RLU/mg protein) with high lung/liver and lung/spleen ratios. GALA/R8 modification and the double-coating design were indispensable for efficient gene expression in the lungs. Despite the fact that NPs prepared with 1-step or 2-step coating have the same lipid amount and composition and the same pDNA dose, the transfection activity was dramatically higher in the lungs in the case of 2-step coating. Surprisingly, 1-step or 2-step coatings had no effect on the amount of nanoparticles that were delivered to the lungs, suggesting that the double-coating strategy substantially improved the efficiency of gene expression at the intracellular level.
  • Yuichi Suzuki, Haruno Onuma, Risa Sato, Yusuke Sato, Akari Hashiba, Masatoshi Maeki, Manabu Tokeshi, Mohammad Enamul Hoque Kayesh, Michinori Kohara, Kyoko Tsukiyama-Kohara, Hideyoshi Harashima
    Journal of controlled release : official journal of the Controlled Release Society 330 61 - 71 2021年02月10日 [査読有り]
     
    The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system has considerable therapeutic potential for use in treating a wide range of intractable genetic and infectious diseases including hepatitis B virus (HBV) infections. While non-viral delivery technologies for the CRISPR/Cas system are expected to have clinical applications, difficulties associated with the clinically relevant synthesis of formulations and the poor efficiency of delivery severely hinder therapeutic genome editing. We report herein on the production of a lipid nanoparticle (LNP)-based CRISPR/Cas ribonucleoprotein (RNP) delivery nanoplatform synthesized using a clinically relevant mixer-equipped microfluidic device. DNA cleavage activity and the aggregation of Cas enzymes was completely avoided under the optimized synthetic conditions. The optimized formulation, which was identified through 2 steps of design of experiments, exhibited excellent gene disruption (up to 97%) and base substitution (up to 23%) without any apparent cytotoxicity. The addition of negative charges to the RNPs by complexing single-stranded oligonucleotide (ssON) significantly enhanced the delivery of both Cas9 and Cpf1 RNPs. The optimized formulation significantly suppressed both HBV DNA and covalently closed circular DNA (cccDNA) in HBV-infected human liver cells compared to adeno-associated virus type 2 (AAV2). These findings represent a significant contribution to the development of CRISPR/Cas RNP delivery technology and its practical applications in genome editing therapy.
  • Yusuke Sato, Takashi Nakamura, Yuma Yamada, Hideyoshi Harashima
    Journal of controlled release : official journal of the Controlled Release Society 330 305 - 316 2021年02月10日 [査読有り][招待有り]
     
    The era of Nanomedicine has arrived with the approval of ONPATTRO™ by the FDA in 2018. Lipid nanoparticle (LNP) technology has succeeded in delivering siRNA to the human liver in genetic diseases and has also been applied to mRNA vaccinations for COVID-19 using a similar LNP technology. In this review, we focus on the current status of new lipids for use in LNP formulations including our original lipids (CL4H6/CL4C6/CL4D6) as well as mechanisms of targeting without a ligand. Clinical applications of nano DDS are moving forward rapidly in the field of cancer immunology since the successful introduction of OPDIVO™ in 2014. Antigen presentation and the maturation of immune cells can be controlled by nano DDS for cancer immunotherapy. YSK12-C4, a newly designed ionizable amino lipid can induce successful immune activation by silencing mRNA in DC and NK cells, which are expected to be evaluated for clinical use. Finally, new cancer therapy by targeting mitochondria involving the use of a MITO-Porter, a membrane fusion-type mitochondrial delivery system, has been introduced. The importance of delivering a photo sensitizer to mitochondria was clearly demonstrated in photodynamic cancer therapy. Clinical applications of MITO-Porters started in collaborative efforts with LUCA Science Co., Ltd. And was established in 2018. The future direction of Nanomedicine is discussed.
  • Seigo Kimura, Ikramy A Khalil, Yaser H A Elewa, Hideyoshi Harashima
    Journal of controlled release : official journal of the Controlled Release Society 330 753 - 764 2021年02月10日 [査読有り]
     
    This study reports on the development of a novel lipid combination that permits the efficient and highly selective delivery of plasmid DNA (pDNA) to immune cells in the spleen. Using DODAP, an ionizable lipid that was previously thought to be inefficient for gene delivery, we show for the first time, that this ignored lipid can be successfully used for efficient and targeted gene delivery in vivo, but only when combined with DOPE, a specific helper lipid. Using certain DODAP and DOPE ratios resulted in the formation of lipid nanoparticles (LNPs) with a ~ 1000-fold higher gene expression, and this expression was specific for the spleen, making it the most spleen-selective system for transfection using pDNA. The developed DODAP/DOPE-LNPs target immune cells in the spleen via receptors for complement C3 and this pathway is critical for efficient gene expression. We hypothesize that the high spleen transfection activity of DODAP/DOPE-LNPs is caused by the promotion of gene expression associated with B cell activation via complement receptors. LNPs encapsulating tumor-antigen encoding pDNA showed both prophylactic and therapeutic anti-tumor effects. The optimized LNPs resulted in the production of different cytokines and antigen-specific antibodies as well as exerting antigen-specific cytotoxic effects. This study revives the use of DODAP in gene delivery and highlights the importance of using appropriate lipid combinations for delivering genes to specific cells.
  • Ryu Hiradate, Ikramy A Khalil, Aya Matsuda, Mika Sasaki, Kyoko Hida, Hideyoshi Harashima
    Journal of controlled release : official journal of the Controlled Release Society 329 665 - 675 2021年01月10日 [査読有り]
     
    Adipose tissue in the body is classified as white adipose tissue (WAT); a fat-accumulating tissue, or brown adipose tissue (BAT); an energy-dissipating tissue. Transforming WAT-to-BAT (browning) is a promising strategy for the treatment of obesity, since it would lead to an increase in energy expenditure. Rosiglitazone (Rosi), an agonist of the peroxisome proliferator-activated receptor γ (PPARγ), is known to be a potent browning inducer in subcutaneous WAT. However, the effectiveness of Rosi has been quite limited because of several off-target effects. The objective of this study was to develop locally administered Rosi-loaded nanoparticles (Rs-NPs) with the ability to target adipocytes to achieve the adipose tissue-specific activation of PPARγ, thus causing the browning of WAT. We prepared dual targeted Rs-NPs that were modified with a specific peptide that targets prohibitin that are expressed in adipocytes, and a cell penetrating peptide for enhancing cellular uptake and controlling intracellular trafficking. The Rs-NPs modified with a single ligand were internalized into mature adipocytes and induced browning activity in vitro but they failed to significantly affect the body weight of the diet-induced obese mice model. The dual-targeted Rs-NPs induced a strong browning activity, both in vitro and in vivo, and successfully inhibited the progression of obesity, as evidenced by the shrinkage of hypertrophied adipocytes without any detectable systemic adverse effects. Meanwhile, free Rosi aggravated hepatic steatosis and did not cause adipose tissue browning nor the inhibition of body weight gain. We conclude that the increased energy expenditure via adipose tissue browning using dual-targeted Rs-NP is a promising strategy for the treatment of obesity and its related metabolic syndrome.
  • Mahmoud M. Abd Elwakil, Tianle Gao, Takuya Isono, Yusuke Sato, Yaser H. A. Elewa, Toshifumi Satoh, Hideyoshi Harashima
    Materials Horizons 2021年 

    Engineered lipomers coming from sustainable sources can efficiently bypass the liver to deliver a genetic message to the lungs after systemic administration without targeting ligands.

  • Niko Kimura, Masatoshi Maeki, Kosuke Sasaki, Yusuke Sato, Akihiko Ishida, Hirofumi Tani, Hideyoshi Harashima, Manabu Tokeshi
    RSC ADVANCES 11 3 1430 - 1439 2021年01月 
    Sub 100 nm-sized lipid nanoparticles (LNPs) have been widely used in drug delivery systems (DDSs). The size of the LNPs is an important parameter for the DDS performance, such as biodistribution and gene silencing using siRNAs. However, the LNPs prepared by the conventional preparation method show a wide size distribution. To improve the LNP size distribution, we developed a microfluidic device, named the iLiNP (TM) device, in a previous study. This device could produce LNPs in the size range of 20 to 150 nm, but the size distribution of the large-sized LNPs needs to be further improved. From the viewpoint of the LNP formation process, a homogeneous and slow rate dilution of ethanol plays an important role in improving the large-size LNP size distribution. In this study, we developed a three-dimensional, symmetrically assembled microfluidic device named the 3D-iLiNP device with the aim of precise size control of large-sized LNPs. We designed the 3D-iLiNP device using a computational fluid dynamics simulation and demonstrated that the 3D-iLiNP device can improve the LNP size distribution. The gene silencing activity of four kinds of siRNA-loaded LNPs was investigated via in vitro and in vivo experiments to elucidate the effect of the LNP size distribution. The results revealed that the LNPs with a size between 90 and 120 nm showed higher gene silencing activity than those with other sizes. The 3D-iLiNP device is expected to improve DDS performance by precisely controlling the size of LNPs.
  • Yuma Yamada, Hideyoshi Harashima
    Methods in molecular biology (Clifton, N.J.) 2275 227 - 245 2021年 [査読有り]
     
    Genetic mutations and defects in mitochondrial DNA (mtDNA) are associated with certain types of mitochondrial dysfunctions, ultimately resulting in the emergence of a variety of human diseases. To achieve an effective mitochondrial gene therapy, it will be necessary to deliver therapeutic agents to the innermost mitochondrial space (the mitochondrial matrix), which contains the mtDNA pool. We recently developed a MITO-Porter, a liposome-based nanocarrier that delivers cargo to mitochondria via a membrane-fusion mechanism. In this chapter, we discuss the methodology used to deliver bioactive molecules to the mitochondrial matrix using a Dual Function (DF)-MITO-Porter, a liposome-based nanocarrier that delivers it cargo by means of a stepwise process, and an evaluation of mtDNA levels and mitochondrial activities in living cells. We also discuss mitochondrial gene silencing by the mitochondrial delivery of antisense RNA oligonucleotide (ASO) targeting mtDNA-encoded mRNA using the MITO-Porter system.
  • Seigo Kimura, Hideyoshi Harashima
    Pharmaceutics 12 12 2020年12月15日 [査読有り][招待有り]
     
    The era of the aging society has arrived, and this is accompanied by an increase in the absolute numbers of patients with neurological disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD). Such neurological disorders are serious costly diseases that have a significant impact on society, both globally and socially. Gene therapy has great promise for the treatment of neurological disorders, but only a few gene therapy drugs are currently available. Delivery to the brain is the biggest hurdle in developing new drugs for the central nervous system (CNS) diseases and this is especially true in the case of gene delivery. Nanotechnologies such as viral and non-viral vectors allow efficient brain-targeted gene delivery systems to be created. The purpose of this review is to provide a comprehensive review of the current status of the development of successful drug delivery to the CNS for the treatment of CNS-related disorders especially by gene therapy. We mainly address three aspects of this situation: (1) blood-brain barrier (BBB) functions; (2) adeno-associated viral (AAV) vectors, currently the most advanced gene delivery vector; (3) non-viral brain targeting by non-invasive methods.
  • Akari Hashiba, Manaya Toyooka, Yusuke Sato, Masatoshi Maeki, Manabu Tokeshi, Hideyoshi Harashima
    Journal of controlled release : official journal of the Controlled Release Society 327 467 - 476 2020年11月10日 [査読有り]
     
    Although great advances have been made in the delivery of short RNAs by lipid nanoparticles (LNPs), the optimal formulation composition and physicochemical properties of LNPs for long RNA (including mRNA) remain unclear. In the present study, we optimized the lipid composition of liver-targeted mRNA-loaded LNPs that were prepared with pH-sensitive cationic lipids that had been previously designed for siRNA delivery through a two stepped design of experiment (DoE). Multiple responses including physicochemical properties, gene expression, and liver-specificity were analyzed in order, not only to understand the role of each formulation parameter, but also to examine parameters that would be difficult to predict. We found that particle size and the PEG-to-phospholipid (PEG/PL) ratio were additional key factors for liver-specific gene expression in addition to the other formulation factors. The optimized formulation showed a better gene expression compared to other lipid formulations from industry leaders. These findings suggest that a "DoE with multiple responses" approach can be used to predict significant parameters and permit optimized formulations to be prepared more efficiently.
  • Yuma Yamada, Yusuke Sato, Takashi Nakamura, Hideyoshi Harashima
    Journal of controlled release : official journal of the Controlled Release Society 327 533 - 545 2020年11月10日 [査読有り][招待有り]
     
    Due to the rapid changes that have occurred in the field of drug discovery and the recent developments in the early 21st century, the role of drug delivery systems (DDS) has become increasingly more important. For the past 20 years, our laboratory has been developing gene delivery systems based on lipid-based delivery systems. One of our efforts has been directed toward developing a multifunctional envelope-type nano device (MEND) by modifying the particle surface with octaarginine, which resulted in a remarkably enhanced cellular uptake and improved intracellular trafficking of plasmid DNA (pDNA). When we moved to in vivo applications, however, we were faced with the PEG-dilemma and we shifted our strategy to the incorporation of ionizable cationic lipids into our system. This resulted in some dramatic improvements over our original design and this can be attributed to the development of a new lipid library. We have also developed a mitochondrial targeting system based on a membrane fusion mechanism using a MITO-Porter, which can deliver nucleic acids/pDNA into the matrix of mitochondria. After the appearance of antibody medicines, Opdivo, an immune checkpoint inhibitor, has established cancer immunology as the 4th strategy in cancer therapy. Our DDS technologies can also be applied to this new field of cancer therapy to cure cancer by controlling our immune mechanisms. The latest studies are summarized in this review article.
  • ミトコンドリア病(Leigh脳症)モデルマウスの心機能の評価とモデルマウス由来心筋前駆細胞のミトコンドリア機能の検討
    佐々木 大輔, 武田 充人, 山田 勇磨, 原島 秀吉
    日本小児循環器学会雑誌 36 Suppl.2 s2 - 358 (NPO)日本小児循環器学会 2020年11月
  • Yuma Yamada, Minako Maruyama, Tomoko Kita, Shin-Ichi Usami, Shin-Ichiro Kitajiri, Hideyoshi Harashima
    Mitochondrion 55 134 - 144 2020年11月 [査読有り]
     
    We report on validating a mitochondrial gene therapeutic strategy using fibroblasts derived from patients with an A1555G point mutation in mitochondrial DNA coding 12S ribosomal RNA (rRNA (12S)). Wild-type rRNA (12S) as a therapeutic RNA was encapsulated in a mitochondrial targeting liposome, a MITO-Porter (rRNA-MITO-Porter), and an attempt was made to deliver the MITO-Porter to mitochondria of the diseased cells. It was confirmed that the rRNA-MITO-Porter treatment significantly decreased the ratio of the mutant rRNA content. Moreover, it was shown that the mitochondrial respiratory activities of the diseased cells were improved as the result of the mitochondrial transfection of the rRNA-MITO-Porter.
  • Saed Abbasi, Yusuke Sato, Kazuaki Kajimoto, Hideyoshi Harashima
    Molecular pharmaceutics 17 10 3773 - 3782 2020年10月05日 [査読有り]
     
    The intravenous administration of drug-loaded nanoparticles (NPs) is needed to achieve passive or active targeting in disease tissues. However, when the loaded drug is a hydrophobic small molecule, the NPs fail to reach adequate plasma drug concentrations mainly because of premature drug release. The pharmacokinetics of such drugs can be controlled by covalent modification, but this approach could compromise the safety or potency of the drug. In this study, we investigated two formulation parameters that could be used to improve the plasma concentrations of unmodified drugs that are loaded in a nanoemulsion (NE), a core-shell type NP. The first parameter is the loading ratio, and the second is the affinity of the drug to the core. Optimized NEs with reduced drug loading and with a high drug-core affinity resulted in a 12.4- and 11.2-fold increase in the plasma retention of curcumin and paclitaxel, respectively. Our strategy for enhancing the drug-core interaction affinity relied on mixing oils and surfactants to achieve cooperativity in noncovalent interactions, such as hydrophobic interactions, hydrogen bonding, and π-π stacking, which was further confirmed by theoretical calculations of interaction affinities. Finally, we report on the development of a cinnamic acid-derived oil-like material as a novel drug vehicle with exceptional solubilizing ability that could be used in intravenous formulations of NEs.
  • リポソームsiRNAデリバリーシステムを用いた腫瘍血管biglycanを標的とした新規血管新生阻害療法
    間石 奈湖, 櫻井 遊, 畠山 浩人, Li Cong, Alam Mohammad T., 菊地 央, 森本 浩史, 森本 真弘, 樋田 泰浩, 原島 秀吉, 樋田 京子
    日本癌学会総会記事 79回 OE16 - 5 2020年10月
  • 核酸医薬とナノメディシン 多機能性エンベロープ型ナノ構造体の開発とナノ医療への展開
    原島 秀吉, 佐藤 悠介, 中村 孝司, 山田 勇磨
    日本癌学会総会記事 79回 ML13 - ML13 2020年10月
  • Nour Shobaki, Yusuke Sato, Yuichi Suzuki, Nana Okabe, Hideyoshi Harashima
    Journal of controlled release : official journal of the Controlled Release Society 325 235 - 248 2020年09月10日 [査読有り]
     
    The tumor-microenvironment contains large numbers of tumor-associated macrophages (TAMs) which are largely M2 phenotypes and are involved in pro-tumorous functions. Targeting TAMs so as to manipulate them and to modify their functions could be a novel immunotherapy for the treatment of cancer. Such a strategy would involve targeting TAMs with short interfering RNA (siRNA) to modify their functions by silencing certain genes that are responsible for their M2 polarization. In this study, a lipid nanoparticle (LNP) formulation was used to target and deliver siRNA to TAMs. The LNP was mainly composed of a novel, pH-sensitive cationic lipid, referred to as the CL4H6 lipid, which had previously been optimized to target hepatocytes. The optimized siRNA-loaded CL4H6-LNPs were selectively and efficiently taken up and showed strong gene silencing activity in TAMs in a human tumor xenograft model in nude mice. Furthermore, an anti-tumor therapeutic response in the same tumor model was obtained by targeting TAMs using the optimized siRNA-loaded CL4H6-LNPs. The anti-tumor therapeutic response was obtained through the silencing of the signal transducer and activator of transcription 3 (STAT3) and hypoxia inducible factor 1 α (HIF-1α), which resulted in an increase in the level of infiltrated macrophage (CD11b+ cells) into the tumor-microenvironment (TME) as well as a tendency to increase the concentration of M1 macrophages (CD169+ cells). The treatment also resulted in reversing the pro-tumorous functions of TAMs -mainly angiogenesis and tumor cell activation-, as evidenced by a decrease in the related gene expression at the mRNA level. This research has promising clinical and pharmaceutical applications for achieving novel macrophage-based cancer immunotherapy.
  • Yuma Yamada, Momo Ito, Manae Arai, Mitsue Hibino, Takao Tsujioka, Hideyoshi Harashima
    International journal of molecular sciences 21 17 2020年09月02日 [査読有り]
     
    Mitochondrial transplantation therapy is an innovative strategy for the treatment of mitochondrial dysfunction. The approach has been reported to be useful in the treatment of cardiac ischemic reperfusion injuries in human clinical trials and has also been shown to be useful in animal studies as a method for treating mitochondrial dysfunction in various tissues, including the heart, liver, lungs, and brain. On the other hand, there is no methodology for using preserved mitochondria. Research into the pharmaceutical formulation of mitochondria to promote mitochondrial transplantation therapy as the next step in treating many patients is urgently needed. In this review, we overview previous studies on the therapeutic effects of mitochondrial transplantation. We also discuss studies related to immune responses that occur during mitochondrial transplantation and methods for preserving mitochondria, which are key to their stability as medicines. Finally, we describe research related to mitochondrial targeting drug delivery systems (DDS) and discuss future perspectives of mitochondrial transplantation.
  • ミトコンドリアDDSを用いたビタミンB1の脳内送達・治療効果の検証
    山田 勇磨, 石丸 拓也, 原島 秀吉
    日本DDS学会学術集会プログラム予稿集 36回 169 - 169 日本DDS学会 2020年08月
  • Yuma Yamada, Reina Munechika, Satrialdi, Fumika Kubota, Yusuke Sato, Yu Sakurai, Hideyoshi Harashima
    Journal of pharmaceutical sciences 109 8 2493 - 2500 2020年08月 [査読有り]
     
    Mitochondrial delivery of an anticancer drug targeting cancer cells would eventually result in cell death. To achieve this, a drug delivery system targeting mitochondria is needed. We recently developed a MITO-Porter, a liposome that delivers its cargo to mitochondria. We reported that such a MITO-Porter could deliver doxorubicin (DOX), an anticancer drug, to mitochondria in OS-RC-2 cells, a drug resistant cancer cell, resulting in inhibiting the cell growth, based in in vitro experiments. Herein, we report on validating the benefit of such a therapeutic strategy for treating drug resistant cancers by the in vivo targeting of mitochondria. We prepared a DOX-MITO-Porter, in which DOX was encapsulated in the MITO-Porter and optimized its retention in blood circulation. When the DOX-MITO-Porter was administered to mice bearing OS-RC-2 cells via tail vein injection, tumor size was significantly decreased, compared to DOX itself and to the DOX-encapsulated polyethylene glycol-modified liposome (DOX-PEG-LP). Intracellular observation confirmed that the DOX-MITO-Porter had accumulated in tumor mitochondria. It was also found a relationship between anti-tumor effect and the mitochondrial function, as indicated by the depolarization of mitochondrial membrane potential. This study provides support for the utility of an in vivo mitochondrial delivery system in drug resistant cancer therapies.
  • Niko Kimura, Masatoshi Maeki, Yusuke Sato, Akihiko Ishida, Hirofumi Tani, Hideyoshi Harashima, Manabu Tokeshi
    ACS applied materials & interfaces 12 30 34011 - 34020 2020年07月29日 [査読有り]
     
    Microfluidic methodologies for preparation of lipid nanoparticles (LNPs) based on an organic solvent injection method enable precise size control of the LNPs. After preparation of LNPs, the organic solvent injection method needs some post-treatments, such as overnight dialysis or direct dilution with a buffer solution. LNP production using the microfluidic-based organic solvent injection method is dominated by kinetics rather than thermodynamics. Kinetics of ethanol removal from the inner and outer membranes of LNPs could induce a structural change in the membrane that could lead to fusion of LNPs. However, the effects of microfluidic post-treatment on the final size of LNPs have not been sufficiently understood. Herein, we investigated the effect of the post-treatment processes on the final product size of LNPs in detail. A simple baffle device and a model lipid system composed of a neutral phospholipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC) and cholesterol were used to produce the LNPs. We demonstrated that flow conditions of the post-treatment diluting the remaining ethanol in the LNP suspension affected the final product size of LNPs. Based on the findings, we developed an integrated baffle device composed of an LNP production region and a post-treatment region for a microfluidic-based LNP production system; this integrated baffle device prevented the undesirable aggregation or fusion of POPC LNPs even for the high-lipid-concentration condition. Finally, we applied our concept to small interfering RNA (siRNA) delivery and confirmed that no significant effects due to the continuous process occurred on the siRNA encapsulation efficiency, biological distribution, and knockdown activity. The microfluidic post-treatment method is expected to contribute to the production of LNPs for practical applications and the development of novel LNP-based nanomedicines.
  • Takashi Nakamura, Koharu Yamada, Yusuke Sato, Hideyoshi Harashima
    International journal of pharmaceutics 587 119652 - 119652 2020年07月17日 [査読有り][通常論文]
     
    Delivering nucleic acid using a non-viral vector is a potent strategy for gene modification and controlling gene expression in immune cell therapy. Since the low-temperature storage (0-4 °C) or cryopreservation of cells are indispensable for performing immune cell therapy, we investigated the interactions between an siRNA-loaded lipid nanoparticle (LNP), a multifunctional envelope-type nanodevice (MEND) containing YSK12-C4 (YSK12-MEND), and human immune cell lines (NK-92 and Jurkat) at low-temperature and its effect on transfection activity. The YSK12-MEND readily bound to the cell membrane of NK-92 cells at low-temperature, but no internalization of the YSK12-MEND by cells was observed, even after returning the temperature to 37 °C. Gene silencing activity was completely impaired. The cause of this inhibition appears to be membrane fusion between the YSK12-MEND and cell membrane at the low-temperature. Collectively, our results suggest that the exposure of siRNA-loaded LNPs to cells at low-temperature should be avoided in defining transfection protocols in immune cell therapy.
  • Takashi Nakamura, Hideyoshi Harashima
    Advanced drug delivery reviews 167 78 - 88 2020年06月06日 [査読有り][招待有り]
     
    It is generally known that the lymph nodes (LNs) are important tissues in cancer immunotherapy. Therefore, delivering immune functional compounds to LNs is a useful strategy for enhancing cancer immunotherapy. Lipid-based nanocarriers have been widely used as delivery systems that target LNs, but lipid nanoparticle (LNP) technology has recently attracted increased interest. High levels of nucleic acids can be efficiently loaded in LNPs, they can be used to actively deliver nucleic acids into the cytoplasm, and they can be produced on an industrial scale. The use of microfluidic devices has been particularly valuable for producing small-sized LNPs, thus paving the way for successful LN targeting. In the review, we focus on the potential of LNP technology for targeting LNs.
  • Eriko Kawamura, Minako Maruyama, Jiro Abe, Akira Sudo, Atsuhito Takeda, Shingo Takada, Takashi Yokota, Shintaro Kinugawa, Hideyoshi Harashima, Yuma Yamada
    Molecular therapy. Nucleic acids 20 687 - 698 2020年06月05日 [査読有り][通常論文]
     
    Here, we report on validating a mitochondrial gene therapy by delivering nucleic acids to mitochondria of diseased cells by a MITO-Porter, a liposome-based carrier for mitochondrial delivery. We used cells derived from a patient with a mitochondrial disease with a G625A heteroplasmic mutation in the tRNAPhe of the mitochondrial DNA (mtDNA). It has been reported that some mitochondrial gene diseases are caused by heteroplasmic mutations, in which both mutated and wild-type (WT) genes are present, and the accumulation of pathological mutations leads to serious, intractable, multi-organ diseases. Therefore, the decrease of the mutated gene rate is considered to be a useful gene therapy strategy. To accomplish this, wild-type mitochondrial pre-tRNAPhe (pre-WT-tRNAPhe), prepared by in vitro transcription, was encapsulated in the MITO-Porter. The pre-WT-tRNAPhe encapsulated in the MITO-Porter was transfected into diseased mitochondrial cells, and the resulting mutant levels were examined by an amplification refractory mutation system (ARMS)-quantitative PCR. The mutation rate of tRNAPhe was decreased, and this therapeutic effect was sustained even on the 8th day after transfection. Furthermore, mitochondrial respiratory activity of the disease cells was increased after the transfection of therapeutic pre-WT-tRNAPhe. These results support the conclusion that the mitochondrial delivery of therapeutic nucleic acids represents a viable strategy for mitochondrial gene therapy.
  • Hideyuki Masuda, Takashi Nakamura, Hideyoshi Harashima
    Journal of pharmaceutical sciences 109 6 1943 - 1950 2020年06月 [査読有り][通常論文]
     
    Interest has developed in the bacillus Calmette-Guerin (BCG) cell wall skeleton (BCG-CWS) as a noninfectious adjuvant. Although BCG-CWS readily undergoes aggregation, in a previous study, we applied it to cancer immunotherapy via intravenous administration by encapsulating the BCG-CWS into nanoparticles (CWS-NPs). The CWS-NPs were taken up by major histocompatibility complex (MHC) class II+ (MHC-II+) cells and induced antigen-specific cytotoxic T lymphocyte (CTL) activity. However, the nature of the contribution of MHC-II+ cells to the CTL response continues to be unclear. In this study, we investigated the relationship between the distribution of CWS-NPs in the spleen and CTL activity. The main MHC-II+ cells that internalized the CWS-NPs were B cells. Decreasing the level of polyethylene glycol modification increased the uptake of CWS-NPs by B cells, leading to an increased CTL activity. A comparison of CWS-NPs with different uptake efficiencies into dendritic cells and B cells suggested that the DCs with internalized CWS-NPs may contribute to CTL activation compared with B cells. We succeeded in enhancing CTL activity by the CWS-NPs, and the findings reported herein should provide important information regarding target cells for the development of CWS-NP.
  • Yuma Yamada, Kana Somiya, Akihiko Miyauchi, Hitoshi Osaka, Hideyoshi Harashima
    Scientific reports 10 1 7511 - 7511 2020年05月05日 
    We report on the validation of a mitochondrial gene therapeutic strategy using fibroblasts from a Leigh syndrome patient by the mitochondrial delivery of therapeutic mRNA. The treatment involves delivering normal ND3 protein-encoding mRNA as a therapeutic RNA to mitochondria of the fibroblasts from a patient with a T10158C mutation in the mtDNA coding the ND3 protein, a component of the mitochondrial respiratory chain complex I. The treatment involved the use of a liposome-based carrier (a MITO-Porter) for delivering therapeutic RNA to mitochondria via membrane fusion. The results confirmed that the mitochondrial transfection of therapeutic RNA by the MITO-Porter system resulted in a decrease in the levels of mutant RNA in mitochondria of diseased cells based on reverse transcription quantitative PCR. An evaluation of mitochondrial respiratory activity by respirometry also showed that transfection using the MITO-Porter resulted in an increase in maximal mitochondrial respiratory activity in the diseased cells.
  • 脂質ナノ粒子の特性がリンパ節送達とリンパ節内分布へ与える影響
    中村 孝司, 河合 美典, 佐藤 悠介, 真栄城 正寿, 渡慶次 学, 原島 秀吉
    日本薬剤学会年会講演要旨集 35年会 171 - 171 (公社)日本薬剤学会 2020年05月
  • MELAS A3243G変異型ミトコンドリアDNAを標的とした遺伝子治療戦略の検証
    山田 勇磨, 宗宮 加奈, 佐々木 大輔, 武田 充人, 原島 秀吉
    日本薬剤学会年会講演要旨集 35年会 169 - 169 (公社)日本薬剤学会 2020年05月
  • Yu Sakurai, Akari Kato, Hideyoshi Harashima
    Biochemical and biophysical research communications 525 2 313 - 318 2020年04月30日 
    For achieving efficient cancer treatment, it is important to elucidate the mechanism responsible for the accumulation of nanoparticles in tumor tissue. Recent studies suggest that nanoparticles are not delivered merely through gaps between tumor endothelial cells. We previously reported that the maturation of the vascular structure by the vascular endothelial cell growth factor receptor 2 (VEGFR2) using a previously developed siRNA delivery technology (RGD-MEND) significantly enhanced the accumulation of nanoparticles in types of cancers that area vessel-rich (renal cell carcinoma). This result was completely inconsistent with the generally accepted theory of the enhanced permeability and retention (EPR) effect. We hypothesized that a caveolin-1 (Cav1)-mediated transcellular route would be involved with the penetration of nanoparticles into tumor vasculature. To reveal the exact mechanism responsible for this enhancement, we observed the delivery of long-circulating liposomes (LPs) after Cav1 was co-suppressed by RGD-MEND with VEGFR2. The enhanced delivery of LPs by siRNA against VEGFR2 (siVEGFR2) was accompanied by the elevated expression of the Cav1 protein. In addition, Cav1 knockdown by siRNA against Cav1 (siCav1) canceled the enhanced delivery of LPs by siVEGFR2. The injection of siCav1 had no effect on the formation of alpha smooth muscle actin or vascular endothelial cell adhesion molecules. These results suggest that a Cav1-induced transcellular route and not a paracellular route, at least partially, contributes to the accumulation of nanoparticles in tumors.
  • Yu Sakurai, Wataru Mizumura, Kenichiro Ito, Kazuhiro Iwasaki, Takayuki Katoh, Yuki Goto, Hiroaki Suga, Hideyoshi Harashima
    Molecular pharmaceutics 17 4 1397 - 1404 2020年04月06日 [査読有り][通常論文]
     
    Peptide modification is a popular strategy for developing an active targeting lipid nanoparticle (LNP). In modifying the surface of an LNP with a peptide, the sequence and structure of the peptide strongly affects the formation of the LNP. Specifically, a peptide with a high hydrophobicity can induce coarsening and aggregation of the LNP. In an attempt to prevent this from occurring, we incorporated monoacyl and diacyl group-conjugated poly(ethylene glycol) (PEG) into a LNP. We previously developed an original LNP, a multifunctional envelope type nanodevice (MEND) modified with an Epi-1 peptide, a ligand with a high affinity for the epithelial cell adhesion molecule (EpCAM). Using this peptide-modified MEND, the efficiency of delivery of a small interfering RNA (siRNA) encapsulated in the MEND was significantly improved. Although increasing the ratio of modification enhanced cellular uptake, the increase also induced aggregation of the LNP, particularly in the case of a large scale preparation. Our results indicate that a monoacyl PEG-lipid can prevent aggregation, even when the LNP is modified with higher molar ratios of peptide, but that this also results in a decrease in delivery efficiency. Moreover, the Epi-1-modified MEND exhibited a strong silencing effect in an ovarian cancer peritoneal dissemination model. Our results suggest that the simple incorporation of a monoacyl derivative into the PEG-lipid resulted in the formation of a peptide-modified LNP with improved characteristics.
  • Mio Maeta, Naoya Miura, Hiroki Tanaka, Takashi Nakamura, Ryo Kawanishi, Yoshifumi Nishikawa, Kenichi Asano, Masato Tanaka, Shinya Tamagawa, Yuta Nakai, Kota Tange, Hiroki Yoshioka, Hideyoshi Harashima, Hidetaka Akita
    Molecular pharmaceutics 17 4 1237 - 1247 2020年04月06日 [査読有り][通常論文]
     
    DNA vaccinations are promising strategies for treating diseases that require cellular immunity (i.e., cancer and protozoan infection). Here, we report on the use of a liposomal nanocarrier (lipid nanoparticles (LNPs)) composed of an SS-cleavable and pH-activated lipidlike material (ssPalm) as an in vivo DNA vaccine. After subcutaneous administration, the LNPs containing an ssPalmE, an ssPalm with vitamin E scaffolds, elicited a higher gene expression activity in comparison with the other LNPs composed of the ssPalms with different hydrophobic scaffolds. Immunization with the ssPalmE-LNPs encapsulating plasmid DNA that encodes ovalbumin (OVA, a model tumor antigen) or profilin (TgPF, a potent antigen of Toxoplasma gondii) induced substantial antitumor or antiprotozoan effects, respectively. Flow cytometry analysis of the cells that had taken up the LNPs in draining lymph nodes (dLNs) showed that the ssPalmE-LNPs were largely taken up by macrophages and a small number of dendritic cells. We found that the transient deletion of CD169+ macrophages, a subpopulation of macrophages that play a key role in cancer immunity, unexpectedly enhanced the activity of the DNA vaccine. These data suggest that the ssPalmE-LNPs are effective DNA vaccine carriers, and a strategy for avoiding their being trapped by CD169+ macrophages will be a promising approach for developing next-generation DNA vaccines.
  • Takashi Nakamura, Minori Kawai, Yusuke Sato, Masatoshi Maeki, Manabu Tokeshi, Hideyoshi Harashima
    Molecular pharmaceutics 17 3 944 - 953 2020年03月02日 [査読有り][通常論文]
     
    Because the lymph node (LN) is a critical organ for inducing immune responses against pathogens and cancers, the transport of immune functional molecules such as antigens and adjuvants to LNs by delivery systems is a useful strategy for the effective outcome of an immune response. The size and charge of a delivery system largely affect the transitivity to and distribution within LN. Although pH-sensitive lipid nanoparticles (LNPs) prepared by microfluidic mixing are the latest delivery system to be applied clinically, the effects of their size and charge on the transitivity to and distribution within LN are currently unknown. We investigated the size and charge effect of LNPs prepared by microfluidic mixing on transitivity to and distribution within LNs. A 30 nm-sized LNP (30-LNP) was efficiently translocated to LNs and was taken up by CD8+ dendritic cells, while the efficiency was drastically decreased in the cases of 100 and 200 nm-sized LNPs. Furthermore, a comparative study between neutral, positively, and negatively charged 30-LNP revealed that the negative 30-LNP moved to the LN more efficiently than the other LNPs. Interestingly, the negative 30-LNP reached the deep cortex, namely, the T cell zone. Our findings provide informative insights for designing LN-targeting LNPs prepared by microfluidic mixing and for the translocation of nanoparticles in LNs.
  • 光増感分子rTPAを搭載したミトコンドリア標的型ナノカプセルの構築および担癌モデルマウスを用いた癌光治療戦略の検証
    山田 勇磨, Satrialdi, 高野 勇太, Biju Vasudevanpillai, 原島 秀吉
    日本薬学会年会要旨集 140年会 26X - am11 (公社)日本薬学会 2020年03月
  • Yuma Yamada, Yutaka Fukuda, Daisuke Sasaki, Minako Maruyama, Hideyoshi Harashima
    Mitochondrion 52 67 - 74 2020年02月22日 [査読有り][通常論文]
     
    The delivery of nucleic acids targeting mutant mtDNA represent a potential strategy for addressing a variety of mitochondria-related diseases. We previously developed a MITO-Porter, a nano carrier that is capable of delivering nanoparticles of nucleic acids to mitochondria of human cells. Here, we report on an investigation of a series of nanoparticles formed with various poly cationic peptides that can release nucleic acids in response to a mitochondrial environment. A significant relationship was found between the number of and the location of arginine and histidine residues in the peptide sequence and the release of nucleic acids in a mitochondrial environment.
  • Satrialdi, Reina Munechika, Vasudevanpillai Biju, Yuta Takano, Hideyoshi Harashima, Yuma Yamada
    Chemical communications (Cambridge, England) 56 7 1145 - 1148 2020年01月23日 [査読有り][通常論文]
     
    The uncontrolled production of reactive oxygen species during photodynamic therapy (PDT) induces oxidative stress. The full potential of PDT is accomplished by delivery of a pi-extended porphyrin-type photosensitizer into mitochondria of tumor cells using a MITO-Porter, a mitochondrial targeting nanodevice. This strategy can be implemented for innovative cancer therapy.
  • Yuma Yamada, Yuta Takano, Satrialdi, Jiro Abe, Mitsue Hibino, Hideyoshi Harashima
    Biomolecules 10 1 2020年01月05日 [査読有り][通常論文]
     
    There have been many reports on the relationship between mitochondrial oxidative stress and various types of diseases. This review covers mitochondrial targeting photodynamic therapy and photothermal therapy as a therapeutic strategy for inducing mitochondrial oxidative stress. We also discuss other mitochondrial targeting phototherapeutic methods. In addition, we discuss anti-oxidant therapy by a mitochondrial drug delivery system (DDS) as a therapeutic strategy for suppressing oxidative stress. We also describe cell therapy for reducing oxidative stress in mitochondria. Finally, we discuss the possibilities and problems associated with clinical applications of mitochondrial DDS to regulate mitochondrial oxidative stress.
  • Niko Kimura, Masatoshi Maeki, Yusuke Note, Yusuke Sato, Akihiko Ishida, Hirofumi Tani, Hideyoshi Harashima, Manabu Tokeshi
    21st International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2017 965 - 966 2020年 
    This paper reports a lipid nanoparticles (LNP) production method and its formation behavior using microfluidic devices with baffle structures. The microfluidic devices showed great mixing efficiency at 500 μL/min, and we achieved 20 nm-sized LNPs production that chaotic micromixers were not able to produce at the same flow rate condition. Additionally, we found that the smaller-sized LNPs/siRNA prepared by baffle structures have higher penetration efficiency than that of the larger-sized LNPs, but all of them showed the gene silencing activity. The microfluidic devices with baffle structures are expected to be a practicable apparatus for DDSs application.
  • Yuma Yamada, Satrialdi, Mitsue Hibino, Daisuke Sasaki, Jiro Abe, Hideyoshi Harashima
    Advanced drug delivery reviews 154-155 187 - 209 2020年 [査読有り][招待有り]
     
    Mitochondria carry out various essential functions including ATP production, the regulation of apoptosis and possess their own genome (mtDNA). Delivering target molecules to this organelle, it would make it possible to control the functions of cells and living organisms and would allow us to develop a better understanding of life. Given the fact that mitochondrial dysfunction has been implicated in a variety of human disorders, delivering therapeutic molecules to mitochondria for the treatment of these diseases is an important issue. To date, several mitochondrial drug delivery system (DDS) developments have been reported, but a generalized DDS leading to therapy that exclusively targets mitochondria has not been established. This review focuses on mitochondria-targeted therapeutic strategies including antioxidant therapy, cancer therapy, mitochondrial gene therapy and cell transplantation therapy based on mitochondrial DDS. A particular focus is on nanocarriers for mitochondrial delivery with the goal of achieving mitochondria-targeting therapy. We hope that this review will stimulate the accelerated development of mitochondrial DDS.
  • Hideyoshi Harashima, Tatsuhiro Ishida
    Advanced drug delivery reviews 154-155 1 - 1 2020年 [査読無し][招待有り]
  • Yusuke Sato, Nana Okabe, Yusuke Note, Kazuki Hashiba, Masatoshi Maeki, Manabu Tokeshi, Hideyoshi Harashima
    Acta Biomaterialia 102 341 - 350 Elsevier {BV} 2020年01月 [査読有り][通常論文]
     
    Despite the fact that small-sized lipid nanoparticles (LNPs) are important for improved tissue penetration and efficient drug delivery, their poor stability and intracellular trafficking significantly hinders their use as potent small-sized LNPs. It has been reported that both the diffusion of lipid components from LNPs and the adsorption of proteins on the surface of LNPs are responsible for their decreased potency. To overcome this issue, we focused on the chemical structure of hydrophobic scaffolds of pH-sensitive cationic lipids with various lengths and shapes. LNPs composed of a pH-sensitive cationic lipid with long, linear scaffolds induced gene silencing in a dose-dependent manner, while LNPs with a classical scaffold length (C18) failed. Replacing the helper lipid from cholesterol to egg sphingomyelin (ESM) resulted in the formation of smaller LNPs with a diameter of similar to 22 nm and enhanced gene silencing activity. Most of the ESMs were located in the outer layer and functioned to stabilize the LNPs. Long, linear scaffolds contributed to immiscibility with phosphocholine-containing lipids including ESM. This contribution was dependent on the scaffold length of pH-sensitive cationic lipids. Although phosphocholine-containing lipids usually inhibit membrane fusion-mediated endosomal escape, long, linear scaffolds contributed to avoiding the inhibitory effect and to enhance the potency of the LNPs. These findings provide useful information needed for the rational design of pH-sensitive cationic lipid structures and the selection of appropriate helper lipids and will facilitate the development of highly potent small-sized LNPs.Statement of significanceDespite the fact that small-sized lipid nanoparticles (LNPs) are important for improved tissue penetration and efficient drug delivery, the size reduction-associated decrease in the stability and intracellular trafficking significantly hinders the development of potent small-sized LNPs. Our limited understanding of the mechanism underlying the reduced potency has also hindered the development of more potent small-sized LNPs. The findings of the present study indicate that long and linear hydrophobic scaffolds of pH-sensitive cationic lipids could overcome the loss of efficiency for nucleic acid delivery. In addition, the long hydrophobic scaffolds led to immiscibility with neutral phospholipids, resulting in efficient endosomal escape. These findings provide useful information needed for the rational design of pH-sensitive cationic lipid structures and will facilitate the development of highly potent small-sized LNPs. (C) 2019 Acta Materialia Inc. Published by Elsevier Ltd.
  • Sakurai Y, Akita H, Harashima H
    International journal of molecular sciences 20 23 2019年11月 [査読有り][通常論文]
  • Nakamura T, Yamada Y, Sato Y, Khalil IA, Harashima H
    Biomaterials 218 119329 - 119329 2019年10月 [査読有り][招待有り]
     
    Nanomedicine promises to play an important role in next generation therapy, including Nucleic acid/Gene therapy. To accomplish this, innovative nanotechnologies will be needed to support nanomedicine by controlling not only the biodistribution but also the intracellular trafficking of macromolecules such as RNA/DNA. A multifunctional envelope-type nano device (MEND) was developed to meet this requirement. We herein provide an update regarding the functions of the MEND system focusing on the introduction of different functional biomaterials that enhance efficiency. The octaarginine (R8) peptide enhances cellular uptake and controls intracellular trafficking to induce synergism in transgene expression. The R8 was also used for developing a MITO-Porter system for mitochondrial targeting. The function of the MITO-Porter system was extended by developing a mitochondrial reporter gene for mitochondrial gene therapy. For efficient in vivo gene delivery, new pH-sensitive lipids have been introduced to achieve controlled biodistribution and to enhance endosomal escape. For example, the CL4H6 lipid exerts a more efficient in vivo gene silencing than that of ONPATTROTM, a preparation that has been approved by the US Food and Drug Administration. We further summarize new technologies that have been successfully applied to cancer immunotherapy leading to the introduction of a new strategy based on the concept of the Cancer-Immunity Cycle.
  • Yamada Y, Fujishita N, Harashima H
    Nucleosides, nucleotides & nucleic acids 1 - 15 2019年10月 [査読有り][通常論文]
  • Yu Sakurai, Akari Kato, Yasuhiro Hida, Junichi Hamada, Nako Maishi, Kyoko Hida, Hideyoshi Harashima
    Journal of pharmaceutical sciences 108 10 3218 - 3224 2019年10月 [査読有り][通常論文]
     
    Malignant pleural mesothelioma (MPM) is a highly aggressive form of cancer, with a median survival of less than 1 year. It is well known that the hyaluronan (HA) receptor CD44 is highly expressed by MPM cells and is reported to be correlated with a poor prognosis. We herein report on the development of a new type if drug delivery system against CD44 that involves the use of lipid nanoparticles (LNPs) equipped with a new type of HA derivative. In this study, we evaluated HA-lipid conjugation (HAL) via the end of the HA molecule through reductive amination, a process that allowed the carboxylate group to remain intact. As a result, the HAL-modified LNP appears to be a potent nanoparticle for dealing with MPM. Surprisingly, the use of a combination of a cationic lipid and HAL had a synergistic effect on cellular uptake in MPM and consequently permitted an anti-cancer drug such as cis-diamminedichloro-platinum(II) (CDDP). Intrapleural injection of CDDP-loaded HAL-LNP (1.5 mg/kg as CDDP) per week significantly suppressed the progression of this type of cancer in an MPM orthotopic model. These results suggest that HAL-modified LNP represents a potent delivery system for MPM cells that express high levels of CD44.
  • Yu Sakurai, Hideyoshi Harashima
    Expert opinion on drug delivery 16 9 915 - 936 2019年09月 
    Introduction: Hyaluronan (HA), a natural polysaccharide, is produced in large amounts by the human body. Since its receptor CD44 is highly expressed in many types of cancers, HA is a promising ligand for cancer-targeting nanoparticles (NPs). Since the enhanced permeability and retention (EPR) effect-based strategy faces difficulties in terms of efficiency in clinical studies, studies focusing on HA-modified NPs that can actively target cancer cells should be prominent for further progress in NP-based cancer treatment. Areas covered: Various materials combined with HA composed of lipid nanoparticles and polymers as well as iron, gold and other metals have been examined. In addition, their cargos have been quite diverse and include small molecule drugs, imaging agents, proteins and nucleic acids. We summarize recent studies on varieties of NPs and describe the properties of HA as a ligand of CD44. Expert opinion: In spite of a number of studies on HA-based NPs, there is few examples of HA-based NPs in clinical. In order to make a progress in clinical study, an evaluation methodology of HA-NPs should be unified and normalized.
  • Endo R, Nakamura T, Kawakami K, Sato Y, Harashima H
    Scientific reports 9 1 11335 - 11335 2019年08月 [査読有り][通常論文]
     
    Cell-based therapy using dendritic cells (DC) represents a potent cancer immunotherapy. However, activated DC express indoleamine 2,3-dioxygenase 1 (IDO1), a counter-regulatory and tolerogenic molecule, leading to the inhibition of T cell activation and the promotion of T cell differentiation into regulatory T cells. Silencing the IDO1 gene in DC by small interfering RNA (siRNA) represents a potent therapeutic strategy. We report on the successful and efficient introduction of a siRNA targeting IDO1 into mouse DCs by a means of a multifunctional envelope-type nanodevice (MEND) containing a YSK12-C4 (YSK12-MEND). The YSK12-C4 has both fusogenic and cationic properties. The YSK12-MEND induced an effective level of gene silencing of IDO1 at siRNA doses in the range of 1-20 nM, a concentration that commercially available transfection reagents are not able to silence. The YSK12-MEND mediated IDO1 silencing had no effect on the characteristic determinants of DC phenotype such as CD11c, CD80 and MHC class II. The silencing of IDO1 in DC by the YSK12-MEND significantly enhanced the antitumor effect against E.G7-OVA tumor. Moreover, a decrease in the numbers of regulatory T cells in the tumor was observed in mice that were treated with the IDO1-silenced DC. The YSK12-MEND appears to be a potent delivery system for IDO1-silenced DC based cancer immunotherapy.
  • Kawamura E, Hibino M, Harashima H, Yamada Y
    Mitochondrion 49 178 - 188 2019年08月 [査読有り][通常論文]
  • Santiwarangkool S, Akita H, Khalil IA, Abd Elwakil MM, Sato Y, Kusumoto K, Harashima H
    Journal of controlled release : official journal of the Controlled Release Society 307 55 - 63 2019年08月 [査読有り][通常論文]
     
    The GALA peptide (WEAALAEALAEALAEHLAEALAEALEALAA) was originally designed to induce the destabilization of endosomal membranes based on its ability to undergo a pH-dependent conformational change from a random coil to an α-helix. We recently found that liposomes modified with GALA peptide (GALA-LPs) extensively accumulate in lung endothelial cells (ECs) after intravenous injection. However, the uptake mechanism of GALA-LPs and their ability to reach alveolar epithelium was unclear. We report herein that GALA-LPs are internalized into ECs via a clathrin-mediated pathway. Surprisingly, GALA-LPs had the ability to pass lung ECs and reach other cells through transcytosis. GALA-LPs were taken up by >70% of lung ECs, while they also accumulated in ~30% of type I alveolar epithelium (ATI). GALA-modified gold nanoparticles were detected in ECs, in the basement membrane and in other cells such as ATI, type II alveolar epithelium (ATII) and alveolar macrophages. Consistent with this result, a significant gene knockdown was achieved in lung epithelium cells using GALA-LPs encapsulating anti-podoplanin siRNA. This indicates that GALA-LPs can be used as a carrier for delivering macromolecules to parenchymal as well as to endothelial cells in the lung. Although caveolae are commonly linked to the transendothelial transport of proteins and antibodies, our data indicate that clathrin-mediated endocytosis might also participate in the transcytosis process.
  • Hibino Mitsue, Yamada Yuma, Fujishita Naoki, Sato Yusuke, Maeki Masatoshi, Tokeshi Manabu, Harashima Hideyoshi
    JOURNAL OF PHARMACEUTICAL SCIENCES 108 8 2668 - 2676 2019年08月 [査読有り][通常論文]
     
    © 2019 A number of drugs that are currently on the market, as well as new candidates for drugs, are poorly water soluble. Because of this, a need exists to develop drug formulations that will permit the expanded use of such drugs. The use of liposomes and lipid nanoparticles for drug delivery has attracted attention as a technique for solubilizing molecules that are poorly water soluble, but this technique faces serious scale-up risks. In this study, we report on attempts to encapsulate Coenzyme Q10 (CoQ10) as a model of a poorly water-soluble drug in an MITO-Porter, a liposome for mitochondrial delivery using a microfluidic device (a CoQ10-MITO-Porter [μ]). The physical properties of the CoQ10-MITO-Porter [μ] including homogeneity, size, and preparation volume were compared with those for a CoQ10-MITO-Porter prepared by the ethanol dilution method (a CoQ10-MITO-Porter [ED]). In the case where a microfluidic device was used, a small-sized CoQ10-MITO-Porter was formed homogeneously, and it was possible to prepare it on a large scale. Intracellular observations using HeLa cells showed that the CoQ10-MITO-Porter [μ] was efficiently internalized by cells to reach mitochondria. These results indicate that the CoQ10-MITO-Porter [μ] represents a potential candidate for use in mitochondrial nanomedicine.
  • Katayama T, Kinugawa S, Takada S, Furihata T, Fukushima A, Yokota T, Anzai T, Hibino M, Harashima H, Yamada Y
    Mitochondrion 49 66 - 72 2019年07月 [査読有り][通常論文]
  • Yuma Yamada, Shinnosuke Daikuhara, Atsushi Tamura, Kei Nishida, Nobuhiko Yui, Hideyoshi Harashima
    Chemical Communications 55 50 7203 - 7206 2019年06月 [査読有り][通常論文]
     
    Failure of autophagy induction results in the accumulation of abnormal mitochondria to cause neurodegenerative diseases. Artificial autophagy activation via the mitochondrial delivery of polyrotaxane with autophagy induced activity is achieved using a MITO-Porter, a nanodevice for mitochondrial delivery. This strategy can be applied to innovative research and therapy.
  • MITO-Porterを用いた治療用mRNA送達によるミトコンドリア遺伝子治療戦略の検証
    山田 勇磨, 宗宮 加奈, 原島 秀吉
    日本薬剤学会年会講演要旨集 34年会 179 - 179 (公社)日本薬剤学会 2019年05月
  • Sato Yusuke, Hashiba Kazuki, Sasaki Kosuke, Maeki Masatoshi, Tokeshi Manabu, Harashima Hideyoshi
    JOURNAL OF CONTROLLED RELEASE 295 140 - 152 2019年02月 [査読有り][通常論文]
     
    Lipid nanoparticles (LNPs) are one of the more promising technologies for efficiently delivering short interfering RNA (siRNA) in vivo. A pH-sensitive cationic lipid that facilitates the targeting of hepatocytes and endosomal escape, strongly influences the availability of siRNA, thus making it a key material for efficient siRNA delivery. A systematic knowledge regarding lipid structure-activity relationships would greatly facilitate the development of sophisticated pH-sensitive cationic lipids for use in siRNA-based therapeutics. The systemic derivatization of a hydrophilic head group and hydrophobic tails of YSK12-C4, a pH-sensitive cationic lipid that was developed in our laboratory, revealed that hydrophilic head significantly affected the apparent pKa of the final product, a key factor in both intrahepatic distribution and endosomal escape. The clogP value of a hydrophilic head group was found to be associated with the apparent pKa of the product. In contrast, the hydrophobic tail structure strongly affected intrahepatic distribution without depending on apparent pKa. A structure-activity relationship study enabled the selection of an adequate combination of a hydrophilic head group and hydrophobic tails and permitted a potent LNP composed of a pH-sensitive cationic lipid CL4H6 (CL4H6-LNPs) to be developed that showed efficient gene silencing activity (50% effective dose: 0.0025 mg/kg), biodegradability and was tolerated. In vivo experiments revealed that the CL4H6-LNPs showed a superior efficiency for endosomal escape, cytosolic release and the RNA-induced silencing for the complex-loading of siRNAs compared to the previously developed LNPs.
  • Niko Kimura, Masatoshi Maeki, Yusuke Sato, Kosuke Sasaki, Akihiko Ishida, Hirofumi Tani, Hideyoshi Harashima, Manabu Tokeshi
    23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2019 368 - 369 2019年 
    This paper describes development of a three-dimensional (3D) baffle mixer device for precise size control of various types of lipid nanoparticles (LNPs) with high encapsulation efficiency of short interfering RNAs (siRNAs). The 3D baffle mixer device achieved more precise size control of various LNPs than that of the conventional micromixer device. In addition, the 3D baffle mixer enabled effective capturing of siRNAs into LNPs without any assistance of electrostatic interaction between lipid molecules and siRNAs. The 3D baffle mixer device is expected to become one of the key platforms for production of novel lipid-based nucleic acid nanocarriers.
  • Two modes of toxicity of lipid nanoparticles containing a pH-sensitive cationic lipid on human A375 and A375-SM melanoma cell lines,
    樋田 京子
    BPB Reports 2 48 - 55 2019年 [査読有り][通常論文]
  • Abbasi S, Kajimoto K, Harashima H
    International journal of pharmaceutics 553 1-2 398 - 407 2018年12月 [査読有り][通常論文]
  • Sakurai Y, Hada T, Kato A, Hagino Y, Mizumura W, Harashima H
    Molecular therapy oncolytics 11 102 - 108 2018年12月 [査読有り][通常論文]
     
    Although metastatic cancer is a major cause of death for cancer patients, no efficacious treatment for metastasis is available. We previously showed that the growth of a primary tumor could be inhibited by the administration of an anti-angiogenic small interfering RNA (siRNA) that is encapsulated in an RGD peptide-modified lipid nanoparticle (RGD-LNP). We herein report on the delivery of siRNA by an RGD-LNP to the vasculature is also effective for treating metastatic tumors. We compared the RGD-LNP with the polyethylene glycol (PEG)ylated LNP (PEG-LNP) in terms of accumulation in a lung-metastasized model. Despite malformed structure of vasculature in the metastasized lung, the accumulation of the PEG-LNP in the metastasized lung was lower than that for the RGD-LNP, which suggests that the delivery strategy based on vascular permeability is not completely effective for targeting metastasis tumors. The systemic injection of the RGD-LNP induced a significant silencing in the metastasized vasculature, but not in the normal lung. In addition, the continuous injection of the RGD-LNP encapsulating siRNA against a delta-like ligand 4 (DLL4) drastically prolonged the overall survival of metastasized model mice. Accordingly, our current findings suggest that vasculature targeting would be more effective than enhanced permeability and retention effect-based therapy for the treatment of metastatic cancer.
  • Tanaka H, Watanabe A, Konishi M, Nakai Y, Yoshioka H, Ohkawara T, Takeda H, Harashima H, Akita H
    Heliyon 4 12 e00959  2018年12月 [査読有り][通常論文]
     
    An mRNA gene therapy represents a potentially promising therapeutic for curing inflammatory diseases. The transient nature of the gene expression of mRNA would be expected to be beneficial for avoiding undesired side effects. Since the mRNA is a vulnerable molecule, a development of a carrier that can deliver the mRNA to the cytoplasm has a high priority. We report herein on the development of a system for delivering mRNA to the inflammatory lesion in a dextran sulfate sodium (DSS)-induced colitis model. We modulated molecular structures of an ionizable lipid, an SS-cleavable and pH-activated lipid-like material (ssPalm). Among the fatty acids investigated, oleic acid scaffolds (ssPalmO) appeared to be more biocompatible than either myristic acid or linoleic acid scaffolds with the colitis model. The structural modification of the hydrophilic head groups from linear tertiary amines to piperazine rings (ssPalmO-Paz4-C2) resulted in a more than 10-fold higher increasing in the transgene activity in inflammatory colon. The most notable observation is that the transgene activity in the inflammatory colon is significantly higher than that in liver, the major clearance organ of lipid nanoparticles. Collectively, the ssPalmO-Paz4-C2 represents a promising material for the delivery of an mRNA to inflammatory lesions.
  • Kawai M, Nakamura T, Miura N, Maeta M, Tanaka H, Ueda K, Higashi K, Moribe K, Tange K, Nakai Y, Yoshioka H, Harashima H, Akita H
    Nanomedicine : nanotechnology, biology, and medicine 14 8 2587 - 2597 2018年11月 [査読有り][通常論文]
     
    Cytoplasmic DNA triggers cellular immunity via activating the stimulator of interferon genes pathway. Since DNA is degradable and membrane impermeable, delivery system would permit cytoplasmic delivery by destabilizing the endosomal membrane for the use as an adjuvant. Herein, we report on the development of a plasmid DNA (pDNA)-encapsulating lipid nanoparticle (LNP). The structural components include an SS-cleavable and pH-activated lipid-like material that mounts vitamin E as a hydrophobic scaffold, and dual sensing motifs that are responsive to the intracellular environment (ssPalmE). The pDNA-encapsulating LNP (ssPalmE-LNP) induced a high interferon-β production in Raw 264.7 cells. The subcutaneous injection of ssPalmE-LNP strongly enhanced antigen-specific cytotoxic T cell activity. The ssPalmE-LNP treatment efficiently induced antitumor effects against E.G7-OVA tumor and B16-F10 melanoma metastasis. Furthermore, when combined with an anti-programmed death 1 antibody, an extensive therapeutic antitumor effect was observed. Therefore, the ssPalmE-LNP is a promising carrier of adjuvants for cancer immunotherapy.
  • Khalil IA, Yamada Y, Harashima H
    Expert opinion on drug delivery 15 11 1053 - 1065 2018年11月 [査読有り][招待有り]
  • Masuda H, Nakamura T, Noma Y, Harashima H
    Molecular pharmaceutics 15 12 5762 - 5771 2018年11月 [査読有り][通常論文]
     
    The intravesical instillation of live Bacillus Calmette-Guerin (BCG) for treating bladder cancer is a powerful cancer immunotherapy. The BCG cell wall skeleton (BCG-CWS) is the main component of the adjuvant, leading to the induction of antitumor immunity. However, the use of live BCG and BCG-CWS is currently limited to local administration because of the infectiousness of live BCG and the insolubility of BCG-CWS. We previously developed a water-dispersible nanoparticle (NP) formulation of BCG-CWS (CWS-NP), which could be used to apply BCG components for use as a systemically injected adjuvant for the treatment of cancers other than bladder cancer. In the present study, we examined the possible use of CWS-NP for cancer immunotherapy, when intravenously administered. The CWS-NP was a highly uniform dispersion and showed no aggregation in serum. The intravenously injected CWS-NP accumulated in the spleen and was efficiently taken up by dendritic cells, leading to their maturation. The coadministration of CWS-NP and ovalbumin (OVA) loaded NP resulted in the generation of OVA-specific cytotoxic T cells and inhibited the growth of E.G7-OVA tumors. These results represent the first findings related to the use of systemically injected CWS-NP as an adjuvant for cancer immunotherapy.
  • Yusuke Sato, Hideki Matsui, Risa Sato, Hideyoshi Harashima
    Journal of Controlled Release 284 179 - 187 2018年08月28日 [査読有り][通常論文]
     
    Lipid nanoparticles (LNPs) are one of the leading technologies for the in vivo delivery of short interfering RNA (siRNA). Cationic lipids are an important component for efficient endosomal escape via membrane fusion followed by release of siRNAs in cytosol where the site of action is located. A high cationic lipid/siRNA charge ratio is usually necessary for maximizing the gene silencing activity of the siRNA-loaded LNPs. However, high levels of cationic lipids are known to cause cytotoxicity through interactions with negatively charged biocomponents. A strategy for solving this dilemma is important, in terms of producing clinically applicable LNPs with a wide therapeutic window. We herein report on the development of LNPs with a high gene silencing activity and a low cationic lipid/siRNA charge ratio, which we refer to as low lipid core-nanoparticles (LLC-NPs). The negative charges of the siRNAs were neutralized by protamines, cationic proteins, to reduce the net dose of cationic lipid, YSK05, which was developed in our laboratory, for endosomal escape, resulting in preserved fusogenic activity and gene silencing activity, both in vitro and in vivo factor VII mouse model. In addition, the LLC-NPs showed an improved hepatotoxicity compared to conventional LNPs, which have a relatively higher cationic lipid/siRNA charge ratio. The concept of the LLC-NPs helps to realize clinically applicable LNPs with a wide therapeutic window and has the potential for use in various applications and for the delivery of different classes of nucleic acid.
  • Abe J, Yamada Y, Harashima H
    Journal of thoracic disease 10 Suppl 18 S2119 - S2121 2018年07月 [査読有り][通常論文]
  • Ryohei Togashi, Hiroki Tanaka, Sakiko Nakamura, Hideo Yokota, Kota Tange, Yuta Nakai, Hiroki Yoshioka, Hideyoshi Harashima, Hidetaka Akita
    Journal of Controlled Release 279 262 - 270 2018年06月10日 [査読有り][通常論文]
     
    Non-viral vectors are considered to be an attractive approach for gene delivery, since an artificial material is less immunogenic and oncogenic compared to a viral vector. We previously reported on the hepatic delivery of plasmid DNA (pDNA) by using lipid-like material (an SS-cleavable and pH-activated lipid-like material: ssPalm) which mounts two hydrophobic scaffolds, proton-accepting motifs (tertiary amines), and a cleavable unit (disulfide bonding). In the present study, we report on an advanced hepatic gene delivery system that uses a new type of ssPalm derivative: ssPalmE-Paz4-C2. The hepatic transgene expression of the intravenously administrated lipid nanoparticle (LNP) that was formed with the ssPalmE-Paz4-C2 (LNPssPalmE-Paz4-C2) was significantly higher than that of conventional LNPs formed with a myristic acid-scaffold ssPalm (LNPssPalmM). However, the LNPssPalmE-Paz4-C2 particle induced a severe innate immune response that involved the production of the pro-inflammatory cytokines (IL-6 and TNFα), intracellular DNA sensor-related cytokine (IL-1β) and interferon (IFNβ), even when a pDNA free from CpG-motifs was encapsulated. The production of the pro-inflammatory cytokines and the DNA sensor-related cytokines is attributed to the combination of vitamin E scaffolds and encapsulated pDNA. The depletion of macrophages by chlodronate-encapsulating liposomes dramatically reduced inflammatory gene expression. Based on the above findings, we conclude that the use of a certain type of non-viral carrier that shows a robust gene expression activity is attended by a risk of eliciting an innate immune response. When a highly hydrophobic derivative of dexamethasone, an anti-inflammatory glucocorticoid compound, was co-loaded to the particle, this inflammatory response was relieved, and gene expression efficiency was enhanced. It is thus concluded that the co-delivery of dexamethasone and pDNA is a promising approach for reducing these risks.
  • Takashi Nakamura, Koharu Yamada, Yuki Fujiwara, Yusuke Sato, Hideyoshi Harashima
    Molecular Pharmaceutics 15 6 2142 - 2150 2018年06月04日 [査読有り][通常論文]
     
    Introducing siRNA into human immune cells by an artificial delivery system continues to be a challenging issue. We previously developed a multifunctional envelope-type nanodevice (MEND) containing the YSK12-C4, a fusogenic cationic lipid, (YSK12-MEND) and succeeded in the efficient delivery of siRNA into human immune cell lines. Significant cytotoxicity, however, was observed at siRNA doses needed for gene silencing in NK-92 cells. NK-92 cells, a unique natural killer (NK) cell line, would be applicable for use in clinical NK therapy. Thus, reducing the cytotoxicity of the YSK12-MEND in NK-92 cells would strengthen the efficacy of NK-92 cell-based therapy. The amount of the YSK12-C4 in the MEND needed to be reduced to reduce the cytotoxicity, because the cytotoxicity was directly associated with the YSK12-C4. In the present study, we decreased the total amount of lipid, including the YSK12-C4, by introducing a core formed by electrostatic interactions of siRNA with a polycation (protamine) (siRNA core), which led to a decrease in cytotoxicity in NK-92 cells. We prepared a YSK12-MEND containing an siRNA core (YSK12-MEND/core) at charge ratios (CR: YSK12-C4/siRNA) of 10, 5, 3, and 2.5 and compared the YSK12-MEND/core with that for a YSK12-MEND (CR16.9). Cell viability was increased by more than 2 times at a CR5 or less. On the other hand, the YSK12-MEND/core (CR5) maintained the same gene silencing efficiency (60%) as the YSK12-MEND. Interestingly, the cellular uptake efficiency and hemolytic activity of the YSK12-MEND/core (CR5) was reduced compared to that for the YSK12-MEND. In calculating the silencing activity per cellular uptake efficiency and hemolytic activity, the value for the YSK12-MEND/core (CR5) was more than 2 times as high as that of the YSK12-MEND. The fact indicates that after endosomal escape, the process can be enhanced by using a YSK12-MEND/core (CR5). Thus, introducing an siRNA core into lipid nanoparticles can be a potent strategy for decreasing cytotoxicity without an appreciable loss of gene silencing activity in NK-92 cells.
  • Niko Kimura, Masatoshi Maeki, Yusuke Sato, Yusuke Note, Akihiko Ishida, Hirofumi Tani, Hideyoshi Harashima, Manabu Tokeshi
    ACS Omega 3 5 5044 - 5051 2018年05月09日 [査読有り][通常論文]
     
    The precise size control of the lipid nanoparticle (LNP)-based nanodrug delivery system (DDS) carriers, such as 10 nm size tuning of LNPs, is one major challenge for the development of next-generation nanomedicines. Size-controlled LNPs would realize size-selective tumor targeting and deliver DNA and RNA to target tumor tissues effectively by passing through the stromal cells. Herein, we developed a baffle mixer device named the invasive lipid nanoparticle production device, or iLiNP device for short, which has a simple two-dimensional microchannel and mixer structure, and we achieved the first reported LNP size tuning at 10 nm intervals in the size range from 20 to 100 nm. In comparison with the conventional LNP preparation methods and reported micromixer devices, our iLiNP device showed better LNP size controllability, robustness of device design, and LNP productivity. Furthermore, we prepared 80 nm sized LNPs with encapsulated small interfering RNA (siRNA) using the iLiNP device these LNPs effectively performed as nano-DDS carriers in an in vivo experiment. We expect iLiNP devices will become novel apparatuses for LNP production in nano-DDS applications.
  • Hiroki Tanaka, Taichi Nakatani, Tomomi Furihata, Kota Tange, Yuta Nakai, Hiroki Yoshioka, Hideyoshi Harashima, Hidetaka Akita
    Molecular pharmaceutics 15 5 2060 - 2067 2018年05月07日 [査読有り][通常論文]
     
    Gene therapy is a promising strategy for curing certain types of brain diseases. Supplementation of therapeutic proteins such as aromatic amino acid decarboxylase (AADC) or nerve growth factor (NGF) have been reported to be successful examples of such treatments. However, there are safety concerns because these systems are based on virus-based gene vectors. A safe and efficient artificial carrier is thus urgently needed as an alternative. In this study, an mRNA based artificial gene carrier was introduced into the mouse brain via intracerebroventricular administration. As a carrier, a lipid nanoparticle (LNP) composed of environmentally sensitive lipid-like materials called an SS-cleavable proton-activated lipid-like material is used. The apolipoprotein E mediated cellular uptake of the lipid nanoparticles is one of the key features for its superior and homogeneous transfection activity compared to commercially available transfection reagents in both in vitro and in vivo situations. Immunostaining of brain specimens suggested that exogenous proteins can be introduced into neuronal cells as well as astrocytes using the mRNA-based gene carrier. This cannot be achieved using DNA-based artificial gene carriers. The findings suggest that a combination of an mRNA and a lipid based delivery system have great promise as a platform for the treatment of brain disorders.
  • 微小化脂質ナノ粒子によるアジュバントのリンパ節送達
    中村 孝司, 河合 美典, 佐藤 悠介, 真栄城 正寿, 渡慶次 学, 原島 秀吉
    日本薬剤学会年会講演要旨集 33年会 193 - 193 (公社)日本薬剤学会 2018年05月
  • Ikramy A. Khalil, Seigo Kimura, Yusuke Sato, Hideyoshi Harashima
    Journal of Controlled Release 275 107 - 116 2018年04月10日 [査読有り][通常論文]
     
    We report on the development of a highly efficient gene delivery system based on synergism between octaarginine (R8), a representative cell penetrating peptide, and YSK05, a recently developed pH-sensitive cationic lipid. Attaching a high density of R8 on the surface of YSK05 nanoparticles (NPs) that contained encapsulated plasmid DNA resulted in the formation of positively charged NPs with improved transfection efficiency. To avoid the development of a net positive charge, we controlled the density and topology of the R8 peptide through the use of a two-step coating methodology, in which the inner lipid coat was modified with a low density of R8 which was then covered with an outer neutral YSK05 lipid layer. Although used in low amounts, the R8 peptide improved cellular uptake and endosomal escape of the DNA encapsulated in YSK05 NPs, which resulted in a high transfection efficiency. The two-step coating design was essential for achieving a high degree of transfection, as evidenced by the low activity of NPs modified with the same amount of R8 in a regular single-coated design. In addition, a high transfection efficiency was not observed when R8 or YSK05 were used alone, which confirms the existence of a synergistic effect between both components. The results of this study indicate that cationic cell penetrating peptides have the ability to improve transfection activities without imparting a net positive charge when used in the proper amount and in conjunction with the appropriate design. This is expected to significantly increase the potential applications of these peptides as tools for augmenting the activity of lipid nanoparticles used in gene delivery.
  • Takuya Ishikawa, Kana Somiya, Reina Munechika, Hideyoshi Harashima, Yuma Yamada
    Journal of Controlled Release 274 109 - 117 2018年03月28日 [査読有り][通常論文]
     
    To achieve mitochondrial gene therapy, developing a mitochondrial transgene expression system that produces therapeutic proteins in mitochondria of disease cells is essential. We previously reported on the design of pCMV-mtLuc (CGG) containing a CMV promotor and a NanoLuc (Nluc) luciferase gene that records adjustments to the mitochondrial codon system, and showed that the mitochondrial transfection of pCMV-mtLuc (CGG) resulted in the efficient production of the Nluc luciferase protein in human HeLa cells. This mitochondrial transfection was achieved using a MITO-Porter, a liposome-based carrier for delivering a cargo to mitochondria via membrane fusion. We report herein that mitochondrial transfection using the MITO-Porter results in mitochondrial transgene expression in G625A fibroblasts obtained from a patient with a mitochondrial disease. We investigated the effect of promoters and the basic structure of pCMV-mtLuc (CGG) on gene expression efficiency, and were able to construct a high performance mitochondrial DNA vector, pCMV-mtLuc (CGG) [hND4] that contains a human mitochondrial endogenous gene. We also constructed an RP/KALA-MITO-Porter composed of the KALA peptide (cell-penetrating peptide) with a mitochondrial RNA aptamer to enhance cellular uptake and mitochondrial targeting. Finally, the mitochondrial transfection of pCMV-mtLuc (CGG) [hND4] in G625A fibroblasts using the RP/KALA-MITO-Porter resulted in strong mitochondrial transgene expression.
  • Yuma Yamada, Hideyoshi Harashima
    Mitochondrial Biology and Experimental Therapeutics 491 - 498 2018年03月21日 [査読有り][通常論文]
     
    Mitochondria are attractive organelles that have the potential to contribute greatly to medical therapy. This organnelle is responsible for a variety of essential functions including ATP production and the regulation of apoptosis, and they have their own genome, mitochondrial DNA (mtDNA). It has recently become evident that a variety of human diseases are associated with mitochondrial dysfunctions caused by mutations and defects in mtDNA. Therefore, the ability to successfully target the mitochondrial genome and to regulate mitochondrial gene expression would contribute to mitochondrial gene therapy for various human diseases. To achieve such an innovative objective, it will be necessary to deliver various cargoes to mitochondria in living cells. However, only a limited number of approaches are available for accomplishing this. In this chapter, we discuss problems associated with mitochondrial delivery systems and mitochondrial gene expression, and propose a strategy for overcoming these problems based our current efforts. To this end, we highlight a MITO-Porter, a mitochondrial delivery system, and show some examples of the regulation of mitochondrial gene expression including mitochondrial RNA knockdown and mitochondrial transgene expression.
  • Masatoshi Maeki, Niko Kimura, Yusuke Sato, Hideyoshi Harashima, Manabu Tokeshi
    Advanced Drug Delivery Reviews 128 84 - 100 2018年03月15日 [査読有り][通常論文]
     
    Lipid-based nanobiomaterials as liposomes and lipid nanoparticles (LNPs) are the most widely used nanocarriers for drug delivery systems (DDSs). Extracellular vesicles (EVs) and exosomes are also expected to be applied as DDS nanocarriers. The performance of nanomedicines relies on their components such as lipids, targeting ligands, encapsulated DNA, encapsulated RNA, and drugs. Recently, the importance of the nanocarrier sizes smaller than 100 nm is attracting attention as a means to improve nanomedicine performance. Microfluidics and lab-on-a chip technologies make it possible to produce size-controlled LNPs by a simple continuous flow process and to separate EVs from blood samples by using a surface marker, ligand, or electric charge or by making a mass or particle size discrimination. Here, we overview recent advances in microfluidic devices and techniques for liposomes, LNPs, and EVs and their applications for DDSs.
  • がん組織における浸潤性向上を目指した極小脂質ナノ粒子の開発
    岡部 奈々, 佐藤 悠介, 真栄城 正寿, 渡慶次 学, 原島 秀吉
    日本薬学会年会要旨集 138年会 4 78 - 78 (公社)日本薬学会 2018年03月
  • Ikramy A. Khalil, Hideyoshi Harashima
    International Journal of Pharmaceutics 538 1-2 179 - 187 2018年03月01日 [査読有り][通常論文]
     
    Because of their ability to translocate different cargos into cells, arginine-rich cell penetrating peptides (CPPs) are promising vehicles for drug and gene delivery. The use of CPP-based carriers, however, is hampered by the lack of specificity and by interactions with negative serum components. Polyethylene glycol (PEG) is used to decrease such non-specific interactions, albeit its use is associated with reduced transfection efficiency. In this study, we describe the development of PEGylated CPP-based gene carrier with an improved targeting and a high transfection activity. The system was prepared by condensing DNA with a polycation followed by coating with a lipid envelope containing the octaarginine (R8) peptide as a model CPP. R8-modified nanoparticles produced high transfection activities, but the efficiency was reduced by PEG shielding. The reduced activity could be fully restored by the addition of a targeting ligand and a pH-sensitive fusogenic peptide. The efficiency of the proposed system is quite high, even in the presence of serum, and shows improved targeting and selectivity. Surprisingly, the effect of the fusogenic peptide was dramatically reduced in the absence of R8. Although shielded, R8 augmented the activity of the fusogenic peptide, suggesting a synergistic effect between the two peptides at the intracellular level.
  • Yamada Y, Tabata M, Abe J, Nomura M, Harashima H
    Journal of pharmaceutical sciences 107 2 647 - 653 2018年02月01日 [査読有り][通常論文]
     
    Patients with type I diabetes, which is caused by the destruction of pancreatic islets, now require regular therapeutic injections of insulin. The use of transgene therapy represents an alternate and potent strategy for the treatment of type I diabetes. However, only a limited number of studies regarding in vivo gene delivery targeting the pancreas and islets have been reported. Here, we report on the possibility of in vivo transgene expression in the pancreas by the intraductal injection of naked plasmid DNA (pDNA). Gene expression activities were detected in the pancreas of mice after the injection of naked pDNA encoding luciferase into the common bile duct. We then investigated the effects of injection dose, volume, and speed on gene delivery and determined the optimal conditions for the delivery of pDNA to the pancreas. Exogenous luciferase mRNA was detected in the pancreatic islets by reverse transcription PCR analysis. Moreover, no injury was detected in the liver, the common bile duct, or the pancreas over time after the injection. These findings indicate that the intraductal injection of naked pDNA promises to be a useful technique for in vivo gene delivery targeted to pancreatic tissue and islets.
  • Shoshiro Yamamoto, Yu Sakurai, Hideyoshi Harashima
    International Journal of Pharmaceutics 536 1 42 - 49 2018年01月30日 [査読有り][通常論文]
     
    Although anti-angiogenic therapy is predicted to be an effective therapy for treating cancer, selectively targeting tumor endothelial cells (TECs), and not normal endothelial cells, remains a major obstacle. Modifying a drug carrier with a targeting ligand is a popular strategy for developing an active-targeting type drug delivery system (DDS). We previously reported that a cyclo(Arg-Gly-Asp-D-Phe-Lys) (cRGD)-equipped liposome that contains encapsulated siRNA (RGD-MEND) achieved an efficient therapeutic outcome in a murine cancer model. To develop a more efficient TEC-targeting DDS, we examined the effect of the length of the polyethylene glycol (PEG) that is used as a peptide-linker on the cholesterol-scaffold, and liposomal composition on the efficiency of delivery of siRNA to cRGD receptor αVβ3 integrin positive cells. An RGD-MEND modified with shorter linker/no-linker, PEG350 or no-PEG, showed a higher cellular uptake in vitro. However, a shorter or no-linker RGD-cholesterol-modified MEND showed no silencing effect despite its high, in vitro silencing efficiency. To examine the possibility that the cholesterol-scaffold ligand was removed from the surface of the RGD-MEND by interactions with serum proteins, the RGD-MEND was incubated in the presence of a 50% serum solution. The cellular uptake of the cholesterol-scaffold ligand was drastically reduced by the incubation in serum. Increasing the cholesterol ratio in the lipid envelope and adding a helper lipid improved the in vivo knockdown efficiency, probably due to an enhanced ligand retention, even in in vivo conditions. The findings reported herein suggest that the lipid composition and the ligand scaffold of the MEND are major factors in successfully developing an efficient active-targeting DDS.
  • Niko Kimura, Masatoshi Maeki, Nana Okabe, Yusuke Sato, Akihiko Ishida, Hirofumi Tani, Hideyoshi Harashima, Manabu Tokeshi
    22nd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2018 3 1404 - 1405 2018年 
    This paper reports a methodology for precise controlling the lipid nanoparticle (LNP) size by stepwise and rapid ethanol dilution using an integrated microfluidic device with baffle mixers. The integrated microfluidic device coupling the LNP synthesis and the post-treatment regions had better size controllability of LNPs than the conventional preparation methods. Additionally, 30 nm-sized siRNA-loaded LNPs prepared by the post-treatment process using the integrated microfluidic device showed great gene-silencing activity and specific intrahepatic biodistribution. The stepwise and rapid ethanol dilution methodology using the integrated microfluidic device provides LNPs with homogeneous size distribution for improving the efficacy of nanomedicines.
  • Yu Sakurai, Hideyoshi Harashima
    Drug Delivery System 33 2 98 - 104 2018年 [査読有り][招待有り]
     
    A discovery of the EPR effect allowed for remarkable progress in a delivery of anti-cancer therapeutics using cancer-targeting nanoparticle. However, recent report on meta-analysis among several clinical trial with patients treated with liposomal doxorubicin revealed that liposomal doxorubicin was not superior to free doxorubicin in terms of therapeutic effect. This difference would be caused by the difference between non-clinical model and clinical model with regard to growth speed, tumor growth speed, tumor microenvironment, endpoint (tumor size vs. over survival). We would like to introduce this report in detail. In addition, to evaluate the effect of tumor microenvironment on the delivery of nanoparticle and to enhance that through remodeling tumor microenvironment, we have recently developed a new strategy that remodeling tumor microenvironment by siRNA delivery to tumor vasculature enhanced the intratumoral distribution of nanoparticles. Specifically in hyperangiogenic cancer, anti-angiogenic therapy by vascular endothelial growth factor receptor 2 (VEGFR2) enhanced the delivery of nanoparticles to tumor tissue, which was inconsistent with previous report. We herein would like to explain this contradiction in terms of tumor microenvironment and introduce our new strategy.
  • Kazuaki Kajimoto, Tatsuhito Katsumi, Takashi Nakamura, Masatoshi Kataoka, Hideyoshi Harashima
    JAOCS, Journal of the American Oil Chemists' Society 95 1 101 - 109 2018年01月01日 [査読有り][通常論文]
     
    In this study, we established a procedure based on the microencapsulation vesicle (MCV) method for preparing surface-modified liposomes, using polyethylene glycol (PEG) and a site-directed ligand, with high entrapment efficiency of cytochrome c (Cyt c). For preparing a water-in-oil (W/O) emulsion, egg phosphatidylcholine and cholesterol were dissolved in organic solvents (O phase) and emulsified by sonication with aqueous solution of Cyt c (W1). Although the dispersion stability of the W1/O emulsion was low when n-hexane was used to dissolve the lipids in the O phase, it was substantially improved by using mixed solvents consisting of n-hexane and other organic solvents, such as ethanol and dichloromethane (DCM). The W1/O emulsion was then added to another water phase (W2) to prepare the W1/O/W2 emulsion. PEG- and/or ligand-modified lipids were introduced into the W2 phase as external emulsifiers not only for stabilizing the W1/O/W2 emulsion but also for modifying the surface of liposomes obtained later. After solvent evaporation and extrusion for downsizing the liposomes, approximately 50% of Cyt c was encapsulated in the liposomes when the mixed solvent consisting of n-hexane and DCM at a volume ratio of 75/25 was used in the O phase. Finally, the fluorescence-labeled liposomes, with a peptide ligand having affinity to the vasculature in adipose tissue, were prepared by the MCV method and intravenously injected into mice. Confocal microscopy showed the substantial accumulation of these liposomes in the adipose tissue vessels. Taken together, the MCV technique, along with solvent optimization, could be useful for generating surface-modified liposomes with high drug entrapment efficiency for targeted delivery.
  • Jiro Abe, Yuma Yamada, Atsuhito Takeda, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 269 177 - 188 2018年01月 [査読有り][通常論文]
     
    It has been reported that transplanting native cells would lack efficiency without producing artificial cell-tissue, due to the exaggerated oxidative stress in doxorubicin-induced cardiomyopathy. We attempted to activate cardiac progenitor cells (CPCs) by delivering resveratrol to mitochondria using a mitochondrial drug delivery system (MITO-Porter system). We first evaluated the viability of H9c2 cells (a cardio myoblast cell line) after doxorubicin treatment, where H9c2 cells were co-cultured with or without the mitochondria activated CPCs (referred to herein as MITO cell). We next evaluated the survival rate of doxorubicin treated mice, with or without the injection of MITO cells into the myocardium. Finally, we examined the molecular mechanism of the cell therapy by detecting oxidative stress and the induction of apoptosis in addition to quantification of the mRNA and protein levels about oxidative phosphorylation (OXPHOS). The MITO cell transplanted mice lived significantly longer than the conventional CPC transplanted ones. Oxidative stress and massive cell death were both significantly reduced in the MITO cell transplanted hearts, in which the expression levels of OXPHOS protein and gene were also higher than the control group. In doxorubicin-induced cardiomyopathy, the transplantation of MITO cells, which possess activated mitochondria, is more efficient compared to conventional CPC transplantation.
  • Joseph W. Nichols, Yu Sakurai, Hideyoshi Harashima, You Han Bae
    JOURNAL OF CONTROLLED RELEASE 267 31 - 46 2017年12月 [査読有り][招待有り]
     
    Navigating intratumoral drug distribution has proven to be one of the most challenging aspects of drug delivery. The barriers are significant and varied; increased diffusional distances, elevated interstitial fluid pressure, regions of dense extracellular matrix and high cell density, and overall heterogeneity. Such a long list imposes significant requirements on nano-sized carriers. Unfortunately, other capabilities are eclipsed by the distribution requirements. A drug can do no good until it reaches its target. Numerous strategies to improve drug distribution have been developed, taking account of various unique characteristics of solid tumors, including some mechanisms that are still not fully understood. Most of these strategies were from small animal tumor models which are our primary tool for understanding cancer physiology. The small animal tumor model is the most versatile and effective means of understanding tumor transport, but its prevalence belies some of its weaknesses. Tumors grown under lab conditions are developed much more quickly than naturally developed cancers, potentially impacting tumor heterogeneity, blood vessel development, extracellular matrix organization, cell diversity, and many other features of structure and physiology that may impact transport. These problems come in addition to the difficulties of making precise measurements within a living tumor. Resolving these problems is best done by improving our analysis methods, and by finding complementary models that can clarify and expound the details. In this review, we will first discuss some of the strategies employed to improve transport and then highlight some of the new models that have recently been developed in the Bae lab and how they may aid in the study of tumor transport in the future.
  • West Kristian D. Paraiso, Hiroki Tanaka, Yusuke Sato, Daiki Shirane, Noriyuki Suzuki, Yasumitsu Ogra, Kota Tange, Yuta Nakai, Hiroki Yoshioka, Hideyoshi Harashima, Hidetaka Akita
    COLLOIDS AND SURFACES B-BIOINTERFACES 160 715 - 723 2017年12月 [査読有り][通常論文]
     
    The use of gold nanorods (AuNRs) that produce heat in response to near infrared (NIR) irradiation is an attractive approach to cancer photothermal therapy. AuNRs are usually prepared by using a highly toxic detergent: cetyltrimethylammonium bromide (CTAB). Thus, the removal of CTAB from the reaction mixture, and further stabilization of the surface of the AuNRs is required. In the present study, AuNRs were encapsulated in a multifunctional envelope-type nano device (AuNR-MEND) formed with an SS-cleavable and pH-activated lipid-like material. In the process of encapsulation, AuNRs were first stabilized with bovine serum albumin (AuNR-BSA), and then further encapsulated in the lipid envelope by the ethanol dilution method. The in vitro photothermal cytotoxicity of AuNR-MEND was further demonstrated on 4T1 breast cancer cells. After NIR radiation, the temperature of the medium was increased to approximately 60 degrees C, and cell viability was drastically decreased to approximately 11%. However, this cytotoxic effect cannot simply be explained by medium heating. It therefore appears that intracellular delivery of the AuNRs is a key factor for achieving a high degree of cytotoxicity. Dose dependent cytotoxicity data revealed that a higher dose of AuNR-MEND resulted in the complete destruction of the cells when they were subjected to NIR irradiation, while the cell survival rate reached a plateau at 30% in the case of AuNR-BSA. Apoptosis was induced after treatment with the nanoparticles. AuNR-MEND showed superior cellular uptake activity over AuNR-BSA. Thus, delivering AuNR by means of functionalized lipid nanoparticles represents a promising approach to induce NIR-triggered apoptosis. (c) 2017 Elsevier B.V. All rights reserved.
  • Yuta Takano, Reina Munechika, Vasudevanpillai Biju, Hideyoshi Harashima, Hiroshi Imahori, Yuma Yamada
    NANOSCALE 9 47 18690 - 18698 2017年12月 [査読有り][通常論文]
     
    It has been known for decades that intracellular redox reactions control various vital functions in living systems, which include the synthesis of biomolecules, the modulation of protein functions, and cell signaling. Although there have been several reports on the control of such functions using DNA and RNA, the non-invasive optical control of biological functions is an important ongoing challenge. In this study, a hybrid of an electron donor-acceptor linked molecule based on a ferrocene(Fc)-porphyrin(ZnP)-fullerene-(C-60) analogue and an elaborately designed nano-carrier, referred to herein as a MITO-Porter, resulted in a successful photoinduced intermolecular electron transfer reaction via the long-lived intramolecular charge separation, leading to site-specific reductive reactions in the mitochondria of living HeLa cells. A Fc-ZnP-C-60 linked molecule, 1-Oct, was designed and prepared for taking advantage of the unique photophysical properties with excellent efficiency (i.e. a long lifetime and a high quantum yield) for photoinduced charge separation. The targeted delivery of 1-Oct to mitochondria was accomplished by using a combination of the Fc-ZnP-C-60 molecule and a drug delivery nano-carrier, MITO-Porter, that was recently established by our group for intracellular cargo delivery. The successful delivery of 1-Oct by the MITO-Porter permitted the optically-controlled generation of O-2(-) in the mitochondria of HeLa cells and the following induction of apoptosis as a cell signalling response was observed in confocal laser microscopy experiments. The obtained results indicate the use of an electron donor-acceptor system such as this can be a promising tool for the non-invasive triggering of redox-coupled cellular activities in living systems.
  • Yuma Yamada, Laila Burger, Eriko Kawamura, Hideyoshi Harashima
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 40 12 2183 - 2190 2017年12月 [査読有り][通常論文]
     
    While Coenzyme Q(10) (CoQ(10)) is thought to be effective for the treatment of a variety of diseases, it limits its cellular uptake. Because of the hydrophobic nature of CoQ(10), it is reasonable to assume that it could be encapsulated within a liposomal carrier. Several reports regarding the packaging of CoQ(10) in liposomes have appeared, but detailed investigations of the preparation of CoQ(10) encapsulated liposomes have not been reported. As a result, information regarding the optimal method of packaging CoQ(10) in liposomes is not available. In this study, several types of liposomes were prepared using different methods and their characteristics were compared. Since CoQ(10) is mainly located in the inner mitochondria! membrane, a liposome that targets mitochondria, a MITO-Porter, was used as a model liposome. It was possible to incorporate high levels of CoQ(10) into the carrier. Transmission electron microscopy analyses showed that an empty MITO-Porter and the CoQ(10)-MITO-Porter were structurally different from one another. Even though significant structural differences were observed, mitochondria! delivery was not affected in mitochondria! disease fibroblast cells, as evidenced by confocal laser scanning microscopy observations. The results reported herein suggest that the CoQ(10)-MITO-Porter might be a suitable candidate for the potential medical therapy of mitochondria-related diseases.
  • Yusuke Sato, Hideki Matsui, Naoki Yamamoto, Risa Sato, Tsubasa Munakata, Michinori Kohara, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 266 216 - 225 2017年11月 [査読有り][通常論文]
     
    Lipid nanoparticles (LNPs) are the leading technology for delivering short interfering RNA (siRNA) in vivo. While numerous attempts to improve the efficiency of siRNA delivery have been reported, only a few studies of the mechanism of LNP-mediated toxicity and attempts to develop safe LNPs in vivo have been reported, in spite of the significance of such systems, in the light of treatment and clinical applications. We herein report on the elucidation of the mechanism of hepatotoxicity following the intravenous injection of a high dose of hepatotropic LNPs. The LNPs accumulated in liver sinusoidal endothelial cells (LSECs), resulting in their activation and the induction of several cytokines related to neutrophils, followed by neutrophilic inflammation. To circumvent this toxic cascade, the LNPs were modified with a hepatocyte-specific ligand, N-acetyl-D-galactosamine (GalNAc), which resulted in a substantial improvement of hepatocyte-specificity and in a dramatic reduction in toxicity. Moreover, modification of the GalNAc-LNPs with polyethyleneglycol abrogated the LNP-associated toxicity without any detectable loss of gene silencing activity in hepatocytes. Finally, we observed that a single injection of the LNPs resulted in a significant reduction of hepatitis B virus (HBV) genomic DNA and their antigens without any sign of toxicity in chimeric mice with humanized livers that had been persistently infected with HBV. These lines of the fact suggest that the newly designed siRNA-loaded LNPs promise to be a useful technology for the treatment of liver diseases.
  • Takashi Nakamura, Hideyoshi Harashima
    Therapeutic Delivery 8 11 987 - 1000 2017年11月01日 [査読有り][通常論文]
     
    Immune checkpoint therapy represents a new, revolutionary type of cancer therapy, but emerging evidence indicates that only a minority of patients will benefit from it. The issue of how to improve and widen the clinical response is a pivotal issue, and combining other types of therapy with immune checkpoint inhibitors is currently under development. A nanotechnology-based drug-delivery system (nano DDS) could be an important contribution to the development of an effective combination therapy. In this document, we review recent findings in the field of tumor immunology, which provide a strategy for an efficient combination therapy, and discuss nano DDS that are associated with cancer immunotherapy and nano DDS strategies based on the immune status in tumor microenvironments.
  • Masatoshi Maeki, Yuka Fujishima, Yusuke Sato, Takao Yasui, Noritada Kaji, Akihiko Ishida, Hirofumi Tani, Yoshinobu Baba, Hideyoshi Harashima, Manabu Tokeshi
    PLOS ONE 12 11 e0187962  2017年11月 [査読有り][通常論文]
     
    Lipid nanoparticles (LNPs) or liposomes are the most widely used drug carriers for nanomedicines. The size of LNPs is one of the essential factors affecting drug delivery efficiency and therapeutic efficiency. Here, we demonstrated the effect of lipid concentration and mixing performance on the LNP size using microfluidic devices with the aim of understanding the LNP formation mechanism and controlling the LNP size precisely. We fabricated microfluidic devices with different depths, 11 mu m and 31 mu m, of their chaotic micromixer structures. According to the LNP formation behavior results, by using a low concentration of the lipid solution and the microfluidic device equipped with the 31 mu m chaotic mixer structures, we were able to produce the smallest-sized LNPs yet with a narrow particle size distribution. We also evaluated the mixing rate of the microfluidic devices using a laser scanning confocal microscopy and we estimated the critical ethanol concentration for controlling the LNP size. The critical ethanol concentration range was estimated to be 60-80% ethanol. Ten nanometer-sized tuning of LNPs was achieved for the optimum residence time at the critical concentration using the microfluidic devices with chaotic mixer structures. The residence times at the critical concentration necessary to control the LNP size were 10, 15-25, and 50 ms time-scales for 30, 40, and 50 nm-sized LNPs, respectively. Finally, we proposed the LNP formation mechanism based on the determined LNP formation behavior and the critical ethanol concentration. The precise size-controlled LNPs produced by the microfluidic devices are expected to become carriers for next generation nanomedicines and they will lead to new and effective approaches for cancer treatment.
  • Naoya Miura, Kota Tange, Yuta Nakai, Hiroki Yoshioka, Hideyoshi Harashima, Hidetaka Akita
    JOURNAL OF PHARMACEUTICAL SCIENCES 106 10 3113 - 3119 2017年10月 [査読有り][通常論文]
     
    The KALA peptide (WEAKLAKALAKALAKHLAKALAKALKA) is an amphiphilic peptide that forms an a-helical structure at physiological pH. We previously reported that, when a plasmid DNA-encapsulating liposomal membrane is modified with the KALA peptide, transgene expression and immune activation are facilitated in bone marrow-derived dendritic cells (BMDCs). However, the minimum unit of the KALA peptide and the importance of its secondary structure for these activities are not completely known at this time. We herein report on the identification of the minimum unit of the KALA peptide (short-KALA) required for activity, as determined by the stepwise removal of "K-A-L-A" units. We evaluated the activities of 4 types of short-KALAs by modifying plasmid DNA-encapsulating multi-functional envelop-type nano devices. Among the peptides tested, a short-KALA3 (WEAKLAKALAKALA) was the shortest KALA peptide that could form an alpha-helical structure, as well as to elicit transgene expression and immune activation in BMDCs. Furthermore, the function of the short-KALA3 as an inducer of cellular uptake was retained, while uptake was completely lost in more shortened versions of KALA (short KALA4), in that transgene expression and immunological activation were both completely lost. These collective data show that the KALA peptide must form an alpha-helical structure to induce cellular uptake in BMDCs. (C) 2017 American Pharmacists Association (R). Published by Elsevier Inc. All rights reserved.
  • Yu Sakurai, Wataru Mizumura, Manami Murata, Tomoya Hada, Shoshiro Yamamoto, Kenichiro Ito, Kazuhiro Iwasaki, Takayuki Katoh, Yuki Goto, Asako Takagi, Michinori Kohara, Hiroaki Suga, Hideyoshi Harashima
    MOLECULAR PHARMACEUTICS 14 10 3290 - 3298 2017年10月 [査読有り][通常論文]
     
    The development of a specific, effective method for the delivery of therapeutics including small molecules and nucleic acids to tumor tissue remains to be solved. Numerous types of lipid nanoparticles (LNPs) have been developed in attempts to achieve this goal. However, LNPs are probably not taken up by target cells because cancer-targeting LNPs are typically modified with poly(ethylene glycol) (PEG), which inhibits the cellular uptake of LNPs, to passively accumulate in tumor tissue via the enhanced permeability and retention (EPR) effect. It would clearly be important to develop a LNP with both a prolonged circulation and cancer-specific efficient uptake for use in an innovative nanodrug delivery system. Herein, we assessed the effect of nonstandard macrocyclic peptides against the epithelial cell adhesion molecule (EpCAM) Epi-1, which was discovered by a random nonstandard peptides integrated discovery (RaPID) system, on the cellular uptake and therapeutics delivery of LNPs. A liposomal siRNA delivery system (MEND) was modified with an Epi-1 lipid-derivative (EpCAM-targeting MEND; ET-MEND). The resulting ET-MEND showed a more than 27-fold increase in cellular uptake in EpCAM-positive cell lines. In the case of negative cells, cellular uptake and the efficiency of the ET-MEND for delivering therapeutics were comparable with those of nonmodified MEND. In addition, when systemically injected, the ET-MEND successfully inhibited gene expression in the tumor tissue at a dose of 0.5 mg siRNA/kg without any obvious toxicity. These results suggest that a combination of a specific peptide ligand can be used to identify a RaPID system and that the use of such a MEND for liposomal drug delivery has the potential for use in developing a system for the efficacious delivery of pharmaceuticals to various cancer cells.
  • Yuma Yamada, Reina Munechika, Eriko Kawamura, Yu Sakurai, Yusuke Sato, Hideyoshi Harashima
    JOURNAL OF PHARMACEUTICAL SCIENCES 106 9 2428 - 2437 2017年09月 [査読有り][通常論文]
     
    Most anticancer drugs are intended to function in the nuclei of cancer cells. If an anticancer drug could be delivered to mitochondria, the source of cellular energy, this organelle would be destroyed, resulting in the arrest of the energy supply and the killing of the cancer cells. To achieve such an innovative strategy, a mitochondrial drug delivery system targeted to cancer cells will be required. We recently reported on the development of a MITO-Porter, a liposome for mitochondrial delivery. In this study, we validated the utility of such a cancer therapeutic strategy by delivering anticancer drugs directly to mitochondria. We succeeded in packaging doxorubicin (DOX) as a model cargo in MITO-Porter to produce a DOX-MITO-Porter. We evaluated cellular toxicity of OS-RC-2 cell, a type of DOX-resistant cancer cell, after delivering DOX to mitochondria using the MITO-Porter system. Cell viability was decreased by the DOX-MITO-Porter treatment, while cell viability was not decreased in the case of naked DOX and a conventional DOX liposomal formulation. We also found a relationship between cellular toxicity and mitochondrial toxicity. The use of a MITO-Porter system for mitochondrial delivery of a toxic agent represents a possible therapeutic strategy for treating drug-resistant cancers. (C) 2017 American Pharmacists Association (R). Published by Elsevier Inc. All rights reserved.
  • Sarochin Santiwarangkool, Hidekata Akita, Taichi Nakatani, Kenji Kusumoto, Hiroki Kimura, Masaru Suzuki, Masaharu Nishimura, Yusuke Sato, Hideyoshi Harashima
    JOURNAL OF PHARMACEUTICAL SCIENCES 106 9 2420 - 2427 2017年09月 [査読有り][通常論文]
     
    A alpha-helical GALA peptide (WEAALAEALAEALAEHLAEALAEALEALAA) has been found to possess dual functions: a pH-dependent inducer of endosomal escape, and a ligand that targets lung endothelium. In the present study, the flexibility of GALA was improved by modifying the edge with polyethylene glycol linker, to increase lung-targeting activity. We first investigated the uptake of the GALA-modified liposomes in which GALA was directly conjugated to the lipid (Cholesterol: GALA/Chol) or the phospholipid-PEG (GALA/PEG(2000)). The liposomes that were modified with GALA/PEG(2000) (GALA/PEG(2000)-LPs) were taken up at a higher level by human lung endothelial cells (HMVEC-L), in comparison with particles that were modified with GALA/Chol (GALA/Chol-LPs). Small-interfering RNA-encapsulating liposomal-based nanocarriers (multifunctional envelope-type nano device: MEND) that were formulated with a vitamin E-scaffold SS-cleavable pH-activated lipid-like material, namely GALA/PEG(2000)-MENDssPalmE were also modified with GALA/PEG(2000). Gene silencing activity in the lung endothelium was then evaluated against an endothelial marker; CD31. In comparison with the unmodified MENDssPalmE, GALA/PEG(2000)-MENDssPalmE exhibited a higher silencing activity in the lung. Optimization of GALA/PEG(2000)-MENDssPalmE resulted in silencing activity in the lung with an ED50 value of 0.21 mg/kg, while non-specific gene silencing in liver was marginal. Collectively, PEGylated GALA is a promising device for use in targeting the lung endothelium. (C) 2017 American Pharmacists Association (R). Published by Elsevier Inc. All rights reserved.
  • Kazuki Hashiba, Yusuke Sato, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 262 239 - 246 2017年09月 [査読有り][通常論文]
     
    Lipid nanoparticles (LNPs) are one of the promising technologies for the in vivo delivery of short interfering RNA (siRNA). Modifying LNPs with polyethyleneglycol (PEG) is widely used to inhibit non-specific interactions with serum components in the blood stream, and is a useful strategy for maximizing the efficiency of active targeting. However, it is a widely accepted fact that PEGylation of the LNP surface strongly inhibits fusion between LNPs and endosomal membranes, resulting in poor cytosolic siRNA delivery, a process that is referred to as the 'PEGdilemma'. In the present study, in an attempt to overcome this problem, siRNA-loaded LNPs were modified with PEG through maleic anhydride, a pH-labile linkage. The in vitro, suppression of cationic charge, stealth function at physiological pH up to 1 h and the rapid desorption of PEG and restoration of fusogenic activity under slightly acidic conditions (within only 2 min) were achieved by PEG modification of the LNPs through maleic anhydride. In vivo, PEG modification through maleic anhydride resulted in a dramatic improvement in the targeting capability of the active targeting of ligand (N-acetyl-D-galactosamine)-modified LNPs to hepatocytes, with an approximately 14-fold increase in gene silencing activity in factor 7 model mice. Taken together, the maleic an-hydride-mediated pH-labile PEGylation of the active targeting LNPs is a useful strategy for achieving the specific and efficient delivery of siRNAs in vivo.
  • Yuma Yamada, Takuya Ishikawa, Hideyoshi Harashima
    BIOMATERIALS 136 56 - 66 2017年08月 [査読有り][通常論文]
     
    Mitochondria have their own gene expression system that is independent of the nuclear system, and control cellular functions in cooperation with the nucleus. While a number of useful technologies for achieving nuclear transgene expression have been reported, only a few have focused on mitochondria. In this study, we validated the utility of an artificial mitochondrial DNA vector with a virus promoter on mitochondrial transgene expression. We designed and constructed pCMV-mtLuc (CGG) that contains a CMV promotor derived from Cytomegalovirus and an artificial mitochondrial genome with a NanoLuc (Nluc) luciferase gene that records adjustments to the mitochondrial codon system. Nluc luciferase activity measurements showed that the pCMV-mtLuc (CGG) efficiently produced the Nluc luciferase protein in human HeLa cells. Moreover, we optimized the mitochondrial transfection of pCMV-mtLuc (CGG) using a MITO-Porter system, a liposome-based carrier for mitochondrial delivery via membrane fusion. As a result, we found that transfection of pCMV-mtLuc (CGG) by MITO-Porter modified with the KALA peptide (cationic amphipathic cell-penetrating peptide) showed a high mitochondrial transgene expression. The developed mitochondrial transgene expression system represents a potentially useful tool for the fields of nanoscience and nanotechnology for controlling the intracellular microenvironment via the regulation of mitochondrial function and promises to open additional innovative research fields of study. (C) 2017 Elsevier Ltd. All rights reserved.
  • Shang-Hsuan Lee, Yusuke Sato, Mamoru Hyodo, Hideyoshi Harashima
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 40 7 1002 - 1009 2017年07月 [査読有り][通常論文]
     
    In the active targeting of a drug delivery system (DDS), the density of the ligand on the functionalized liposome determines its affinity for binding to the target. To evaluate these densities on the surface of different sized liposomes, 4 liposomes with various diameters (188, 137, 70, 40 nm) were prepared and their surfaces were modified with fluorescently labeled ligand lipid conjugates by the post-insertion method. Each liposomal mixture was fractionated into a series of fractions using size exclusion chromatography (SEC), and the resulting liposome fractions were precisely analyzed and the surface ligand densities calculated. The data collected using this methodology indicate that the density of the ligand on a particle is greatly dependent on the size of the liposome. This, in turn, indicates that smaller lip osomes (75-40nm) tend to possess higher densities. For developing active targeting systems, size and the density of the ligands are two important and independent factors that can affect the efficiency of a system as it relates to medical use.
  • リンパ節内樹状細胞を標的とした極小ナノキャリアシステムの開発
    河合 美典, 中村 孝司, 佐藤 悠介, 真栄城 正寿, 原島 秀吉
    日本DDS学会学術集会プログラム予稿集 33回 175 - 175 日本DDS学会 2017年06月
  • オートファジー誘導能を有する超分子構造ポリマー搭載ミトコンドリアDDSの構築
    大工原 伸之輔, 山田 勇磨, 田村 篤志, 西田 慶, 由井 伸彦, 原島 秀吉
    日本DDS学会学術集会プログラム予稿集 33回 173 - 173 日本DDS学会 2017年06月 [査読有り][通常論文]
  • Shoshiro Yamamoto, Akari Kato, Yu Sakurai, Tomoya Hada, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 251 1 - 10 2017年04月 [査読有り][通常論文]
     
    The vascular endothelial growth factor (VEGF)-mediated enhancement in vascular permeability is considered to be a major factor in tumor-targeting delivery via the enhanced permeability and retention (EPR) effect. We previously reported that the silencing of the endothelial VEGF receptor (VEGFR2) by a liposomal siRNA system (RGD-MEND) resulted in an enhanced intratumoral distribution of polyethylene glycol (PEG)-modified liposomes (LPs) in a renal cell carcinoma, a type of hypervascularized cancer, although the inhibition of VEGF signaling would be expected to decrease the permeability of the tumor vasculature. We herein report that the enhancement in the intratumoral distribution of LPs by VEGFR2 inhibition was dependent on the vascular type of the tumor (stroma vessel type; SV and tumor vessel type; TV). In the case of TV-type tumors (renal cell carcinoma and hepatocellular carcinoma), inhibiting VEGFR2 improved intratumoral distribution, while no effectwas found in the case of SV-type tumors (colorectal cancer). Moreover, through a comparison of the intratumoral distribution of LPs with a variety of physical properties (100 nmvs 400 nm, neutral vs negative vs positive), VEGFR2 inhibition was found to alter the tumor microenvironment, including heparan sulfate proteoglycans (HSPGs). In addition, the results regarding the effect of the size of nanoparticles indicated that VEGFR2 inhibition improved the penetration of nanoparticles through the vesselwall, but not via permeability, suggesting the involvement of an unknownmechanism. Our findings suggest that a combination of anti-angiogenic therapy and delivery via the EPR effect would be useful in certain cases, and that altering the tumor microenvironment by VEGFR2 blockade has a drastic effect on the intratumoral distribution of nanoparticles. (C) 2017 Elsevier B.V. All rights reserved.
  • Naoya Miura, Hidetaka Akita, Naho Tateshita, Takashi Nakamura, Hideyoshi Harashima
    MOLECULAR THERAPY 25 4 1003 - 1013 2017年04月 [査読有り][通常論文]
     
    For a successful anti-cancer vaccine, antigen presentation on the major histocompatibility complex (MHC) class I is a requirement. To accomplish this, an antigen must be delivered to the cytoplasm by overcoming the endosome/lysosome. We previously reported that a lipid nanoparticle modified with a KALA peptide (WEAKLAKALAKALAKHLAKALAICALICA), an alpha-helical cationic peptide, permits the encapsulated pDNA to be efficiently delivered to the cytoplasm in bone marrow derived dendritic cells (BMDCs). Herein, we report on the use of KALA-modified liposomes as an antigen carrier, in an attempt to induce potent antigen-specific cellular immunity. The subcutaneous injection of KALA-modified ovalbumin (OVA)-encapsulating liposomes (KALA-OVA-LPs) elicited a much more potent OVA-specific cytotoxic T lymphocyte activity and anti-tumor effect in comparison with particles that were modified with octa-arginine (R8), a cell-penetrating peptide (R8-OVA-LPs). In addition, the numbers of OVA-specific CD8(+) T cells were increased by immunization the KALAOVA-LPs. The treatment of BMDCs with KALA-OVA-LPs induced a substantial MHC class I antigen presentation. Furthermore, the acidic pH-dependent membrane destabilization activity of KALA-OVA-LPs strongly suggests that they are able to escape from endosomes/lysosomes and thereby deliver their cargos to the cytoplasm. Collectively, the KALAmodified liposome is a potential antigen delivery platform for use as a protein vaccine.
  • Mika Nishihara, Genki N. Kanda, Tetsuya Suzuki, Shin'ichiro Yamakado, Hideyoshi Harashima, Hiroyuki Kamiya
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 123 3 277 - 280 2017年03月 [査読有り][通常論文]
     
    Histone acetylation is associated with the activation of genes on chromosomes. Transgene expression from plasmid DNA might be increased by the acetylation of histones bound to plasmid DNA. To examine this hypothesis, we employed a positive feedback system, using a fusion protein of the sequence-specific DNA binding domain of yeast GAL4 and the histone acetyltransferase (HAT) domain of mouse CREB-binding protein (GAL4-HAT), in which GAL4-HAT promotes its own expression as well as that of a reporter gene product (luciferase). The activator plasmid DNA carrying the gene encoding GAL4-HAT was introduced into mouse Hepal-6 cells, together with the reporter plasmid DNA, by lipofection. Significantly increased luciferase expression was observed by the co-introduction of the activator plasmid DNA. Moreover, the acetylation of histones bound to the reporter plasmid DNA was enriched by the activator plasmid DNA. These results indicated that the GAL4-HAT system is useful for enhanced transgene expression. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.
  • Hiroki Tanaka, Sho Oasa, Masataka Kinjo, Kota Tange, Yuta Nakai, Hideyoshi Harashima, Hidetaka Akita
    COLLOIDS AND SURFACES B-BIOINTERFACES 151 95 - 101 2017年03月 [査読有り][通常論文]
     
    Lipids functionalized with tertiary amines (ionizable lipids) for a pH-dependent positive charge have been developed extensively as a carrier material for delivering nucleic acids. We previously developed an SS-cleavable proton-activated lipid-like material (ssPalm) as a component of a functionalized lipid envelope structure of a nanoparticle that encapsulated plasmid DNA and short interfering RNA. In this study, we report on the unique characteristics of such an ionizable lipid: the formation of a nano-sized emulsion (ave. 40nm) via pH-triggered self-emulsification in the absence of a cargo (nucleic acids). The particle has a neutral charge at physiological pH and is stabilized by helper lipids and polyethyleneglycol (PEG)-conjugated lipids. The generalized polarization of 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan), which indicates the surface polarity caused by the invasion of water onto the surface, changes dynamically in response to pH and temperature, while the fluidity of the intra-particle compartment, as measured by the fluorescence anisotropy of 1,6-Diphenyl-1,3,5-hexatriene (DPH), is not affected. Even when the particle contains a high density of PEG on the surface, it shows a high fusogenecity to negatively charged liposomes in response to an acidic pH to a higher degree than a conventional cationic lipid. These characteristics suggest that the ssPalm particle possesses unique properties for delivering lipophilic drugs across the biomembrane. (C) 2016 Elsevier B.V. All rights reserved.
  • Takashi Nakamura, Yosuke Noma, Yu Sakurai, Hideyoshi Harashima
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 40 2 234 - 237 2017年02月 [査読有り][通常論文]
     
    Intravesical drug delivery by cationic liposomes (Cat-LPs) represents a potent nanotechnology for enhancing therapeutic effects against bladder disorders. However, preventing the aggregation of Cat-LPs in urine poses a significant barrier. We report on an examination of the effect of modifying liposomes with polyethylene glycol (PEG) lipids to prevent Cat-LPs from aggregating in human urine. Although Cat-LPs underwent significant aggregation in human urine, introducing 5 mol% of PEG2k lipid or 2 mol% of PEG5k lipid completely inhibited the aggregation of the Cat-LPs. When 2 mol% of PEG2k lipids were introduced, the lipid structures of 1,2-distearoly-sn-glycero-3-phosphoethanolamine (DSPE) and 1,2-distearoyl-sn-glycerol (DSG) greatly prevented aggregation compared with cholesterol. By contrast, when Cat-LPs, after incubation in urine, were exposed to bladder cancer cells, only introducing cholesteryl-PEG into the Cat-LPs showed a significant enhancement in cellular uptake. These results offer the potential for incorporating cholesteryl-PEG into Cat-LPs for achieving both stability in urine and effective cellular uptake.
  • Yuma Yamada, Hideyoshi Harashima
    Journal of the Society of Powder Technology, Japan 54 3 158 - 166 2017年 [査読有り][通常論文]
     
    A single cell contains a variety of organelles, including a nucleus, mitochondria, the golgi apparatus and others. If it were possible to prepare a nano craft that could specifically target a specific organelle, it would open a new field of research directed toward therapeutic treatments for a variety of diseases. We recently developed a new concept that we refer to as “Programmed Packaging”, by which we succeeded in creating a multifunctional envelope-type nano device (MEND) as a non-viral vector. Our attempts to target mitochondria are discussed here, mainly focusing on a MITO-Porter, a MEND for delivering cargoes to mitochondria. A variety of human diseases, including various neurodegenerative disorders, ischemic heart disease, diabetes and cancer have been reported to be associated with mitochondrial dysfunction. Because of this, mitochondrial therapy would be expected to be useful and productive in the treatment of such diseases. Our findings regarding mitochondrial drug delivery systems that are directed toward mitochondrial nano medicine development are summrized herein.
  • Yusuke Sato, Yu Sakurai, Kazuaki Kajimoto, Takashi Nakamura, Yuma Yamada, Hidetaka Akita, Hideyoshi Harashima
    MACROMOLECULAR BIOSCIENCE 17 1 2017年01月 [査読有り][通常論文]
     
    Nanomedicines promise to extend drug therapy from small molecular compounds to proteins/nucleic acids/genes. Multifunctional envelope-type nanodevices (MENDs) have been developed for delivering such molecules to the site of action. The YSK-MEND contains new types of pH-responsive cationic lipids to efficiently deliver siRNA to hepatocytes via receptor-mediated endocytosis and use in treating hepatitis C and B in model mice. The RGD ligand is introduced to target tumor endothelial cells (TEC) and RGD-MEND is able to send siRNA to TEC to regulate the function of tumor microenvironments. The MITO-Porter is also developed to target mitochondria via membrane fusion. Antisense oligo RNA in the MITO-Porter permits the knock down of mitochondrial function. Finally, the ssPalms is designed based on a new concept of pH-dependent protonation in endosomes and cleavage of SS bonds in the reducing conditions in cytosol. These new technologies promise to stimulate the use of Nanomedicines in the future.
  • Yuma Yamada, Hideyoshi Harashima
    Handbook of Experimental Pharmacology 240 457 - 472 2017年 [査読有り][通常論文]
     
    Mitochondria are attractive organelles that have the potential to contribute greatly to medical therapy, the maintenance of beauty and health, and the development of the life sciences. Therefore, it would be expected that the further development of mitochondrial drug delivery systems (DDSs) would exert a significant impact on the medical and life sciences. To achieve such an innovative objective, it will be necessary to deliver various cargoes to mitochondria in living cells. However, only a limited number of approaches are available for accomplishing this. We recently proposed a new concept for mitochondrial delivery, a MITO-Porter, a liposome-based carrier that introduces macromolecular cargoes into mitochondria via membrane fusion. To date, we have demonstrated the utility of mitochondrial therapeutic strategy by MITOPorter using animal models of diseases. We also showed that the mitochondrial delivery of antisense oligo-RNA by the MITO-Porter results in mitochondrial RNA knockdown and has a functional impact on mitochondria. Here, we summarize the current state of mitochondrial DDS focusing on our research and show some examples of mitochondrial functional regulations using mitochondrial DDS.
  • Samir Mitragotri, Twan Lammers, You Han Bae, Steven Schwendeman, Stefaan De Smedt, Jean-Christophe Leroux, Dan Peer, Ick Chan Kwon, Hideyoshi Harashima, Akihiko Kikuchi, Yu-Kyoung Oh, Vladmir Torchilin, Wim Hennink, Justin Hanes, Kinam Park
    JOURNAL OF CONTROLLED RELEASE 246 183 - 184 2017年01月 [査読有り][通常論文]
  • Yu Sakurai, Tomoya Hada, Hideyoshi Harashima
    JOURNAL OF BIOMATERIALS SCIENCE-POLYMER EDITION 28 10-12 1086 - 1096 2017年 [査読有り][通常論文]
     
    While a number of siRNA delivery systems have been developed, the methods used in their preparation are not suitable for large-scale production. We herein report on methodology for the large-scale preparation of liposomal siRNA using a fluidic device and tangential flow filtration (TFF). A number of studies have appeared on the use of fluidic devices for preparing and purifying liposomes, but no systematic information regarding appropriate membrane type of commercially available apparatus is available. The findings reported herein indicate that, under optimized conditions, a microfluidic device and TFF can be used to produce siRNA lipid nanoparticles with the same characteristics as traditional ones'. The in vivo silencing efficiency of these lipid nanoparticles in the liver was comparable to laboratory-produced nanoparticles. In addition, con-focal laser scanning microscopy analyses revealed that they accumulated in the liver accumulation at the same levels as particles produced by batch-type and continuous-type procedures. This methodology has the potential to contribute to the advancement of this process from basic research to clinical studies of liposomal DDS.
  • Yu Sakurai, Tomoya Hada, Shoshiro Yamamoto, Akari Kato, Wataru Mizumura, Hideyoshi Harashima
    Molecular Therapy 24 12 2090 - 2099 2016年12月01日 [査読有り][通常論文]
     
    A number of nano drug delivery systems have recently been developed for cancer treatment, most of which are based on the enhanced permeability and retention effect. The advantages of the enhanced permeability and retention effect can be attributed to immature vasculature. Herein we evaluated the intratumoral distribution of lipid nanoparticles when the VEGF receptor 2 on tumor endothelial cells was inhibited by liposomal siRNA. VEGF receptor 2 inhibition resulted in an increase in intratumoral distribution and therapeutic efficacy despite the maturation of the tumor vasculature. A small molecule inhibitor against matrix metalloproteinase and macrophage depletion cancelled the improvement in the distribution of the lipid nanoparticles, suggesting that remodeling of tumor microenvironment played a role in the facilitated intratumoral distribution via the down-regulation of VEGF receptor 2. Accordingly, our results suggest that the enhanced permeability and retention effect is dependent, not only on the structure of the tumor vasculature, but also on the dynamics of the tumor microenvironment including extracellular matrix remodeling. Regulating the tumor microenvironment and the extracellular matrix by delivering tumor endothelial cell-targeting siRNA could potentiate the enhanced permeability and retention effect-based strategy.
  • Yusuke Sato, Takashi Nakamura, Yuma Yamada, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 244 Pt B 194 - 204 2016年12月 [査読有り][通常論文]
     
    Successful nanomedicines should be based on sound drug delivery systems (DDS) the permit intracellular trafficking as well as the biodistribution of cargos to be controlled. We have been developing new types of DDS that are multifunctional envelope-type nano devices referred to as MENDs. First, we will focus the in vivo delivery of siRNA to hepatocytes using a YSK-MEND which is composed of pH-responsive cationic lipids. The YSK-MEND is capable of inducing efficient silencing activity in hepatocytes and can be used to cure mice that are infected with hepatitis C or B. The YSK-MEND can also be applied to cancer immunotherapy through the activation of immune cells by delivering different compounds such as cyclic-di-GMP, siRNA or alpha-galactosylceramide as a lipid antigen. The findings indicate that, as predicted, these compounds, when encapsulated in the YSK-MEND, can be delivered to the site of action and induced immune activation through different mechanisms. Finally, a MITO-Porter, amembrane fusion-based delivery system to mitochondria, is introduced as an organelle targeting DDS and a new strategy for cancer therapy is proposed by delivering gentamicin to mitochondria of cancer cells. These new technologies are expected to extend the therapeutic area of Nanomedicine by increasing the power of DDS, especially from the view point of controlled intracellular trafficking. (C) 2016 Elsevier B.V. All rights reserved.
  • Sakurai Y, Hada T, Yamamoto S, Kato A, Mizumura W, Harashima H
    Molecular therapy : the journal of the American Society of Gene Therapy 24 12 2090 - 2099 2016年12月 [査読有り][通常論文]
     
    A number of nano drug delivery systems have recently been developed for cancer treatment, most of which are based on the enhanced permeability and retention effect. The advantages of the enhanced permeability and retention effect can be attributed to immature vasculature. Herein we evaluated the intratumoral distribution of lipid nanoparticles when the VEGF receptor 2 on tumor endothelial cells was inhibited by liposomal siRNA. VEGF receptor 2 inhibition resulted in an increase in intratumoral distribution and therapeutic efficacy despite the maturation of the tumor vasculature. A small molecule inhibitor against matrix metalloproteinase and macrophage depletion cancelled the improvement in the distribution of the lipid nanoparticles, suggesting that remodeling of tumor microenvironment played a role in the facilitated intratumoral distribution via the downregulation of VEGF receptor 2. Accordingly, our results suggest that the enhanced permeability and retention effect is dependent, not only on the structure of the tumor vasculature, but also on the dynamics of the tumor microenvironment including extracellular matrix remodeling. Regulating the tumor microenvironment and the extracellular matrix by delivering tumor endothelial cell-targeting siRNA could potentiate the enhanced permeability and retention effect-based strategy.
  • Shang-Hsuan Lee, Yusuke Sato, Mamoru Hyodo, Hideyoshi Harashima
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 39 12 1983 - 1994 2016年12月 [査読有り][通常論文]
     
    The surface topology of ligands on liposomes is an important factor in active targeting in drug delivery systems. Accurately evaluating the density of anchors and bioactive functional ligands on a liposomal surface is critical for ensuring the efficient delivery of liposomes. For evaluating surface ligand density, it is necessary to clarify that on the ligand-modified liposomal surfaces, some anchors are attached to ligands but some are not. To distinguish between these situations, a key parameter, surface anchor density, was introduced to specify amount of total anchors on the liposomal surface. Second, the parameter reaction yield was introduced to identify the amount of ligand-attached anchors among total anchors, since the conjugation efficiency is not always the same nor 100%. Combining these independent parameters, we derived: incorporation ratio=surface anchor densityxreaction yield. The term incorporation ratio defines the surface ligand density. Since the surface anchor density represents the density of polyethylene glycol (PEG) on the surfaces in most cases, it also determines liposomal function. It is possible to accurately characterize various PEG and ligand densities and to define the surface topologies. In conclusion, this quantitative methodology can standardize the liposome preparation process and qualify the modified liposomal surfaces.
  • Takashi Nakamura, Moeka Kuroi, Yuki Fujiwara, Shota Warashina, Yusuke Sato, Hideyoshi Harashima
    SCIENTIFIC REPORTS 6 37849  2016年11月 [査読有り][通常論文]
     
    Gene silencing by small interfering RNA (siRNA) is useful for analyzing the functions of human immune cells. However, the transfection of siRNA to human immune cells is difficult. Here, we used a multifunctional envelope-type nanodevice (MEND) containing YSK12-C4 (YSK12-MEND) to efficiently introduce siRNA to human immune cell lines, Jurkat, THP-1, KG-1 and NK92. The YSK12-MEND was transfected to human immune cell lines at a siRNA dose range of 1-30 nM, resulting that maximum gene silencing efficiencies at the mRNA level in Jurkat, THP-1, KG-1 and NK92 were 96%, 96%, 91% and 75%, respectively. The corresponding values for Lipofectamine RNAiMAX (RNAiMAX) were 37%, 56%, 43% and 19%, respectively. The process associated with cellular uptake played a role in effective gene silencing effect of the YSK12-MEND. The small size and high non-aggregability of the YSK12MEND were advantageous for the cellular internalization of siRNA to immune cell lines. In the case of RNAiMAX, a drastic increase in particles size was observed in the medium used, which inhibited cellular uptake. The YSK12-MEND reported in herein appears to be appropriate for delivering siRNA to human immune cells, and the small particle size and non-aggregability are essential properties.
  • Garima Shrivastava, Mamoru Hyodo, Shige H. Yoshimura, Hidetaka Akita, Hideyoshi Harashima
    NUCLEIC ACID THERAPEUTICS 26 5 286 - 298 2016年10月 [査読有り][通常論文]
     
    An active targeting drug delivery system that targets the nucleus could solve the problem of the treatment of genetic disorders through gene delivery, but it has met with limited success. The purpose of this study was to establish an RNA aptamer-modified nucleus-targeting liposomal carrier system referred to as NupApt-liposomes. RNA aptamers against the Nup358 protein are prepared using a newly established Protein SELEX method. After confirming aptamer binding to the recombinant protein, an aptamer-lipid conjugate (Apt-PEG-DSPE) was prepared. Aptamer-modified liposomes and simple polyethylene glycol (PEG) liposomes were prepared to check its ability to bind to isolated nuclei. Confocal studies indicated that the aptamer-modified liposomes had the ability to bind to isolated nuclei, whereas PEG-liposomes showed only weak binding. Confocal laser scanning microscopy studies of inhibition assays also supported the above conclusion. The dissociation constant of the Nucleoporin358-specific aptamer referred to as NupApt01 and NupApt02 were 36 and 70nM, respectively. Finally, with aptamer-modified liposomes, gene expression studies showed a two times better gene expression in NupApt-liposome-treated nuclei in comparison to that of PEG-liposomes. This represents the first artificial RNA aptamer-modified liposomes promoting the specific binding of a nanocarrier to the nucleus, thus improving gene expression in comparison to PEG-liposomes.
  • Kazuaki Kajimoto, Erina Suemitsu, Yusuke Sato, Yu Sakurai, Hideyoshi Harashima
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 39 10 1653 - 1661 2016年10月 [査読有り][通常論文]
     
    Lipin1, a bifunctional protein, regulates fatty acid utilization in the triglyceride biosynthesis pathway. In the current study, using a liver-specific in vivo short interfering RNA (siRNA) delivery system, we examined the pathological and physiological roles of hepatic Lipin1 in the development of insulin resistance and the maintenance of systemic energy homeostasis. Liver-specific silencing of Lipin1 expression was achieved by the systemic administration of siRNA against Lpin1 mRNA (siLpin1)-loaded lipid nanoparticles (LNPs) to wild type mice at 3-4 d intervals for 25 d. The siLpin1-treated mice showed normal blood glucose levels and insulin sensitivity, however, triglyceride (TG) levels were reduced in liver and peripheral blood of them. The knockdown of hepatic Lipin1 in mice led to marked decrease in adipose tissue mass and adipocyte diameters in epididymal and inguinal fat depots without the undesired silencing of Lipin1 in adipose tissue. In summary, we report for the first time that the down-regulation of hepatic Lipin1 expression leads to less adiposity as well as a decrease in TG level in the liver and blood circulation, without any alterations in the glucose tolerance and blood glucose levels. Our findings may provide new insights into the physiological roles of hepatic Lipin1 in systemic energy homeostasis.
  • ミトコンドリア薬物送達システムを用いた心筋炎に対する新たな治療戦略
    阿部 二郎, 山田 勇磨, 武田 充人, 原島 秀吉
    日本小児循環器学会雑誌 32 Suppl.1 s1 - 208 (NPO)日本小児循環器学会 2016年07月 [査読有り][通常論文]
  • Ayaka Watanabe, Hiroki Tanaka, Yu Sakurai, Kota Tange, Yuta Nakai, Tatsuya Ohkawara, Hiroshi Takeda, Hideyoshi Harashima, Hidetaka Akita
    INTERNATIONAL JOURNAL OF PHARMACEUTICS 509 1-2 118 - 122 2016年07月 [査読有り][通常論文]
     
    Taking advantage of the enhanced permeation and retention (EPR) effect is a promising approach for delivering macromolecules or nanoparticles to tumors. Recent studies revealed that this strategy is also applicable for targeting other pathological lesions (i.e. inflammatory disease). In the present study, we report the optimal size of a nanoparticle for allowing the higher accumulation of a particle in an inflammatory lesion using a dextran sulfate sodium (DSS)-induced colitis model. As a nanoparticle platform, we utilized a SS-cleavable and pH-activated lipid-like material (ssPalm), that can be used to produce particles in a variety of sizes ranging from 50 nm to 180 nm while using the same lipid composition. In healthy mice, particle accumulation remained low regardless of size. In contrast, the accumulation in inflammatory colon tissue was enhanced depending on the progress of the inflammation. In this situation, the apparent uptake clearance accumulation of a mid-sized particle (113 nm on average) was higher than that for smaller and larger (54 nm and 183 nm in average, respectively) ones. Therefore, controlling particle size is an important parameter for the extensive targeting of inflammatory lesion. (C) 2016 Elsevier B.V. All rights reserved.
  • Golam Kibria, Hiroto Hatakeyama, Yusuke Sato, Hideyoshi Harashima
    INTERNATIONAL JOURNAL OF PHARMACEUTICS 509 1-2 178 - 187 2016年07月 [査読有り][通常論文]
     
    The PEGylated liposomal (PEG-LP) Doxorubicin, PEG-LP (DOX), with a diameter of around 100 nm, accumulates in tumors via the enhanced permeability and retention (EPR) effect, and is used clinically for the treatment of several types of cancer. However, there are a number of tumor types that are resistant to DOX. We report herein on a unique anti-tumor effect of PEG-LP (DOX) in a DOX-resistant tumor xenograft model. PEG-LP (DOX) failed to suppress the growth of the DOX-resistant tumors (ex. non-small cell lung cancer, H69AR; renal cell carcinoma, OSRC-2) as observed in the xenograft model. Unexpectedly, tumor growth was suppressed in a DOX-resistant breast cancer (MDA-MB-231) xenograft model. We investigated the mechanism by which PEG-LP (DOX) responses differ in different drug resistant tumors. In hyperpermeable OSRC-2 tumors, PEG-LP was distributed to deep tumor tissues, where it delivers DOX to drug-resistant tumor cells. In contrast, extracellular matrix (ECM) molecules such as collagen, pericytes, cancer-associated fibroblasts render MDA-MB-231 tumors hypopermeable, which limits the extent of the penetration and distribution of PEG-LP, thereby enhancing the delivery of DOX to the vicinity of the tumor vasculature. Therefore, a remarkable anti-angiogenic effect with a preferential suppression in tumor growth is achieved. Based on the above findings, it appears that the response of PEG-LP (DOX) to drug-resistant tumors results from differences in the tumor microenvironment. (C) 2016 Elsevier B.V. All rights reserved.
  • Saed Abbasi, Kazuaki Kajimoto, Hideyoshi Harashima
    International Journal of Nanomedicine 11 2685 - 2694 2016年06月08日 [査読有り][通常論文]
     
    15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) has a dual action of stimulating anti-inflammation and anti-proliferation when exogenously administered at high doses. However, at lower doses, it can be toxic inducing opposite actions, ie, stimulation of both inflammation and cell proliferation. This biphasic phenomenon of 15d-PGJ2 is believed to be due to its multitarget behavior. In this study, we provide a strategy for controlling such biphasic pharmacodynamics by separating its dual actions while retaining the beneficial one by using a nanoemulsion (NE). The 15d-PGJ2 was encapsulated in the NE composed of triolein/distearoyl phosphatidylcholine/Tween 80 at a high encapsulation ratio (> 83%). Furthermore, NE enhanced drug retention by slowing down its release rate, which was, unconventionally, inversely dependent on the total surface area of the NE system. Next, focusing on the biphasic effect on cell proliferation, we found that the 15d-PGJ2-loaded slow-release NE showed only a dose-dependent inhibition of the viability of a mouse macrophage cell line, RAW264.7, although a fast-release NE as well as free 15d-PGJ2 exerted a biphasic effect. The observed slow-release kinetics are believed to be responsible for elimination of the biphasic pharmacodynamics of 15d-PGJ2 mainly for two reasons: 1) a high proportion of 15d-PGJ2 that is retained in the NE was delivered to the cytosol, where proapoptotic targets are located and 2) 15d-PGJ2 was able to bypass cell membrane-associated targets that lead to the induction of cellular proliferation. Collectively, our strategy of eliminating the 15d-PGJ2-induced biphasic pharmacodynamics was based on the delivery of 15d-PGJ2 to its desired site of action, excluding undesired sites, on a subcellular level.
  • プラスミドDNA結合ヒストンの特異的アセチル化による外来遺伝子発現上昇
    紙谷 浩之, 西原 実香, 神田 元紀, 鈴木 哲矢, 山門 振一郎, 原島 秀吉
    日本DDS学会学術集会プログラム予稿集 32回 153 - 153 日本DDS学会 2016年06月
  • Tomohito Tanoi, Takafumi Tamura, Naoki Sano, Ken Nakayama, Kiyoshi Fukunaga, Yun-Wen Zheng, Afsana Akhter, Yu Sakurai, Yasuhiro Hayashi, Hideyoshi Harashima, Nobuhiro Ohkohchi
    HEPATOLOGY RESEARCH 46 7 697 - 706 2016年06月 [査読有り][通常論文]
     
    Aim: Apoptosis is associated with various types of hepatic disorders. We have developed a novel cell-transfer drug delivery system (DDS) using a multifunctional envelope-type nano device that targets liver sinusoidal endothelial cells (LSECs). The purpose of this study was to determine the efficacy of the novel DDS containing siRNA at suppressing apoptosis in LSECs. Methods: Bax siRNA was transfected into a sinusoidal endothelial cell line (M1) to suppress apoptosis induced by an anti-Fas antibody and staurosporine. C57BL/6J mice were divided into three groups: (i) a control group, only intravenous saline; (ii) a nonselective group, injections of siRNA sealed in the nonselective DDS; and (iii) an LSEC-transfer efficient group, injections of siRNA sealed in an LSEC-transfer efficient DDS. Hepatic cell apoptosis was induced by an anti-Fas antibody. Results: Bax siRNA had an anti-apoptotic effect on M1 cells. Serum alanine aminotransferase was reduced in the LSEC-transfer efficient group, as were cleaved caspase-3 and the number of terminal deoxynucleotidyl transferase dUTP nick end labeling positive hepatocytes. Silver impregnation staining indicated that the sinusoidal space was maintained in the LSEC-transfer efficient group but not in the other groups. Electron microscopy showed that the LSECs were slightly impaired, although the sinusoidal structure was maintained in the LSEC-transfer efficient group. Conclusion: Hepatocyte apoptosis was reduced by the efficient suppression of LSEC apoptosis with a novel DDS. Protecting the sinusoidal structure by suppressing LSEC damage will be an effective treatment for acute liver failure.
  • Hiroyuki Kamiya, Mana Ito, Kosuke Nishi, Hideyoshi Harashima
    JOURNAL OF BIOTECHNOLOGY 228 52 - 57 2016年06月 [査読有り][通常論文]
     
    A novel in vivo selection method for active deoxyribonucleoside kinase proteins is described here. A pool of randomly mutated genes for deoxyribonucleoside kinase from Drosophila melanogaster (Dm-dNK) was prepared and inserted into an expression vector. Enzymatically active mutants were selected by repeated cycles, including (i) introduction into Escherichia coli, (ii) treatment of the E. coli pool with a mutagenic deoxyribonucleoside (2-hydroxy-dA), and (iii) selection of antibiotic-resistant colonies resulting from mutations by phosphorylated 2-hydroxy-dA and the subsequent isolation of the plasmid DNAs. The ratio of the resistant colonies increased by two orders of magnitude from the first cycle to the fifth cycle, and then reached a plateau. Fifteen Dm-dNK mutants selected after the seventh and eighth evolution cycles were actually active in vivo. Moreover, one of the mutant proteins was as active as the wild type protein in vitro. These results indicate that this novel in vivo evolution method was useful and that similar strategies would be applicable to other deoxyribonucleoside kinases. In addition, the distribution of mutated amino acids suggests important residues/regions in the Dm-dNK protein. (C) 2016 Elsevier B.V. All rights reserved.
  • Yusuke Sato, Yusuke Note, Masatoshi Maeki, Noritada Kaji, Yoshinobu Baba, Manabu Tokeshi, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 229 48 - 57 2016年05月 [査読有り][通常論文]
     
    Because nanoparticles with diameters less than 50 nm penetrate stromal-rich tumor tissues more efficiently, the synthesis of small-sized nanoparticles encapsulating short interfering RNA (siRNA) is important in terms of realizing novel siRNA medicine for the treatment of various cancers. Lipid nanoparticles (LNPs) are the leading systems for the delivery of siRNA in vivo. Limit size LNPs were successfully synthesized using a microfluidic mixing technique. However, the physicochemical properties and potential for in vivo siRNA delivery of the limit-size LNPs have not been examined in detail. In the present study, we prepared LNPs with different diameters from 32 to 67 nm using a microfluidic mixing devise and examined the physicochemical properties of the particles and the potential for their use in delivering siRNA in vitro and in vivo to liver. Reducing the size of the LNPs causes poor-packing and an increased surface area, which result in their instability in serum. Moreover, it was revealed that the ability of endosomal escape (cytosolic siRNA release) of the smaller LNPs is subject to inhibition by serum compared to that of larger counterparts. Taken together, an increase in packing and avoiding the adsorption of serum components are key strategies for the development of next-generation highly potent and small-sized LNPs. (C) 2016 Elsevier B.V. All rights reserved.
  • Yuma Yamada, Ryo Furukawa, Hideyoshi Harashima
    JOURNAL OF PHARMACEUTICAL SCIENCES 105 5 1705 - 1713 2016年05月 [査読有り][通常論文]
     
    It has been reported that the use of mitochondrial RNA aptamers including RNase P (RP) results in the selective mitochondrial delivery of endogenous and exogenous RNAs. The issue of whether these aptamers would be useful ligands for the mitochondrial targeting of a nanoparticle has not been demonstrated to date because nanocarriers modified with these RNA aptamers are insufficiently internalized by cells. We report here on the development of a dual-ligand liposomal system composed of octaarginine (R8), a device that enhances cellular uptake, and an RP aptamer for mitochondrial targeting to permit a nanocarrier to be efficiently delivered to mitochondria. Surprisingly, the cellular uptake of the R8-modified nanocarrier was facilitated by modification with an RP aptamer. The optimal composition of a nanocarrier needed for efficient cellular uptake and mitochondrial targeting was determined. In a confocal laser scanning microscopy analysis, the dual-ligandemodified nanocarrier was found to result in effective mitochondrial targeting through an ATP-dependent pathway and was much more effective than a single-ligand R8-modified nanocarrier. This is the first report of the regulation of intracellular trafficking by a mitochondrial RNA aptamer-modified nanocarrier system. (C) 2016 American Pharmacists Association (R). Published by Elsevier Inc. All rights reserved.
  • Kyoko Hida, Nako Maishi, Yu Sakurai, Yasuhiro Hida, Hideyoshi Harashima
    ADVANCED DRUG DELIVERY REVIEWS 99 Pt B 140 - 147 2016年04月 [査読有り][通常論文]
     
    To date anti-angiogenic therapy has been used for cancer therapy widely, yielding promising results. However, it has been elucidated that current anti-angiogenic drug has several issues to be solved, such as side-effects and drug resistance. It has been reported that tumor endothelial cells (TECs) differ from normal counterparts. In addition, it was shown that the TECs are heterogeneous according to the malignancy status of tumor. The development of novel strategy for targeting tumor vasculature is required. Recently, we have developed an active targeting system, which targets TECs specifically. In this review, we will discuss how TECs in tumor vasculature are heterogeneous and offer new perspectives on a drug delivery system, which can target heterogeneous tumor blood vessels from a viewpoint of personalized medicine. (C) 2015 Elsevier B.V. All rights reserved.
  • Yusuke Sato, Hiroto Hatakeyama, Mamoru Hyodo, Hideyoshi Harashima
    MOLECULAR THERAPY 24 4 788 - 795 2016年04月 [査読有り][通常論文]
     
    While a variety of short interfering RNA (siRNA) delivery compounds have been developed, a deep understanding of the key parameters that determine the quality of siRNA delivery are not known with certainty. Therefore, an understanding of the factors required for the efficient, selective, and safe delivery of siRNA is a great challenge for successful siRNA delivery. Herein, we report on the development of two pH-sensitive cationic lipids and their use in examining the impact of the acid dissociation constant (pK(a)) value, lipase sensitivity and the size of lipid nanoparticles on the biodistribution, and efficiency and cell specificity of gene silencing in the liver. An increase in the pK(a) value resulted in a significant change in the intrahepatic localization of siRNA and gene-silencing efficiency in hepatocytes and liver sinusoidal endothelial cells (LSECs). The sensitivity of the pH-sensitive cationic lipid to lipases was a major factor in achieving hepatocyte-specific gene silencing. Increasing the particle size can improve the LSEC specificity of gene silencing. As a consequence, we succeeded in developing both a highly efficient, hepatocyte-specific formulation, and the most efficacious LSEC-targeted formulation reported to date. These findings will facilitate the development of more sophisticated siRNA delivery systems.
  • Asako Yamada, Asako Mitsueda, Mahadi Hasan, Miho Ueda, Susumu Hama, Shota Warashina, Takashi Nakamura, Hideyoshi Harashima, Kentaro Kogure
    Biomaterials Science 4 3 439 - 447 2016年03月01日 [査読有り][通常論文]
     
    Membrane fusion is a rational strategy for crossing intracellular membranes that present barriers to liposomal nanocarrier-mediated delivery of plasmid DNA into the nucleus of non-dividing cells, such as dendritic cells. Based on this strategy, we previously developed nanocarriers consisting of a nucleic acid core particle coated with four lipid membranes [Akita, et al., Biomaterials, 2009, 30, 2940-2949]. However, including the endosomal membrane and two nuclear membranes, cells possess three intracellular membranous barriers. Thus, after entering the nucleus, nanoparticles coated with four membranes would still have one lipid membrane remaining, and could impede cargo delivery. Until now, coating a core particle with an odd number of lipid membranes was challenging. To produce nanocarriers with an odd number of lipid membranes, we developed a novel coating method involving lipid nano-discs, also known as bicelles, as a material for packaging DNA in a carrier with an odd number of lipid membranes. In this procedure, bicelles fuse to form an outer coating that resembles a patchwork quilt, which allows the preparation of nanoparticles coated with only three lipid membranes. Moreover, the transfection activity of dendritic cells with these three-membrane nanoparticles was higher than that for nanoparticles coated with four lipid membranes. In summary, we developed novel nanoparticles coated with an odd number of lipid membranes using the novel "patchwork-packaging method" to deliver plasmid DNA into the nucleus via membrane fusion.
  • Hidetaka Akita, Takahiro Fujiwara, Sarochin Santiwarangkool, Nazir Hossen, Kazuaki Kajimoto, Ayman El-Sayed, Yasuhiko Tabata, Hideyoshi Harashima
    SMALL 12 9 1212 - 1221 2016年03月 [査読有り][通常論文]
     
    The ultimate goal in the area of drug-delivery systems is the development of a nanoparticle that can penetrate the endothelial cell monolayer for the targeting of tissue parenchyma. In the present study, we identify a transcytosis-targeting peptide (TTP) that permits polyethyleneglycol (PEG)-modified liposomes (PEG-LPs) to penetrate through monolayers of brain-derived endothelial cells. These endothelial cells were layered on a gelatin nanofiber sheet, a nanofiber meshwork that allows the evaluation of transcellular transport of nanosized particles (ca. 100 nm). Systematic modification of the sequences results in the identification of the consensus sequence of TTP as L(R/K) QZZZL, where Z denotes hydrophilic amino acids (R/K/S and partially D). The TTP-modified liposomes are bound on the heparin sulfate proteoglycan, and are then taken up via lipid raft-mediated endocytosis. Subsequent intracellular imaging of the particles reveals a unique intracellular sorting of TTP-modified PEG liposomes (TTP-PEG-LPs); namely the TTP-LPs are not localized with the lysosomes, whereas this co-localization is dominant in the unmodified PEG liposomes (PEG-LPs). The in vivo endothelial penetration of liposomes in adipose tissue is conferred by the dual modification of the particles with TTP and tissue-targeting ligands. This technology promises innovations in intravenously available delivery system to tissue parenchyma.
  • Naoki Yamamoto, Yusuke Sato, Tsubasa Munakata, Masakazu Kakuni, Chise Tateno, Takahiro Sanada, Yuichi Hirata, Shuko Murakami, Yasuhito Tanaka, Kazuaki Chayama, Hiroto Hatakeyama, Mamoru Hyodo, Hideyoshi Harashima, Michinori Kohara
    JOURNAL OF HEPATOLOGY 64 3 547 - 555 2016年03月 [査読有り][通常論文]
     
    Background & Aims: Antiviral agents including entecavir (ETV) suppress the replication of the hepatitis B virus (HBV) genome in human hepatocytes, but they do not reduce the abundance of viral proteins. The present study focused on effectively reducing viral protein levels. Methods: We designed siRNAs (HBV-siRNA) that target consensus sequences in HBV genomes. To prevent the emergence of escaped mutant virus, we mixed three HBV-siRNAs (HBV-siRNAmix); the mixture was encapsulated in a novel pH-sensitive multifunctional envelope-type nanodevice (MEND), a hepatocyte-specific drug delivery system. Coagulation factor 7 siRNA was used to assess delivery and knockdown efficiencies of MEND/siRNA treatments in mice. The potency of MEND/HBV-siRNAmix was evaluated in primary human hepatocytes and in chimeric mice with humanized liver persistently infected with HBV. Results: Effective knockdown of targets, efficient delivery of siRNA, and liver-specific delivery were each observed with MEND. MEND/HBV-siRNA caused efficient reduction of HBsAg and HBeAg in vitro and in vivo. However, ETV treatment did not efficiently reduce HBsAg or HBeAg when compared with a single MEND/HBV-siRNAmix treatment. Furthermore, the suppressive effects of a single dose of MEND/HBV-siRNAmix persisted for 14 days in vitro and in vivo. Conclusion: We demonstrated that MEND/HBV-siRNA controlled HBV more efficiently than did ETV. Furthermore, the effect of a single dose of MEND/HBV-siRNA persisted for a long time. These results indicated that MEND/HBV-siRNA may be a promising novel HBV treatment that is more effective than reverse transcriptase inhibitors. (C) 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
  • Zhang T, Kim DH, Xiao X, Lee S, Gong Z, Muzumdar R, Calabuig-Navarro V, Yamauchi J, Harashima H, Wang R, Bottino R, Alvarez-Perez JC, Garcia-Ocaña A, Gittes G, Dong HH
    Endocrinology 157 3 1055 - 1070 2016年03月 [査読有り][通常論文]
     
    beta-Cell compensation is an essential mechanism by which beta-cells increase insulin secretion for overcoming insulin resistance to maintain euglycemia in obesity. Failure of beta-cells to compensate for insulin resistance contributes to insulin insufficiency and overt diabetes. To understand the mechanism of beta-cell compensation, we characterized the role of forkhead box O1 (FoxO1) in beta-cell compensation in mice under physiological and pathological conditions. FoxO1is a key transcription factor that serves as a nutrient sensor for integrating insulin signaling to cell metabolism, growth, and proliferation. We showed that FoxO1 improved beta-cell compensation via 3 distinct mechanisms by increasing beta-cell mass, enhancing beta-cell glucose sensing, and augmenting beta-cell antioxidative function. These effects accounted for increased glucose-stimulated insulin secretion and enhanced glucose tolerance in beta-cell-specific FoxO1-transgenic mice. When fed a high-fat diet, beta-cell-specific FoxO1-transgenic mice were protected from developing fat-induced glucose disorder. This effect was attributable to increased beta-cell mass and function. Furthermore, we showed that FoxO1 activity was up-regulated in islets, correlating with the induction of physiological beta-cell compensation in high-fat-induced obese C57BL/6J mice. These data characterize FoxO1 as a pivotal factor for orchestrating physiological adaptation of beta-cell mass and function to overnutrition and obesity.
  • Shota Warashina, Takashi Nakamura, Yusuke Sato, Yuki Fujiwara, Mamoru Hyodo, Hiroto Hatakeyama, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 225 183 - 191 2016年03月 [査読有り][通常論文]
     
    Applying small interfering RNA (siRNA) to dendritic cell (DC) based therapy represents a potential candidate for cancer immunotherapy. However, delivering siRNA to DCs is a challenging issue for non-viral vectors. To date, only viral vectors have achieved efficient gene silencing in DCs. We report herein that a novel cationic lipid, YSK12-C4, when loaded in a nanoparticle with siRNA (YSK12-C4 multifunctional envelope type nano device [YSK12-MEND]), greatly facilitated gene silencing in mouse DCs. The use of the YSK12-MEND resulted in a gene silencing efficiency in excess of 90%, with a median effective dose (ED50) of 1.5 nM, whereas the maximum gene silencing efficiency of Lipofectamine RNAiMAX was less than 60% and the ED50 was 25 nM. Furthermore, suppressor of cytokine signaling 1, an immune suppressive molecule in DCs, silenced in the mouse DC by the YSK12-MEND showed a drastic enhancement in cytokine production, resulting in the significant suppression of tumor growth when it was applied to DC-based therapy against a mouse lymphoma. These results clearly indicate that YSK12-MEND overcomes the obstacle associated with non-viral vectors and can be considered to be a promising non-viral vector for siRNA delivery to DCs, thus accelerating DC-based therapies with siRNA. (C) 2016 Elsevier B.V. All rights reserved.
  • Jiro Abe, Yuma Yamada, Hideyoshi Harashima
    JOURNAL OF PHARMACEUTICAL SCIENCES 105 2 734 - 740 2016年02月 [査読有り][通常論文]
     
    Mitochondria in human cancer cells have been implicated in cancer cell proliferation, invasion, metastasis, and even drug-resistance mechanisms, making them a potential target organelle for the treatment of human malignancies. Gentamicin (GM), an aminoglycoside drug (AG), is a small molecule that functions as an antibiotic and has ototoxic and nephrotoxic characteristics. Thus, the delivery of GM to mitochondria in cancer cells would be an innovative anticancer therapeutic strategy. In this study, we attempted mitochondrial delivery of GM in HeLa cells derived from a human cervical cancer. For the mitochondrial delivery, we used MITO-Porter, a liposomal nanocarrier for mitochondrial delivery via membrane fusion. We first encapsulated GM in the aqueous phase of the carrier to construct GM-MITO-Porter. Flow cytometry analysis and fluorescent microscopy observations permitted us to confirm that the GMeMITO-Porter was efficiently taken up by HeLa cells and accumulated in mitochondria, whereas naked GM was not taken up by the cells. Moreover, cell viability assays using HeLa cells showed that the GMeMITO-Porter induced strong cytotoxic effects related to mitochondrial disorder. This finding is the first report of the mitochondrial delivery of an AG to cancer cells for cancer therapeutic strategy. (C) 2016 American Pharmacists Association (R). Published by Elsevier Inc. All rights reserved.
  • Hiroto Hatakeyama, Hideyoshi Harashima
    Drug Delivery System 31 4 293 - 299 2016年 [査読有り][通常論文]
     
    Gene and nucleic acid therapy are expected to play a major role in the next generation of medicine. We recently developed a multifunctional envelope-type nano device (MEND) for use as a novel non-viral gene delivery system. PEGylation is a useful method for achieving a longer circulation time for delivery of the MEND to a tumor via the EPR effect. However, PEGylation strongly inhibits cellular uptake and endosomal escape, which results significant loss of activity for the delivery system. For successful gene delivery for cancer treatment, the crucial issue associated with the use of PEG, the "PEG dilemma" must be solved. Here, we describe the development and applications of MEND, and discuss strategies for overcoming the PEG dilemma, based on the manipulation of intracellular trafficking using functional devices such as cleavable PEG systems, endosomal fusogenic peptides, and pH-sensitive lipids.
  • Saed Abbasi, Kazuaki Kajimoto, Hideyoshi Harashima
    INTERNATIONAL JOURNAL OF NANOMEDICINE 11 2685 - 2694 2016年 [査読有り][通常論文]
     
    15-Deoxy-Delta 12,14-prostaglandin J2 (15d-PGJ2) has a dual action of stimulating anti-inflammation and anti-proliferation when exogenously administered at high doses. However, at lower doses, it can be toxic inducing opposite actions, ie, stimulation of both inflammation and cell proliferation. This biphasic phenomenon of 15d-PGJ2 is believed to be due to its multitarget behavior. In this study, we provide a strategy for controlling such biphasic pharmacodynamics by separating its dual actions while retaining the beneficial one by using a nanoemulsion (NE). The 15d-PGJ2 was encapsulated in the NE composed of triolein/distearoyl phosphatidylcholine/Tween 80 at a high encapsulation ratio (> 83%). Furthermore, NE enhanced drug retention by slowing down its release rate, which was, unconventionally, inversely dependent on the total surface area of the NE system. Next, focusing on the biphasic effect on cell proliferation, we found that the 15d-PGJ2-loaded slow-release NE showed only a dose-dependent inhibition of the viability of a mouse macrophage cell line, RAW264.7, although a fast-release NE as well as free 15d-PGJ2 exerted a biphasic effect. The observed slow-release kinetics are believed to be responsible for elimination of the biphasic pharmacodynamics of 15d-PGJ2 mainly for two reasons: 1) a high proportion of 15d-PGJ2 that is retained in the NE was delivered to the cytosol, where proapoptotic targets are located and 2) 15d-PGJ2 was able to bypass cell membrane-associated targets that lead to the induction of cellular proliferation. Collectively, our strategy of eliminating the 15d-PGJ2-induced biphasic pharmacodynamics was based on the delivery of 15d-PGJ2 to its desired site of action, excluding undesired sites, on a subcellular level.
  • Sakurai Y, Hada T, Harashima H
    Methods in molecular biology (Clifton, N.J.) 1364 63 - 69 2016年 [査読有り][通常論文]
  • Sato Y, Harashima H, Kohara M
    Methods in molecular biology (Clifton, N.J.) 1364 71 - 78 2016年 [査読有り][通常論文]
  • Asako Yamada, Asako Mitsueda, Mahadi Hasan, Miho Ueda, Susumu Hama, Shota Warashina, Takashi Nakamura, Hideyoshi Harashima, Kentaro Kogure
    BIOMATERIALS SCIENCE 4 3 439 - 447 2016年 [査読有り][通常論文]
     
    Membrane fusion is a rational strategy for crossing intracellular membranes that present barriers to liposomal nanocarrier-mediated delivery of plasmid DNA into the nucleus of non-dividing cells, such as dendritic cells. Based on this strategy, we previously developed nanocarriers consisting of a nucleic acid core particle coated with four lipid membranes [Akita, et al., Biomaterials, 2009, 30, 2940-2949]. However, including the endosomal membrane and two nuclear membranes, cells possess three intracellular membranous barriers. Thus, after entering the nucleus, nanoparticles coated with four membranes would still have one lipid membrane remaining, and could impede cargo delivery. Until now, coating a core particle with an odd number of lipid membranes was challenging. To produce nanocarriers with an odd number of lipid membranes, we developed a novel coating method involving lipid nano-discs, also known as bicelles, as a material for packaging DNA in a carrier with an odd number of lipid membranes. In this procedure, bicelles fuse to form an outer coating that resembles a patchwork quilt, which allows the preparation of nanoparticles coated with only three lipid membranes. Moreover, the transfection activity of dendritic cells with these three-membrane nanoparticles was higher than that for nanoparticles coated with four lipid membranes. In summary, we developed novel nanoparticles coated with an odd number of lipid membranes using the novel "patchwork-packaging method" to deliver plasmid DNA into the nucleus via membrane fusion.
  • Abdelmegeed H, Nakamura T, Harashima H
    Journal of pharmaceutical sciences 105 1 250 - 256 2016年01月 [査読有り][通常論文]
     
    Alpha-galactosylceramide (GC) represents a potentially new class of adjuvant because GC strongly induces interferon (IFN) gamma production from natural killer T (NKT) cells, leading to the induction of strong antitumor immunity. Interleukin (IL)-12 is another stimulating signal that induces IFN-gamma production by NKT cells. We report herein on an investigation of the effect of recombinant IL-12 on NKT cell activation, when used in combination with GC-loaded octaarginine modified liposomes (GC-Lip). IFN-gamma production from splenocytes simulated with GC-Lip was dose dependently enhanced in the presence of IL-12 in vitro. In contrast, IFN-gamma production in vivo was enhanced at a low dose of IL-12. Enhanced IFN-gamma production was observed in the case of low doses (0.5 mu g and 2.5 mu g) of GC-Lip but not a high dose (5 mu g), that is, the IL-12 combination enhanced NKT cell activation at a 10-fold lower GC dose. The use of the above combination also enhanced the expansion of the NKT cell population. These findings indicate that in vivo IFN-gamma production is inversely correlated with the dose of IL-12 during dual signal stimulation of NKT cells via both GC-Lip and IL-12, indicating that the dose of GC-Lip can be reduced without weakening NKT cell activation. (C) 2016 American Pharmacists Association (R). Published by Elsevier Inc. All rights reserved.
  • Hiroki Tanaka, Yusuke Sato, Hideyoshi Harashima, Hidetaka Akita
    EXPERT OPINION ON DRUG DELIVERY 13 7 1015 - 1027 2016年 [査読有り][通常論文]
     
    Introduction: The development of gene and nucleic acid-based medication is one of the ultimate strategies in the research field of personalized medicine. For the desired function of a gene or siRNA, these molecules need to be delivered to the appropriate organelle (i.e. nucleus and cytoplasm, respectively). Areas covered: The topics covered herein are rational design in order to control the pharmacokinetics, intracellular trafficking and release (decondensation or decapsulation) of the intended material. Since the endosome and cytoplasm are acidic (endosome) and reducing (cytoplasm) environments, respectively, a large variety of the materials have been developed that induce destabilization of endosome via its protonation, or are spontaneously collapsed in the cytoplasm. Finally, we propose materials (SS-cleavable and pH-activated lipid-like materials: ssPalm) that mount these sensing motifs, i.e., a positive charging unit in response to the acid environment (tertiary amines) and a cleavage unit (disulfide bonding) that is responsive to an reducing environment, respectively. Expert opinion: Currently, the main target of the nanocarrier-mediated siRNA delivery systems is liver. The targeting of non-hepatic tissue is the next challenge. In this case, the design of neutral particle with well-organized intracellular trafficking, as well as an identification of the promising ligand is needed.
  • Hiroyuki Kamiya, Natsuki Nishigaki, Akihiro Ikeda, Seiya Yukawa, Yukiko Morita, Yoshimichi Nakatsu, Teruhisa Tsuzuki, Hideyoshi Harashima
    NUCLEOSIDES NUCLEOTIDES & NUCLEIC ACIDS 35 7 379 - 388 2016年 [査読有り][通常論文]
     
    A 5-tailed duplex (TD) DNA corrects a base-substitution mutation. In this study, the effects of insertion and deletion (indel) mismatches distant from the target position on the gene correction were examined. Three target plasmid DNAs with and without indel mismatches approximate to 330 bases distant from the correction target position were prepared, and introduced into HeLa cells together with the TD. The indel mismatches improved the gene correction efficiency and specificity without sequence conversions at the indel mismatch site. These results suggested that the gene correction efficiency and specificity are increased when an appropriate second mismatch is introduced into the TD fragment.
  • Yukari Yasuzaki, Yuma Yamada, Takuya Ishikawa, Hideyoshi Harashima
    MOLECULAR PHARMACEUTICS 12 12 4311 - 4320 2015年12月 [査読有り][通常論文]
     
    For successful mitochondrial transgene expression, two independent processes, i.e., developing a mitochondrial gene delivery system and construction of DNA vector to achieve mitochondrial gene expression, are required. To date, very few studies dealing with mitochondrial gene delivery have been reported and, in most cases, transgene expression was not validated, because the construction of a reporter DNA vector for mitochondrial gene expression is the bottleneck. In this study, mitochondrial transgene expression by the in vivo mitochondrial gene delivery of an artificial mitochondrial reporter DNA vector via hydrodynamic injection is demonstrated. In the procedure, a large volume of naked plasmid DNA (pDNA) is rapidly injected. We designed and constructed pHSP-mtLuc (CGG) as a mitochondrial reporter DNA vector that possesses a mitochondrial heavy strand promoter (HSP) and an artificial mitochondrial genome with the reporter NanoLuc (Nluc) luciferase gene that records adjustments to the mitochondrial codon system. We delivered the pDNA into mouse liver mitochondria by hydrodynamic injection, and detected exogenous mRNA in the liver using reverse transcription PCR analysis. The hydrodynamic injection of pHSP-mtLuc (CGG) resulted in the expression of the Nluc luciferase protein in liver and skeletal muscle. Our mitochondrial transgene expression reporter system would contribute to mitochondrial gene therapy and further studies directed at mitochondrial molecular biology.
  • Naoki Sano, Takafumi Tamura, Naoyuki Toriyabe, Takeshi Nowatari, Ken Nakayama, Tomohito Tanoi, Soichiro Murata, Yu Sakurai, Mamoru Hyodo, Kiyoshi Fukunaga, Hideyoshi Harashima, Nobuhiro Ohkohchi
    WORLD JOURNAL OF GASTROENTEROLOGY 21 45 12778 - 12786 2015年12月 [査読有り][通常論文]
     
    AIM: To investigate the cytoprotective effects in hepatic ischemia-reperfusion injury, we developed a new formulation of hyaluronic acid (HA) and sphingosine 1-phophate. METHODS: We divided Sprague-Dawley rats into 4 groups: control, HA, sphingosine 1-phosphate (S1P), and HA-S1P. After the administration of each agent, we subjected the rat livers to total ischemia followed by reperfusion. After reperfusion, we performed the following investigations: alanine aminotransferase (ALT), histological findings, TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining, and transmission electron microscopy (TEM). We also investigated the expression of proteins associated with apoptosis, hepatoprotection, and S1P accumulation. RESULTS: S1P accumulated in the HA-S1P group livers more than S1P group livers. Serum ALT levels, TUNEL-positive hepatocytes, and expression of cleaved caspase-3 expression, were significantly decreased in the HA-S1P group. TEM revealed that the liver sinusoidal endothelial cell (LSEC) lining was preserved in the HA-S1P group. Moreover, the HA-S1P group showed a greater increase in the HO-1 protein levels compared to the S1P group. CONCLUSION: Our results suggest that HA-S1P exhibits cytoprotective effects in the liver through the inhibition of LSEC apoptosis. HA-S1P is an effective agent for hepatic ischemia/reperfusion injury.
  • Hideki Matsui, Yusuke Sato, Hiroto Hatakeyama, Hidetaka Akita, Hideyoshi Harashima
    INTERNATIONAL JOURNAL OF PHARMACEUTICS 495 1 171 - 178 2015年11月 [査読有り][通常論文]
     
    Macrophages are key contributors to various inflammatory diseases. Therefore, the development of an efficient in vivo short interference RNA (siRNA) system that can be delivered to macrophages represents a novel treatment strategy for addressing these disorders. It was recently revealed that peritoneal macrophages (PEMs) are involved in several diseases including ovarian cancer, and are now recognized as a promising drug target. We report herein on the use of pH-sensitive cationic YSK05-MENDs as siRNA carriers and on the impact of both the size of the YSK05-MENDs and their administration routes for the efficient targeting PEMs to achieve a high level of gene silencing activity. The size of the YSK05-MENDs had a dramatic effect on their specificity for PEMs when administered intravenously, but not for intraperitoneal injection. Also, significant gene silencing was achieved by an intraperitoneal administration of the YSK05-MEND at a dose in the single digit mg/kg range. To our knowledge, this is the most efficacious method for siRNA delivery for gene silencing in PEMs in vivo reported to date. These findings enabled us to investigate the complex function of PEMs through several gene silencing simultaneously. (C) 2015 Elsevier B.V. All rights reserved.
  • Takashi Nakamura, Yuki Fujiwara, Shota Warashina, Hideyoshi Harashima
    INTERNATIONAL JOURNAL OF PHARMACEUTICS 494 1 271 - 277 2015年10月 [査読有り][通常論文]
     
    The delivery of small interfering RNA (siRNA) to dendritic cells (DCs) is a challenging issue for siRNA-loaded lipid nanoparticles. The cause of this difficulty is unknown. The findings reported herein indicate that the rate-limiting step in gene silencing using siRNA-loaded lipid nanoparticles in DCs, as evidenced by a quantitative analysis of each process in siRNA delivery between mouse bone marrow derived DC (BMDC) and other cell lines, was not associated with the actual delivery of siRNA. A gene silencing of only 50% was observed in BMDC, even when a high dose was used. Contrary to our expectation, the interval between cellular uptake and the delivery of siRNA to the cytosol was not responsible for the low gene silencing. Meanwhile, a drastic difference was found in the relationship between the efficiency of gene silencing and the amount of intracellular intact siRNA. This fact indicates that the processes after cytosolic delivery of siRNA, namely the intracellular pharmacodynamics (PD) of siRNA, appear to be the rate-limiting step in gene silencing in BMDC. The findings reported here demonstrate the importance of the intracellular PD of siRNA delivered to cytosol in the development of siRNA delivery systems for gene silencing in DCs. (C) 2015 Elsevier B.V. All rights reserved.
  • Takashi Nakamura, Hiroko Miyabe, Mamoru Hyodo, Yusuke Sato, Yoshihiro Hayakawa, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 216 149 - 157 2015年10月 [査読有り][通常論文]
     
    Malignant melanomas escape immunosurveillance via the loss/down-regulation of MHC-I expression. Natural killer (NK) cells have the potential to function as essential effector cells for eliminating melanomas. Cyclic di-GMP (c-di-GMP), a ligand of the stimulator of interferon genes (STING) signal pathway, can be thought of as a new class of adjuvant against cancer. However, it is yet to be tested, because technologies for delivering c-di-GMP to the cytosol are required. Herein, we report that c-di-GMP efficiently activates NK cells and induces antitumor effects against malignant melanomas when loaded in YSK05 lipid containing liposomes, by assisting in the efficient delivery of c-di-GMP to the cytosol. The intravenous administration of c-di-GMP encapsulated with-in YSK05-liposomes (c-di-GMP/YSK05-Lip) into mice efficiently induced the production of type I interferon (IFN) as well as the activation of NK cells, resulting in a significant antitumor effect in a lung metastasis mouse model using B16-F10. This antitumor effect was dominated by NK cells. The infiltration of NK cells was observed in the lungs with B16-F10 melanomas. These findings indicate that the c-di-GMP/YSK05-Lip induces MHC-I nonrestricted antitumor immunity mediated by NK cells. Consequently, c-di-GMP/YSK05-Lip represents a potentially new adjuvant system for use in immunotherapy against malignant melanomas. (C) 2015 Elsevier B.V. All rights reserved.
  • Hidetaka Akita, Yuki Noguchi, Hiroto Hatakeyama, Yusuke Sato, Kota Tange, Yuta Nakai, Hideyoshi Harashima
    ACS BIOMATERIALS SCIENCE & ENGINEERING 1 9 834 - 844 2015年09月 [査読有り][通常論文]
     
    A lipid nanoparticle (LNP) composed of a series of SS-cleavable and pH-activated lipid-like materials (ssPalm) was previously developed as a platform of a gene delivery system. A tertiary amine and disulfide bonding were employed to destabilize the endosomal membrane and for intracellular collapse. We report herein on the development of a hepatocyte-targeting siRNA carrier by the molecular tuning of the hydrophobic scaffold, and tertiary amine structures. The gene knockdown activity against a hepatocyte-specific marker (factor VII: FVII) was improved when a more fat-soluble vitamin (vitamin E) was employed as a hydrophobic scaffold. Moreover, to allow the tertiary amines to accept protons by sensing a slight change in endosomal acidification, its structural flexibility was minimized by fixing it in a piperidine structure, and the distance between the surface of the particle to the ternary amine was increased. As a result, the pK(a) value was increased to the approximately 6.18 depending on its distance, while the pK(a) reached plateau when the tertiary amine was linked by an excess number of linear carbon chains. The pH-dependent membrane destabilization activity, as assessed by a hemolysis assay, was increased in parallel with the pK(a) value. Moreover, the gene knockdown activity was improved in parallel with hemolytic activity. Finally, further optimization of the lipid/siRNA ratio, and the use of chemically (2'-fluoro) modified siRNA synergistically improved the gene knockdown efficacy to an effective dose (ED50) of 0.035 mg/kg. The developed ssPalm represents a promising platform for use as a hepatocyte-targeting siRNA carrier.
  • Yuma Yamada, Kohei Nakamura, Jiro Abe, Mamoru Hyodo, Sanae Haga, Michitaka Ozaki, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 213 86 - 95 2015年09月 [査読有り][通常論文]
     
    We herein report on a mitochondrial therapeutic effect based on the delivery of coenzyme Q(10) (CoQ(10)), an anti-oxidant, to in vivo mitochondria using a MITO-Porter, a liposome-based mitochondrial delivery system that functions via membrane fusion. To evaluate the effects, we used a mouse liver ischemia/reperfusion injury (I/R injury) model, in which mitochondrial reactive oxygen species are overexpressed. We packaged CoQ(10) in the lipid phase of a MITO-Porter and optimized the mitochondrial fusogenic activities to produce the CoQ(10)-MITO-Porter. A histological observation of the carriers in the liver by confocal laser scanning microscopy was done and the accumulation of the carrier labeled with a radio isotope in the liver confirmed that the CoQ(10)-MITO-Porter was delivered to liver mitochondria via systemic injection. These analytical results permitted us to optimize the compositions of the CoQ(10)-MITO-Porter so as to permit it to efficiently accumulate in mouse liver mitochondria. Finally, we applied the optimized CoQ(10) -MITO-Porter to mice via tail vein injection, and hepatic I/R injury was then induced, followed by measuring serum alanine aminotransferase (ALT) levels, a marker of liver injury. We confirmed that the use of the CoQ(10)-MITO-Porter resulted in a significant decrease in serum ALT levels, indicating that in vivo mitochondrial delivery of the CoQ(10) via MITO-Porter prevents I/R injury in mice livers. This provides a demonstration of the potential use of such a delivery system in mitochondrial therapies. (C) 2015 Elsevier B.V. All rights reserved.
  • Yuma Yamada, Yutaka Fukuda, Hideyoshi Harashima
    MITOCHONDRION 24 50 - 55 2015年09月 [査読有り][通常論文]
     
    To achieve mitochondrial gene therapy, therapeutic molecules need to be transported through the outer and inner membranes of mitochondria into the innermost space (mitochondrial matrix), which contains the mtDNA pool. We previously reported on the construction of a MITO-Porter with a high fusogenic activity for the mitochondrial outer membrane for delivering molecules to the mitochondria of human cells. Here, we report on an investigation of a fusogenic lipid composition for the inner membrane, and an analysis of the fusogenic compositions for the outer and inner membranes. A significant relationship was found between fusion activity and the mitochondrial delivery of nucleic acids. (C) 2015 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
  • Yuma Yamada, Sandra Milena Vergara Perez, Mai Tabata, Jiro Abe, Yukari Yasuzaki, Hideyoshi Harashima
    JOURNAL OF PHARMACEUTICAL SCIENCES 104 9 2845 - 2854 2015年09月 [査読有り][通常論文]
     
    The transduction of antibodies into living cells would represent a major contribution to both basic and applied biomedical fields, as currently available methods suffer from limitations such as low-uptake efficiency and endosomal entrapment. In this study, a liposome-based carrier was designed to overcome these issues. Liposomes were modified with octaarginine (R8), a cell penetrating peptide and GALA, a pH-sensitive fusogenic peptide. The presence of R8 enhanced the cellular uptake of antibodies, whereas GALA reduced endosomal entrapment, resulting in antibodies being released into the cytosol within 30 min. Moreover, compared with commercially available reagents for delivering antibodies, our system was superior in both cellular uptake and endosomal escape. In addition, specific antibodies delivered by R8-GALA liposomes were found to be associated with their epitope, confirming the preservation of functionality. This system for the efficient and high-speed cytosolic delivery of an antibody provides a valuable tool that can be useful in basic and applied research for analyzing the expression and function of intracellular molecules. (c) 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:2845-2854, 2015
  • Yu Sakurai, Takashi Matsuda, Tomoya Hada, Hideyoshi Harashima
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 38 8 1185 - 1191 2015年08月 [査読有り][通常論文]
     
    Plasmid DNA (pDNA) is expected to be a new class of medicine for treating currently incurable diseases. To deliver these nucleic acids, we developed a liposomal delivery system we have called a multifunctional envelope-type nano device (MEND). In this report, we demonstrate that a MEND containing a pH-sensitive cationic lipid, YSK05 (YSK-MEND), efficiently delivered pDNA via systemic injection, and that its expression was highly dependent on the encapsulation state of the pDNA. In the preparation, the pH, ionic strength, and sodium chloride (NaCl) concentration of the lipid/pDNA mixture strongly affected the encapsulation efficiency of pDNA. Additionally, the transgene expression of luciferase in the liver by the injected YSK-MEND was dependent on the encapsulation state of pDNA rather than the nature of the YSK-MEND. Confocal laser scanning microscopy findings revealed that injection of the YSK-MEND led to homogenous gene expression in the liver compared to injection via the hydrodynamic tail vein (HTV). Concerning the safety of the YSK-MEND, a transient increase in the activity of liver enzymes was observed. However, no significant adverse events were observed. Taken together, the YSK-MEND represents a potentially attractive therapy for the treatment of various hepatic diseases.
  • Nakamura T, Kuroi M, Harashima H
    Molecular pharmaceutics 12 8 2791 - 2799 2015年08月 [査読有り][通常論文]
     
    Alpha-galactosylceramide (GC), a lipid antigen present on CD1d molecules, is a unique adjuvant that enables a strong antitumor effect to be induced via activation of natural killer T cells. We previously reported that a liposomal formulation of GC significantly enhanced GC presentation via CD1d and antitumor immunity. However, the influence of the intracellular fate of liposomes controlled by the lipid composition on GC presentation using GC-loaded liposomes (GC-Lip) remains unclear. In this study, we prepared a GC-Lip formulation by incorporating dioleoyl-phosphatidylethanolamine (DOPE)/cholesterol, egg phosphatidylcholine (EPC)/cholesterol, and distearoyl phosphocholine (DSPC)/cholesterol, and investigated the relationship between the intracellular trafficking of GC-Lip and GC presentation in antigen-presenting cells. When GC-Lip was prepared using DOPE, a fusogenic lipid, the endosomal escape of liposomes was enhanced, resulting in a decrease in GC presentation of CD1d, compared to the EPC based GC-Lip (EPC/GC-Lip). The stability of liposomes in endosomes/lysosomes had no influence on GC presentation. The DSPC based GC-Lip (DSPC/GC-Lip) induced GC presentation without any detectable degradation in liposomal structure, although the EPC/GC-Lip induced GC presentation with degradation of liposomal structure. The efficiency of GC presentation between EPC/GC-Lip and DSPC/GC-Lip was comparable. These GC presentations that were independent of the degradation of liposomes were dominated by saposins, sphingolipid activator proteins. Our findings reveal that GC presentation on CD1d from the fluid liposomes involves the action of saposins, regardless of whether liposome degradation occurs. This insight can be of use in terms of developing GC-Lip formulation for efficient GC presentation.
  • Furukawa R, Yamada Y, Kawamura E, Harashima H
    Biomaterials 57 107 - 115 2015年07月 [査読有り][通常論文]
     
    Mitochondrial genome-targeting nucleic acids are promising therapeutic candidates for treating mitochondrial diseases. To date, a number of systems for delivering genetic information to the cytosol and the nucleus have been reported, and several successful gene therapies involving gene delivery targeted to the cytosol and the nucleus have been reported. However, much less progress has been made concerning mitochondrial gene delivery systems, and mitochondrial gene therapy has never been achieved. Here, we report on the mitochondrial delivery of an antisense RNA oligonucleotide (ASO) to perform mitochondrial RNA knockdown to regulate mitochondrial function. Mitochondrial delivery of the ASO was achieved using a combination of a MITO-Porter system, which contains mitochondrial fusogenic lipid envelopes for mitochondrial delivery via membrane fusion and D-arm, a mitochondrial import signal of tRNA to the matrix. Mitochondrial delivery of the ASO induces the knockdown of the targeted mitochondria-encoded mRNA and protein, namely cytochrome c oxidase subunit II, a component of the mitochondrial respiratory chain. Furthermore, the mitochondrial membrane potential was depolarized by the down regulation of the respiratory chain as the result of the mitochondrial delivery of ASO. This finding constitutes the first report to demonstrate that the nanocarrier-mediated mitochondrial genome targeting of antisense RNA effects mitochondrial function. (C) 2015 Elsevier Ltd. All rights reserved.
  • Yasuhiro Hayashi, Hiroto Hatakeyama, Kazuaki Kajimoto, Mamoru Hyodo, Hidetaka Akita, Hideyoshi Harashima
    BIOCONJUGATE CHEMISTRY 26 7 1266 - 1276 2015年07月 [査読有り][通常論文]
     
    A paradigm shift has occurred in the field of drug delivery systems (DDS), one being intracellular targeting, and the other, active targeting. An important aspect of intracellular targeting involves delivering nucleic acids such as siRNA/pDNA rather than small molecular compounds, since the mechanism responsible for their entering a target cell is usually via endocytosis, and the efficiency of endosomal escape is a critical factor in determining the functional activities of siRNA/pDNA. A multifunctional envelope-type nano device (MEND) was developed to control the intracellular trafficking of nano carriers containing siRNA/pDNA. An octaarginine (R8) modified MEND was developed to achieve this. Considerable progress has been made in active targeting to selective tissue vasculature such as tumor, adipose tissue, and the lung where endothelial barrier is tight against nanoparticles with diameters larger than 50 nm. A dual-ligand system is proposed to enhance active targeting ability by virtue of a synergistic interaction between a selective ligand and a cell penetrating ligand. Prohibitin targeted nanoparticles (PTNP) were developed to target endothelial cells in adipose tissue, which deliver apoptotic peptides/proteins to the adipose vasculature. Lung endothelial cells can be targeted by means of the GALA peptide, which is usually used to enhance endosomal escape. These active targeting systems can induce pharmacological effects in in vivo conditions. Finally, a novel strategy for producing an original ligand has been developed, especially for the tumor vasculature. This progress in DDS promises to extend the area of nanomedicine as a breakthrough technology.
  • Kumiko Sakai-Kato, Nobuhiro Nishiyama, Masato Kozaki, Takeshi Nakanishi, Yoshihiro Matsuda, Mai Hirano, Hiroyuki Hanada, Shigeru Hisada, Hiroshi Onodera, Hideyoshi Harashima, Yasuhiro Matsumura, Kazunori Kataoka, Yukihiro Goda, Haruhiro Okuda, Toru Kawanishi
    JOURNAL OF CONTROLLED RELEASE 210 76 - 83 2015年07月 [査読有り][通常論文]
     
    Block copolymer micelles are nanoparticles formed from block copolymers that comprise a hydrophilic polymer such as poly(ethylene glycol) and a poorly soluble polymer such as poly(amino acids). The design of block copolymer micelles is intended to regulate the in vivo pharmacokinetics, stability, and distribution profiles of an entrapped or block copolymer-linked active substance. Several block copolymer micelle products are currently undergoing clinical development; however, a major challenge in the development and evaluation of such products is identification of the physicochemical properties that affect the properties of the drug product in vivo. Here we review the overall in vitro and in vivo characteristics of block copolymer micelle products with a focus on the products currently under clinical investigation. We present examples of methods suitable for the evaluation of the physicochemical properties, non-clinical pharmacokinetics, and safety of block copolymer micelle products. (C) 2015 Elsevier B.V. All rights reserved.
  • Hidetaka Akita, Taichi Nakatani, Kimiko Kuroki, Katsumi Maenaka, Kota Tange, Yuta Nakai, Hideyoshi Harashima
    INTERNATIONAL JOURNAL OF PHARMACEUTICS 490 1-2 142 - 145 2015年07月 [査読有り][通常論文]
     
    Efficient DNA carriers are needed as a gene medication for curing brain disorders. In the present study, the function of a neutral lipid envelope-type nanoparticle (LNP) encapsulating pDNA was evaluated after intracerebroventricular administration. The lipid envelope was composed of a series of SS-cleavable and pH-activated lipid like materials (ssPalm) including myristic acid, vitamin A and vitamin E in the hydrophobic scaffold (LNPssPalmM, LNPssPalmA, LNPssPalmE, respectively). The LNPssPalmA and LNPssPalmE were extensively distributed in the corpus callosum, and then gene expression occurred mainly astrocytes in this region, while not in LNPssPalmM. The recombinant human ApoE3-dependent enhancement of the uptake into an astrocyte-derived cell line (KT-5) was observed in LNPssPalmA and LNPssPalmE. Thus, ApoE in the brain plays a key role in the cellular uptake of these particles by astrocytes, and this uptake is dependent on the structure of the hydrophobic scaffold. (C) 2015 Elsevier B.V. All rights reserved.
  • Yuma Yamada, Masahiro Hashida, Hideyoshi Harashima
    BIOMATERIALS 52 189 - 198 2015年06月 [査読有り][通常論文]
     
    The cellular uptake pathway for a gene vector is an important factor in transgene expression. We previously constructed an original gene vector, multifunctional envelope-type nano device (MEND). The use of octaarginine (R8), a cell-penetrating peptide dramatically enhanced the transfection activity of the MEND since efficient cellular uptake via macropinocytosis, while the R8 should overcome its poor cell selectivity. Here we prepared an R8-MEND equipped with GALA (a peptide for endosomal escape) (R8/ GALA-MEND) coated with hyaluronic acid (HA) (HA-R8/GALA-MEND), a natural ligand for cancer cells overexpressing CD44. We investigated the cellular uptake pathway of the HA-R8/GALA-MEND and the R8/GALA-MEND using HCT116 cells overexpressing CD44. Both carriers were taken up by cells mainly via macropinocytosis, whereas only the HA-R8/GALA-MEND was partially internalized into cells via a CD44-mediated pathway. Investigation of transgene expression showed that the HA-R8/GALA-MEND had a high transfection activity in HCT116 cells via both macropinocytotic and CD44-mediated pathways. On the other hand, the value for the HA-R8/GALA-MEND was significantly decreased compared with the value for the R8/GALA-MEND in NIH3T3 cells (CD44-negative cells). These findings indicate that the HA-coating controls the intracellular pathway for R8-modified nanocarriers, and that a CD44-mediated pathway is an important route for transgene expression. (C) 2015 Elsevier Ltd. All rights reserved.
  • Virtu Calabuig-Navarro, Jun Yamauchi, Sojin Lee, Ting Zhang, Yun-Zi Liu, Kelsey Sadlek, Gina M. Coudriet, Jon D. Piganelli, Chun-Lei Jiang, Rita Miller, Mark Lowe, Hideyoshi Harashima, H. Henry Dong
    JOURNAL OF BIOLOGICAL CHEMISTRY 290 25 15581 - 15594 2015年06月 [査読有り][通常論文]
     
    Background: Excessive endogenous glucose production is a major contributing factor for fasting hyperglycemia in diabetes. Results: FoxO6 deficiency attenuates hepatic gluconeogenesis and protects against fat-induced glucose disorder in mice. Conclusion: FoxO6 plays a significant role in regulating gluconeogenesis in the liver. Significance: FoxO6 is a potential therapeutic target for improving glucose metabolism in diabetes. Excessive endogenous glucose production contributes to fasting hyperglycemia in diabetes. FoxO6 is a distinct member of the FoxO subfamily. To elucidate the role of FoxO6 in hepatic gluconeogenesis and assess its contribution to the pathogenesis of fasting hyperglycemia in diabetes, we generated FoxO6 knock-out (FoxO6-KO) mice followed by determining the effect of FoxO6 loss-of-function on hepatic gluconeogenesis under physiological and pathological conditions. FoxO6 depletion attenuated hepatic gluconeogenesis and lowered fasting glycemia in FoxO6-KO mice. FoxO6-deficient primary hepatocytes were associated with reduced capacities to produce glucose in response to glucagon. When fed a high fat diet, FoxO6-KO mice exhibited significantly enhanced glucose tolerance and reduced blood glucose levels accompanied by improved insulin sensitivity. These effects correlated with attenuated hepatic gluconeogenesis in FoxO6-KO mice. In contrast, wild-type littermates developed fat-induced glucose intolerance with a concomitant induction of fasting hyperinsulinemia and hyperglycemia. Furthermore, FoxO6-KO mice displayed significantly diminished macrophage infiltration into liver and adipose tissues, correlating with the reduction of macrophage expression of C-C chemokine receptor 2 (CCR2), a factor that is critical for regulating macrophage recruitment in peripheral tissues. Our data indicate that FoxO6 depletion protected against diet-induced glucose intolerance and insulin resistance by attenuating hepatic gluconeogenesis and curbing macrophage infiltration in liver and adipose tissues in mice.
  • Hidetaka Akita, Dai Kurihara, Marco Schmeer, Martin Schleef, Hideyoshi Harashima
    PHARMACEUTICS 7 2 64 - 73 2015年06月 [査読有り][通常論文]
     
    The nuclear transfer process is one of the critical rate-limiting processes in transgene expression. In the present study, we report on the effect of compaction and the size of the DNA molecule on nuclear transfer efficiency by microinjection. A DNA/protamine complex-or variously-sized naked DNA molecules were injected into the cytoplasm or nucleus of synchronized HeLa cells. To evaluate the nuclear transfer process, a nuclear transfer score (NT score), calculated based on transgene expression after cytoplasmic microinjection divided by that after nuclear microinjection, was employed. The compaction of DNA with protamine decreased the NT score in comparison with the injection of naked DNA when the N/P ratio was increased to >2.0. Moreover, when naked DNA was microinjected, gene expression increased in parallel with the size of the DNA in the following order: minicircle DNA (MC07. CMV-EGFP; 2257 bp) > middle-sized plasmid DNA (pBS-EGFP; 3992 bp) > conventional plasmid DNA (pcDNA3.1-EGFP; 6172 bp), while the level of gene expression was quite comparable among them when the DNAs were injected into the nucleus. The above findings suggest that the intrinsic size of the DNA molecule is a major determinant for nuclear entry and that minicircle DNA has a great advantage in nuclear transfer.
  • Hiroyuki Kamiya, Daiki Yamazaki, Eri Nakamura, Tetsuaki Makino, Miwako Kobayashi, Ichiro Matsuoka, Hideyoshi Harashima
    CHEMICAL RESEARCH IN TOXICOLOGY 28 4 621 - 628 2015年04月 [査読有り][通常論文]
     
    8-Oxo-7,8-dihydroguanine (G(O), 8-hydroxyguanine) in DNA is one of the most important oxidatively damaged bases and causes G:C -> T:A substitution mutations. The Werner syndrome protein (WRN) is a cancer-related RecQ DNA helicase and plays many roles in DNA replication and repair. To examine the relationships between G(O)-induced mutations and WRN, shuttle plasmid DNA containing a G(O):C pair in the supF gene was transfected into human U2OS cells, in which WRN was knocked down. The plasmid DNA replicated in the knockdown cells was introduced into an Escherichia coli indicator strain. The knockdown of WRN increased the mutant frequency of the G(O)-plasmid DNA. Unexpectedly, however, the WRN knockdown only slightly enhanced the targeted G:C -> T:A mutation. Instead, base-substitution mutations at various positions were more frequently detected, with statistical significance. The results obtained in this study suggested that the reduction of the cancer-related WRN induced action-at-a-distance mutagenesis by the G(O):C pair in human cells. In addition, the WRN knockdown decreased the G(O):A-induced A:T -> C:G mutations, suggesting that WRN may enhance the mutations caused by G(O) in the nucleotide pool.
  • Hidetaka Akita, Ryohei Ishiba, Ryohei Togashi, Kota Tange, Yuta Nakai, Hiroto Hatakeyama, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 200 97 - 105 2015年02月 [査読有り][通常論文]
     
    A renal cell carcinoma (RCC) is one of the refractory tumors, since it readily acquires resistance against chemotherapy. Thus, alternative therapeutic approaches such as obstructing the neovasculature are needed. We previously reported on the development of a plasmid DNA (pDNA)-encapsulating liposomal nanoparticle (LNP) as a hepatic gene delivery system that is applicable to systemic administration. The key molecular component is a SS-cleavable and pH-activated lipid-like material (ssPalm) that mounts dual sensing motifs (ternary amines and disulfide bonding) that are responsive to the intracellular environment. The main purpose of the present study was to expand its application to a tumor-targeting gene delivery systemin mice bearing tumors established from a RCC (OS-RC-2). When the modification of the surface of the particle is optimized for the polyethyleneglycol (PEG), stability in the blood circulation is improved, and consequently tumor-selective gene expression can be achieved. Furthermore, gene expression in the tumor was increased slightly when the hydrophobic scaffold of the ssPalm was replaced from the conventionally used myristic acid (ssPalmM) to alpha-tocopherol succinate (ssPalmE). Moreover, tumor growth was significantly suppressed when the completely CpG-free pDNA encoding the solute form of VEGFR (fms-like tyrosine kinase-1: sFlt-1) was used, especially when it was delivered by the LNP formed with ssPalmE(LNPssPalmE). Thus, the PEG-modified LNPssPalmE is a promising gene carrier for the cancer gene therapy of RCC. (C) 2014 Elsevier B.V. All rights reserved.
  • Naoya Miura, Sharif M. Shaheen, Hidetaka Akita, Takashi Nakamura, Hideyoshi Harashima
    NUCLEIC ACIDS RESEARCH 43 3 1317 - 1331 2015年02月 [査読有り][通常論文]
     
    Technologies that delivery antigen-encoded plasmid DNA (pDNA) to antigen presenting cell and their immune-activation are required for the success of DNA vaccines. Here we report on an artificial nanoparticle that can achieve these; a multifunctional envelope-type nanodevice modified with KALA, a peptide that forms alpha-helical structure at physiological pH (KALA-MEND). KALA modification and the removal of the CpG-motifs from the pDNA synergistically boosted transfection efficacy. In parallel, transfection with the KALA-MEND enhances the production of multiple cytokines and chemokines and co-stimulatory molecules via the Toll-like receptor 9-independent manner. Endosome-fusogenic lipid envelops and a long length of pDNA are essential for this immune stimulation. Furthermore, cytoplasmic dsDNA sensors that are related to the STING/TBK1 pathway and inflammasome are involved in IFN-beta and IL-1 beta production, respectively. Consequently, the robust induction of antigen-specific cytotoxic T-lymphoma activity and the resulting prophylactic and therapeutic anti-tumor effect was observed in mice that had been immunized with bone marrow-derived dendritic cells ex vivo transfected with antigen-encoding pDNA. Collectively, the KALA-MEND possesses dual functions; gene transfection system and immune-stimulative adjuvant, those are both necessary for the successful DNA vaccine.
  • Yuma Yamada, Hideyoshi Harashima
    Mitochondrial Medicine 2 123 - 133 2015年01月29日 [査読有り][通常論文]
     
    Genetic mutations and defects in mitochondrial DNA (mtDNA) are associated with certain types of mitochondrial dysfunction, ultimately resulting in the occurrence of a variety of human diseases. For an effective mitochondrial gene therapy, it will be necessary to deliver therapeutic agents to the innermost mitochondrial space (the mitochondrial matrix), which contains the mtDNA pool. We recently developed a MITO-Porter, a liposome-based nano-carrier that delivers cargo to mitochondria via a membrane-fusion mechanism. Using propidium iodide, as a probe to detect mtDNA, we were able to confirm that the MITO-Porter delivered cargoes to mitochondrial matrices in living cells. More recently, we constructed a Dual Function (DF)-MITO-Porter, a liposome-based nanocarrier for mitochondrial delivery via a stepwise process. In this chapter, we describe the methodology used to deliver bioactive molecules to the mitochondrial matrix using the above DF-MITO-Porter, and the evaluation of mtDNA levels and mitochondrial activities in living cells.
  • Tomoya Hada, Yu Sakurai, Hideyoshi Harashima
    Pharmaceutics 7 3 320 - 333 2015年 [査読有り][通常論文]
     
    In recent years, anti-angiogenic therapy has attracted much interest because it is a versatile approach to treating most types of tumors, and therefore would be expected to be applicable for various cancers. Severe adverse events in patients treated with currently available anti-angiogenic therapeutics have, however, been reported, and these are caused by their inhibitory effects in normal tissue. To achieve an efficient anti-angiogenic therapy with minimal toxicity, a drug delivery system (DDS) specific to tumor endothelial cells (TECs) is needed. Cyclic RGD (cRGD) is a well-known ligand against αVβ3 integrin that is expressed at high levels in the cell surface of TECs. To address this issue, we previously developed a cyclic RGD-equipped liposomal DDS (RGD-MEND) in which small interfering RNA (siRNA) was encapsulated. However, in the previous study, details of the preparation steps were not thoroughly examined. In this paper, to produce the most efficient delivery of therapeutic TECs, we explored optimum preparation conditions and components of the RGD-MEND. The cellular uptake and silencing ability of the RGD-MEND were investigated as a function of ligand density, poly(ethyleneglycol) linker length, and llipid composition. As a result, a knockdown efficiency that was five-fold higher than that of the previously reported one (ED50, from 4.0 to 0.75 mg/kg) was achieved.
  • Masatoshi Maeki, Tatsuyoshi Saito, Yusuke Sato, Takao Yasui, Noritada Kaji, Akihiko Ishida, Hirofumi Tani, Yoshinobu Baba, Hideyoshi Harashima, Manabu Tokeshi
    RSC ADVANCES 5 57 46181 - 46185 2015年 [査読有り][通常論文]
     
    Formation behavior of lipid nanoparticles (LNPs) in microfluidic devices with a staggered herringbone micromixer (SHM) structure was investigated. The fundamental role for SHMs in LNP formation was demonstrated by determining such factors as the limiting SHM cycle numbers and the effect of flow rate. The SHM cycle numbers and the position of the first SHM were as significant as factors as the flow rate condition for producing the small-size LNPs.
  • Yu Sakurai, Kazuaki Kajimoto, Hiroto Hatakeyama, Hideyoshi Harashima
    EXPERT OPINION ON DRUG DELIVERY 12 1 41 - 52 2015年01月 [査読有り][通常論文]
     
    Introduction: Data reported during the last decade of the twentieth century indicate that passive targeting is an efficient strategy for delivering nanocarrier systems to tumor tissues. The focus of this review is on active targeting as a next-generation strategy for extending the capacity of a drug delivery system (DDS). Areas covered: Tumor vasculature targeting was achieved using arginine-glycine-aspartic acid, asparagine-glycine-arginine and other peptides, which are well-known peptides, as ligand against tumor vasculature. An efficient system for delivering small interfering RNA to the tumor vasculature involved the use of a multifunctional envelope-type nanodevice based on a pH-modified cationic lipid and targeting ligands. The active-targeting system was extended from tumor delivery to adipose tissue delivery, where endothelial cells are tightly linked and are impermeable to nanocarriers. In mice, prohibitin-targeted nanoparticles can be used to successfully deliver macromolecules to induce anti-obese effects. Finally, the successful delivery of nanocarriers to adipose tissue in obese mice via the enhanced permeability and retention-effect is reported, which can be achieved in tumor tissue. Expert opinion: Unlike tumor tissues, only a few reports have appeared on how liposomal carriers accumulate in adipose tissues after systemic injection. This finding, as well as active targeting to the adipose vasculature, promises to extend the capacity of DDS to adipose tissue. Since the site of action of nucleic acids is the cytosol, the intracellular trafficking of carriers and their cargoes as well as cellular uptake must be taken into consideration.
  • Seiichi Sato, Kai Li, Takeshi Kameyama, Takaya Hayashi, Yuji Ishida, Shuko Murakami, Tsunamasa Watanabe, Sayuki Iijima, Yu Sakurai, Koichi Watashi, Susumu Tsutsumi, Yusuke Sato, Hidetaka Akita, Takaji Wakita, Charles M. Rice, Hideyoshi Harashima, Michinori Kohara, Yasuhito Tanaka, Akinori Takaoka
    IMMUNITY 42 1 123 - 132 2015年01月 [査読有り][通常論文]
     
    Host innate recognition triggers key immune responses for viral elimination. The sensing mechanism of hepatitis B virus (HBV), a DNA virus, and the subsequent downstream signaling events remain to be fully clarified. Here we found that type III but not type I interferons are predominantly induced in human primary hepatocytes in response to HBV infection, through retinoic acid-inducible gene-I (RIG-I)-mediated sensing of the 5'-epsilon region of HBV pregenomic RNA. In addition, RIG-I could also counteract the interaction of HBV polymerase (P protein) with the 5'-epsilon region in an RNA-binding dependent manner, which consistently suppressed viral replication. Liposome-mediated delivery and vector-based expression of this e region-derived RNA in liver abolished the HBV replication in human hepatocyte-chimeric mice. These findings identify an innate-recognition mechanism by which RIG-I dually functions as an HBV sensor activating innate signaling and to counteract viral polymerase in human hepatocytes.
  • Takashi Nakamura, Masafumi Fukiage, Yoshiteru Suzuki, Ikuya Yano, Jun Miyazaki, Hiroyuki Nishiyama, Hideyuki Akaza, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 196 161 - 167 2014年12月 [査読有り][通常論文]
     
    We previously reported on the development of a water soluble formulation of the cell wall skeleton of BCG (BCG-CWS), a major immune active center of BCG, by encapsulating it into a nanoparticle (CWS-NP). The CWS-NP allowed us to clarify the machinery associated with the BCG mediated anti-bladder tumor effect, especially the roles of bladder cancer cells and dendritic cells (DCs) in the initial step, which remains poorly understood. We show herein that the internalization of BCG-CWS by bladder cancer cells, but not DCs, is indispensable for the induction of an antitumor effect against bladder cancer. Tumor growth was significantly inhibited in mice that had been inoculated with mouse bladder cancer (MBT-2) cells containing internalized BCG-CWS. On the other hand, the internalization of BCG-CWS by DCs had only a minor effect on inducing an antitumor effect against MBT-2 tumors. This was clarified for the first time by using the CWS-NP. This finding provides insights into our understanding of the role of bladder cancer cells and DCs in BCG therapy against bladder cancer. (C) 2014 Elsevier B.V. All rights reserved.
  • Golam Kibria, Hiroto Hatakeyama, Kosuke Akiyama, Kyoko Hida, Hideyoshi Harashima
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 37 12 1926 - 1935 2014年12月 [査読有り][通常論文]
     
    Multi-drug resistance (MDR) of cancers to chemotherapy including doxorubicin (DOX) is mediated by several factors. To design an effective therapy for the treatment of chemotherapy-resistant cancers, it is essential to explore the elements responsible for mediating MDR. However, exploring these factors in detail in a wide range of tumor types is challenging as several critical analytical steps are involved. Here, we demonstrated the way of exploring the factors mediating MDR in the tumor types without performing the analysis at the molecular level of cells. The sensitivities of 15 different types of cancer cells to DOX were evaluated, and the role of P-glycoprotein (P-gp), one of the major efflux-pumps, was explored. A correlation curve was developed between the intracellular amounts of DOX and the sensitivities of cells, and, based on this correlation, the cells were classified in response to the involvement of P-gp that mediates MDR. P-gp plays an active role in mediating MDR of cancer cells where a correlation between the sensitivities of cells and the accumulated DOX exists. In contrast, in cells that show a resistance to DOX but whose sensitivities are independent of the amount of accumulated drug, it was reasonably presumed that mechanisms other than P-gp are likely to be involved in mediating MDR. Based on the correlation between the availability of a drug and cell sensitivity, it would be reasonable to explore the factors governing cancer MDR, which is essential in designing an effective therapeutic approach for treating chemotherapy-resistant cancers using chemotherapeutic drugs.
  • M. Hyodo, Y. Sakurai, H. Akita, H. Harashima
    JOURNAL OF CONTROLLED RELEASE 193 316 - 323 2014年11月 [査読有り][通常論文]
     
    We report on the development of a multifunctional envelope-type nano device (MEND) based on our packaging concept "Programmed packaging" to control not only intracellular trafficking but also the biodistribution of encapsulated compounds such as nucleic acids/proteins/peptides. Our strategy for achieving this is based on molecular mechanisms of cell biology such as endocytosis, vesicular trafficking, etc. In this review, we summarize the concept of programmed packaging and discuss some of our recent successful examples of using MENDs. Systematic evolution of ligands by exponential enrichment (SELEX) was applied as a new methodology for identifying a new ligand toward cell or mitochondria. The delivery of siRNA to tumors and the tumor vasculature was achieved using pH sensitive lipid (YSK05), which was newly designed and optimized under in vivo conditions. The efficient delivery of pDNA to immune cells such as dendritic cells has also been developed using the KALA ligand, which can be a breakthrough technology for DNA vaccine. Finally, ss-cleavable and pH-activated lipid-like surfactant (ssPalm) which is a lipid like material with pH-activatable and SS-cleavable properties is also introduced as a proof of our concept. (C) 2014 Elsevier B.V. All rights reserved.
  • Afsana Akhter, Yasuhiro Hayashi, Yu Sakurai, Noritaka Ohga, Kyoko Hida, Hideyoshi Harashima
    INTERNATIONAL JOURNAL OF PHARMACEUTICS 475 1-2 227 - 237 2014年11月 [査読有り][通常論文]
     
    The specific delivery of a gene to liver sinusoidal endothelial cells (LSEC) could become a useful strategy for treating various liver diseases associated with such cells. We previously reported that the accumulation of KLGR peptide modified liposomes through liver sinusoidal blood vessels was enhanced after an intravenous administration. Here, we report on an attempt to develop an LSEC targeted nanocarrier system to deliver siRNA for the successful knockdown of LSEC specific gene expression. The system involved the development of a multifunctional envelop-type nano device (MEND) modified with the KLGR peptide for siRNA delivery targeting LSEC. Our developed carrier successfully lowered specific gene expression in LSEC. An in vivo study showed that at a lower density of ligand at the surface of the MEND resulted in the highest knockdown of gene expression in LSEC. This is the first report of the successful delivery of siRNA to LSECs. Further experiments suggest that not only a higher endosomal escape efficiency into the cytosol but also the uptake mechanism as a function of ligand density are two important factors to be considered for targeting LSEC. (C) 2014 Elsevier B.V. All rights reserved.
  • Garima Shrivastava, Mamoru Hyodo, Mst Naznin Ara, Hideyoshi Harashima
    NUCLEOSIDES NUCLEOTIDES & NUCLEIC ACIDS 33 11 697 - 708 2014年11月 [査読有り][通常論文]
     
    Carbonic anhydrases (CA) or carbonate dehydratases are a family of enzymes that catalyze the rapid interconversion of carbon dioxide and water to bicarbonate. CA I is the most abundant protein in the cytosol and has been reported to the partially associated with a number of fatal diseases. A newly established Systematic Evolution of Ligands by EXponential enrichment (SELEX) method referred to as Protein-SELEX was used to select RNA aptamers against the human erythrocyte CA I (CA I) protein. After five rounds of selection and counter selection the specific binding of the 6th cycle in vitro transcribed RNA library to CA I was detected by an Electrophoretic Mobility Shift Assay (EMSA). Three Specific sequences were identified as binding candidates after cloning and sequence analysis and one of the selected CA I specific RNA aptamers, CAapt1, was used to confirm specific binding and the Kd values were determined using an EMSA. The CAapt1 RNA aptamer showed no affinity towards any other protein and in comparison to the "0" cycle library, a significant enrichment was obtained. This methodology permitted us to successfully investigate the ssRNA aptamer CAapt1 for CA I protein.
  • Mst Naznin Ara, Takashi Matsuda, Mamoru Hyodo, Yu Sakurai, Noritaka Ohga, Kyoko Hida, Hideyoshi Harashima
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 37 11 1742 - 1749 2014年11月 [査読有り][通常論文]
     
    We describe herein the development of a high affinity and specific DNA aptamer as a new ligand for use in liposomal nanoparticles to target cultured mouse tumor endothelial cells (mTECs). Active targeted nanotechnology based drug delivery systems are currently of great interest, due to their potential for reducing side effects and facilitating the delivery of cytotoxic drugs or genes in a site specific manner. In this study, we report on a promising aptamer candidate AraHH036 that shows selective binding towards mTECs. The aptamer does not bind to normal cells, normal endothelial cells or tumor cells. Therefore, we synthesized an aptamer-polyethylene glycol (PEG) lipid conjugate and prepared aptamer based liposomes (ALPs) by the standard lipid hydration method. First, we quantified the higher capacity of ALPs to internalize into mTECs by incubating ALPs containing 1 mol%, 5 mol% and 10 mol% aptamer of total lipids and compared the results to those for unmodified PEGylated liposomes (PLPs). A confocal laser scanning microscope (CLSM) uptake study indicated that the ALPs were taken up more efficiently than PLPs. The measured K-d value of the ALPs was 142 nM. An intracellular trafficking study confirmed that most of the rhodamine labeled ALPs were taken up and co-localized with the green lysotracker, thus confirming that they were located in lysosomes. Finally, using an aptamer based proteomics approach, the molecular target protein of the aptamer was identified as heat shock protein 70 (HSP70). The results suggest that these ALPs offer promise as a new carrier molecule for delivering anti-angiogenesis drugs to tumor vasculature.
  • Kenji Kusumoto, Hidetaka Akita, Sarochin Santiwarangkool, Hideyoshi Harashima
    INTERNATIONAL JOURNAL OF PHARMACEUTICS 473 1-2 144 - 147 2014年10月 [査読有り][通常論文]
     
    We previously reported that a multifunctional envelope-type nano device (MEND) modified with a GALA peptide (GALA/MEND) exerted dual functions; effective targeting the pulmonary endothelium and endosomal escape. The GALA/MEND containing encapsulated siRNA was originally prepared by the film coated hydration method (GALA/MENDHyd). However, an ethanol dilution method was found to be more appropriate for scaling up the preparation of this liposomal nanoparticle. In this study, we report on the preparation of a GALA/MEND based on the principal of the ethanol dilution (GALA/MENDEto). The gene knockdown efficiency of the MENDHyd and MENDEt0H without GALA-modification was equivalent regardless of the method used in the preparation. The GALA/MENDEt0H induced more efficient gene silencing in the pulmonary endothelium (ED50; approximately 0.17 mg siRNA/kg) compared to the GALA/ MENDHyd. The GALA/MENDEt0H escaped from endosomes more rapidly than GALA/MENDHyd, while the pharmacokinetics and lung accumulation of GALA/MENDEtcni and GALA/MENDHyd were comparable after i.v. administration. Collectively, the ethanol dilution method improves the function of the GALA/MEND as a lung-targeting siRNA carrier. (C) 2014 Elsevier B.V. All rights reserved.
  • Kazuaki Kajimoto, Yusuke Sato, Takashi Nakamura, Yuma Yamada, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 190 593 - 606 2014年09月 [査読有り][通常論文]
     
    Nanomedicine is expected to be a basic technology for using nucleic acids as a drug, in which treating the cause of diseases represent the ultimate therapy. However, a sophisticated delivery system is required for efficient delivery of RNA/DNA, since these compounds need precise control of intracellular trafficking as well as biodistribution. Here we report on the use of a multifunctional envelope-type nano device (MEND) which is capable of intracellular trafficking such as endosomal escape, delivery to mitochondria, as well as active targeting to selective tissues/cells in vivo. In this review, we focused on the controlled intracellular trafficking of antigens for advanced immunotherapy, and then introduced a mitochondrial delivery system as an organelle targeting system for unmet medical needs. We also provide a successful in vivo delivery of siRNA to the liver based on a newly designed pH-responsive cationic lipid. Finally we will discuss an important role of an active targeting system using a peptide ligand to adipose vasculature. These progresses in drug delivery system will break through the barriers exist in our body, tissues and cells and open a window for future Nanomedicine. (C) 2014 Elsevier B.V. All rights reserved.
  • Yukari Yasuzaki, Yuma Yamada, Yutaka Fukuda, Hideyoshi Harashima
    Pharmaceuticals 7 8 881 - 893 2014年08月21日 [査読有り][通常論文]
     
    Mitochondrial gene therapy and diagnosis have the potential to provide substantial medical benefits. However, the utility of this approach has not yet been realized because the technology available for mitochondrial gene delivery continues to be a bottleneck. We previously reported on mitochondrial gene delivery in skeletal muscle using hydrodynamic limb vein (HLV) injection. HLV injection, a useful method for nuclear transgene expression, involves the rapid injection of a large volume of naked plasmid DNA (pDNA). Moreover, the use of a condensed form of pDNA enhances the nuclear transgene expression by the HLV injection. The purpose of this study was to compare naked pDNA and condensed pDNA for mitochondrial association in skeletal muscle, when used in conjunction with HLV injection. PCR analysis showed that the use of condensed pDNA rather than naked pDNA resulted in a more effective mitochondrial association with pDNA, suggesting that the physicochemical state of pDNA plays a key role. Moreover, no mitochondrial toxicities in skeletal muscle following the HLV injection of condensed pDNA were confirmed, as evidenced by cytochrome c oxidase activity and mitochondrial membrane potential. These findings have the potential to contribute to the development for in vivo mitochondrial gene delivery system. © 2014 by the authors licensee MDPI, Basel, Switzerland.
  • Masami Ukawa, Hidetaka Akita, Yasuhiro Hayashi, Ryohei Ishiba, Kota Tange, Masaya Arai, Kazuhiro Kubo, Yuriko Higuchi, Kazunori Shimizu, Satoshi Konishi, Mitsuru Hashida, Hideyoshi Harashima
    ADVANCED HEALTHCARE MATERIALS 3 8 1222 - 1229 2014年08月 [査読有り][通常論文]
  • Yu Sakurai, Hiroto Hatakeyama, Hidetaka Akita, Hideyoshi Harashima
    MOLECULAR PHARMACEUTICS 11 8 2713 - 2719 2014年08月 [査読有り][通常論文]
     
    It is well-known that renal cell carcinomas (RCCs) are resistant to classical cytotoxic anticancer drugs. Therefore, facilitating the impact of anticancer drugs by altering the cell phenotype should be a useful strategy for circumventing this. We developed a multifunctional envelopetype nanodevice (MEND) as an in vivo carrier of siRNA to tumor tissues. We previously reported that a MEND containing YSKOS (YSK-MEND) efficiently delivered siRNA in RCC-bearing mice. We herein report on a combination therapy involving the use of siRNA-mediated specific gene knockdown and cytotoxic drug doxorubicin (DOX), and an advantage of YSK-MEND as an investigation tool for in vivo function of a gene. si-PLK1 encapsulated within YSK-MEND was prepared using the tertiary butanol dilution method. The in vitro cellular viability under the exposure of DOX was compared between OS-RC-2 cells with and without si-PLK1 transfection. In an in vivo study, tumor-bearing mice were systemically injected with YSK-MEND and DOX-loaded liposomes. The combination of DOX and si-PLK1 drastically reduced tumor growth rate, and apoptotic cells were observed. In an in vitro study, PLKI knockdown increased G2/M cell population and reduced the expression of cyclin B1 (CCNB1) mRNA. CCNBI suppression by si-PLK1 encapsulated in YSK-MEND was also observed in the in vivo experiments. A combination of DOX and anti-polo-like kinase 1 siRNA (si-PLK1) resulted in a measurable delay in OS-RC-2 tumor growth. This result suggests that the combination of si-PLKI delivery and doxorubicin by YSK-MEND holds potential for RCC therapy via cell CCNB1 regulation.
  • Md. Nazir Hossen, Kazuaki Kajimoto, Ryo Tatsumi, Mamoru Hyodo, Hideyoshi Harashima
    JOURNAL OF DRUG TARGETING 22 7 600 - 609 2014年08月 [査読有り][通常論文]
     
    We previously developed a ligand-targeted liposome, referred to as a prohibitin-targeted nanoparticle (PTNP), for specifically delivering encapsulated drugs into vascular endothelial cells in adipose tissue. In this study, we explored the critical factors for the successful development and application of ligand-targeted nanocarriers through comparative assessments of PTNP prepared by the reverse-phase evaporation (REV) and lipid film hydration (HYD) methods with reference to physicochemical characteristics and in vivo and in vitro behavior. The in vivo delivery and therapeutic properties of HYD-PTNP were dramatically inferior to those of REV-PTNP, although the size, zeta-potential, fixed aqueous layer thickness and surface ligand density of the two preparations were similar. Circular dichroism spectral analyses revealed that the irreversible alteration in ligand conformation was caused by the organic solvent used to prepare the thin lipid film. In addition, perturbation of the ligand by the organic solvent resulted in a reduced internalization of PTNP into adipose endothelial cells. Alteration of the ligand conformation did not appear to affect the physicochemical characteristics of nanocarriers. Therefore, appropriate handling of ligands and appropriate evaluation of their conformations are critical for the successful development and application of such targeted nanocarriers.
  • Mst. Naznin Ara, Mamoru Hyodo, Noritaka Ohga, Kosuke Akiyama, Kyoko Hida, Yasuhiro Hida, Nobuo Shinohara, Hideyoshi Harashima
    CANCER MEDICINE 3 4 825 - 834 2014年08月 [査読有り][通常論文]
     
    The identification of a specific biomarker involves the development of new clinical diagnostic tools, and an in-depth understanding of the disease at the molecular level. When new blood vessels form in tumor cells, endothelial cell production is induced, a process that plays a key role in disease progression and metastasis to distinct organs for solid tumor types. The present study reports on the identification of a new biomarker on primary cultured mouse tumor endothelial cells (mTECs) using our recently developed high-affinity DNA aptamer AraHH001 (K-d = 43 nmol/L) assisted proteomics approach. We applied a strategy involving aptamer-facilitated biomarker discovery. Biotin-tagged AraHH001 was incubated with lysates of mTECs and the aptamer-proteins were then conjugated with streptavidin magnetic beads. Finally, the bound proteins were separated by sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. We identified troponin T via matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, the molecular target of aptamer AraHH001, and its presence was confirmed by measuring mRNA, protein levels, western blot, immunostaining, a gel shift assay of AraHH001 with troponin T. We first report here on the discovery of troponin T on mTECs, a promising and interesting diagnostic tool in the development of antiangiogenic therapy techniques the involves the targeting of the tumor vasculature.
  • Yuma Yamada, Mai Tabata, Yukari Yasuzaki, Masatoshi Nomura, Atsushi Shibata, Yuta Ibayashi, Yosuke Taniguchi, Shigeki Sasaki, Hideyoshi Harashima
    BIOMATERIALS 35 24 6430 - 6438 2014年08月 [査読有り][通常論文]
     
    Pancreatic beta cells secrete insulin in response to glucose levels and thus are involved in controlling blood glucose levels. A line of pancreatic beta cells "MIN6" has been used in studies related to the function of beta cells and diabetes therapy. Regulating gene expression in MIN6 cells could accelerate these studies, but an efficient method for the transfection of nucleic acids targeted to MINE cells is required. We report here on a liposome-based carrier targeted to pancreatic beta cells (Multifunctional envelope-type nano device for pancreatic beta cells, beta-MEND). We identified a lipid composition for use in preparing the beta-MEND, which permits the particles to be efficiently internalized into MIN6, as evidenced by flow cytometry analyses. Intracellular observation by confocal laser scanning microscopy showed that the beta-MEND efficiently delivered the oligo nucleic acids to the cytosol of MINE cells. Moreover, using a beta-MEND encapsulating a 2'-O-Methyl RNA complementary to a microRNA that suppresses insulin secretion, the knockdown of the targeted microRNA and an up-regulation of insulin secretion were observed in MINE. Thus, the beta-MEND holds promise as an efficient system for delivering nucleic acids targeted to MIN6 and can contribute to research and therapy aimed at diabetes. (C) 2014 Elsevier Ltd. All rights reserved.
  • Mst Naznin Ara, Takashi Matsuda, Mamoru Hyodo, Yu Sakurai, Hiroto Hatakeyama, Noritaka Ohga, Kyoko Hida, Hideyoshi Harashima
    BIOMATERIALS 35 25 7110 - 7120 2014年08月 [査読有り][通常論文]
     
    The objective of this study was to construct our recently developed aptamer-modified targeted liposome nano-carrier (Apt-PEG-LPs) system to target primary cultured mouse tumor endothelial cells (mTEC), both in vitro and in vivo. We first synthesized an aptamer-polyethylene glycol 2000-distearoyl phosphoethanolamine (Apt-PEG(2000)-DSPE). The conjugation of the Apt-PEG(2000)-DSPE was confirmed by MALDI-TOF mass spectroscopy. A lipid hydration method was used to prepare Apt-PEG-LPs, in which the outer surface of the PEG-spacer was decorated with the aptamer. Apt-PEG-LPs were significantly taken up by mTECs. Cellular uptake capacity was observed both quantitatively and qualitatively using spectrofluorometry, and confocal laser scanning microscopy (CLSM), respectively. In examining the extent of localization of aptamer-modified liposomes that entered the cells, approximately 39% of the Apt-PEG-LPs were not co-localized with lysotracker, indicating that they had escaped from endosomes. The uptake route involved a receptor mediated pathway, followed by clathrin mediated endocytosis. This Apt-PEG-LP was also applied for in vivo research whether this system could target tumor endothelial cells. Apt-PEG-LP and PEG(5000)-DSPE modified Apt-PEG-LP (Apt/PEG(5000)-LP) were investigated by human renal cell carcinoma (OS-RC-2 cells) inoculating mice using CLSM. Apt-PEG-LP and Apt/PEG(5000)-LP showed higher accumulation on tumor vasculature compared to PEG-LP and the co-localization efficacy of Apt-PEG-LP and Apt/PEG(5000)-LP on TEC were quantified 16% and 25% respectively, which was also better than PEG-LP (3%). The findings suggest that this system is considerable promise for targeting tumor endothelial cells to deliver drugs or genes in vitro and in vivo. (C) 2014 Elsevier Ltd. All rights reserved.
  • Takashi Nakamura, Kouhei Ono, Yoshiteru Suzuki, Rumiko Moriguchi, Kentaro Kogure, Hideyoshi Harashima
    MOLECULAR PHARMACEUTICS 11 8 2787 - 2795 2014年08月 [査読有り][通常論文]
     
    Exogenous antigen proteolysis by proteasomes and amino peptidases is essential for the production of mature major histocompatibility complex class 1 (MHC-I) peptides to induce cross-presentation. We report here that when liposomes are modified with octaarginine (R8-Lip), a type of cell-penetrating peptide, the production of the mature MIC-I peptide is enhanced by promoting the C-terminal trimming of the antigen peptide. The efficiency of cross-presentation of ovalbumin (OVA) using the R8-Lip was dramatically higher than that by octalysine modified liposomes (K8-Lip) in mouse bone-marrow derived dendritic cells (BMDCs), although the physical characters of both liposomes were comparable. In this study, we investigated the mechanism responsible for the enhancement in cross-presentation by R8-Lip. Although the efficiencies of cellular uptake, endosomal escape, proteolysis of OVA and DC maturation between the two systems were essentially the same, an analysis of peptide trimming to SIINFEKL (mature MHC-I peptide of OVA) by using R8-Lip and K8-Lip encapsulating peptides of various length dearly indicates that the use of R8-Lip enhances the efficiency of the C-terminal cleavage of antigen-derived peptides. This finding provides a new strategy for achieving efficient cross-presentation by using R8 peptide and arginine-rich peptides. Moreover, this result may contribute to the development of a new paradigm regarding the machinery associated with antigen peptide production.
  • Yuri Tawaraya, Mamoru Hyodo, Mst Naznin Ara, Yuma Yamada, Hideyoshi Harashima
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 37 8 1411 - 1415 2014年08月 [査読有り][通常論文]
     
    The use of mitochondria-based systematic evolution of ligands by exponential enrichment (SELEX) was explored. Mitochondria were isolated from rat liver and confirmed intact by respiratory control index. Isolated mitochondria and a 2'-F RNA random library were mixed and the bound RNAs collected. The counter selection was applied with nucleus and unbound RNAs were collected. After 7 rounds of selection, two sequences (Mitomer1 and Mitomer2) were verified to bind to mitochondria and the truncated Mitomer2 (short Mitomer2) showed better binding to isolated mitochondria than Mitomer1.
  • プラスミドDNA特異的ヒストン修飾による導入遺伝子発現上昇
    西原 実香, 神田 元紀, 山門 振一郎, 原島 秀吉, 紙谷 浩之
    日本DDS学会学術集会プログラム予稿集 30回 165 - 165 日本DDS学会 2014年07月
  • Ryosuke Suzuki, Yuma Yamada, Eriko Kawamura, Hideyoshi Harashima
    JOURNAL OF NANOPARTICLE RESEARCH 16 8 2014年07月 [査読有り][通常論文]
     
    Controlling the number of lipid bilayers, the lamellarity, in a liposome is a major factor in the in vivo/in vitro pharmacokinetics of drug delivery using liposome-based nanocarriers. Findings reported in a previous study indicated that a mono-cationic detergent (MCD) could be useful in controlling liposomal size via interaction with the lipid envelope. Here, we investigated controlling the lamellarity of the liposomal gene vector by MCD, using a multifunctional envelope-type nano device (MEND). The MEND consisted of a condensed plasmid DNA core and lipid envelopes. The size of the MCD-contained MEND (MCD-MEND) decreased as a function of the amount of MCD, indicating that MCD can be used to control the number of the lipid bilayers. We also developed a triple-layered MEND (TL-MEND) by packaging a di-lamellar MEND into an MCD-containing lipid bilayer. We hypothesized that the TL-MEND would efficiently deliver a gene to the nucleus, when the outer single bilayer fused with the plasma membrane and the inner double membranes then fused with the nuclear double membranes. Transfection assays showed that the TL-MEND had a high transfection activity in JAWS II cells, non-dividing cells. These results indicate that MCD has the potential for enhancing the gene delivery by controlling liposomal lamellarity.
  • Hiroko Miyabe, Mamoru Hyodo, Takashi Nakamura, Yusuke Sato, Yoshihiro Hayakawa, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 184 20 - 27 2014年06月 [査読有り][通常論文]
     
    Cyclic dinucleotides are of importance in the field of microbiology and immunology. They function as second messengers and are thought to participate in the signal transduction of cytosolic DNA immune responses. One such dinucleotide, cyclic di-GMP (c-di-GMP), stimulates the immune system. It is thought that c-di-GMP is recognized by ATP dependent RNA helicase (DDX41) in the cytosol, forms a complex with the Stimulator of interferon genes protein (STING), triggers a signal via the tank binding kinase 1-interferon regulatory factor 3 (TBK1-IRF3) pathway and induces the production of type I interferons. Therefore c-di-GMP can be thought of as a new class of adjuvant. However, because c-di-GMP contains two phosphate groups, this prevents its use as an adjuvant because it cannot pass through the cell membrane, even though the target molecule of c-di-GMP is located in the cytoplasm. Our group has been developing a series of liposomal drug delivery systems and recently investigated YSK05 which is a synthetic, pH sensitive lipid that has a high fusogenicity. We utilized this lipid as a carrier to transport c-di-GMP into the cytosol to then use c-di-GMP as an adjuvant. Based on screening experiments, YSK05/POPE/cholesterol = 40/25/35 was found to induce IFN-beta in Raw264.7 cells. The induction of IFN-beta from c-di-GMP liposomes was inhibited by adding BX795, a TBK1 inhibitor, indicating that the production of IFN-beta caused the activation of the STING-TBK1 pathway. C-di-GMP liposomes also showed significantly higher levels of expression of CD80, CD86 and MHC class I. The c-di-GMP/YSK05 liposome facilitated antigen specific cytotoxic T cell activity and the inhibition of tumor growth in a mouse model. These findings indicate that c-di-GMP/YSK05 liposomes could be used, not only to transfer c-di-GMP to the cytosol and induce an innate immune system but also as a platform for investigating the mechanism of immune sensing with cyclic dinucleotides in vitro and in vivo. (C) 2014 Elsevier B.V. All rights reserved.
  • Yuki Hattori, Daisuke Morita, Nagatoshi Fujiwara, Daiki Mori, Takashi Nakamura, Hideyoshi Harashima, Sho Yamasaki, Masahiko Sugita
    JOURNAL OF BIOLOGICAL CHEMISTRY 289 22 15405 - 15412 2014年05月 [査読有り][通常論文]
     
    Background: A host receptor has not yet been identified for glycerol monomycolate (GroMM), an immunostimulatory lipid of mycobacteria. Results: GroMM recognition occurred in cell transfectants expressing human, but not mouse Mincle. Human Mincle transgenic mice acquired the ability to respond to GroMM. Conclusion: GroMM is a ligand for human Mincle. Significance: The molecular basis underlying the innate immune recognition of GroMM has been elucidated. An array of lipidic compounds that constitute the cell wall of mycobacteria is recognized by host receptors. Examples include trehalose dimycolate (TDM), which is a major surface-exposed glycolipid of mycobacteria, that interacts with the macrophage inducible C-type lectin, Mincle, and exerts its highly potent adjuvant functions. Recent evidence has suggested that glycerol monomycolate (GroMM), another mycolate-containing lipid species produced by mycobacteria, can stimulate innate immune cells; however, its specific host receptors have yet to be identified. We here demonstrated that cell transfectants expressing human Mincle (hMincle) reacted to both TDM and GroMM, while those expressing mouse Mincle (mMincle) only reacted to TDM and failed to recognize GroMM. Studies using domain swap chimeras confirmed that the ectodomain of hMincle, but not that of mMincle, interacted with GroMM, and site-directed mutagenesis analyses revealed that short stretches of amino acid residues at positions 174-176 and 195-196 were involved in GroMM recognition. To further substantiate the differential recognition of GroMM by hMincle and mMincle, hMincle transgenic/mMincle knock-out mice (i.e. hMincle(+) mice) were established and compared with non-transgenic mice (i.e. mMincle(+) mice). We showed that macrophages derived from hMincle(+) mice were activated by GroMM and produced inflammatory cytokines, whereas those derived from mMincle(+) mice did not exhibit any reactivity to GroMM. Furthermore, local inflammatory responses were elicited in the GroMM-injected skin of hMincle(+), but not mMincle(+) mice. These results demonstrated that GroMM is a unique ligand for hMincle that is not recognized by mMincle.
  • Genki N. Kanda, Shiho Miyamoto, Miwako Kobayashi, Ichiro Matsuoka, Hideyoshi Harashima, Hiroyuki Kamiya
    INTERNATIONAL JOURNAL OF PHARMACEUTICS 464 1-2 27 - 33 2014年04月 [査読有り][通常論文]
     
    The transience of transgene expression is a major obstacle in the development of nonviral vectors. The CpG-free and pLIVE plasmids reportedly achieve long-term transgene expression in mouse liver. In this work, the anti-silencing elements within these plasmids were studied. The effects of plasmid that was being silenced on transgene expression from the CpG-free plasmid and those of transgene expression at early time points on silencing were also examined. The results suggested that the backbone sequence of the CpG-free plasmid and the 3' untranslated region of the albumin gene of the pLIVE plasmid contribute to durable expression. In addition, no influence of the silencing of another plasmid on the duration of CpG-free plasmid expression or of transgene expression at early time points on silencing was detected. (C) 2014 Elsevier B.V. All rights reserved.
  • Mina Tamaru, Hidetaka Akita, Kazuaki Kajimoto, Yusuke Sato, Hiroto Hatakeyama, Hideyoshi Harashima
    INTERNATIONAL JOURNAL OF PHARMACEUTICS 465 1-2 77 - 82 2014年04月 [査読有り][通常論文]
     
    A disorder in the brain endothelium is thought to be closely related to the pathophysiology of brain diseases. A method for delivering nucleic acids (i.e. short interference RNA; siRNA) to the brain endothelium should be an attractive strategy for curing brain disorders. A liposornal nanoparticle containing a proton ionizable amino lipid was recently developed as a carrier of encapsulated siRNA. The aim of this study was to evaluate the utility of apolipoprotein E (ApoE) as a targeting ligancl for mouse brain endothelial cells (MBEC4 cells). The cellular uptake of the ApoE-modified nanoparticles was gradually increased in an ApoE-density dependent mariner. Furthermore, the ApoE-modified nanoparticles were taken up via both clathrin and caveolae mediated endocytosis, thus permitting them to avoid lysosomal degradation. Finally, endogenous gene silencing in MBEC4 cells was efficiently achieved depending on the ApoE-modification. Collectively, the ApoE-modified nanoparticle is a promising carrier for delivering nucleic acids to the brain endothelium. (c) 2014 Elsevier B.V. All rights reserved.
  • Tsunamasa Watanabe, Hiroto Hatakeyama, Chiho Matsuda-Yasui, Yusuke Sato, Masayuki Sudoh, Asako Takagi, Yuichi Hirata, Takahiro Ohtsuki, Masaaki Arai, Kazuaki Inoue, Hideyoshi Harashima, Michinori Kohara
    SCIENTIFIC REPORTS 4 4750  2014年04月 [査読有り][通常論文]
     
    The development of RNA interference (RNAi)-based therapy faces two major obstacles: selecting small interfering RNA (siRNA) sequences with strong activity, and identifying a carrier that allows efficient delivery to target organs. Additionally, conservative region at nucleotide level must be targeted for RNAi in applying to virus because hepatitis C virus (HCV) could escape from therapeutic pressure with genome mutations. In vitro preparation of Dicer-generated siRNAs targeting a conserved, highly ordered HCV 5' untranslated region are capable of inducing strong RNAi activity. By dissecting the 5'-end of an RNAi-mediated cleavage site in the HCV genome, we identified potent siRNA sequences, which we designate as Dicer-hunting siRNAs (dh-siRNAs). Furthermore, formulation of the dh-siRNAs in an optimized multifunctional envelope-type nano device inhibited ongoing infectious HCV replication in human hepatocytes in vivo. Our efforts using both identification of optimal siRNA sequences and delivery to human hepatocytes suggest therapeutic potential of siRNA for a virus.
  • Yasuhiro Hayashi, Erina Suemitsu, Kazuaki Kajimoto, Yusuke Sato, Afsana Akhter, Yu Sakurai, Hiroto Hatakeyama, Mamoru Hyodo, Noritada Kaji, Yoshinobu Baba, Hideyoshi Harashima
    MOLECULAR THERAPY-NUCLEIC ACIDS 3 e154  2014年03月 [査読有り][通常論文]
     
    Over the past decade, considerable advances have been made in the discovery of gene targets in metabolic diseases. However, in vivo studies based on molecular biological technologies such as the generation of knockout mice and the construction of short hairpin RNA vectors require considerable effort and time, which is a major limitation for in vivo functional analysis. Here, we introduce a liver-specific nonviral small interfering RNA (siRNA) delivery system into rapid and efficient characterization of hepatic gene targets in metabolic disease mice. The comparative transcriptome analysis in liver between KKAy diabetic and normal control mice demonstrated that the expression of monoacylglycerol O-acyltransferase 1 (Mogat1), an enzyme involved in triglyceride synthesis and storage, was highly elevated during the disease progression. The upregulation of Mogat1 expression in liver was also found in other genetic (db/db) and diet-induced obese mice. The silencing of hepatic Mogat1 via a liver-specific siRNA delivery system resulted in a dramatic improvement in blood glucose levels and hepatic steatosis as well as overweight with no apparent overall toxicities, indicating that hepatic Mogat1 is a promising therapeutic target for metabolic diseases. The integrated approach with transcriptomics and nonviral siRNA delivery system provides a blueprint for rapid drug discovery and development.
  • Hafiz Muhammad Ali, Andrei Maksimenko, Giorgia Urbinati, Hubert Chapuis, Mouna Raouane, Didier Desmaele, Hayashi Yasuhiro, Hideyoshi Harashima, Patrick Couvreur, Liliane Massaad-Massade
    THYROID 24 2 327 - 338 2014年02月 [査読有り][通常論文]
     
    Background:RET/PTC1 is the most prevalent type of gene rearrangement found in papillary thyroid carcinoma (PTC). Previously, we introduced a new noncationic nanosystem for targeted RET/PTC1 silencing by efficient delivery of small interfering RNA (siRNA) using the squalenoylation approach. With the aim of improving these results further, we designed new squalenoyl nanostructures consisting of the fusogenic peptide GALA-cholesterol (GALA-Chol) and squalene (SQ) nanoparticles (NPs) of siRNA RET/PTC1. Methods: The siRNA RET/PTC1-SQ bioconjugate was synthesized. The corresponding NPs were prepared with or without GALA-Chol by nanoprecipitation and then characterized for their size and zeta potential. The effects of NPs on BHP 10-3 SCmice and TPC-1 cell viability (MTT assay), gene and protein silencing (reverse transcription-quantitative polymerase chain reaction [rt-qPCR], Western blot), and cellular uptake (fluorescent microscopy) were studied. In vivo gene silencing efficiency of siRNA RET/PTC1-SQ NPs was assessed by administration in nude mice via either intratumoral (i.t.) or intravenous (i.v.) routes. Tumor growth was followed for 19 days. Tumors were then collected, and RET/PTC1 gene and protein inhibitions were assessed by RT-qPCR and Western blot. Results: The combination of siRNA RET/PTC1-SQ bioconjugate and GALA-Chol leads to stable NPs of approximate to 200nm diameter. In vitro, the results revealed that combining GALA-Chol with siRNA RET/PTC1-SQ NPs decreased cell viability, enhanced cellular internalization, and induced gene silencing efficiency in both human PTC (BHP 10-3 SCmice and TPC-1) cell lines. On the contrary, in vivo, the siRNA RET/PTC1-SQ GALA-Chol NPs were not found to be efficient either in gene silencing or in tumor growth inhibition, compared to siRNA RET/PTC1-SQ NPs both via i.t. and i.v. routes (p<0.001). Conclusions: Conversely to siRNA RET/PTC1-SQ NPs, the siRNA RET/PTC1-SQ GALA-Chol NPs are efficient in vitro but not in vivo. Finally, NPs of siRNA RET/PTC1-SQ were found to be efficient silencers of the RET/PTC1 fusion oncogene in in vivo applications even at a concentration lower than used in a previously published study.
  • Hiroki Tanaka, Hidetaka Akita, Ryohei Ishiba, Kota Tange, Masaya Arai, Kazuhiro Kubo, Hideyoshi Harashima
    BIOMATERIALS 35 5 1755 - 1761 2014年02月 [査読有り][通常論文]
     
    Biomembranes and cytoplasm, a diffusion-limited region for nanoparticles are critical barriers to be overcome for the successful gene delivery. We herein report on a neutral, and intracellularly degradable lipid nanoparticle (LNP), containing encapsulated plasmid DNA (pDNA) that can be effectively delivered to the nucleus. A key material component in this particle is a vitamin A-scaffold SS-cleavable ProtonActivated Lipid-like Material ((SS)Palm(A)), which contains tertiary amine groups as proton sponge units that can respond to the acidic pH in endosomes, disulfide bonding for programmed collapse in the cytoplasm, and retinoic acid (RA) as a hydrophobic unit for assembly into LNP. LNP prepared using ssPalmA (LNPpalmA) exhibited a 15-fold higher gene expression activity compared to particles prepared with a simple acyl chain (myristoyl group)-scaffold one (LNPpalmM). Intracellular imaging studies revealed that LNPPalmA unexpectedly showed excessive endosome-disruptive characteristics. Furthermore, the decapsulation of pDNA slowly, but successively occurred in parallel with pen-nuclear accumulation. Nuclear targeting was blocked in the presence of native RA. Collectively, LNPpalmA is an intelligent particle that passes through the cytoplasm in particle form with the aid of the intrinsic nuclear transport system of RA, and thereafter releases its encapsulated pDNA for effective gene expression. (C) 2013 Elsevier Ltd. All rights reserved.
  • Takashi Nakamura, Masafumi Fukiage, Megumi Higuchi, Akihiro Nakaya, Ikuya Yano, Jun Miyazaki, Hiroyuki Nishiyama, Hideyuki Akaza, Toshihiro Ito, Hiroyuki Hosokawa, Toshinori Nakayama, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 176 44 - 53 2014年02月 [査読有り][通常論文]
     
    The Mycobacterium bovis Bacille Calmette-Guerin cell wall skeleton (BCG-CWS) could be used to replace live BCG as a bladder cancer drug. However, because BCG-CWS is poorly soluble, has a strong-negative charge, very high molecular weight and heterogeneity in size of tens of mu m, it cannot be used in such an application. We report herein on the development of a novel packaging method that permits BCG-CWS to be encapsulated into 166 nm-sized lipid particles. The BCG-CWS encapsulated nano particle (CWS-NP) has a high uniformity and can be easily dispersed. Thus, it has the potential for use as a packaging method that would advance the scope of applications of BCG-CWS as a bladder cancer drug. In a functional evaluation, CWS-NP was efficiently taken up by mouse bladder tumor (MBT-2) cells in vitro and inhibited tumor growth in mice bearing MBT-2 tumors. Moreover, intravesically administered CWS-NP showed significant antitumor effects in a rat model with naturally developed bladder cancer. An enhancement in Th1 differentiation by CWS-NP was also confirmed in human T cells. In conclusion, CWS-NP represents a promising delivery system for BCG-CWS for clinical development as a potent bladder cancer drug. (C) 2013 Elsevier B. V. All rights reserved.
  • 原島 秀吉
    薬剤学 74 1 1 - 1 The Academy of Pharmaceutical Science and Technology, Japan 2014年
  • Harashima Hideyoshi, Hidetaka Akita, Kumiko Sakai-Kato, Takehiko Ishii, Yasuhiro Matsumura, Kazunori Kataoka
    Drug Delivery System 29 3 217 - 225 2014年 [査読有り][通常論文]
     
    Nanomaterials often have physical, chemical, or biological properties that are different from those of bulk materials. These properties may have potential impacts on a variety of products, and nanotechnology application to pharmaceuticals is a typical such example. Because the unique size-specific interaction with biological systems or biodistribution may have significant impacts on the efficacy and safety of nanomedicines, regulatory science researches of nanomedicines are required. We have been conducting the formulation study, and nonclinical pharmacokinetics, pharmacodynamics, toxicology, and clinical studies of the polymeric micelles and lipid nanoparticles which are designed to ensure high stability in vivo and to optimize the pharmacokinetics. In this review, we introduce our initiatives for regulatory science researches of nanomedicines.
  • Mina Tamaru, Hidetaka Akita, Taichi Nakatani, Kazuaki Kajimoto, Yusuke Sato, Hiroto Hatakeyama, Hideyoshi Harashima
    INTERNATIONAL JOURNAL OF NANOMEDICINE 9 4267 - 4276 2014年 [査読有り][通常論文]
     
    An innovative drug delivery technology is urgently needed to satisfy unmet medical needs in treating various brain disorders. As a fundamental carrier for plasmid DNA or nucleic acids, we developed a liposomal nanoparticle (multifunctional envelope-type nano device [MEND]) containing a proton-ionizable amino lipid (YSK-MEND). Here we report on the impact of apolipoprotein E (ApoE) modification on the function of YSK-MEND in terms of targeting brain cells. The cellular uptake and function of YSK-MEND encapsulating short interference RNA or plasmid DNA were significantly improved as a result of ApoE modification in mouse neuron-derived cell lines (Neuro-2a and CAD). Intracerebroventricular administration of ApoE-modified YSK-MEND (ApoE/YSK-MEND) encapsulating plasmid DNA also resulted in higher transgene expression in comparison with YSK-MEND that was not modified with ApoE. Moreover, observation of fluorescence-labeled ApoE/YSK-MEND and expression of mCherry (fluorescence protein) derived from plasmid DNA indicated that this carrier might be useful for delivering and conferring transgene expression in neural stem cells and/or neural progenitor cells. Thus, this system may be a useful tool for the treatment of neurodegenerative disease.
  • Hiroto Hatakeyama, Manami Murata, Yusuke Sato, Mayumi Takahashi, Noriaki Minakawa, Akira Matsuda, Hideyoshi Harashima
    Journal of Controlled Release 173 1 43 - 50 2014年 [査読有り][通常論文]
     
    Efficient delivery continues to be a challenge in microRNA (miRNA) therapeutics. We utilized a pH-sensitive multifunctional envelope-type nano device (MEND) containing a pH-sensitive lipid YSK05 (YSK05-MEND) to regulate liver specific miRNA-122 (miR-122). Anti-microRNA oligonucleotides including 2′-OMe and phosphorothioate modifications against miR-122 (AMO122) were encapsulated in the YSK05-MEND. Despite the lower uptake, the YSK05-MEND showed a higher activity in liver cancer cells than Lipofectamine2000 (LFN2k) due to efficient endosomal escape. Cytotoxicity was minimal at 100 nM of AMO122 in YSK05-MEND treated cells, but LFN2k showed toxicity at 50 nM. When mice were administrated with free AMO122, it was eliminated via the kidney due to its molecular weight, and lesser amounts were detected in the liver. Conversely, the YSK05-MEND delivered higher amounts of the AMO122 to the liver. Systemic administration of YSK05-MEND induced the knockdown of miR-122 and an increase in target genes in the liver, and a subsequent reduction in plasma cholesterol at a dose of 1 mg AMO/kg while free AMO122 showed no activity at the same dose. The effect of AMO122 delivered by YSK05-MEND persisted for over 2 weeks. These results suggest that YSK05-MEND is a promising system for delivering AMOs to the liver. © 2013 Elsevier B.V.
  • Masami Ukawa, Hidetaka Akita, Yasuhiro Hayashi, Ryohei Ishiba, Kota Tange, Masaya Arai, Kazuhiro Kubo, Yuriko Higuchi, Kazunori Shimizu, Satoshi Konishi, Mitsuru Hashida, Hideyoshi Harashima
    Advanced Healthcare Materials 3 8 1222 - 1229 2014年 [査読有り][通常論文]
     
    Charge-neutralized lipid envelope-type nanoparticles formed with SS-cleavable and pH-activated lipid-like materials (ssPalm) accumulate rapidly in the liver without forming aggregates in the blood circulation, and result in a liver-specific gene expression for a long duration (> 2 weeks) with neither immunological responses nor hepatotoxicity after intraveneous administration, when it carries pDNA free from CpG-motifs. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
  • Hiroyuki Kamiya, Tetsuya Suzuki, Hideyoshi Harashima
    Genes and Environment 36 2 65 - 68 2014年 [査読有り][通常論文]
     
    The loss of the p53 tumor suppressor protein causes genomic instability. The double strand break is the most severe form of DNA damage, and homologous recombination (HR) acts as a defense against it. In this study, the effects of p53 reduction on HR were investigated, using plasmid DNA in human U2OS cells. The plasmid DNA was designed to contain two inactivated kanamycin resistance genes that reconstitute the functional gene after HR. The p53 protein was knocked-down by siRNA, and then linearized plasmid DNA was introduced into the knockdown cells. The knockdown of p53 reduced the frequency of short tract gene conversion in the plasmid. These results suggested that p53 may enhance the HR of episomal DNA in human cells. © The Japanese Environmental Mutagen Society.
  • Yu Sakurai, Hiroto Hatakeyama, Yusuke Sato, Mamoru Hyodo, Hidetaka Akita, Noritaka Ohga, Kyoko Hida, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 173 110 - 118 2014年01月 [査読有り][通常論文]
     
    Angiogenesis is one of crucial processes associated with tumor growth and development, and consequently a prime target for cancer therapy. Although tumor endothelial cells (TECs) play a key role in pathological angiogenesis, investigating phenotypical changes in neovessels when a gene expression in TEC is suppressed is a difficult task. Small interfering RNA (siRNA) represents a potential agent due to its ability to silence a gene of interest. We previously developed a system for in vivo siRNA delivery to cancer cells that involves a liposomal-delivery system, a MEND that contains a unique pH-sensitive cationic lipid, YSK05 (YSK-MEND). In the present study, we report on the development of a system that permits the delivery of siRNA to TECs by combining the YSK-MEND and a ligand that is specific to TECs. Cyclo(Arg-Gly-Asp-D-Phe-Lys) (cRGD) is a well-known ligand to alpha(V)beta(3) integrin, which is selectively expressed at high levels in TECs. We incorporated cRGD into the YSK-MEND (RGD-MEND) to achieve an efficient gene silencing in TECs. Quantitative RT-PCR and the 5' rapid amplification of cDNA ends PCR indicated that the intravenous injection of RGD-MEND at a dose of 4.0 mg/kg induced a significant RNAi-mediated gene reduction in TEC but not in endothelial cells of other organs. Finally, we evaluated the therapeutic potency of the RGD-MEND encapsulating siRNA against vascular endothelial growth factor receptor 2. A substantial delay in tumor growth was observed after three sequential RGD-MEND injections on alternate days. In conclusion, the RGD-MEND represents a new approach for the characterization of TECs and for us in anti-angiogenic therapy. (C) 2013 Elsevier B.V. All rights reserved.
  • Golam Kibria, Hiroto Hatakeyama, Hideyoshi Harashima
    ARCHIVES OF PHARMACAL RESEARCH 37 1 4 - 15 2014年01月 [査読有り][通常論文]
     
    Multidrug resistance (MDR), the principal mechanism by which many cancers develop resistance to chemotherapy, is one of the major obstacles to the successful clinical treatment of various types of cancer. Several key regulators are responsible for mediating MDR, a process that renders chemotherapeutic drugs ineffective in the internal organelles of target cells. A nanoparticulate drug delivery system (DDS) is a potentially promising tool for circumventing such MDR, which can be achieved by targeting tumor cells themselves or tumor endothelial cells that support the survival of MDR cancer cells. The present article discusses key factors that are responsible for MDR in cancer cells, with a specific focus on the application of DDS to overcome MDR via the use of chemotherapy or macromolecules.
  • Hiroto Hatakeyama, Manami Murata, Yusuke Sato, Mayumi Takahashi, Noriaki Minakawa, Akira Matsuda, Hideyoshi Harashima
    Journal of Controlled Release 173 1 43 - 50 2014年 [査読有り][通常論文]
     
    Efficient delivery continues to be a challenge in microRNA (miRNA) therapeutics. We utilized a pH-sensitive multifunctional envelope-type nano device (MEND) containing a pH-sensitive lipid YSK05 (YSK05-MEND) to regulate liver specific miRNA-122 (miR-122). Anti-microRNA oligonucleotides including 2′-OMe and phosphorothioate modifications against miR-122 (AMO122) were encapsulated in the YSK05-MEND. Despite the lower uptake, the YSK05-MEND showed a higher activity in liver cancer cells than Lipofectamine2000 (LFN2k) due to efficient endosomal escape. Cytotoxicity was minimal at 100 nM of AMO122 in YSK05-MEND treated cells, but LFN2k showed toxicity at 50 nM. When mice were administrated with free AMO122, it was eliminated via the kidney due to its molecular weight, and lesser amounts were detected in the liver. Conversely, the YSK05-MEND delivered higher amounts of the AMO122 to the liver. Systemic administration of YSK05-MEND induced the knockdown of miR-122 and an increase in target genes in the liver, and a subsequent reduction in plasma cholesterol at a dose of 1 mg AMO/kg while free AMO122 showed no activity at the same dose. The effect of AMO122 delivered by YSK05-MEND persisted for over 2 weeks. These results suggest that YSK05-MEND is a promising system for delivering AMOs to the liver. © 2013 Elsevier B.V.
  • Hiroto Hatakeyama, Manami Murata, Yusuke Sato, Mayumi Takahashi, Noriaki Minakawa, Akira Matsuda, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 173 43 - 50 2014年01月 [査読有り][通常論文]
     
    Efficient delivery continues to be a challenge in microRNA (miRNA) therapeutics. We utilized a pH-sensitive multifunctional envelope-type nano device (MEND) containing a pH-sensitive lipid YSK05 (YSK05-MEND) to regulate liver specific miRNA-122 (miR-122). Anti-microRNA oligonucleotides including 2'-OMe and phosphorothioate modifications against miR-122 (AMO122) were encapsulated in the YSK05-MEND. Despite the lower uptake, the YSK05-MEND showed a higher activity in liver cancer cells than Lipofectamine2000 (LFN2k) due to efficient endosomal escape. Cytotoxicity was minimal at 100 nM of AMO122 in YSK05-MEND treated cells, but LFN2k showed toxicity at 50 nM. When mice were administrated with free AMO122, it was eliminated via the kidney due to its molecular weight, and lesser amounts were detected in the liver. Conversely, the YSK05-MEND delivered higher amounts of the AMO122 to the liver. Systemic administration of YSK05-MEND induced the knockdown of miR-122 and an increase in target genes in the liver, and a subsequent reduction in plasma cholesterol at a dose of 1 mg AMO/kg while free AMO122 showed no activity at the same dose. The effect of AMO122 delivered by YSK05-MEND persisted for over 2 weeks. These results suggest that YSK05-MEND is a promising system for delivering AMOs to the liver. (C) 2013 Elsevier B.V. All rights reserved.
  • Yuma Yamada, Hideyoshi Harashima
    Methods in Molecular Biology 1141 57 - 66 2014年 [査読有り][通常論文]
     
    Various types of mitochondrial dysfunctions have been implicated in a variety of human diseases. This suggests that mitochondria would be promising therapeutic drug targets and mitochondrial therapy would be expected to be useful for the treatment of various diseases. We have already reported the development of a MITO-Porter, a liposome-based nano-carrier that delivers its cargo to mitochondria via a membrane-fusion mechanism. In our strategy for delivering cargos to mitochondria using a MITO-Porter, the carriers must fuse with the organelle membrane. Here we report on methodology for screening various types of lipid envelopes that have the potential for fusing with a mitochondrial membrane. The method involves monitoring the cancellation of fluorescence resonance energy transfer (FRET) and evaluating membrane fusion between the carriers and mitochondria in living cells by FRET analysis using a spectral imaging fluorescent microscopy system. © 2014 Springer Science+Business Media, New York.
  • Kazuaki Kajimoto, Yoshitaka Minami, Hideyoshi Harashima
    FEBS OPEN BIO 4 602 - 610 2014年 [査読有り][通常論文]
     
    The fatty acid binding protein 4 (FABP4), one of the most abundant proteins in adipocytes, has been reported to have a proinflammatory function in macrophages. However, the physiological role of FABP4, which is constitutively expressed in adipocytes, has not been fully elucidated. Previously, we demonstrated that FABP4 was involved in the regulation of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) production in 3T3-L1 adipocytes. In this study, we examined the effects of FABP4 silencing on the oxidative and endoplasmic reticulum (ER) stress in 3T3-L1 adipocytes. We found that the cellular reactive oxygen species (ROS) and 8-nitro-cyclic GMP levels were significantly elevated in the differentiated 3T3-L1 adipocytes transfected with a small interfering RNA (siRNA) against Fabp4, although the intracellular levels or enzyme activities of antioxidants including reduced glutathione (GSH), superoxide dismutase (SOD) and glutathione S-transferase A4 (GSTA4) were not altered. An in vitro evaluation using the recombinant protein revealed that FABP4 itself functions as a scavenger protein against hydrogen peroxide (H2O2). FABP4-knockdown resulted in a significant lowering of cell viability of 3T3-L1 adipocytes against H2O2 treatment. Moreover, four kinds of markers related to the ER stress response including the endoplasmic reticulum to nucleus signaling 1 (Ern1), the signal sequence receptor alpha (Ssr1), the ORM1-like 3 (Ormdl3), and the spliced X-box binding protein 1 (Xbp1s), were all elevated as the result of the knockdown of FABP4. Consequently, FABP4 might have a new role as an antioxidant protein against H2O2 and contribute to cytoprotection against oxidative and ER stress in adipocytes. (C) 2014 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
  • Tamaru M, Akita H, Nakatani T, Kajimoto K, Sato Y, Hatakeyama H, Harashima H
    International journal of nanomedicine 9 4267 - 4276 2014年 [査読有り][通常論文]
  • Yusuke Sato, Takashi Nakamura, Yuma Yamada, Hidetaka Akita, Hideyoshi Harashima
    NONVIRAL VECTORS FOR GENE THERAPY LIPID- AND POLYMER-BASED GENE TRANSFER 88 139 - 204 2014年 [査読有り][通常論文]
     
    It is anticipated that nucleic acid medicines will be in widespread use in the future, since they have the potential to cure diseases based on molecular mechanisms at the level of gene expression. However, intelligent delivery systems are required to achieve nucleic acid therapy, since they can perform their function only when they reach the intracellular site of action. We have been developing a multifunctional envelope-type nanodevice abbreviated as MEND, which consists of functional nucleic acids as a core and lipid envelope, and can control not only biodistribution but also the intracellular trafficking of nucleic acids. In this chapter, we review the development and evolution of the MEND by providing several successful examples, including the R8-MEND, the KALA-MEND, the MITO-Porter, the YSK-MEND, and the PALM.
  • Mayumi Takahashi, Naoki Yamada, Hiroto Hatakeyama, Manami Murata, Yusuke Sato, Noriaki Minakawa, Hideyoshi Harashima, Akira Matsuda
    Nucleic Acids Research 41 22 10659 - 10667 2013年12月 [査読有り][通常論文]
     
    MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression post-transcriptionally. Previous studies, which characterized miRNA function, revealed their involvement in fundamental biological processes. Importantly, miRNA expression is deregulated in many human diseases. Specific inhibition of miRNAs using chemically modified anti-miRNA oligonucleotides (AMOs) can be a potential therapeutic strategy for diseases in which a specific miRNA is overexpressed. 2′-O-Methyl (2′-OMe)-4′-thioRNA is a hybrid type of chemically modified oligonucleotide, exhibiting high binding affinity to complementary RNAs and high resistance to nuclease degradation. Here, we evaluate 2′-OMe-4′-thioribonucleosides for chemical modification on AMOs. Optimization of the modification pattern using a variety of chemically modified AMOs that are perfectly complementary to mature miR-21 revealed that the uniformly 2′-OMe-4′-thioribonucleoside-modified AMO was most potent. Further investigation showed that phosphorothioate modification contributed to long-term miR-122 inhibition by the 2′-OMe-4′- thioribonucleoside-modified AMO. Moreover, systemically administrated AMOs to mouse using a liposomal delivery system, YSK05-MEND, showed delivery to the liver and efficient inhibition of miR-122 activity at a low dose in vivo. © 2013 The Author(s).
  • Mayumi Takahashi, Naoki Yamada, Hiroto Hatakeyama, Manami Murata, Yusuke Sato, Noriaki Minakawa, Hideyoshi Harashima, Akira Matsuda
    NUCLEIC ACIDS RESEARCH 41 22 10659 - 10667 2013年12月 [査読有り][通常論文]
     
    MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression post-transcriptionally. Previous studies, which characterized miRNA function, revealed their involvement in fundamental biological processes. Importantly, miRNA expression is deregulated in many human diseases. Specific inhibition of miRNAs using chemically modified anti-miRNA oligonucleotides (AMOs) can be a potential therapeutic strategy for diseases in which a specific miRNA is overexpressed. 2'-O-Methyl (2'-OMe)-4'-thioRNA is a hybrid type of chemically modified oligonucleotide, exhibiting high binding affinity to complementary RNAs and high resistance to nuclease degradation. Here, we evaluate 2'-OMe-4'-thioribonucleosides for chemical modification on AMOs. Optimization of the modification pattern using a variety of chemically modified AMOs that are perfectly complementary to mature miR-21 revealed that the uniformly 2'-OMe-4'-thioribonucleoside modified AMO was most potent. Further investigation showed that phosphorothioate modification contributed to long-term miR-122 inhibition by the 2'-OMe-4'-thioribonucleoside-modified AMO. Moreover, systemically administrated AMOs to mouse using a liposomal delivery system, YSK05-MEND, showed delivery to the liver and efficient inhibition of miR-122 activity at a low dose in vivo.
  • Shinya Iida, Hiroyuki Kamiya, Akihiro Nakaya, Yasuhiro Hayashi, Akihiro Sawada, Noritada Kaji, Yoshinobu Baba, Hideyoshi Harashima
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 36 12 2009 - 2011 2013年12月 [査読有り][通常論文]
     
    Expression of the asparagine synthetase gene is dependent on extracellular glucose concentration. This gene was knocked-down in livers of male Balb/c mice by a hydrodynamic tail vein injection of small interfering RNA (siRNA) against the gene. This knockdown resulted in a significant decrease in plasma glucose concentration. These results suggested that asparagine synthetase is a novel protein that regulates plasma glucose levels.
  • Eriko Kawamura, Yuma Yamada, Yukari Yasuzaki, Mamoru Hyodo, Hideyoshi Harashima
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 116 5 634 - 637 2013年11月 [査読有り][通常論文]
     
    This study focused on the intracellular observation of nanocarriers modified with a mitochondrial targeting signal peptide (MTS). The nanocarriers showed an efficient cellular uptake, and the MTS had a positive effect on their mitochondrial targeting. This is the first report of an intracellular observation of nanocarriers modified with MTS. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.
  • Asako Mitsueda, Yuri Shimatani, Masahiro Ito, Takashi Ohgita, Asako Yamada, Susumu Hama, Astrid Graslund, Staffan Lindberg, Ulo Langel, Hideyoshi Harashima, Ikuhiko Nakase, Shiroh Futaki, Kentaro Kogure
    BIOPOLYMERS 100 6 698 - 704 2013年11月 [査読有り][通常論文]
     
    Development of novel devices for effective nucleotide release from nanoparticles is required to improve the functionality of nonviral delivery systems, because decondensation of nucleotide/polycation complexes is considered as a key step for cytoplasmic delivery of nucleotides. Previously, PepFect6 (PF6) comprised chloroquine analog moieties and a stearylated cell-penetrating peptide to facilitate endosomal escape and cellular uptake, respectively, was developed as a device for efficient siRNA delivery. As PF6 contains bulky chloroquine analog moieties, the polyplexes are expected to be loose structure, which facilitates decondensation. In the present study, siRNA was electrostatically condensed by PF6, and the PF6/siRNA complexes were coated with lipid membranes. The surface of the nanoparticles encapsulating the PF6/siRNA core (PF6-NP) was modified with PF6 for endosomal escape (PF6/PF6-NP). The RNAi effect of PF6/PF6-NP was compared with those of stearylated cell-penetrating peptide octaarginine (R8)-modified PF6-NP, R8-modified nanoparticles encapsulating the R8/siRNA core (R8-NP) and PF6-modified R8-NP. Nanoparticles encapsulating the PF6 polyplex, especially PF/PF-NP, showed a significant knockdown effect on luciferase activity of B16-F1 cells stably expressing luciferase. siRNA was widely distributed within the cytoplasm after transfection of the nanoparticles encapsulating the PF6 polyplex, while siRNA encapsulated in the R8-presenting nanoparticles was localized within the nuclei. Thus, the siRNA distribution was dependent on the manner of peptide-modification. In conclusion, we have successfully developed PF6/PF6-NP exhibiting a potent RNAi effect resulting from high cellular uptake, efficient endosomal escape and decondensation of the polyplexes based on the multifunctional cell penetrating peptide PF6. PF6 is therefore a useful pluripotential device for siRNA delivery. (C) 2013 Wiley Periodicals, Inc.
  • Akhter A, Hayashi Y, Sakurai Y, Ohga N, Hida K, Harashima H
    International journal of pharmaceutics 456 1 195 - 201 Elsevier science bv 2013年11月 [査読有り][通常論文]
     
    Liver dysfunction is associated with a variety of liver diseases, including viral or alcoholic hepatitis, fibrosis, cirrhosis, and portal hypertension. A targeted drug delivery system would be very useful in the treatment of these diseases. We herein describe the development of a system comprised of a new peptide-lipid conjugate for the efficient delivery of molecules to LEC. The RLTRKRGLK sequence (3359-3367), which mediates the association of LDL with arterial CSPG and an LDL receptor, was utilized as a ligand for achieving this goal. The peptide modified PEG-LPs (RLTR-PEG-LPs) were efficiently taken up by primary liver endothelial cells (liver ECs) and other types of cells. In vivo biodistribution and confocal microscopy analysis showed that RLTR-PEG-LPs became widely accumulated in LECs within a short time. Distribution of RLTR-PEG-LPs was greatly reduced with a pretreatment of unlabeled RLTR-PEG-LPs, not cationic LPs, indicating that the sequence is important for LECs. The findings indicate that a reverse sequence of RLTR (KLGR) modified PEG-LPs (KLGR-PEG-LP) did the same pattern compared with RLTR-PEG- LPs, suggesting that the RKR or RXXR sequence might be essential for LECs targeting. Collectively RLTR-PEG-LPs and KLGR-PEG-LPs have the potential for delivering drugs to LECs. (C) 2013 Elsevier B.V. All rights reserved.
  • Hidetaka Akita, Soichiro Ishii, Naoya Miura, Sharif Mohammad Shaheen, Yasuhiro Hayashi, Takashi Nakamura, Noritada Kaji, Yoshinobu Baba, Hideyoshi Harashima
    BIOMATERIALS 34 35 8979 - 8990 2013年11月 [査読有り][通常論文]
     
    Technologies for the transfection of antigen-encoding genes into the dendritic cells, and subsequent immune-activation are both prerequisites for a successful DNA vaccine. We herein report on the density-dependent enhancement of transgene expression by the simple modification by stearyl-conjugated KALA, an alpha-helical peptide (STR-KALA), onto a lipid envelope-type nanoparticle (the R8-MEND, an octaarginine-modified multifunctional envelope-type nano device). The enhanced transgene expression in the KALA-modified R8-MEND (R8/KALA-MEND) cannot be explained by cellular uptake and nuclear delivery efficacy. Thus, the post-nuclear delivery process (i.e. transcription), but not intracellular trafficking processes attributed the enhanced transfection efficacy. Microarray analyses revealed that transfection with the R8/KALA-MEND resulted in a greater perturbation in host genes expression in comparison with the R8-MEND and that this effect was time-dependent. Further pathway analyses in the category of transcription-related genes and a gene ontology analysis indicated that the R8/KALA-MEND stimulated the expression of transcription factors that are closely related to immune-activation (i.e. NF-kB and STAT). Inhibition of the transfection efficacy by blockage of the STAT pathways revealed that the enhanced transcription activity is the result of immune-stimulation. Collectively, the R8/KALA-MEND mounts a "switch-on" function that triggers signal transduction forward to the immune-stimulation analogous to an adjuvant, and consequently elicits active transcription. (C) 2013 Elsevier Ltd. All rights reserved.
  • Eriko Kawamura, Yuma Yamada, Hideyoshi Harashima
    MITOCHONDRION 13 6 610 - 614 2013年11月 [査読有り][通常論文]
     
    To achieve mitochondrial therapy, we previously reported on the use of an octaarginine (R8) modified Dual Function (DF)-MITO-Porter for delivering molecules to mitochondria in living cells. In this study, using isolated mitochondria, homogenates and living cells, we evaluated the utility of mitochondrial targeting functional peptides as a ligand for delivering carriers. The S2 peptide modified carrier showed a high mitochondrial targeting activity in homogenates and living cells. In addition, the S2 peptide had a lower cell toxicity compared to R8 modified liposomes. The S2 peptide represents a potentially useful moiety for constructing an efficient and safe mitochondrial delivery system. (C) 2013 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
  • Daisuke Morita, Ayumi Miyamoto, Yuki Hattori, Takaya Komori, Takashi Nakamura, Tatsuhiko Igarashi, Hideyoshi Harashima, Masahiko Sugita
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 441 1 108 - 113 2013年11月 [査読有り][通常論文]
     
    Trehalose 6,6'-dimycolate (TDM) is a major glycolipid of the cell wall of mycobacteria with remarkable adjuvant functions. To avoid detection by the host innate immune system, invading mycobacteria down-regulate the expression of TDM by utilizing host-derived glucose as a competitive substrate for their mycolyltransferases; however, this enzymatic reaction results in the concomitant biosynthesis of glucose monomycolate (GMM) which is recognized by the acquired immune system. GMM-specific, CD1-restricted T cell responses have been detected in the peripheral blood of infected human subjects and monkeys as well as in secondary lymphoid organs of small animals, such as guinea pigs and human CD1-transgenic mice. Nevertheless, it remains to be determined how tissues respond at the site where GMM is produced. Here we found that rhesus macaques vaccinated with Mycobacterium bovis bacillus Calmette Guerin mounted a chemokine response in GMM-challenged skin that was favorable for recruiting T helper (Th)1 T cells. Indeed, the expression of interferon-gamma, but not Th2 or Th17 cytokines, was prominent in the GMM-injected tissue. The GMM-elicited tissue response was also associated with the expression of monocyte/macrophage-attracting CC chemokines, such as CCL2, CCL4 and CCL8. Furthermore, the skin response to GMM involved the up-regulated expression of granulysin and perforin. Given that GMM is produced primarily by pathogenic mycobacteria proliferating within the host, the Th1-skewed tissue response to GMM may function efficiently at the site of infection. (C) 2013 Elsevier Inc. All rights reserved.
  • Yamada Y, Suzuki R, Harashima H
    Cancers 5 4 1413 - 1425 2013年11月 [査読有り][通常論文]
  • Ryohei Togashi, Hidetaka Akita, Hideyoshi Harashima
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 116 4 528 - 531 2013年10月 [査読有り][通常論文]
     
    We report on the technical advance of linearized pDNA (pDNA(linear)) above the circular one (pDNA(circ)) for preparation of small-sized DNA/polycation complexes (DPC) at a low pH. Also, the resistance of the DPC formed with pDNA(linear) against poly-L-asparagine indicates that effective ion-pairing occurred between the pDNA(linear) and polycations. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.
  • Md. Nazir Hossen, Kazuaki Kajimoto, Hidetaka Akita, Mamoru Hyodo, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 171 2 104 - 112 2013年10月 [査読有り][通常論文]
     
    Antiangiogenesis has been the focus of a new strategy for the treatment of obesity. However, little is known regarding the issue of whether targeting angiogenesis by nanoparticle-targeted therapeutic is advantageous or not in debugging the co-morbidity associated with diet-induced obesity (DIO) and the metabolic syndrome. We report herein on the positive effect of prohibitin (an adipose vascular marker)-targeted nanoparticle (PTNP) encapsulated in a proapoptotic peptide [(D)(KLAKLAK)(2), KLA] on DIO and dysfunctional adipose tissue, a major mediator of the metabolic syndrome, as evidenced by ectopic fat deposition. The systemic injection of DIO mice with a low dose of KLA-PTNP, rather than a bioconjugate composed of the same targeting peptide and KLA (Adipotide) resulted in a reduction in body weight, as evidenced by a significant decrease in serum leptin levels, in parallel with an antiobesity effect on dysfunctional adipose cells, including adipocytes and macrophages. In addition, the KLA-PTNP treatment resulted in a reduction in ectopic fat deposits in liver and muscle with the lipolytic action of elevated serum adiponectin, with no detectable hepatoxicity. Notably, drug delivery via PTNP that had accumulated in obese fat via the enhanced permeability and retention effect was enhanced by multivalent active targeting and cytoplasmic delivery into adipose endothelial cells via escaping from endosomes/lysosomes. Thus, vascular-targeted nanotherapy has the potential to contribute to the control of adipose function and ectopic fat deposition associated with obesity and the metabolic syndrome. (C) 2013 Elsevier B. V. All rights reserved.
  • Yuma Yamada, Hideyoshi Harashima
    Mitochondrion 13 5 526 - 532 2013年09月 [査読有り][通常論文]
     
    The focus of this study was on the development of a nano carrier targeted to mitochondria, a promising therapeutic drug target. We synthesized a lipid derivative that is conjugated with a mitochondrial targeting signal peptide (MTS), which permits the selective delivery of certain types of proteins to mitochondria. We then explored the use of an innovative technology in which MTS and the MITO-Porter were integrated. The latter is a liposome that delivers cargos to mitochondria via membrane fusion. The results indicate that the combination of MTS and the MITO-Porter would be useful for selective mitochondrial delivery via membrane fusion. © 2012 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
  • Yuma Yamada, Masahiro Hashida, Yasuhiro Hayashi, Mai Tabata, Mamoru Hyodo, Mst Naznin Ara, Noritaka Ohga, Kyoko Hida, Hideyoshi Harashima
    JOURNAL OF PHARMACEUTICAL SCIENCES 102 9 3119 - 3127 2013年09月 [査読有り][通常論文]
     
    Dysfunctional sinusoidal liver endothelial cells (LECs) are associated with liver diseases, such as liver fibrosis, cirrhosis, and portal hypertension. Because of this, gene therapy targeted to LECs would be a useful and productive strategy for directly treating these diseases at the level of genes. Here, we report on the development of a transgene vector that specifically targets LECs. The vector is a liposome-based gene vector coated with hyaluronic acid (HA). HA is a natural ligand for LECs and confers desirable properties on particles, rendering them biodegradable, biocompatible, and nonimmunogenic. In this study, we constructed HA-modified carriers, and evaluated cellular uptake and transfection activity using cultured LECs from KSN nude mice (KSN-LECs). Cellular uptake analyses showed that KSN-LECs recognized the HA-modified carriers more effectively than skin endothelial cells. The transfection assay indicated that the efficient gene expression in KSN-LECs, using the HA-modified carriers, required an adequate lipid composition and a functional device to control intracellular trafficking. This finding contributes to our overall knowledge of transgene expression targeted to LECs. (C) 2013 Wiley Periodicals, Inc. and the American Pharmacists Association
  • Kenji Kusumoto, Hidetaka Akita, Taichi Ishitsuka, Yu Matsumoto, Takahiro Nomoto, Ryo Furukawa, Ayman El-Sayed, Hiroto Hatakeyama, Kazuaki Kajimoto, Yuma Yamada, Kazunori Kataoka, Hideyoshi Harashima
    ACS NANO 7 9 7534 - 7541 2013年09月 [査読有り][通常論文]
     
    A system that permits the delivery of cargoes to the lung endothelium would be extraordinarily useful in terms of curing a wide variety of lung-related diseases. This study describes the development of a multifunctional envelope-type nanodevice (MEND) that targets the lung endothelium, delivers its encapsulated siRNA to the cytoplasm, and eradicates lung metastasis. The key to the success can be attributed to the presence of a surface-modified GALA peptide that has dual functions: targeting the sialic acid-terminated sugar chains on the pulmonary endothelium and subsequently delivering the encapsulated cargoes to the cytosol via endosomal membrane fusion, analogous to the influenza virus. The active targeting of MENDs without the formation of large aggregates was verified by intravital real-time confocal laser scanning microscopy in living lung tissue. The GALA-modified MEND is a promising carrier that opens a new generation of therapeutic approaches for satisfying unmet medical needs in curing lung diseases.
  • Hidetaka Akita, Ryohei Ishiba, Hiroto Hatakeyama, Hiroki Tanaka, Yusuke Sato, Kota Tange, Masaya Arai, Kazuhiro Kubo, Hideyoshi Harashima
    Advanced Healthcare Materials 2 8 1120 - 1125 2013年08月 [査読有り][通常論文]
     
    SS-cleavable proton-activated lipid-like material (ssPalm) functions as a key element in a lipid nanoparticle in which pDNA is encapsulated. The ssPalm contains dual sensing motifs that can respond to the intracellular environment a proton-sponge unit (tertiary amines) that functions in response to an acidic environment (endosome/lysosome), and disulfide bonding that can be cleaved in a reducing environment (cytosol). © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
  • Kibria G, Hatakeyama H, Ohga N, Hida K, Harashima H
    Biomaterials 34 22 5617 - 5627 2013年07月 [査読有り][通常論文]
     
    Size of the liposomes (LPs) specially governs its biodistribution. In this study, LPs were developed with controlled sizes, where variation in LP size dictates the ligand receptor interaction, cellular internalization and its distribution within the tumor microenvironment. The therapeutic efficacies of doxorubicin (DOX)-loaded RGD modified small size (similar to 100 nm in diameter, dnm) and large size (similar to 300 dnm) PEGylated LPs (RGD-PEG-LPs) were compared to that of Doxil (a clinically used DOX-loaded PEG-LP, similar to 100 dnm) in DOX resistant OSRC-2 (Renal cell carcinoma, RCC) tumor xenografts. Doxil, which accumulated in tumor tissue via the enhanced permeability and retention (EPR) effect, failed to suppress tumor growth. Small size RGD-PEG-LP, that targets the tumor endothelial cells (TECs) and extravasates to tumor cells, failed to provide anti-tumor effect. Large size RGD-PEG-LP preferentially targets the TECs via minimization of the EPR effect, and significantly reduced the tumor growth, which was exerted through its strong anti-angiogenic activity on the tumor vasculature rather than having a direct effect on DOX resistant RCC. The prepared large size RGD-PEG-LP that targets the TECs via interacting with Integrin alpha v beta 3, is a potentially effective and alternate therapeutic strategy for the treatment of DOX resistant tumor cells by utilizing DOX, in cases where Doxil is ineffective. (C) 2013 Elsevier Ltd. All rights reserved.
  • Ayman El-Sayed, Hideyoshi Harashima
    MOLECULAR THERAPY 21 6 1118 - 1130 2013年06月 [査読有り][通常論文]
     
    The ideal nonviral vector delivers its nucleic acid cargo to a specific intracellular target. Vectors enter cells mainly through endocytosis and are distributed to various intracellular organelles. Recent advances in microscopy, lipidomics, and proteomics confirm that the cell membrane is composed of clusters of lipids, organized in the form of lipid raft domains, together with non-raft domains that comprise a generally disordered lipid milieu. The binding of a nonviral vector to either region can determine the pathway for its endocytic uptake and subsequent intracellular itinerary. Given this model of the cell membrane structure, endocytic pathways should be reclassified in relation to lipid rafts. In this review, we attempt to assess the currently recognized endocytic pathways in mammalian cells. The endocytic pathways are classified in relation to the membrane regions that make up the primary endocytic vesicles. This review covers the well-recognized clathrin-mediated endocytosis (CME), phagocytosis, and macropinocytosis in addition to the less addressed pathways that take place in lipid rafts. These include caveolae-mediated, flotillin-dependent, GTPase regulator associated with focal adhesion kinase-1 (GRAF1)-dependent, adenosine diphosphate-ribosylation factor 6 (Arf6)-dependent, and RhoA-dependent endocytic pathways. We summarize the regulators associated with each uptake pathway and methods for interfering with these regulators are discussed. The fate of endocytic vesicles resulting from each endocytic uptake pathway is highlighted.
  • Hiroto Hatakeyama, Hidetaka Akita, Hideyoshi Harashima
    Biological and Pharmaceutical Bulletin 36 6 892 - 899 2013年06月 [査読有り][通常論文]
     
    Gene and nucleic acid therapy is expected to play a major role in the next generation of agents for cancer treatment. We have recently developed a multifunctional envelope-type nano device (MEND) for use as a novel nonviral gene delivery system. The modification of polyethyleneglycol (PEG), i.e., PEGylation, is a useful method for achieving a longer circulation time for the delivery of MEND to a tumor via the enhanced permeability and retention (EPR) effect. However, PEGylation strongly inhibits cellular uptake and endosomal escape, which results in significant loss of activity of the delivery system. For successful nucleic acid delivery for cancer treatment, the crucial problem associated with the use of PEG, i.e., the "PEG dilemma" must be resolved. In this review, we describe the development and applications of MEND and discuss various strategies for overcoming the PEG dilemma based on the manipulation of both pharmacokinetics and intracellular trafficking of cellular uptake and endosomal release. To increase cellular uptake, target ligands including proteins, peptides, antibodies and aptamers that recognize molecules specifically expressed on tumors are first introduced. Second, cleavable PEG systems are described. The cleavage of PEG from carriers was achieved in response to the intracellular environment as well as the tumor microenvironment, which improvs cellular uptake and endosomal escape. Then, endosomal fusogenic peptides are discussed. Finally, pH-sensitive liposomes using pH-sensitive lipids are described. © 2013 The Pharmaceutical Society of Japan.
  • Ryohei Togashi, Hideyoshi Harashima, Hiroyuki Kamiya
    Journal of Gene Medicine 15 6-7 242 - 248 2013年06月 [査読有り][通常論文]
     
    Background: Transgene expression from plasmid DNA is dependent on the expression efficiency per plasmid and the amount of intranuclear plasmid. In the present study, intranuclear dispositions of two types of plasmid DNAs (i.e. the pCpGfree and pLIVE plasmids) that maintain transgene expression in mouse liver were analyzed. In addition, the relationship between transgene expression and plasmid stability in the nucleus was examined. Methods: First, the pCpGfree and pLIVE plasmid DNAs, bearing the mouse secreted alkaline phosphatase (Seap) gene, were administered into mouse liver by the hydrodynamics-based method. Next, various Seap-plasmid DNAs containing different promoters, upstream and downstream sequences, and backbones were injected into mice, and both SEAP expression and plasmid DNA amounts were monitored for 28days. Results: At the 14- and 28-day time points, the amount of the pCpGfree plasmid DNA was one order of magnitude less than that of the pLIVE plasmid. Meanwhile, the expression efficiency per plasmid was one order of magnitude more efficient for the pCpGfree plasmid DNA. Moreover, the administration of various Seap-plasmid DNAs revealed that negative correlations exist between plasmid stability and SEAP expression level. Conclusions: The results obtained suggest that the pCpGfree plasmid is unstable from the viewpoint of quantity and maintains transgene expression by its high expression efficiency and also that transgene expression negatively affects the stability of plasmid DNA. © 2013 John Wiley & Sons, Ltd.
  • Kazuaki Kajimoto, Md. Nazir Hossen, Hideyoshi Harashima
    NANOMEDICINE 8 5 671 - 673 2013年05月 [査読有り][通常論文]
  • Md. Nazir Hossen, Kazuaki Kajimoto, Hidetaka Akita, Mamoru Hyodo, Taichi Ishitsuka, Hideyoshi Harashima
    MOLECULAR THERAPY 21 3 533 - 541 2013年03月 [査読有り][通常論文]
     
    Because the functional apoptosis-initiating protein, cytochrome C (CytC) is rapidly cleared from the circulation (t(1/2) (half-life): 4 minutes), it cannot be used for in vivo therapy. We report herein on a hitherto unreported strategy for delivering exogenous CytC as a potential and safe antiobesity drug for preventing diet-induced obesity, the most common type of obesity in humans. The functional activity of CytC encapsulated in prohibitin (a white fat vessel-specific receptor)-targeted nanoparticles (PTNP) was evaluated quantitatively, as evidenced by the observations that CytC-loaded PTNP causes apoptosis in primary adipose endothelial cells in a dose-dependent manner, whereas CytC alone did not. The delivery of a single dose of CytC through PTNP into the circulation disrupted the vascular structure by the targeted apoptosis of adipose endothelial cells in vivo. Intravenous treatment of CytC-loaded PTNP resulted in a substantial reduction in obesity in high-fat diet (HFD) fed wild-type (wt) mice, as evidenced by the dose-dependent prevention of the percentage of increase in body weight and decrease in serum leptin levels. In addition, no detectable hepatotoxicity was found to be associated with this prevention. Thus, the finding highlights the promising potential of CytC for use as an antiobesity drug, when delivered through a nanosystem.
  • Yuma Yamada, Kohei Nakamura, Ryo Furukawa, Eriko Kawamura, Takuya Moriwaki, Kenji Matsumoto, Katsuhiro Okuda, Mitsuru Shindo, Hideyoshi Harashima
    JOURNAL OF PHARMACEUTICAL SCIENCES 102 3 1008 - 1015 2013年03月 [査読有り][通常論文]
     
    The fact that mitochondrial dysfunction has been implicated in a variety of human diseases suggests that they would be expected as a target organelle for these diseases. Bongkrekic acid (BKA) is a chemical that functions as a ligand of the adenine nucleotide translocator and is known to potently inhibit the mitochondrial permeability transition that is associated with apoptosis. Thus, delivering it to mitochondria would be an innovative therapy for the treatment of mitochondrial diseases that are largely associated with apoptosis. Here, we report on the use of a MITO-Porter, an innovative nanocarrier for mitochondrial delivery via mitochondrial membrane fusion, for delivering BKA to mitochondria. We first constructed a BKAMITO-Porter, in which BKA is contained in lipid envelopes of a MITO-Porter. We then confirmed that the BKAMITO-Porter was efficiently internalized into cells and is delivered to mitochondria, similar to a conventional MITO-Porter. Moreover, we evaluated the antiapoptosis effect of the BKAMITO-Porter in HeLa cells by measuring caspase 3/7 activity. The findings confirmed that the BKAMITO-Porter showed a strong antiapoptosis effect compared with naked BKA. The results reported here demonstrate its potential for the use in therapies aimed at mitochondrial diseases, as a mitochondrial medicine candidate. (c) 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:10081015, 2013
  • Hiroyuki Kamiya, Shiho Miyamoto, Hitomi Goto, Genki N. Kanda, Miwako Kobayashi, Ichiro Matsuoka, Hideyoshi Harashima
    International Journal of Pharmaceutics 441 1-2 146 - 150 2013年01月30日 [査読有り][通常論文]
     
    Plasmid DNA was chromatinized with core histones (H2A, H2B, H3, and H4) in vitro and was delivered into mouse liver by hydrodynamics-based administration. Transgene expression from the chromatinized plasmid DNA was more efficient than that from plasmid DNA delivered in the naked form. The use of acetylation-enriched histones isolated from cells treated with a histone deacetylase inhibitor (trichostatin A) seemed to be more effective. These results indicated that chromatinized plasmid DNA is useful for efficient transgene expression in vivo. © 2012 Elsevier B.V. All rights reserved.
  • Daisuke Morita, Yuki Hattori, Takashi Nakamura, Tatsuhiko Igarashi, Hideyoshi Harashima, Masahiko Sugita
    INFECTION AND IMMUNITY 81 1 311 - 316 2013年01月 [査読有り][通常論文]
     
    Human CD1b molecules contain a maze of hydrophobic pockets and a tunnel capable of accommodating the unusually long, branched acyl chain of mycolic acids, an essential fatty acid component of the cell wall of mycobacteria. It has been accepted that CD1b-bound mycolic acids constitute a scaffold for mycolate-containing (glyco) lipids stimulating CD1b-restricted T cells. Remarkable homology in amino acid sequence is observed between human and monkey CD1b molecules, and indeed, monkey CD1b molecules are able to bind glucose monomycolate (GMM), a glucosylated species of mycolic acids, and present it to specific human T cells in vitro. Nevertheless, we found, unexpectedly, that Mycobacterium bovis bacillus Calmette-Guerin (BCG)-vaccinated monkeys exhibited GMM-specific T cell responses that were restricted by CD1c rather than CD1b molecules. GMM-specific, CD1c-restricted T cells were detected in the circulation of all 4 rhesus macaque monkeys tested after but not before vaccination with BCG. The circulating GMM-specific T cells were detected broadly in both CD4(+) and CD8(+) cell populations, and upon antigenic stimulation, a majority of the GMM-specific T cells produced both gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha), two major host protective cytokines functioning against infection with mycobacteria. Furthermore, the GMM-specific T cells were able to extravasate and approach the site of infection where CD1c(+) cells accumulated. These observations indicate a previously inconceivable role for primate CD1c molecules in eliciting T cell responses to mycolate-containing antigens.
  • Naoyuki Toriyabe, Yasuhiro Hayashi, Hideyoshi Harashima
    Biomaterials 34 4 1337 - 1343 2013年01月 [査読有り][通常論文]
     
    Emerging evidence indicates that the efficiency of siRNA loading into an RNA-induced silencing complex (RISC) is a major factor in gene silencing at low doses. In particular, the release of siRNA from components delivered to the cytoplasm could be a first step for achieving maximum gene knockdown effect in siRNA delivery vector. To test this hypothesis, we used a stearylated-octahistidine (STR-H8) as a pH responsive polycation that facilitates the efficient release of siRNA into the cytoplasm, while a stearylated-octaarginine (STR-R8) was used as a conventional cationic polycation. As a fundamental structure, we used octaarginine (R8) and GALA, as a pH-sensitive fusogenic peptide, modified lipid envelope-type nanoparticles (R8/GALA-MENDSUV), as reported previously. When STR-H8/siRNA condensed complexes were loaded in the R8/GALA-MENDSUV, the luciferase knockdown effect was significantly increased compared to STR-R8/siRNA condensed complexes in time and dose dependent manners. Quantification of the released siRNA from the condensed complexes demonstrated that only the STR-H8/siRNA released significant levels of siRNA at pH = 7.4, the pH of cytoplasmic, compared with STR-R8/siRNA condensed complexes. In addition, imaging studies indicated that STR-H8/siRNA facilitated siRNA release. Collectively, these data reveal the importance of the controlled release of siRNA to the cytoplasm. © 2012 Elsevier Ltd.
  • Takashi Nakamura, Rumiko Moriguchi, Kentaro Kogure, Hideyoshi Harashima
    INTERNATIONAL JOURNAL OF PHARMACEUTICS 441 1-2 476 - 481 2013年01月 [査読有り][通常論文]
     
    In a previous study, we reported on the efficient delivery of an antigen to the cytosol and a specific-antigen presentation on MHC class I in dendritic cells by rationally controlling the intracellular trafficking of ovalbumin (OVA), a model antigen, with stearylated octaarginine-modified liposomes (R8-Lip/OVA). However, no significant difference in antitumor effects against E.G7-OVA, OVA expressed lymphoma, was observed between R8-Lip/OVA and an electrostatic complex of R8 and OVA (R8/OVA-Com). In this study, we hypothesized that use of adjuvants clarified the difference in immune responses between R8-Lip/OVA and R8/OVA-Com, and selected polyinosine-polycytidylic acid (polyI:C) as an adjuvant. Cytotoxic T lymphocyte (CTL) activity of the polyI:C and OVA encapsulated R8-Lip (R8-Lip/PIC/OVA) was drastically enhanced compared to R8-Lip/OVA and complete Freund's adjuvant with OVA. Moreover, the incorporation of polyI:C clearly was critical for the difference in antitumor effects and CTL activities between R8-Lip/OVA and R8/OVA-Com. These findings suggest that the carriers that are incorporated polyI:C has a great influence on the induction of cellular immunity in vivo. (C) 2012 Elsevier B. V. All rights reserved.
  • Hidetaka Akita, Kaoru Enoto, Hiroki Tanaka, Hideyoshi Harashima
    Molecular Therapy 21 2 309 - 317 2013年 [査読有り][通常論文]
     
    Previous studies showed that the cytoplasmic transport of nanoparticles to the nucleus is driven by a vesicular sorting system. Artificial approaches for targeting a microtubule-associating motor complex is also a challenge. We describe herein the development of a liposomal nanoparticle, the surface of which is modified with stearylated octa-arginine (STR-R8), and a dynein light chain (LC8)-associated peptide derived from an African swine fever virus protein p54 (p54149-161) with polyethyleneglycol (PEG) as a spacer (p54 149-161-PEG/R8-liposomal nanoparticles (LNPs)). The p54 149-161-PEG/R8-LNPs preferentially gain access to the nucleus, resulting in a one-to two-order of magnitude higher transfection activity in comparison with p54149-161-free nanoparticles (PEG/R8-LNPs). Further studies of particle tracking in HeLa cells stably expressing green fluorescent protein (GFP)-tagged tubulin (GFP/Tub-HeLa) indicate that p54149-161 stimulated the transport of nanoparticles along fibrous tubulin structures. Moreover, a part of the p54149-161-PEG/R8-LNPs appeared to undergo quasi-straight transport without sharing the tracks corresponding to PKH67, the plasma membrane of which had been prestained with a marker just before transfection, while corresponding movement was never observed in the case of PEG/R8-LNPs. These findings suggest that a portion of the p54 149-161-modified nanoparticles can use microtubule-dependent transport without the need for an assist by a vesicular sorting system. © The American Society of Gene & Cell Therapy.
  • Yu Sakurai, Hiroto Hatakeyama, Yusuke Sato, Mamoru Hyodo, Hidetaka Akita, Hideyoshi Harashima
    Molecular Therapy 21 6 1195 - 1203 2013年 [査読有り][通常論文]
     
    Small interfering RNA (siRNA) would be predicted to function as a cancer drug, but an efficient siRNA delivery system is required for clinical development. To address this issue, we developed a liposomal siRNA carrier, a multifunctional envelope-type nanodevice (MEND). We previously reported that a MEND composed of a pH-sensitive cationic lipid, YSK05, showed significant knockdown in both in vitro and in tumor tissue by intratumoral injection. Here, we report on the development of an in vivo siRNA delivery system that is delivered by systemic injection and an analysis of the pharmacokinetics of an intravenously administered siRNA molecule in tumor tissue. Tumor delivery of siRNA was quantified by means of stem-loop primer quantitative reverse transcriptase PCR (qRT-PCR) method. PEGylation of the YSK-MEND results in the increase in the accumulation of siRNA in tumor tissue from 0.0079% ID/g tumor to 1.9% ID/g tumor. The Administration of the MEND (3 mg siRNA/kg body weight) showed about a 50% reduction in the target gene mRNA and protein. Moreover, we verified the induction of RNA interference by 5′ RACE-PCR method. The collective results reported here indicate that an siRNA carrier was developed that can deliver siRNA to a target cell in tumor tissue through an improved siRNA bioavailability. © The American Society of Gene & Cell Therapy.
  • Nakamura T, Yamazaki D, Yamauchi J, Harashima H
    Journal of controlled release : official journal of the Controlled Release Society 171 2 216 - 224 2013年 [査読有り][通常論文]
     
    Alpha-galactosylceramide (αGC), a lipid antigen present on CD1d molecules, is predicted to have clinical applications as a new class of adjuvant, because αGC strongly activates natural killer T (NKT) cells which produce large amounts of IFN-γ. Here, we incorporated αGC into stearylated octaarginine-modified liposomes (R8-Lip), our original delivery system developed for vaccines, and investigated the effect of nanoparticulation. Unexpectedly, the systemic administered R8-Lip incorporating αGC (αGC/R8-Lip) failedtoimprove the immune responses mediated by αGC compared with soluble αGCin vivo, although αGC/R8-Lip drastically enhanced αGC presentation on CD1d in antigen presenting cells invitro. Thus, we optimized the αGC/R8-Lipin vivoto overcome this inverse correlation. In optimization in vivo, we found that size control ofliposome and R8-modification were critical for enhancing the production of IFN-γ. The optimization led to the accumulation of αGC/R8-Lip in the spleen and a positive therapeutic effect against highly malignant B16 melanoma cells. The optimized αGC/R8-Lip also enhanced αGC presentation on CD1d in antigen presenting cells and resulted in an expansion in the population of NKT cells. Herein, we show that R8-Lip is a potent delivery system, and size control and R8-modification in liposomal construction are promising techniques for achieving systemic αGC therapy. © 2013 The Authors.
  • Kuraya D, Watanabe M, Koshizuka Y, Ogura M, Yoshida T, Asahi Y, Kamachi H, Nakamura T, Harashima H, Ozaki M, Umezawa K, Matsushita M, Yamashita K, Todo S
    Transplantation 96 5 445 - 453 2013年 [査読有り][通常論文]
     
    BACKGROUND: Pancreatic islet transplantation (PITx) is an attractive treatment option for restoring appropriate glucose homeostasis in type 1 diabetes patients. Although islet grafts can successfully engraft after PITx, large numbers of islet grafts are required mainly because immune reactions, including inflammation, destroy islet grafts. In these processes, nuclear factor (NF)-κB plays a central role. We hypothesized that the inhibition of NF-κB activation would ameliorate inflammatory responses after PITx and aid successful engraftment. METHODS: To test this hypothesis, a newly developed NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), was used on a syngeneic mouse PITx model. One hundred seventy-five islets from C57BL/6 (B6) mice were transplanted into streptozotocin-induced diabetic B6 mice. The recipient mice were administered DHMEQ for 1, 2, or 3 days after PITx. The underlying mechanisms of DHMEQ on islet graft protection were investigated in an in vitro coculture model of pancreatic islets and macrophages. RESULTS: With a vehicle treatment, only 11.1% of the islet-recipients achieved normoglycemia after PITx. In sharp contrast, DHMEQ treatment markedly improved the normoglycemic rate, which was associated with the suppression of serum high mobility group complex-1 (HMGB1) and proinflammatory cytokines, including tumor necrosis factor-α, monocyte chemoattractant protein-1, macrophage inflammatory protein-1β, interleukin-1β, and interleukin-6, after PITx. In a murine macrophage-like cell line, DHMEQ inhibited HMGB1-driven activation and proinflammatory cytokine secretion and further prevented death isolated islets after coculture with these activated macrophages. CONCLUSIONS: Inhibition of NF-κB activation by DHMEQ after PITx reduces the HMGB1-triggered proinflammatory responses and results in engraftment of transplanted islets even with fewer islet grafts. Copyright © 2013 by Lippincott Williams & Wilkins.
  • Yukari Yasuzaki, Yuma Yamada, Tsutomu Kanefuji, Hideyoshi Harashima
    Journal of Controlled Release 172 3 805 - 811 2013年 [査読有り][通常論文]
     
    Mitochondrial genetic disorders are a major cause of mitochondrial diseases. It is therefore likely that mitochondrial gene therapy will be useful for the treatment of such diseases. Here, we report on the possibility of mitochondrial gene delivery in skeletal muscle using hydrodynamic limb vein (HLV) injection. The HLV injection procedure, a useful method for transgene expression in skeletal muscle, involves the rapid injection of a large volume of naked plasmid DNA (pDNA) into the distal vein of a limb. We hypothesized that the technique could be used to deliver pDNA not only to nuclei but also to mitochondria, since cytosolic pDNA that is internalized by the method may be able to overcome mitochondrial membrane. We determined if pDNA could be delivered to myofibrillar mitochondria by HLV injection by PCR analysis. Mitochondrial toxicity assays showed that the HLV injection had no influence on mitochondrial function. These findings indicate that HLV injection promises to be a useful technique for in vivo mitochondrial gene delivery. © 2013 Elsevier B.V.
  • Yusuke Sato, Hiroto Hatakeyama, Mamoru Hyodo, Hidetaka Akita, Hideyoshi Harashima
    YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 132 12 1355 - 1363 2012年12月 [査読有り][通常論文]
     
    The development of a carrier for the delivery of siRNA is a factor in the realization of RNA interference (RNAi) therapeutics. Modification of siRNA carriers with polyethylene glycol, i.e., PEGylation, is a general strategy for stabilizing a particle in the blood stream and delivering it to tissue or cells. However, it is well-known that, when a carrier is modified by PEGylation, it results in a significant inhibition of both cellular uptake and the endosomal escape process. In a previous study, we reported on the development of a multifunctional envelope-type nano device (MEND) for delivering siRNA and peptide-based functional devices for overcoming the effects conferred by PEGylation and succeeded in the delivery of siRNA to tumor tissue. In this study, we noticed that the pH-sensitive property, changing from neutral to cationic in response to a decrease in pH, could avoid the inhibition caused by PEGylation and succeeded in synthesizing a pH-sensitive cationic lipid, YSK05. The YSK05-MEND had a higher fusogenicity and potency for endosomal escape than other MENDs containing conventional cationic lipids. The PEGylated YSK05-MEND induced efficient gene silencing and avoided the inhibition of endosomal escape caused by PEGylation followed by optimization of the lipid composition. Furthermore, the intratumoral injection of the PEGylated YSK05-MEND resulted in a more efficient gene silencing compared with MENDs containing conventional cationic lipids. Thus, the YSK05-MEND is a promising siRNA carrier for avoiding the inhibition in intracellular trafficking caused by PEGylation both in vitro and in vivo.
  • Ryo Furukawa, Yuma Yamada, Hideyoshi Harashima
    YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 132 12 1389 - 1398 2012年12月 [査読有り][通常論文]
     
    Gene therapy is an attractive strategy, for not only targeting nuclear genome, but the mitochondrial genome as well. Human mitochondrial DNA (mtDNA) encodes 13 subunits of the electron transport chain, 22 tRNAs, and 2 rRNAs and their mutations cause a wide range of mitochondrial diseases. Each cell contains hundreds to thousands of mtDNAs, and in the case of a diseased cell, the mitochondrion possesses both mutant mtDNA and wild-type mtDNA. It is generally accepted that the disease phenotype appears when the proportion of the pathogenic mutant mtDNA exceeds a certain threshold. Therefore, the suppression of mutant mtDNA or supplementing wild-type mtDNA will control the onset of mitochondrial disease. To achieve the transfection of an exogenous therapeutic gene to the mitochondrial matrix where mtDNA is transcribed and translated, it is necessary to transfer cargos through mitochondrial outer and inner membranes. Several methods have been examined for mitochondrial transfection, but a universal, wide-ranging transfection technique has yet not been established. We recently developed a mitochondrial targeting delivery system, namely the MITO-Porter. The MITO-Porter is liposomal nanocarrier with a mitochondrial fusogenic lipid composition. We reported that the MITO-Porter could deliver chemical compounds and proteins to the mitochondrial matrix via membrane fusion. In this review, we report (1) on the pharmacological enhancement of lecithinized superoxide dismutase (PC-SOD) using MITO-Porter, (2) the transcription activation of exogenous DNA by mitochondrial transcription factor A (TFAM), and (3) perspectives on a mitochondrial targeting device.
  • Mst. Naznin Ara, Mamoru Hyodo, Noritaka Ohga, Kyoko Hida, Hideyoshi Harashima
    PLOS ONE 7 12 e50174  2012年12月 [査読有り][通常論文]
     
    The present study used a spontaneous cell-based SELEX method (Systemic Evolution of Ligands by EXponential Enrichment) to produce DNA aptamers that specifically bind to cell surface proteins or biomarkers produced by primary cultured mouse tumor endothelial cells (mTECs). In solid tumors, new blood vessels are formed through an angiogenesis process, and this plays a critical role in cancer development as well as metastasis. To combat angiogenesis, an appropriate diagnosis and a molecular-level understanding of the different cancer types are now a high priority. The novel DNA aptamer AraHH001, developed in this study, binds specifically to mTECs with high affinity in the nano-molar range, but does not bind to normal skin endothelial cells (skin-ECs). The selected DNA aptamer was also found to bind to cultured human tumor endothelial cells (hTECs), isolated from a clinical patient with a renal carcinoma. The aptamer AraHH001 showed significant anti-angiogenesis activity by inhibiting tube formation by mTECs on matrigel. Interestingly, a confocal laser scanning microscopy examination of in vitro cellular uptake revealed that AraHH001 was assimilated by mTECs, and became co-localized in acidic compartments, as detected by labeling with Lysotracker Red. Therefore, the development of a specific DNA aptamer that binds to mTECs, as reported here for the first time, holds great promise not only as a therapeutic aptamer but also as a targeted molecular probe that appears to play a major role in angiogenesis, and for the development of a targeted new drug delivery system. Citation: Ara MN, Hyodo M, Ohga N, Hida K, Harashima H (2012) Development of a Novel DNA Aptamer Ligand Targeting to Primary Cultured Tumor Endothelial Cells by a Cell-Based SELEX Method. PLoS ONE 7(12): e50174. doi:10.1371/journal.pone.0050174
  • Okamura T, Katayama T, Obinata C, Iso Y, Chiba Y, Kobayashi H, Yamada Y, Harashima H, Minami M
    Journal of neuroscience research 90 11 2127 - 2133 2012年11月 [査読有り][通常論文]
     
    Chemokines are potent chemoattractants for immune and hematopoietic cells. In the central nervous system, chemokines play an important role in inflammatory responses through activation of infiltrating leukocytes and/or resident glial cells. We previously demonstrated that N-methyl-D-aspartate (NMDA)-evoked neuronal injury induced astrocytic production of monocyte chemoattractant protein-1 (MCP-1, CCL2) via sustained activation of extracellular signal-regulated kinase (ERK) in rat organotypic slice cultures. In the present study, we examined mRNA expression and protein production of macrophage inflammatory protein-1a (MIP-1a, CCL3) induced by NMDA-evoked neuronal injury in the slice cultures. MIP-1a mRNA expression was transiently increased by NMDA treatment in a concentration-dependent manner. Double-fluorescence immunohistochemistry revealed that MIP-1a was produced predominantly in microglia. Depletion of microglial cells from the slice cultures by pretreatment with liposome-encapsulated clodronate abrogated the increase in MIP-1a mRNA expression after NMDA treatment. NMDA-induced MIP-1a mRNA expression was partially but significantly inhibited by the c-Jun N-terminal kinase inhibitor SP600125; conversely, the p38 mitogen-activated protein (MAP) kinase inhibitor SB203580 enhanced it. U0126, a MAP kinase/ERK kinase inhibitor, did not affect mRNA expression. These results, combined with our previous findings, demonstrate that NMDA-evoked neuronal injury differentially induces MIP-1a and MCP-1 production in microglia and astrocytes, respectively, through activation of different intracellular signaling pathways. (c) 2012 Wiley Periodicals, Inc.
  • Yusuke Sato, Hiroto Hatakeyama, Yu Sakurai, Mamoru Hyodo, Hidetaka Akita, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 163 3 267 - 276 2012年11月 [査読有り][通常論文]
     
    Modification of liposomal siRNA carriers with polyethylene glycol, i.e., PEGylation, is a generally accepted strategy for achieving in vivo stability and delivery to tumor tissue. However, PEGylation significantly inhibits both cellular uptake and the endosomal escape process of the carriers. In a previous study, we reported on the development of a multifunctional envelope-type nano device (MEND) for siRNA delivery and peptide-based functional devices for overcoming the limitations and succeeded in the efficient delivery of siRNA to tumors. In this study, we synthesized a pH-sensitive cationic lipid, YSK05, to overcome the limitations. The YSK05-MEND had a higher ability for endosomal escape than other MENDs containing conventional cationic lipids. The PEGylated YSK05-MEND induced efficient gene silencing and overcame the limitations followed by optimization of the lipid composition. Furthermore, the intratumoral administration of the YSK05-MEND resulted in a more efficient gene silencing compared with MENDs containing conventional cationic lipids. Collectively, these data confirm that YSK05 facilitates the endosomal escape of the MEND and thereby enhances the efficacy of siRNA delivery into cytosol and gene silencing. (c) 2012 Elsevier B.V. All rights reserved.
  • Kazuaki Kajimoto, Shiharu Takayanagi, Shun Sasaki, Hidetaka Akita, Hideyoshi Harashima
    ENDOCRINOLOGY 153 11 5629 - 5636 2012年11月 [査読有り][通常論文]
     
    The fatty acid-binding protein 4 (FABP4) is believed to play an important role in maintaining glucose and lipid homeostasis. However, the physiological functions of FABP4 in adipocytes have not been fully elucidated because of difficulties associated with the effective transfection of small interfering RNA (siRNA) to differentiated adipocytes. The aim of this study was to clarify the physiological roles of FABP4 in adipocytes by establishing an efficient, universal technique for endogenous gene silencing in fully differentiated 3T3-L1 cells. Confocal-based three-dimensional observations demonstrated that, in traditionally cultured 3T3-L1 cells, multilayers of undifferentiated cells were formed. As a result, small interfering RNA failed to reach many of the differentiated cells. To solve this problem, we developed a reliable method, denoted as density-based separation followed by replating of enriched adipocytes in a monolayer (DREAM) and, using the developed method, succeeded in a significant knockdown of FABP4. Loss-of-function analyses revealed that FABP4 regulates the expression of IL-6 and vascular endothelial growth factor (VEGF) mediated by the protease-activated receptor 1 (PAR1), a thrombin receptor, in adipocytes. In addition, the basal IL-6 production was partially suppressed by PAR1 knockdown. Moreover, we also demonstrated that IL-6 stimulates the proliferation of primary endothelial cells isolated from murine adipose tissue. These findings indicate that FABP4 may have a crucial role in modulating IL-6 and vascular endothelial growth factor as angiogenesis inducers stimulated by the cellular action of thrombin on adipocytes via PAR1. These findings promise to be helpful for developing an understanding of physiological counterparts with respect to the inflammatory and angiogenic properties of adipose tissue. (Endocrinology 153: 5629-5636, 2012)
  • Md Nazir Hossen, Kazuaki Kajimoto, Hidetaka Akita, Mamoru Hyodo, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 163 2 101 - 110 2012年10月 [査読有り][通常論文]
     
    We previously reported that nanoparticles (NPs) modified with a prohibitin- homing peptide ligand via a short PEG(2) (kDa)-spacer could deliver its pay-load into the cytoplasm of endothelial cells in murine adipose tissue and escape from endosomes/lysosomes in vitro. We herein report, for the first time, on a dual-targeting strategy for mediating the enhanced targeting activity of NPs to adipose endothelial cells in diet-induced obesity (DIO). The targeted accumulation of prohibitin- targeted nanoparticles (PTNP), modified with a peptide ligand via a long PEG-linker, was significantly increased in white fat vessels of normal healthy mice compared to the other non-PEGylated targeted NPs, whereas the undesired accumulation of PTNP in the liver was considerably reduced. These results demonstrate that the PEGylation of targeted NPs is a critical factor in maximizing the in vivo targeted delivery of NPs and can be attributed to a significant decrease in recognition by the reticuloendothelial system. After systemic administration to DIO mice, PTNP exclusively accumulated in both adipose vessels and angiogenic clusters of obese fat cells. Surprisingly, PEGylated NPs with no active targeting moieties also accumulated in these clusters, demonstrating that the nanoscaled carriers passively accumulate in clusters via a mechanism similar to that for the enhanced permeability and retention effect, as has been well established in tumor targeting. Therefore, the enhanced delivery of PTNP appears to be mediated by both passive accumulation to angiogenic regions and active recognition by endothelial cells. Thus, the systemic administration of a proapoptotic peptide with the delivery via PTNP significantly reduced the body weight of DIO mice, as evidenced by the targeted ablation of adipose endothelial cells. These findings are potentially useful in terms of the design and development of vascular-targeted nanotherapy in the effective control of obesity. (c) 2012 Elsevier B.V. All rights reserved.
  • Yuma Yamada, Hideyoshi Harashima
    YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 132 10 1111 - 1118 2012年10月 [査読有り][通常論文]
     
    Mitochondrial dysfunction has been implicated in a variety of human diseases, including cancer and neurodegenerative disorders. Effective medical therapies for such diseases will ultimately require the targeted delivery of therapeutic agents to mitochondria. This will likely be achieved through innovations in the areas of the nanotechnology of intracellular trafficking. Mitochondrial delivery systems for a variety of cargoes have been repored to date. However, only a limited number of approaches are available for delivering macromolecules directly to mitochondria. We previously reported on the construction of a MITO-Porter, a liposome-based carrier that introduces macromolecular cargos into mitochondria via membrane fusion. Using the green fluorescence protein as a model macromolecule in conjunction with analysis by confocal laser scanning microscopy, we were able to confirm the mitochondrial delivery of a macromolecule by the MITO-Porter. Moreover, we reported that the Dual Function MITO-Porter (DF-MITO-Porter) could efficiently deliver cargo to mitochondria, through endosomal and mitochondrial membranes via step-wise membrane fusion. Here, We will present our findings on the development of our mitochondrial drug delivery system, and discuss our attempts regarding mitochondrial gene delivery and therapy. Finally, We will discuss the potential use of mitochondrial drug delivery systems in mitochondrial medicine.
  • Genki N. Kanda, Ryohei Togashi, Hideyoshi Harashima, Hiroyuki Kamiya
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 35 9 1534 - 1538 2012年09月 [査読有り][通常論文]
     
    A positive feedback system, using GAL4-vp16 (a fusion protein of yeast GAL4 and herpes simplex virus vp16) as an activator and firefly luciferase as a reporter, maintained luciferase expression for 7 d in mice. However, the luciferase expression decreased after 7 d, and this phenomenon could be caused by immunoreactions against these exogenous proteins. This hypothesis was examined by the following three strategies, designed to avoid the putative immunoreactions: (i) use of the endogenous secreted alkaline phosphatase (SEAP) protein as a reporter, (ii) replacement of vp16 with endogenous transcription factors, and (iii) insertion of the target sequence of microRNA expressed in cells of hematopoietic origin, to suppress GAL4-vp16 expression in antigen-presenting cells. The results obtained in this study suggested that silencing would be induced by mechanism(s) besides immunoreactions against reporter and activator proteins.
  • S. Fukunaga, G. Kanda, J. Tanase, H. Harashima, T. Ohyama, H. Kamiya
    Gene Therapy 19 8 828 - 835 Nature Publishing 2012年08月 [査読有り][通常論文]
     
    The intranuclear disposition of plasmid DNA is extremely important for transgene expression. The interactions between the plasmid DNA and the histone proteins are one of the keys for controlling the disposition. In this study, the effects of a left-handedly curved sequence (20-40 repeated AT tracts) on transgene expression from a plasmid were examined in vivo. A naked luciferase plasmid with the curved sequence was delivered into mouse liver by a hydrodynamics-based injection, and the luciferase activities were quantitated at various time points. Interestingly, transgene expression was markedly increased by the addition of the curved sequence. An analysis of the nucleosome positions near the left-handedly curved sequence suggested that the sequence functions as an acceptor of the histone core and allows nucleosome sliding, resulting in transcriptional activation. These results suggested that the designed curved DNA sequences could control transgene expression from plasmid DNAs in vivo. © 2012 Macmillan Publishers Limited All rights reserved.
  • Yasuhiro Hayashi, Yuki Noguchi, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 161 3 757 - 762 2012年08月 [査読有り][通常論文]
     
    A rational development of an efficient siRNA delivery system is important for streamlining the RNAi-based drug development process. However, a huge gap frequently exists between in vitro and in vivo activity, which is the rate-limiting step for developing versatile nanoparticles. We report herein on a remarkable non-linearity in pharmacokinetics (PK), but not pharmacodynamics (PD) using octaarginine (R8) modified lipid nanoparticles in mice. A quantitative study of siRNA molecules between cultured cells and mouse liver revealed a high correlation between intracellular siRNA molecules and their RNAi activities, indicating that there was no significant difference in the efficiency in PD. In contrast, a remarkable difference was observed in the non-linearity in PK. Quantitative analysis of the time profile for siRNA showed that the percentage of siRNA accumulation in mice was severely decreased with decreasing input dose compared to in vitro data. These unexpected data reveal an important clue to bridging the gap between in vitro and in vivo activity. (c) 2012 Elsevier B.V. All rights reserved.
  • Mayumi Takahashi, Chisato Nagai, Hiroto Hatakeyama, Noriaki Minakawa, Hideyoshi Harashima, Akira Matsuda
    Nucleic Acids Research 40 12 5787 - 5793 2012年07月 [査読有り][通常論文]
     
    Chemically modified siRNAs are expected to have resistance toward nuclease degradation and good thermal stability in duplex formation for in vivo applications. We have recently found that 2′-OMe-4′-thioRNA, a hybrid chemical modification based on 2′-OMeRNA and 4′-thioRNA, has high hybridization affinity for complementary RNA and significant resistance toward degradation in human plasma. These results prompted us to develop chemically modified siRNAs using 2′-OMe-4′-thioribonucleo sides for therapeutic application. Effective modification patterns were screened with a luciferase reporter assay. The best modification pattern of siRNA, which conferred duration of the gene-silencing effect without loss of RNAi activity, was identified. Quantification of the remaining siRNA in HeLa-luc cells using a Heat-in-Triton (HIT) qRT-PCR revealed that the intracellular stability of the siRNA modified with 2′-OMe-4′-thioribonucleosides contributed significantly to the duration of its RNAi activity. © The Author(s) 2012. Published by Oxford University Press.
  • Ikuhiko Nakase, Hidetaka Akita, Kentaro Kogure, Astrid Graslund, Ulo Langel, Hideyoshi Harashima, Shiroh Futaki
    ACCOUNTS OF CHEMICAL RESEARCH 45 7 1132 - 1139 2012年07月 [査読有り][通常論文]
     
    Over the last 20 years, researchers have designed or discovered peptides that can permeate membranes and deliver exogenous molecules inside a cell. These peptides, known as cell-penetrating peptides (CPPs), typically consist of 6-30 residues, including HIV TAT peptide, penetratin, oligoarginine, transportan, and TP10. Through chemical conjugation or noncovalent complex formation, these structures successfully deliver bioactive and membrane-impermeable molecules into cells. CPPs have also gained attention as an attractive vehicle for the delivery of nucleic add pharmaceuticals (NAPs), including genes/plasmids, short oligonucleotides, and small interference RNAs and their analogues, due to their high internalization efficacy, low cytotoxicity, and flexible structural design. In this Account, we survey the potential of CPPs for the design and optimization of NAP delivery systems. First, we describe the impact of the N-terminal stearylation of CPPs. Endocytic pathways make a major contribution to the cellular uptake of NAPS. Stearylation at the N-terminus of CPPs with stearyl-octaarginine (R8), stearyl-(RxR)(4), and stearyl-TP10 prompts the formation of a self-assembled core shell nanoparticle with NAPS, a compact structure that promotes cellular uptake. Researchers have designed modifications such as the addition of trifluoromethylquinoline moieties to lysine residues to destabilize endosomes, as exemplified by PepFect 6, and these changes further improve biological responsiveness. Alternatively, stearylation also allows implantation of CPPs onto the surface of liposomes. This feature facilitates "programmed packaging" to establish multifunctional envelope-type nanodevices (MEND). The R8-MEND showed high transfection efficiency comparable to that of adenovirus in non-dividing cells. Understanding the cellular uptake mechanisms of CPPs will further improve CPP-mediated NAP delivery. The cellular uptake of CPPs and their NAP complex involves various types of endocytosis. Macropinocytosis, a mechanism which is also activated in response to stimuli such as growth factors or viruses, is a primary pathway for arginine-rich CPPs because high cationic charge density promotes this endocytic pathway. The use of larger endosomes (known as macropinosomes) rather than clathrin- or caveolae-mediated endocytosis has been reported in macropinocytosis which would also facilitate the endocytosis of NAP nanoparticles into cells.
  • Katsuma Kitazoe, Yeon-Su Park, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Kentaro Kogure, Hideyoshi Harashima, Yoshinobu Baba
    PLOS ONE 7 6 e39057  2012年06月 [査読有り][通常論文]
     
    Multifunctional envelope-type nanodevices (MENDs) are very promising non-viral gene delivery vectors because they are biocompatible and enable programmed packaging of various functional elements into an individual nanostructured liposome. Conventionally MENDs have been fabricated by complicated, labor-intensive, time-consuming bulk batch methods. To avoid these problems in MEND fabrication, we adopted a microfluidic chip with a chaotic mixer array on the floor of its reaction channel. The array was composed of 69 cycles of the staggered chaotic mixer with bas-relief structures. Although the reaction channel had very large Peclet numbers (>10(5)) favorable for laminar flows, its chaotic mixer array led to very small mixing lengths (<1.5 cm) and that allowed homogeneous mixing of MEND precursors in a short time. Using the microfluidic chip, we fabricated a double-lamellar MEND (D-MEND) composed of a condensed plasmid DNA core and a lipid bilayer membrane envelope as well as the D-MEND modified with trans-membrane peptide octaarginine. Our lab-on-a-chip approach was much simpler, faster, and more convenient for fabricating the MENDs, as compared with the conventional bulk batch approaches. Further, the physical properties of the on-chip-fabricated MENDs were comparable to or better than those of the bulk batch-fabricated MENDs. Our fabrication strategy using microfluidic chips with short mixing length reaction channels may provide practical ways for constructing more elegant liposome-based non-viral vectors that can effectively penetrate all membranes in cells and lead to high gene transfection efficiency.
  • Yuma Yamada, Taku Nomura, Hideyoshi Harashima, Atsushi Yamashita, Nobuhiko Yui
    Biomaterials 33 15 3952 - 3958 2012年05月 [査読有り][通常論文]
     
    A quantitative comparison between nuclear DNA release from carriers and their transfection activity would be highly useful for improving the effectiveness of non-viral gene vectors. We previously reported that, for condensed DNA particles, a close relationship exists between the efficiency of DNA release and transfection activity, when biocleavable polyrotaxanes (DMAE-SS-PRX), in which the cationic density can be easily controlled. In this study, we first investigated the efficiencies of DNA release from condensed DNA particles with various types of DMAE-SS-PRX. The findings indicate that an optimal cationic density in DMAE-SS-PRX exists for DNA release. We then packaged condensed DNA particles in a multifunctional envelope-type nano device (MEND), and evaluated their transfection activities. The results showed that the transfection activity was increased and this increase was, to some extent, dependent on the efficiency of the DNA release. However, transfection activity decreased, when the value for the efficiency of DNA release was higher than a certain value. An investigation of the fate of intranuclear DNA indicated that a very high efficiency of DNA release has a positive influence on transcription, however, it would inhibit the post-transcription process nuclear mRNA export, translation and related processes. Such information provides a new viewpoint for the development of cationic polymer-based vectors. © 2012 Elsevier Ltd.
  • Yuma Yamada, Taku Nomura, Hideyoshi Harashima, Atsushi Yamashita, Nobuhiko Yui
    BIOMATERIALS 33 15 3952 - 3958 2012年05月 [査読有り][通常論文]
     
    A quantitative comparison between nuclear DNA release from carriers and their transfection activity would be highly useful for improving the effectiveness of non-viral gene vectors. We previously reported that, for condensed DNA particles, a close relationship exists between the efficiency of DNA release and transfection activity, when biocleavable polyrotaxanes (DMAE-SS-PRX), in which the cationic density can be easily controlled. In this study, we first investigated the efficiencies of DNA release from condensed DNA particles with various types of DMAE-SS-PRX. The findings indicate that an optimal cationic density in DMAE-SS-PRX exists for DNA release. We then packaged condensed DNA particles in a multifunctional envelope-type nano device (MEND), and evaluated their transfection activities. The results showed that the transfection activity was increased and this increase was, to some extent, dependent on the efficiency of the DNA release. However, transfection activity decreased, when the value for the efficiency of DNA release was higher than a certain value. An investigation of the fate of intranuclear DNA indicated that a very high efficiency of DNA release has a positive influence on transcription, however, it would inhibit the post-transcription process; nuclear mRNA export, translation and related processes. Such information provides a new viewpoint for the development of cationic polymer-based vectors. (C) 2012 Elsevier Ltd. All rights reserved.
  • Vasudevanpillai Biju, Abdulaziz Anas, Hidetaka Akita, Edakkattuparambil Sidharthan Shibu, Tamitake Itoh, Hideyoshi Harashima, Mitsuru Ishikawa
    ACS NANO 6 5 3776 - 3788 2012年05月 [査読有り][通常論文]
     
    Protection of genes against enzymatic degradation and overcoming of cellular barriers are critical for efficient gene delivery. The effectiveness of gene delivery by nonviral vectors depends mostly on the extent of DNA packaging or condensation. We show that Forster resonance energy transfer (FRET)-mediated photodecomposition of undesired acceptors In doubly labeled plasmid DNA (pDNA) and FRET recovery after acceptor photodecomposition (FRET-RAP) are effective methods for the detection of DNA condensation and decondensation. Our hypothesis is that undesired acceptors within the Forster distance of highly-photostable donors in precondensed DNA can be selectively photodecomposed by FRET. We Investigate this hypothesis by the random labeling of pcDNA3.1-GL3 and pUC18DNA with quantum dots (QDs) as the energy donor and AlexaFluor594 or Cy5 as the acceptor. At first, the random labeling generates efficient FRET, also called intrinsic FRET, in precondensed DNA, which prevents us from decoding any changes in the FRET efficiency during DNA condensation. Next, we suppressed the intrinsic FRET by the FRET-mediated photodecomposition of acceptors within the Forster distance of QDs. Conversely, many acceptors kept intact beyond the Forster distance provide us with high FRET efficiency during the condensation of pDNA using protamine. Further, the FRET efficiency is significantly decreased during the decondensation of DNA using heparan sulfate and glutathione. The random labeling of DNA using excess acceptors around photostable donors followed by the FRET-mediated photodecomposition of undesired acceptors can be a promising method for not only the sensitive detection of DNA condensation by FRET but also the customization of biomolecular sensors.
  • Kaoru Kigasawa, Moeko Miyashita, Kazuaki Kajimoto, Kiyoshi Kanamura, Hideyoshi Harashima, Kentaro Kogure
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 35 5 781 - 785 2012年05月 [査読有り][通常論文]
     
    Superoxide dismutase (SOD) is a potent antioxidant agent that protects against UV-induced skin damage. However, its high molecular weight is a significant obstacle for efficient delivery into the skin through the stratum corneum and development of antioxidant activity. Recently, we developed a non-invasive transfollicular delivery system for macromolecules using a combination of liposomes and iontophoresis, that represents promising technology for enhancing transdermal administration of charged drugs (IJP, 403, 2011, Kajimoto et al.). In this study, in rats we attempted to apply this system to intradermal delivery of SOD for preventing UV-induced skin injury. SOD encapsulating in cationic liposomes was subjected to anodal iontophoresis. After iontophoretic treatment, the liposomes were diffused widely in the viable skin layer around hair follicles. In contrast, passive diffusion failed to transport liposomes efficiently into the skin. Iontophoretic delivery of liposomes encapsulating SOD caused a marked decrease in the production of oxidative products, such as malondialdehyde, hexanoyl lysine, and 8-hydroxi-2-deoxyguanosine, in UV-irradiated skin. These findings suggested that functional SOD can be delivered into the skin using a combination of iontophoresis and a liposomal system. In conclusion, we succeeded in developing an efficient intradermal SOD delivery system, that would be useful for delivery of other macromolecules.
  • Yuma Yamada, Masahiro Hashida, Taku Nomura, Hideyoshi Harashima, Yuichi Yamasaki, Kazunori Kataoka, Atsushi Yamashita, Ryo Katoono, Nobuhiko Yui
    ChemPhysChem 13 5 1161 - 1165 2012年04月10日 [査読有り][通常論文]
     
    Ratios that matter: Investigations of nanoparticle formation using polyrotaxane as a polycation reveal that the formations of pDNA and siRNA nanoparticles with polycations are dependent on the nitrogen/phosphate ratio and the molar ratio, respectively. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
  • Kumiko Kawasaki, Momoko Yoneyama, Naoko Murata-Kamiya, Hideyoshi Harashima, Chojiro Kojima, Yutaka Ito, Hiroyuki Kamiya, Masaki Mishima
    Biomol NMR Assign 6 1 1-4  2012年04月 [査読有り][通常論文]
     
    大腸菌Orf135蛋白質のNMR信号帰属
  • Yuma Yamada, Masahiro Hashida, Taku Nomura, Hideyoshi Harashima, Yuichi Yamasaki, Kazunori Kataoka, Atsushi Yamashita, Ryo Katoono, Nobuhiko Yui
    CHEMPHYSCHEM 13 5 1161 - 1165 2012年04月 [査読有り][通常論文]
  • Kenji Kusumoto, Hidetaka Akita, Ayman El-Sayed, Hideyoshi Harashima
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 35 4 445 - 448 2012年04月 [査読有り][通常論文]
     
    The use of polyethylene glycol (PEG)-modified lipids (PEG-lipids) as a component of cationic liposomes impairs the cytoplasmic delivery of the encapsulated cargos by reducing endosomal escape. While this results in a loss of gene expression of encapsulated plasmid DNA, PEG-modification is useful in that it permits the formation of small, stabilized particles. In the present study, the dilemma associated with the use of PEG was overcome by modifying liposomes with stearylated INF7 (STR-INF7), a membrane fusion-independent destabilizer of endosomes, and substituting hydrophobic lipid-anchors in the PEG-lipid. The cationic liposomes modified with a series of PEG-lipids showed a drastically impaired transgene expression. However, the incorporation of STR-INF7 recovered the gene expression, and this was found to be mainly dependent on the type of PEG lipid-anchor used. Of note, the fold increase in transfection activity was highest in cholesterol-anchored PEG (>100-fold), whose enhanced endosomal escape was followed by imaging techniques. These data suggest that the structure of lipid-anchors in PEG affects the action of the peptides for inducing of endosomal escape.
  • Ayman El-Sayed, Tomoya Masuda, Hidetaka Akita, Hideyoshi Harashima
    JOURNAL OF PHARMACEUTICAL SCIENCES 101 2 879 - 882 2012年02月 [査読有り][通常論文]
     
    We previously reported on a stearylated INF7 peptide (str-INF7), which enhances the endosomal escape of an octaarginine (R8)-modified liposomal particle encapsulating plasmid DNA (pDNA) in a fusion-independent manner. This study examined whether this peptide derivative enhanced the endosomal escape and gene expression of PEGylated liposomes encapsulating pDNA. We used a PEGylated, R8-modified multifunctional envelope-type nanodevice (R8MEND) as a model for PEGylated liposomes. Polyethylene glycol 2000 (PEG2000) attached to two different anchors, distearoylphosphatidylethanolamine (DSPEPEG) or dimyristoylphosphatidylethanolamine (DMPEPEG), was used to modify the R8MEND in the presence or absence of two different concentrations of str-INF7. Modification of the PEGylated R8MEND with str-INF7 resulted in luciferase gene expression levels in HeLa cells that were 73-fold and 24-fold higher than the corresponding value for an unmodified MEND in the case of DSPEPEG and DMPEPEG, respectively. The endosomal escape of the PEGylated R8MEND was improved by str-INF7, as confirmed by confocal laser scanning microscopy. Furthermore, modification with str-INF7 enhanced the hepatic gene expression of the R8MEND modified with DSPEPEG and DMPEPEG by 95-fold and 1885-fold, respectively, after intravenous injection in mice. Collectively, these data demonstrate that str-INF7 can be a useful device for enhancing the endosomal escape even for PEGylated liposomes encapsulating pDNA. (C) 2011 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:879882, 2012
  • Yuma Yamada, Hideyoshi Harashima
    BIOMATERIALS 33 5 1589 - 1595 2012年02月 [査読有り][通常論文]
     
    Mitochondrial dysfunction has been implicated in a variety of human diseases. It is now well accepted that mutations and defects in the mitochondrial genome form the basis of these diseases. Therefore, mitochondrial gene therapy and diagnosis would be expected to have great medical benefits. To achieve such a strategy, it will be necessary to deliver therapeutic agents into mitochondria in living cells. We report here on an approach to accomplish this via the use of a Dual Function (DF)-MITO-Porter, aimed at the mitochondrial genome, so-called mitochondrial DNA (mtDNA). The DF-MITO-Porter, a nano carrier for mitochondrial delivery, has the ability to penetrate the endosomal and mitochondrial membranes via step-wise membrane fusion. We first constructed a DF-MITO-Porter encapsulating DNase 1 protein as a bioactive cargo. It was expected that mtDNA would be digested, when the DNase I was delivered to the mitochondria. We observed the intracellular trafficking of the carriers, and then measured mitochondrial activity and mtDNA-levels after the delivery of DNase I by the DF-MITO-Porter. The findings confirm that the DF-MITO-Porter effectively delivered the DNase 1 into the mitochondria, and provides a demonstration of its potential use in therapies that are selective for the mitochondrial genome. (C) 2011 Elsevier Ltd. All rights reserved.
  • Daisuke Shigenaka, Masami Ukawa, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Hidetaka Akita, Hideyoshi Harashima, Yoshinobu Baba
    Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012 1546 - 1548 2012年 [査読有り][通常論文]
     
    Multifunctional envelope-type nanodevices (MEND) developed by Harashima et al. is one of the novel non-viral DNA vectors expected as a safe gene delivery system. Our group has already attained fast and easy preparation of MEND by using the microfluidic device. However, purification of MEND should be performed in a high-throughput manner because prepared MEND contains impurities such as plasmid-cationic polymer complex. In this paper, we report a novel purification method for MEND by free flow electrophoresis based on microfluidic device, which purification principle is based on the zeta potential difference between MEND and impurities.
  • Ryo Furukawa, Yuma Yamada, Yuichi Matsushima, Yu-ichi Goto, Hideyoshi Harashima
    FEBS OPEN BIO 2 145 - 150 2012年 [査読有り][通常論文]
     
    For successful mitochondrial transgene expression, an optimal packaging exogenous DNA is an important issue. We report herein on the effects of DNA packaged with mitochondrial transcription factor A (TFAM), which packages mitochondrial DNA (mtDNA), on the transcription process. Our initial findings indicated that the transcription of the TFAM/DNA complex was activated, when the complex was formed at an optimal ratio. We also found that TFAM has a significant advantage over protamine, a nuclear DNA packaging protein, from the viewpoint of transcription efficiency. This result indicates that TFAM can be useful packaging protein for exogenous DNA to achieve mitochondrial transgene expression. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.
  • Genki Kanda, Hiroshi Ochiai, Hideyoshi Harashima, Hiroyuki Kamiya
    JOURNAL OF BIOTECHNOLOGY 157 1 7 - 11 2012年01月 [査読有り][通常論文]
     
    The positive feedback system using a fusion protein of the sequence-specific DNA binding domain of yeast GAL4 and the transcription activation domain of herpes simplex virus VP16 (GAL4-VP16), in which GAL4-VP16 promotes its own expression as well as that of a reporter gene product, is useful for efficient transgene expression from plasmid DNA. In this study, the transcription activation domains of endogenous proteins, instead of VP16, were fused to the GAL4 DNA binding domain, and the positive feedback systems employing the novel fusion proteins were examined. Plasmid DNAs encoding the transcription factors were introduced into mouse Hepa 1-6 cells by electroporation and lipofection. Among CREB-binding protein (226-460), sterol regulatory element-binding protein-1 (1-140), p53 (1-70), and Med15 (9-73), the CREB-binding protein functioned efficiently as an activator. These results indicated that the GAL4-CREB-binding protein is useful for enhanced transgene expression by the positive feedback system. (C) 2011 Elsevier B.V. All rights reserved.
  • Yuma Yamada, Hidetaka Akita, Hideyoshi Harashima
    NANOMEDICINE: INFECTIOUS DISEASES, IMMUNOTHERAPY, DIAGNOSTICS, ANTIFIBROTICS, TOXICOLOGY AND GENE MEDICINE 509 301 - 326 2012年 [査読有り][通常論文]
     
    A single cell contains a variety of organelles. Included among these organelles are the nucleus that regulates the central dogma, mitochondria that function as an energy plant, the Golgi apparatus that determines the destination of endogenous protein, and others. If it were possible to prepare a nano craft that could specifically target a specific organelle, this would open a new field of research directed toward therapy for various diseases. We recently developed a new concept of "Programmed Packaging," by which we succeeded in creating a multifunctional envelope-type nano device (MEND) as a nonviral gene-delivery system. Our attempts to target certain organelles (nucleus and mitochondria) are described here, mainly focusing on the construction of a tetra-lamellar MEND (T-MEND), and on methods for screening the organelle-specific fusogenic envelope. The critical structural elements of the T-MEND include an organelle-specific membrane-fusogenic inner envelope and a cellular membrane-fusogenic outer envelope. The resulting T-MEND can be utilized to overcome intracellular membrane barriers, since it involves stepwise membrane fusion. To deliver cargos into a target organelle in our strategy, the carriers must fuse with the organelle membrane. Therefore, we screened a series of lipid envelopes that have the potential for fusing with an organelle membrane by monitoring the inhibition of fluorescence resonance energy transfer and identified the optimal lipid conditions for nuclear and mitochondrial membrane fusion. Finally, we describe the delivery of a bioactive molecule targeted to the nucleus and mitochondria in living cells, demonstrating that this system can be useful for targeting various organelles.
  • Ikramy A. Khalil, Yasuhiro Hayashi, Ryoichi Mizuno, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 156 3 374 - 380 2011年12月 [査読有り][通常論文]
     
    We previously reported that octaarginine peptide modified liposomes (R8-liposomes) largely accumulated in the liver after intravenous administration and that this is dependent on the R8-density. We report herein on the development of a Multifunctional Envelope-type Nano Device modified with R8 and GALA, as a pH-sensitive fusogenic peptide (R8-GALA-MEND) for liver gene delivery. An R8-MEND encapsulating pDNA prepared using two different cores (negatively or positively charged pDNA/polyethylene imine condensed particles) failed to produce a high gene expression in the liver. Modification with GALA dramatically increased gene expression particularly in the liver only in the case of a negative core R8-MEND. Quantification of the number of gene copies delivered to liver cells and nuclei revealed that the amount of pDNA was significantly higher in the case of positive core R8-MENDs, regardless of the absence or presence of GALA. However, gene expression efficiencies per nucleus-delivered pDNA were much higher in the case of the negative core R8-MEND, especially the R8-GALA-MEND suggesting that the substantial improvement in gene expression can be explained by an improved gene expression efficiency per pDNA in the presence of GALA. A comparative study between the developed R8-GALA-MEND and a similar system containing DOTAP, a commonly used cationic lipid, instead of R8 showed that gene expression of the R8-GALA-MEND was 29 times higher than that of the DOTAP-GALA-MEND and is more selective for the liver. Collectively, these results suggest that the combination of a negatively charged core system and GALA modification of the R8-MEND is useful system for efficiently delivering genes to the liver. (C) 2011 Elsevier B.V. All rights reserved.
  • Miyazaki J, Kawai K, Kojima T, Oikawa T, Joraku A, Shimazui T, Nakaya A, Yano I, Nakamura T, Harashima H, Akaza H
    BJU international 108 9 1520 - 1526 2011年11月 [査読有り][通常論文]
     
    OBJECTIVE To conduct a preclinical evaluation of the ability of natural killer cells to cytolyze bladder cancer cells that were modified to show enhanced expression of natural-killer group 2, member D (NKG2D) ligands by R8-liposome-bacillus Calmette-Guein (BCG)-cell wall skeleton (CWS) treatment. MATERIALS AND METHODS The T24 cells and RT-112 cells were co-cultured with R8-liposome-BCG-CWS and BCG for 2, 4, or 6 h, and then the surface expression of NKG2D ligands was analyzed using TaqMan real-time quantitative RT-PCR. Peripheral blood mononuclear cells were obtained with a conventional preparation kit, and then lymphokine-activated killer (LAK) cells were generated from these purified peripheral blood mononuclear cells via interleukin-2 stimulation. The anti-tumour effect of LAK cells against untreated and R8-liposome-BCG-CWS co-cultured with cells of the human bladder cancer cell lines T24 and RT-112 was analyzed using the cytotoxic WST-8 assay method at 4 h of culture at various effector/target (E : T) ratios. RESULTS Major histocompatibility complex class I-related chain B (MICB) expression was increased approximate to 1.5-fold on T24 cells and RT-112 cells with BCG. UL-16-binding protein (ULBP) 1 expression was also increased approximate to 1.5-fold on T24 cells and RT-112 cells with BCG. R8-liposome-BCG-CWS increased the surface expression of MICB 2.2-fold on T24 cells but did not increase it significantly on RT-112 cells. ULBP1 expression was increased approximate to 2.2-fold on RT-112 cells, although no differences were observed between the expression of ULBP2 and 3 with R8-liposome-BCG-CWS. T24 cells that were co-cultured with R8-liposome-BCG-CWS showed an approximate to 1.3-fold increase in sensitivity to cytolysis by LAK cells at an E : T ratio of 4 and RT-112 cells showed an approximate to 1.4-fold increase at an E : T ratio of 2. CONCLUSIONS In the present study, the induction of surface NKG2D ligands by R8-liposome-BCG-CWS rendered cancer cells more susceptible to cytolysis by LAK cells. T24 cells and RT-112 cells, even when cultured singly in the absence of immune cells, can directly respond to R8-liposome-BCG-CWS. The results obtained in the present study may therefore indicate a novel adoptive immunotherapy against bladder cancers.
  • Takao Arimori, Haruhiko Tamaoki, Teruya Nakamura, Hiroyuki Kamiya, Shinji Ikemizu, Yasumitsu Takagi, Toru Ishibashi, Hideyoshi Harashima, Mutsuo Sekiguchi, Yuriko Yamagata
    NUCLEIC ACIDS RESEARCH 39 20 8972 - 8983 2011年11月 [査読有り][通常論文]
     
    Human NUDT5 (hNUDT5) hydrolyzes various modified nucleoside diphosphates including 8-oxo-dGDP, 8-oxo-dADP and ADP-ribose (ADPR). However, the structural basis of the broad substrate specificity remains unknown. Here, we report the crystal structures of hNUDT5 complexed with 8-oxo-dGDP and 8-oxo-dADP. These structures reveal an unusually different substrate-binding mode. In particular, the positions of two phosphates (alpha and beta phosphates) of substrate in the 8-oxo-dGDP and 8-oxo-dADP complexes are completely inverted compared with those in the previously reported hNUDT5-ADPR complex structure. This result suggests that the nucleophilic substitution sites of the substrates involved in hydrolysis reactions differ despite the similarities in the chemical structures of the substrates and products. To clarify this hypothesis, we employed the isotope-labeling method and revealed that 8-oxo-dGDP is attacked by nucleophilic water at P beta, whereas ADPR is attacked at P alpha. This observation reveals that the broad substrate specificity of hNUDT5 is achieved by a diversity of not only substrate recognition, but also hydrolysis mechanisms and leads to a novel aspect that enzymes do not always catalyze the reaction of substrates with similar chemical structures by using the chemically equivalent reaction site.
  • Hiroshi Ochiai, Hideyoshi Harashima, Hiroyuki Kamiya
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 112 5 432 - 434 2011年11月 [査読有り][通常論文]
     
    The effects of a genetic insulator (chicken cHS4) on transgene expression in a positive feedback system using the GAL4-VP16 activator and luciferase reporter plasmids were examined. The introduction of cHS4 enhanced luciferase expression in mouse liver, indicating that this system would be useful for efficient transgene expression. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.
  • Naoko Abe, Hiroshi Abe, Chisato Nagai, Mitsuru Harada, Hiroto Hatakeyama, Hideyoshi Harashima, Takahito Ohshiro, Mizuki Nishihara, Kazuhiro Furukawa, Mizuo Maeda, Satoshi Tsuneda, Yoshihiro Ito
    BIOCONJUGATE CHEMISTRY 22 10 2082 - 2092 2011年10月 [査読有り][通常論文]
     
    RNA interference (RNAi) is one of the most promising new approaches for disease therapy. The design of a dumbbell shaped nanocircular RNA allows it to act as a short interfering RNA (siRNA) precursor. To optimize the,design, we studied the relationship between the nanostructure and RNAi activity by synthesizing various RNA - dumbbells. An RNA dumbbell with a 23 bp stem and 9-nt loops was the most potent. Sequence analysis by mass spectrometry showed that Dicer could edit RNA dumbbells to siRNA species. The reaction offered the slow release of siRNA species, which conferred prolonged RNAi activity. Introduction of DNA into the loop position significantly stabilized the dumbbell in biological fluid without any loss of RNAi activity. In-depth pharmacological evaluation was performed by introducing dumbbells into HeLa cells that stably express : the target luciferase gene. The dumbbells provided a rapid silencing effect and retained this effect for a longer time even at a lower concentration than that at which standard siRNA completely lost RNAi activity. We conclude that an RNA dumbbell with DNA the most promising design for in vivo applications for RNA medicine.
  • Mika Hori, Tetsuya Suzuki, Noriaki Minakawa, Akira Matsuda, Hideyoshi Harashima, Hiroyuki Kamiya
    MUTATION RESEARCH-FUNDAMENTAL AND MOLECULAR MECHANISMS OF MUTAGENESIS 714 1-2 11 - 16 2011年09月 [査読有り][通常論文]
     
    8-Oxo-7,8-dihydroguanine (8-hydroxyguanine) is oxidized more easily than normal nucleobases, which can produce spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh). These secondary oxidation products of 8-oxo-7,8-dihydroguanine are highly mutagenic when formed within DNA. To evaluate the mutagenicity of the corresponding oxidation products of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-hydroxy-2'- deoxyguanosine 5'-triphosphate) in the nucleotide pool, Escherichia coli cells deficient in the mutT gene were treated with H2O2, and the induced mutations were analyzed. Moreover, the 2'-deoxyriboside 5'-triphosphate derivatives of Sp and Gh were also introduced into competent E. coli cells. The H2O2 treatment of mutT E. coli cells resulted in increase of G:C -> T:A and A:T -> T:A mutations. However, the incorporation of exogenous Sp and Gh 2'-deoxyribonucleotides did not significantly increase the mutation frequency. These results suggested that the oxidation product(s) of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate induces G:C -> T:A and A:T -> T:A mutations, and that the 2'-deoxyriboside 5'-triphosphate derivatives of Sp and Gh exhibit quite weak mutagenicity, in contrast to the bases in DNA. (C) 2011 Elsevier BM. All rights reserved.
  • Yukiko Morita, Hiroyuki Tsuchiya, Hideyoshi Harashima, Hiroyuki Kamiya
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 34 9 1465 - 1468 2011年09月 [査読有り][通常論文]
     
    Tailed duplex (TD) DNAs, prepared by annealing an oligonucleotide to a several-hundred-base single-stranded (ss) DNA fragment, correct a base-substitution mutation with high efficiency. In the present study, the abilities of TD fragments to correct single-base insertion and deletion mutations were examined, using hygromycin-resistance and enhanced green fluorescent protein fusion (Hyg-EGFP) genes inactivated by +G and -C frameshift mutations. The 5'-TD and 3'-TD DNA fragments were co-transfected with plasmid DNA containing the inactivated Hyg-EGFP gene into CHO-K1 cells, and the gene correction efficiencies were determined by introducing the plasmid DNA recovered from the transfected cells into Escherichia coli cells. In contrast to their efficiencies for the substitution mutation, the gene correction abilities of the TD fragments were relatively low. The correction efficiencies by the TD fragments were apparently higher than that by a ss DNA fragment, one of the DNA fragments employed for gene correction. These results suggest that the TD fragments have the potential to correct frameshift mutations, although further improvement is required.
  • Sharif M. Shaheen, Hidetaka Akita, Takashi Nakamura, Shota Takayama, Shiroh Futaki, Atsushi Yamashita, Ryo Katoono, Nobuhiko Yui, Hideyoshi Harashima
    Biomaterials 32 26 6342 - 6350 2011年09月 [査読有り][通常論文]
     
    DNA vaccines are a new-generation vaccines that elicit an immunological response against a wide-variety of antigens with frequent mutations. However, an effective non-viral vector for genetically engineered DNA to dendritic cells is yet to be developed. We previously reported that an octaarginine (R8)-modified tetra-lamellar multi-functional envelope-type nano device (R8-T-MEND) increases transfection efficiency in dendritic cell cultures (JAWS II). The critical structural elements of the R8-T-MEND are a DNA-polycation condensed core coated with two nuclear membrane-fusogenic inner envelopes, and two endosome-fusogenic outer envelopes. While the gene expression was drastically enhanced by R8-T-MEND, antigen presentation using an epitope-encoding plasmid DNA remains an obstacle for future non-viral vectors in DNA vaccinations. In the present study, we upgraded the function of R8-T-MEND by improving the membrane-fusion processes with endosome- and nuclear membranes by incorporating the KALA peptide, and by reducing the charge ratio (+/-), in an attempt to accelerate intra-nuclear decondensation. The resulting KALA-modified T-MEND (R8/KALA-T-MEND) showed an approximately 20-fold higher transgene expression compared with the conventional R8-T-MEND in JAWS II, and exceeded that of Lipofectamine PLUS, a commercially available transfection reagent. Furthermore, significant antigen presentation of a specific epitope (SIINFEKL) was observed for the R8/KALA-T-MEND but was not detected for the conventional T-MEND or Lipofectamine PLUS when an ovalbumin (OVA)-encoding plasmid DNA was transfected. It thus appears that the R8/KALA-T-MEND has the potential for use as a vector in DNA vaccinations. © 2011 Elsevier Ltd.
  • Sharif M. Shaheen, Hidetaka Akita, Takashi Nakamura, Shota Takayama, Shiroh Futaki, Atsushi Yamashita, Ryo Katoono, Nobuhiko Yui, Hideyoshi Harashima
    BIOMATERIALS 32 26 6342 - 6350 2011年09月 [査読有り][通常論文]
     
    DNA vaccines are a new-generation vaccines that elicit an immunological response against a wide-variety of antigens with frequent mutations. However, an effective non-viral vector for genetically engineered DNA to dendritic cells is yet to be developed. We previously reported that an octaarginine (R8)-modified tetra-lamellar multi-functional envelope-type nano device (R8-T-MEND) increases transfection efficiency in dendritic cell cultures (JAWS II). The critical structural elements of the R8-T-MEND are a DNA-polycation condensed core coated with two nuclear membrane-fusogenic inner envelopes, and two endosome-fusogenic outer envelopes. While the gene expression was drastically enhanced by R8-T-MEND, antigen presentation using an epitope-encoding plasmid DNA remains an obstacle for future non-viral vectors in DNA vaccinations. In the present study, we upgraded the function of R8-T-MEND by improving the membrane-fusion processes with endosome- and nuclear membranes by incorporating the KALA peptide, and by reducing the charge ratio (+/-), in an attempt to accelerate intra-nuclear decondensation. The resulting KALA-modified T-MEND (R8/KALA-T-MEND) showed an approximately 20-fold higher transgene expression compared with the conventional R8-T-MEND in JAWS II, and exceeded that of Lipofectamine PLUS, a commercially available transfection reagent. Furthermore, significant antigen presentation of a specific epitope (SIINFEKL) was observed for the R8/KALA-T-MEND but was not detected for the conventional T-MEND or Lipofectamine PLUS when an ovalbumin (OVA)-encoding plasmid DNA was transfected. It thus appears that the R8/KALA-T-MEND has the potential for use as a vector in DNA vaccinations. (C) 2011 Elsevier Ltd. All rights reserved.
  • Shota Warashina, Takashi Nakamura, Hideyoshi Harashima
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 34 8 1348 - 1351 2011年08月 [査読有り][通常論文]
     
    In a previous report, we described the development of lipid envelope-type nanoparticles (MEND) modified with octaarginine (R8) and a pH-sensitive fusogenic peptide (GALA) for delivering short interference RNA (siRNA) to mouse dendritic cells (DCs). A20 was recently reported to be a negative regulator of the toll-like receptor and the tumor necrosis factor receptor signaling pathways. Although A20 would be expected to be a useful target for boosting the effects of adjuvants in DC immunotherapy, limited information is available regarding the use of A20-silenced DC by an original non-viral vector. In this study, we loaded anti-A20 siRNA into a MEND and investigated the gene knockdown activity in DC and the immunological functions of A20-silenced DC. The use of a MEND resulted in a significant A20 knockdown effect, and the A20-silenced DC resulted in an enhanced production of proinflammatory molecules, after lipopolysaccharide (LPS) stimulation. The expression of co-stimulatory molecules by LPS stimulation was also increased in the A20-silenced DC. The findings reported herein show that a MEND loaded with anti-A20 siRNA is a potent non-viral vector that has the ability to enhance the adjuvant effect of LPS in DC.
  • Yu Sakurai, Hiroto Hatakeyama, Yusuke Sato, Hidetaka Akita, Kentaro Takayama, Sachiko Kobayashi, Shiroh Futaki, Hideyoshi Harashima
    BIOMATERIALS 32 24 5733 - 5742 2011年08月 [査読有り][通常論文]
     
    An siRNA that specifically silences the expression of mRNA is a potential therapeutic agent for dealing with many diseases including cancer. However, the poor cellular uptake and bioavailability of siRNA remains a major obstacle to clinical development. For efficient delivery to tumor tissue, the pharmacokinetics and intracellular trafficking of siRNA must be rigorously controlled. To address this issue, we developed a liposomal siRNA carrier, a multi-functional nano device (MEND). We describe herein an approach for systemic siRNA delivery to tumors by combining the MEND system with shGALA, a fusogenic peptide. In cultured cell experiments, shGALA-modification enhanced the endosomal escape of siRNA encapsulated in a polyethylene glycol modified MEND (PEG-MEND), resulting in an 82% knockdown of the target gene. In vivo systemic administration clarified that the shGALA-modified MEND (shGALA-MEND) showed 58% gene silencing in tumor tissues at a dose of 4 mg of siRNA/kg body weight. In addition, a significant inhibition of tumor growth was observed only for the shGALA-MEND and no somatic or hepatic toxicity was observed. Given the above data, this peptide-modified delivery system, a shGALA-MEND has great potential for the systemic delivery of therapeutic siRNA aimed at cancer therapy. (C) 2011 Elsevier Ltd. All rights reserved.
  • Naoyuki Toriyabe, Yasuhiro Hayashi, Mamoru Hyodo, Hideyoshi Harashima
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 34 7 1084 - 1089 2011年07月 [査読有り][通常論文]
     
    Hyaluronic acid (HA) is a naturally-occurring ligand that can be useful for targeting liver endothelial cells. We describe herein the development of a new HA-lipid conjugate for the efficient delivery of liposomes to liver endothelial cells. When free HA coated cationic liposomes were injected into mice, their accumulation in the liver was significantly decreased depending on the content of free HA, while accumulation in the lung was not significantly changed. When cationic liposomes modified with HA-stearylamine (HA-SA conjugate) were injected in mice, liver accumulation was increased depending on the amount of HA-SA conjugate and accumulation in the lung was drastically reduced, compared to non-modified liposomes. Confocal imaging analyses showed that HA-SA modified liposomes were accumulated to a greater extent along with blood vessels than non-modified liposomes, suggesitng that HA-SA modified liposomes are distributed in endothelial cells in the liver. Collectively, these findings indicate that an HA-SA conjugate is a useful material that can be used to modify liposomes and for delivering bioactive liposomal cargoes to liver endothelial cells.
  • Hidetaka Akita, Tomoya Masuda, Takashi Nishio, Kenichi Niikura, Kuniharu Ijiro, Hideyoshi Harashima
    MOLECULAR PHARMACEUTICS 8 4 1436 - 1442 2011年07月 [査読有り][通常論文]
     
    The successful control of intracellular trafficking (i.e., endosomal escape and nuclear delivery) is prerequisite for the development of a gene delivery system. In the present study, we developed an in vivo hepatic gene delivery system using a plasmid DNA (pDNA) -encapsulating lipid envelope-type nanoparticle, to which we refer as a multifunctional envelope-type nanodevice (MEND). The critical structural elements of the MEND are a DNA/protamine condensed core coated with lipid bilayers including serum-resistant cationic lipids. Intravenous administration of bare MEND represents minimal transfection activity. For the surface modification of functional devices, hydrophobic moieties were chemically attached, which are shed in the spontaneous orientation outward from the MEND surface by anchoring to the lipid bilayers. Modification of the pH-dependent fusogenic peptide GALA as an endosome escape induced transfection activity by 1 and 2 orders of magnitude. In an attempt to induce the nuclear delivery of pDNA, maltotriose, a recently characterized nuclear localization signal, was additionally modified. As a result, transfection activity further enhanced by 1 order of magnitude, and it reached to the higher level obtained for a conventional lipoplex and an in vivo jetPEI-Gal, with less hepatic toxicity. The data show that the combination of GALA and maltotriose results in a highly potent functional device that shows an enhanced endosomal escape and nuclear delivery in vivo.
  • Jun Miyazaki, Hiroyuki Nishiyama, Ikuya Yano, Akihiro Nakaya, Hideyasu Kohama, Koji Kawai, Akira Joraku, Takashi Nakamura, Hideyoshi Harashima, Hideyuki Akaza
    ANTICANCER RESEARCH 31 6 2065 - 2071 2011年06月 [査読有り][通常論文]
     
    Background: The present gold standard for bladder cancer is Mycobacterium bovis bacillus Calmette-Guerin (BCG) immunotherapy, but serious side-effects are common. We previously reported that C3H/HeN mice vaccinated with a mixture of MBT-2 cells and artificial BCG, octaarginine-modified liposomes incorporating the cell wall of BCG (R8-liposome-BCG-CW), significantly inhibited growth of R8-liposome-BCG-CW pretreated MBT-2 cells. Our aim was to determine if a non-live bacterial agent could be as efficacious as live BCG in a model of bladder cancer. We investigated the suppressive effect of liposome-incorporating cell wall skeleton (BCG-CWS) on N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced urinary bladder carcinogenesis in rats. Materials and Methods: F344 rats were fed with BBN and sodium ascorbate for 8 weeks, after which all rats were confirmed to have excreted atypical epithelial cells in the urine. Rats were administered BCG-CW(1.0 mg/rat) or R8-liposome-BCG-CWS (0.1 or 1.0 mg/rat) intravesically once/week for 8 weeks from week 28 to 35 of the experimental protocol. Results: Rats receiving R8-liposome-BCG-CWS intravesically showed significantly inhibited numbers of tumors, especially those of simple hyperplasia, in comparison with the control rats. Conclusion: R8-liposome-BCG-CWS administration had inhibitory effects on rat bladder carcinogenesis. These results may indicate a novel adoptive immunotherapy against bladder cancers.
  • Hiroto Hatakeyama, Hidetaka Akita, Erika Ito, Yasuhiro Hayashi, Motoi Oishi, Yukio Nagasaki, Radostin Danev, Kuniaki Nagayama, Noritada Kaji, Hiroshi Kikuchi, Yoshinobu Baba, Hideyoshi Harashima
    BIOMATERIALS 32 18 4306 - 4316 2011年06月 [査読有り][通常論文]
     
    Previously, we developed a multifunctional envelope-type nano device (MEND) for efficient delivery of nucleic acids. For tumor delivery of a MEND, PEGylation is a useful method, which confers a longer systemic circulation and tumor accumulation via the enhanced permeability and retention (EPR) effect. However, PEGylation inhibits cellular uptake and subsequent endosomal escape. To overcome this, we developed a PEG-peptide-DOPE (PPD) that is cleaved in a matrix metalloproteinase (MMP)-rich environment. In this study, we report on the systemic delivery of siRNA to tumors by employing a MEND that is modified with PPD (PPD-MEND). An in vitro study revealed that PPD modification accelerated both cellular uptake and endosomal escape, compared to a conventional PEG modified MEND. To balance both systemic stability and efficient activity, PPD-MEND was further co-modified with PEG-DSPE. As a result, the systemic administration of the optimized PPD-MEND resulted in an approximately 70% silencing activity in tumors, compared to non-treatment. Finally, a safety evaluation showed that the PPD-MEND showed no hepatotoxicity and innate immune stimulation. Furthermore, in a DNA microarray analysis in liver and spleen tissue, less gene alternation was found for the PPD-MEND compared to that for the PEGunmodified MEND due to less accumulation in liver and spleen. (C) 2011 Elsevier Ltd. All rights reserved.
  • Yuki Hattori, Isamu Matsunaga, Takaya Komori, Tetsuo Urakawa, Takashi Nakamura, Nagatoshi Fujiwara, Kenji Hiromatsu, Hideyoshi Harashima, Masahiko Sugita
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 409 2 304 - 307 2011年06月 [査読有り][通常論文]
     
    Dynamic changes in the lipid composition of the cell wall occur in pathogenic mycobacteria that are often intended for adaptation to the host environment. Dormant mycobacteria should have evolved efficient maneuvers for cohabitation, allowing the microbes to persist for years within the host. Glycerol monomycolate (GroMM) has been implicated as a specific immune target in human individuals with latent, but not active, tuberculosis, but the in vivo response to GroMM and the relevance of it to latent infection remain poorly understood. Here, we immunized guinea pigs with bacillus Calmette-Guerin (BCG) expressing high levels of GroMM and then, monitored skin reactions at the site of challenge with GroMM-containing liposome. We found that BCG-immunized guinea pigs mounted enhanced skin reactions to GroMM with prominent local infiltration by eosinophils. Consistent with this, GroMM-stimulated lymph node cells upregulated the expression of T helper (Th)2-type cytokines, such as interleukin (IL)-5 and IL-10, that could potentially counteract the microbe-eliminating Th1-type cytokine response. On the basis of these observations, we predict that the host response to GroMM produced by dormant mycobacteria would contribute to their long-term survival in the host. (C) 2011 Elsevier Inc. All rights reserved.
  • Takaya Komori, Takashi Nakamura, Isamu Matsunaga, Daisuke Morita, Yuki Hattori, Hirotaka Kuwata, Nagatoshi Fujiwara, Kenji Hiromatsu, Hideyoshi Harashima, Masahiko Sugita
    JOURNAL OF BIOLOGICAL CHEMISTRY 286 19 16800 - 16806 2011年05月 [査読有り][通常論文]
     
    Delayed-type hypersensitivity (DTH) is marked by high levels of protein antigen-specific T cell responses in sensitized individuals. Recent evidence has revealed a distinct pathway for T cell immunity directed against glycolipid antigens, but DTH to this class of antigen has been undetermined and difficult to prove due to their insolubility in aqueous solutions. Here, glucose monomycolate (GMM), a highly hydrophobic glycolipid of the cell wall of mycobacteria, was dispersed in aqueous solutions in the form of octaarginine-modified liposomes and tested for its ability to elicit cutaneous DTH responses in bacillus Calmette-Guerin (BCG)-immunized guinea pigs. After an intradermal challenge with the GMM liposome, a significant skin induration was observed in BCG-immunized, but not mock-treated, animals. The skin reaction peaked at around 2 days with local infiltration by mononuclear cells, and therefore, the response shared basic features with the classical DTH to protein antigens. Lymph node T cells from BCG-immunized guinea pigs specifically increased IFN-gamma transcription in response to the GMM liposome, and this response was completely blocked by antibodies to CD1 lipid antigen-presenting molecules. Finally, whereas the T cells increased transcription of both T helper (Th) 1-type (IFN-gamma and TNF-alpha) and Th2-type (IL-5 and IL-10) cytokines in response to the purified protein derivative or tuberculin, their GMM-specific response was skewed to Th1-type cytokine production known to be critical for protection against tuberculosis. Thus, our study reveals a novel form of DTH with medical implications.
  • Sharif M. Shaheen, Hidetaka Akita, Atsushi Yamashita, Ryo Katoono, Nobuhiko Yui, Vasudevanpillai Biju, Mitsuru Ishikawa, Hideyoshi Harashima
    Nucleic Acids Research 39 7 e48 - e48 2011年04月 [査読有り][通常論文]
     
    Recent studies indicate that controlling the nuclear decondensation and intra-nuclear localization of plasmid DNA (pDNA) would result in an increased transfection efficiency. In the present study, we established a technology for imaging the nuclear condensation/decondensation status of pDNA in nuclear subdomains using fluorescence resonance energy transfer (FRET) between quantum dot (QD)-labeled pDNA as donor, and rhodamine-labeled polycations as acceptor. The FRET-occurring pDNA/polycation particle was encapsulated in a nuclear delivery system a tetra-lamellar multifunctional envelope-type nano device (T-MEND), designed to overcome the endosomal membrane and nuclear membrane via step-wise fusion. Nuclear subdomains (i.e. heterochromatin and euchromatin) were distinguished by Hoechst33342 staining. Thereafter, Z-series of confocal images were captured by confocal laser scanning microscopy. pDNA in condensation/decondensation status in heterochromatin or euchromatin were quantified based on the pixel area of the signals derived from the QD and rhodamine. The results obtained indicate that modulation of the supra-molecular structure of polyrotaxane (DMAE-ss-PRX), a condenser that is cleaved in a reductive environment, conferred euchromatin-preferred decondensation. This represents the first demonstration of the successful control of condensation/decondensation in specific nuclear sub-domain via the use of an artificial DNA condenser. © 2011 The Author(s).
  • Sharif M. Shaheen, Hidetaka Akita, Atsushi Yamashita, Ryo Katoono, Nobuhiko Yui, Vasudevanpillai Biju, Mitsuru Ishikawa, Hideyoshi Harashima
    NUCLEIC ACIDS RESEARCH 39 7 E48 - U108 2011年04月 [査読有り][通常論文]
     
    Recent studies indicate that controlling the nuclear decondensation and intra-nuclear localization of plasmid DNA (pDNA) would result in an increased transfection efficiency. In the present study, we established a technology for imaging the nuclear condensation/decondensation status of pDNA in nuclear subdomains using fluorescence resonance energy transfer (FRET) between quantum dot (QD)-labeled pDNA as donor, and rhodamine-labeled polycations as acceptor. The FRET-occurring pDNA/polycation particle was encapsulated in a nuclear delivery system; a tetra-lamellar multifunctional envelope-type nano device (T-MEND), designed to overcome the endosomal membrane and nuclear membrane via step-wise fusion. Nuclear subdomains (i.e. heterochromatin and euchromatin) were distinguished by Hoechst33342 staining. Thereafter, Z-series of confocal images were captured by confocal laser scanning microscopy. pDNA in condensation/decondensation status in heterochromatin or euchromatin were quantified based on the pixel area of the signals derived from the QD and rhodamine. The results obtained indicate that modulation of the supra-molecular structure of polyrotaxane (DMAE-ss-PRX), a condenser that is cleaved in a reductive environment, conferred euchromatin-preferred decondensation. This represents the first demonstration of the successful control of condensation/decondensation in specific nuclear sub-domain via the use of an artificial DNA condenser.
  • Diky Mudhakir, Hidetaka Akita, Hideyoshi Harashima
    REACTIVE & FUNCTIONAL POLYMERS 71 3 340 - 343 2011年03月 [査読有り][通常論文]
     
    It was recently reported that liposomes modified with octaarginine (R8) and its analogue peptide (IRQRRRR: IRQ) are taken up by NIH3T3 cells by unique pathways, macropinocytosis and caveolae-mediated endocytosis, respectively. This study evaluated the topology of these peptides as it relates to the uptake routes of liposomes, where they are modified either directly on the surface, or on the edge of a polyethylene glycol (PEG) spacer. The uptake mechanism of peptide-modified liposomes and peptide-modified PEG-liposomes was investigated by confocal laser scanning microscopy. To determine the contribution of clathrin-mediated endocytosis, macropinocytosis and caveolar endocytosis to the uptake of liposomes, uptake was evaluated in the presence of some specific inhibitors of these processes. The uptake pathway changed from macropinocytosis to clathrin-mediated endocytosis when R8 was modified on the edge of a PEG spacer, indicating that the flexible display of R8 impaired the induction of macropinocytosis. However, the contribution of caveolae-mediated endocytosis increased when IRQ was conjugated to the distal end of the PEG chain, suggesting that flexible surface display enhanced IRQ recognition by the specific molecules in the caveolae. The present results demonstrate that topology control by the ligand affects the contribution of the entry pathway, depending on the uptake mechanism. (C) 2010 Elsevier Ltd. All rights reserved.
  • Hiroto Hatakeyama, Hidetaka Akita, Hideyoshi Harashima
    ADVANCED DRUG DELIVERY REVIEWS 63 3 152 - 160 2011年03月 [査読有り][通常論文]
     
    Gene and nucleic acid therapy are expected to play a major role in the next generation of medicine. We recently developed a multifunctional envelope-type nano device (MEND) for use as a novel non-viral gene delivery system. Poly(ethylene glycol) (PEG)ylation is a useful method for achieving a longer circulation time for delivery of the MEND to a tumour via the enhanced permeability and retention (EPR) effect. However, PEGylation strongly inhibits cellular uptake and endosomal escape, which results in significant loss of activity for the delivery system. For successful gene delivery for cancer treatment, the crucial issue associated with the use of PEG, the 'PEG dilemma' must be addressed. In this review, we describe the development and applications of MEND, and discuss strategies for overcoming the PEG dilemma, based on the manipulation of intracellular trafficking of cellular uptake and endosomal release using functional devices such as specific ligands, cleavable PEG systems and endosomal fusogenic/disruptic peptides. (C) 2010 Elsevier B.V. All rights reserved.
  • Kaoru Kigasawa, Kazuaki Kajimoto, Takashi Nakamura, Susumu Hama, Kiyoshi Kanamura, Hideyoshi Harashima, Kentaro Kogure
    JOURNAL OF CONTROLLED RELEASE 150 3 256 - 265 2011年03月 [査読有り][通常論文]
     
    Oligodeoxynucleotides containing unmethylated cytosine-phosphate-guanosine motifs (CpG-ODN) possess immunostimulatory effects and potential antitumor activity. Since the skin is an easily available site of administration of CpG-ODN due to its accessibility and the presence of abundant antigen presenting cells, it is expected that the application of CpG-ODN to the skin would induce systemic immune response and antitumor activity. However, it is difficult to deliver hydrophilic macromolecules including CpG-ODN through the skin. We have previously demonstrated that small interfering RNA (siRNA) was efficiently delivered into rat epidermis by iontophoresis. In this report, we investigate the effect of transdermal iontophoretic delivery of CpG-ODN on the induction of immune responses and antitumor activity against B16F1 melanoma in mice. Iontophoresis promoted CpG-ODN delivery into the epidermis and dermis. Furthermore, iontophoretic delivery of CpG-ODN to the skin induced the expression of proinflammatory and Th1-type cytokines in the skin and draining lymph node. Finally, transdermal iontophoretic delivery of CpG-ODN led to antitumor activity against B16F1 melanoma. Interestingly, the CpG-ODN administration site is not restricted to the tumor area. In conclusion, CpG-ODN delivered transdermally induced potent antitumor activity, and our system is expected to serve as a simple and noninvasive approach for cancer immunotherapy. (C) 2011 Elsevier B.V. All rights reserved.
  • Diky Mudhakir, Hidetaka Akita, Hideyoshi Harashima
    4TH NANOSCIENCE AND NANOTECHNOLOGY SYMPOSIUM (NNS2011): AN INTERNATIONAL SYMPOSIUM 1415 2011年 [査読有り][通常論文]
     
    The novel IRQ peptide is one of cell penetrating peptides (CPPs) that has ability to induce endosomal escape. It has been demonstrated that IRQ ligand had ability to facilitate an escape of liposomes encapsulating siRNA from the endosomes presumably by fusion-independent mechanism [1,2]. In the present study, we attempted to modulate the intracellular trafficking of IRQ-modified nano-carrier in term of escaping process by changing the lipid composition. The peptide was attached to the terminal end of maleimide group of polyethylene glycol-modified liposomes (IRQ-PEG-Lip). The liposomes were composed of DOTAP, DOPE and cholesterol and it was labeled by water soluble sulphorhodamine B (Sr-B). The escape of PEG-coated liposomes was then observed by confocal laser scanning microscope after the endosomes were stained with Lysosensor. The results exhibited that IRQ-PEG-Lip was escaped from endosomal compartment after 1 h transfection when 40% of DOPE was incorporated into the nanostructure comparing to that of PEG-Lip. These results are consistent with the previous results that the IRQ facilitates endosomal escape via independent-mechanism. However, IRQ-PEG-Lip were then completely co-localized in the acidic compartment when density of DOPE was reduced approximately 20%. These results indicated that the utilizing of DOPE is important for the escape process even in the presence of hydrophilic PEG polymer. In conclusion, the regulation of endosomal escape ability of the PEGylated-IRQ nano-carrier was induced by fusion-independent manner as well as fusogenic lipid.
  • Diky Mudhakir, Erdal Tan, Hidetaka Akita, Hideyoshi Harashima
    4TH NANOSCIENCE AND NANOTECHNOLOGY SYMPOSIUM (NNS2011): AN INTERNATIONAL SYMPOSIUM 1415 2011年 [査読有り][通常論文]
     
    To obtain an efficient delivery of macromolecule such as siRNA, a sophisticated design of vector is needed. It must be retained to the active uptake by reticuloendothelial system (RES) in the bloodstream and it can release its content inside the cellular target site. In this study, we attempted to design a vector which accommodates the needs. siRNA was encapsulated in the liposomes and its surface was shielded with polyethylene glycol (PEG) moiety to avoid recognizing by RES system. To facilitate cellular internalization, newly synthetizing peptide was attached to tip end of PEG moiety. As results, blood concentration of PEG-modified liposomes was higher than that of unmodified PEG. Results by confocal laser scanning microscopy studies exhibited PEG-coated nanodevice can escape from endosomes in both two types PEG used, shorter and longer one. However, prominent cytosolic release of siRNA was only shown by the use of shorter PEG type. These results were in line with gene silencing study in which shorter PEGylated nanostructure achieved dramatic levels of transgene expression study. In conclusion, shorter PEG modified-liposomes encapsulating siRNA could be utilized as a device for in vivo use.
  • Ryo Furukawa, Yuma Yamada, Mitsuko Takenaga, Rie Igarashi, Hideyoshi Harashima
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 404 3 796 - 801 2011年01月 [査読有り][通常論文]
     
    The anti-oxidant enzyme superoxide dismutase (SOD) has the potential for use as a therapeutic agent in the treatment of various diseases caused by reactive oxygen species. However, achieving this would be difficult without a suitable delivery system for SOD. We previously reported that PC-SOD, in which four molecules of a phosphatidylcholine (PC) derivative were covalently bound to each dimer of recombinant human CuZnSOD, was a high affinity for the cell membrane [14]. Here, we show that an octaarginine (R8) modified liposome equipped with PC-SOD (R8-LP (PC-SOD)) enhances its anti-oxidant effect. High-density R8-modified liposomes can stimulate macropinocytosis and are taken up efficiently by cells as demonstrated in a previous study [21]. Flow cytometry analyses showed that R8-LP (PC-SOD) was taken up by cells more efficiently than PC-SOD. Moreover, R8-LP (PC-SOD) liposomes were found to scavenge superoxide anions (O(2)(-)) very efficiently. These results suggest that the efficient cytosolic delivery of PC-SOD by R8-modified liposomes would enhance the anti-oxidant effects of PC-SOD. (C) 2010 Elsevier Inc. All rights reserved.
  • Kazuaki Kajimoto, Masahiko Yamamoto, Misuzu Watanabe, Kaoru Kigasawa, Kiyoshi Kanamura, Hideyoshi Harashima, Kentaro Kogure
    INTERNATIONAL JOURNAL OF PHARMACEUTICS 403 1-2 57 - 65 2011年01月 [査読有り][通常論文]
     
    Iontophoresis is a promising technique for enhancing transdermal administration of charged drugs. However, conventional iontophoresis is not sufficient for effective delivery of large, hydrophilic, or electrically neutral molecules. In this study, we utilized charged liposomes as carriers, focused on a transfollicular route for delivery of the liposomes, and optimized iontophoretic conditions and lipid composition for this method in both in vitro and in vivo conditions. As a result, we identified the optimum condition (lipid composition: DOTAP/EPC/Chol = 2:2:1, current supply: 0.45 mA/cm(2), duration: 1 h) for effective iontophoretic delivery of aqueous solution, which cannot be transferred into the skin without charged liposomes. We also examined the pharmacological effects of iontophoresis of liposomes encapsulating insulin (INS-lipo) using a rat model of type I diabetes. Interestingly, iontophoresis of INS-lipo onto a diabetes rat skin resulted in a gradual decrease in blood glucose levels, with levels reaching 20% of initial values at 18 h after administration. These lower blood glucose levels were maintained for up to 24 h. Significant amount of insulin were also detected in plasma 18 h after iontophoresis of INS-lipo. We succeeded in developing a non-invasive and persistent transfollicular drug delivery system that used a combination of liposomes and iontophoresis. (C) 2010 Elsevier B.V. All rights reserved.
  • Katsuma Kitazoe, Jun Wang, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Kentaro Kogure, Hideyoshi Harashima, Yoshinobu Baba
    LAB ON A CHIP 11 19 3256 - 3262 2011年 [査読有り][通常論文]
     
    Multifunctional envelope-type gene delivery nanodevices (MENDs) are promising non-viral vectors for gene therapy. Though MENDs remain strong in prolonged exposure to blood circulation, have low immunogenic response, and are suitable for gene targeting, their fabrication requires labor-intensive processes. In this work, a novel approach has been developed for rapid fabrication of MENDs by a touch-and-go lipid wrapping technique in a polydimethylsiloxane (PDMS)/glass microfluidic device. The MEND was fabricated on a glass substrate by introduction of a condensed plasmid DNA core into microfluidic channels that have multiple lipid bilayer films. The principle of the MEND fabrication in the microfluidic channels is based on electrostatic interaction between the condensed plasmid DNA cores and the coated lipid bilayer films. The constructed MEND was collected off-chip and characterized by dynamic light scattering. The MEND was constructed within 5 min with a narrow size distribution centered around 200 nm diameter particles. The size of the MEND showed strong dependence on flow velocity of the condensed plasmid DNA core in the microfluidic channels, and thus, could be controlled to provide the optimal size for medical applications. This approach was also proved possible for fabrication of a MEND in multiple channels at the same time. This on-chip fabrication of the MEND was very simple, rapid, convenient, and cost-effective compared with conventional methods. Our results strongly indicated that MENDs fabricated with our microfluidic device have a good potential for medical use. Moreover, MENDs fabricated by this microfluidic device have a great potential for clinical use because the devices are autoclavable and all the fabrication steps can be completed inside closed microfluidic channels without any external contamination.
  • Hidetaka Akita, Kentaro Kogure, Rumiko Moriguchi, Yoshio Nakamura, Tomoko Higashi, Takashi Nakamura, Satoshi Serada, Minoru Fujimoto, Tetsuji Naka, Shiroh Futaki, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 149 1 58 - 64 2011年01月 [査読有り][通常論文]
     
    We previously developed octaarginine (R8)-modified lipid envelope-type nanoparticles for siRNA delivery (R8-MEND). Herein, we report on their ex vivo siRNA delivery to primary mouse bone marrow-derived dendritic cells (BMDCs) for potential use as a cancer vaccine. Quantitative imaging analysis of the intracellular trafficking of siRNA revealed that the dissociation process, as well as the rate of endosomal escape limits the siRNA efficiency of the prototype R8-MEND, prepared by the hydration method (R8-MENDhydo). Successful endosomal escape was achieved by using a pH-dependent fusogenic peptide (GALA) modified on a lipid mixture that was optimized for endosomal fusion. Furthermore, a modified protocol for the preparation of nanoparticles, mixing the siRNA/STR-R8 complex and small unilamellar vesicles (R8/GALA-MENDSUV), results in a more homogenous, smaller particle size, and results in a more efficient intracellular dissociation. Gene knockdown of the suppressor of cytokine signaling 1 (SOCS1), a negative-feedback regulator of the immune response in BMDCs resulted in an enhanced phosphorylation of STAT1, and the production of proinflammatory cytokines. Moreover, SOCS1-silenced BMDCs were more potent in suppressing tumor growth. Collectively, these results show that siRNA loaded in R8/GALA-MENDSUV efficiently suppresses endogenous gene expression and consequently enhances dendritic cell-based vaccine potency in vivo. (C) 2010 Elsevier B.V. All rights reserved.
  • Hideyoshi Harashima, Akihiko Kikuchi, Kazunori Kataoka
    JOURNAL OF CONTROLLED RELEASE 149 1 1 - 1 2011年01月 [査読有り][通常論文]
  • (Review) Application of a fusiogenic peptide GALA for intracellular delivery
    Ikuhiko Nakase, Kentaro Kogure, Hideyoshi Harashima, Shiroh Futaki
    Methods in Molecular Biology 683 525 - 533 2011年 [査読有り][通常論文]
  • 内在性転写活性化因子を用いた細胞系における外来遺伝子発現の活性化
    神田 元紀, 落合 浩史, 原島 秀吉, 紙谷 浩之
    日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 83回・33回 1P - 1247 (公社)日本生化学会 2010年12月
  • 2種類の「発現持続型」プラスミドの核内挙動の違い
    冨樫 亮平, 神田 元紀, 紙谷 浩之, 原島 秀吉
    日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 83回・33回 1P - 1248 (公社)日本生化学会 2010年12月
  • Hitoshi Shiku, Daisuke Okazaki, Junya Suzuki, Yasufumi Takahashi, Tatsuya Murata, Hidetaka Akita, Hideyoshi Harashima, Kosuke Ino, Tomokazu Matsue
    FEBS LETTERS 584 18 4000 - 4008 2010年09月 [査読有り][通常論文]
     
    mRNA from single cells was quantified using real-time RT-PCR after recording the address and reporter protein activity with chemiluminescence, fluorescence, and electrochemical techniques, using luciferase, green fluorescent protein, and secreted alkaline phosphatase. mRNA copy number ranging from below 103 to 107 in single cells showed a lognormal distribution for both externally introduced reporter genes and internally expressed genes. The fluctuation in the gene expression decreased with the increase of the number of cells picked but did not decrease with the increase of mRNA copy number per cell. We found that the correlation coefficients for mRNA and protein expression in logarithmic plot at single-cell level were much lower than 1.00. (c) 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  • Masami Ukawa, Hidetaka Akita, Tomoya Masuda, Yasuhiro Hayashi, Tomohiro Konno, Kazuhiko Ishihara, Hideyoshi Harashima
    BIOMATERIALS 31 24 6355 - 6362 2010年08月 [査読有り][通常論文]
     
    We previously reported that modification of GALA peptide on the surface of liposomes enhanced fusion with endosomal membrane, and cytoplasmic release of encapsulated macromolecules. We report herein that an additional coating of GALA-modified liposomes with 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer resulted in a two order of magnitude enhancement in the transfection activity of encapsulating plasmid DNA (pDNA). Quantification of the delivered gene copies in whole cells and isolated nuclei revealed that the increase of transfection activity can be attributed to improved efficiencies in cellular uptake and post-nuclear delivery processes. Imaging studies revealed that the intracellular dissociation of pDNA from the lipid envelope is enhanced by GALA modification and further coating with MPC polymer in a stepwise manner. The MPC polymer-coating decreased the zeta-potential of GALA-modified liposomes, suggesting that it assisted in the functional display of negatively charged GALA on the cationic liposomes by providing shielding from mutual electrostatic interactions. Collectively, these data indicate that MPC polymer-coating induced the fusogenic activity of the GALA-modified envelope with endosomes, leading to a more effective cytoplasmic release pDNA. The extensive fusion of the lipid envelope may also reduce electrostatic interactions between mRNA and cationic lipid components, thereby resulting in an enhancement in the translation process. (C) 2010 Elsevier Ltd. All rights reserved.
  • Yuma Yamada, Taku Nomura, Hideyoshi Harashima, Atsushi Yamashita, Ryo Katoono, Nobuhiko Yui
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 33 7 1218 - 1222 2010年07月 [査読有り][通常論文]
     
    It has been believed that nuclear gene delivery is the most important process for gene expression, and various non-viral vectors are currently being developed with this assumption. However, some of our earlier studies revealed a surprising difference in transfection activity between viral and non-viral vectors: this difference is largely due to the result of the intranuclear disposition of DNA rather than its delivery to the nucleus (Hama S. et al. (2006), Quantitative comparison of intracellular trafficking and nuclear transcription between adenoviral and lipoplex systems. Mol. Then, 13, 786-794). Here; we report on some direct evidence that demonstrates the importance of the release of intranuclear DNA on transfection activity. The data show that transfection activity can be substantially enhanced by integrating a multifunctional envelope-type nano device (MEND) and a biocleavable polyrotaxane (DMAE-SS-PRX) as an artificial condenser. Our integration system showed significantly higher transfection activity compared to conventional gene delivery system. Moreover, this system provides a strong support for our hypothesis that intranuclear DNA disposition plays a critical role in gene expression for non-viral vectors.
  • Yusuke Sato, Hiroto Hatakeyama, Hideyoshi Harashima
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 33 7 1246 - 1249 2010年07月 [査読有り][通常論文]
     
    The delivery of nucleic acids to cancer cells represents a potentially useful strategy. Previously, we developed a multifunctional envelope-type nano device (MEND) for the efficient delivery of plasmid DNA. In addition, we successfully delivered short interference RNA (siRNA) into cytoplasm using a MEND which contains siRNA particles that were produced using stearyl octaarginine (STR-R8). In the present study, to achieve further gene silencing activity compared with STR-R8, various additional polycations were screened. We used protamine and 10 different polypeptides containing random sequence of basic amino acids. The ability of these polycations to form nano particles with siRNA were evaluated by measuring the size and zeta-potential of produced nano particles, and as a consequence, 6 of the polycations were selected for further evaluation. We then prepared MENDs containing the particles. The lipid composition of the MEND consisted of dioleoylphosphatidyl ethanolamine (DOPE)/phosphatidic acid (PA) (7/2). For cellular uptake and endosomal escape, the MEND was modified with PPD (polyethylene glycol (PEG)-peptide-DOPE), STR-R8 and GALA, pH-sensitive fusogenic peptide. The resulting MEND had a diameter of 120-170 nm and a zeta-potential of 15-25 mV. The MEND was transfected into HeLa cells stably expressing luciferase and the silencing activity of the polycations was compared. Most of the polycations failed to knockdown luciferase activity. However, the polypeptide containing ornithine and tryptophan (Orn/Trp) induced a higher knockdown than STR-R8. In addition, Orn/Trp induced a silencing effect at lower doses than STR-R8, as evidenced by dose-response data. In conclusion, the findings suggest that Orn/Trp is a superior polycation to STR-R8 for siRNA delivery.
  • Hiroshi Ochiai, Hideyoshi Harashima, Hiroyuki Kamiya
    MOLECULAR PHARMACEUTICS 7 4 1125 - 1132 2010年07月 [査読有り][通常論文]
     
    The two-step transcriptional amplification (TSTA) system, using artificial transcription factors, effectively enhances transgene expression. In this study, a TSTA system-based positive feedback system was developed to achieve efficient and persistent transgene expression. A fusion protein of the sequence-specific DNA binding domain of yeast GAL4 and the transcriptional activation domain of herpes simplex virus VP16 (GAL4-VP16) was used as an "activator" to amplify the expression of the luciferase "reporter" gene. It was found that the introduction of five tandem copies of the GAL4 recognition sequence (G5) into both the upstream and downstream regions of the expression cassette synergistically enhanced the transgene expression. The upstream and downstream G5 sequences were introduced into the expression cassette of the activator itself, and into that of the reporter, to form the positive feedback loop that enabled continuous activator expression. This positive feedback system maintained the expression levels of the reporter for 4 days in HeLa cells and for a week in mouse liver, while those from the usual plasmids decreased by 30- and 50-fold, respectively. These results constitute the first evidence that the positive feedback system is a useful method for long-term transgene expression in cultured cells and in vivo. This system would be applicable to gene therapy, in vivo imaging, and biotechnology.
  • Yukari Yasuzaki, Yuma Yamada, Hideyoshi Harashima
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 397 2 181 - 186 2010年06月 [査読有り][通常論文]
     
    Mitochondria are are the principal producers of energy in cells of higher organisms. It was recently reported that mutations and defects in mitochondrial DNA (mtDNA) are associated with various mitochondrial diseases including a variety of neurodegenerative and neuromuscular diseases. Therefore, an effective mitochondrial gene therapy and diagnosis would be expected to have great medical benefits. To achieve this, therapeutic agents need to be delivered into the innermost mitochondrial space (mitochondrial matrix), which contains the mtDNA pool. We previously reported on the development of MITO-Porter, a liposome-based carrier that introduces macromolecular cargos into mitochondria via membrane fusion. In this study, we provide a demonstration of mitochondrial matrix delivery and the visualization of mitochondrial genes (mtDNA) in living cells using the MITO-Porter. We first prepared MITO-Porter containing encapsulated propidium iodide (PI), a fluorescent dye used to stain nucleic acids to detect mtDNA. We then confirmed the emission of red-fluorescence from PI by conjugation with mtDNA, when the carriers were incubated in the presence of isolated rat liver mitochondria. Finally, intracellular observation by confocal laser scanning microscopy clearly verified that the MITO-Porter delivered PI to the mitochondrial matrix. (C) 2010 Elsevier Inc. All rights reserved.
  • Hidetaka Akita, Kaoru Enoto, Tomoya Masuda, Hiroyuki Mizuguchi, Tomomi Tani, Hideyoshi Harashima
    MOLECULAR THERAPY 18 5 955 - 964 2010年05月 [査読有り][通常論文]
     
    It is previously reported that octaarginine (R8)-modified liposome (R8-Lip) was taken up via macropinocytosis, and subsequently delivered to the nuclear periphery. In the present study, we investigated the mechanism for the cytoplasmic transport of R8-Lips, comparing with that for adenovirus. Treatment with microtubule-disruption reagent (nocodazole) inhibited the transfection activity of plasmid DNA (pDNA)-encapsulating R8-Lip more extensively than that of adenovirus. The directional transport of R8-Lips along green fluorescent protein (GFP)-tagged microtubules was observed; however, the velocity was slower than those for adenovirus or endosomes that were devoid of R8-Lips. These directional motions were abrogated in R8-Lips by nocodazole treatment, whereas adenovirus continued to undergo random motion. This finding suggests that the nuclear access of R8-Lip predominantly involves microtubule-dependent transport, whereas an apparent diffusive motion is also operative in nuclear access of adenovirus. Furthermore, quantum dot-labeled pDNA underwent directional motion concomitantly with rhodamine-labeled lipid envelopes, indicating that the R8-Lips were subject to microtubule-dependent transport in the intact form. Dual particle tracking of carriers and endosomes revealed that R8-Lip was directionally transported, associated with endosomes, whereas this occurs after endosomal escape in adenovirus. Collectively, the findings reported herein indicate that vesicular transport is a key factor in the cytoplasmic transport of R8-Lips.
  • Tetsuya Suzuki, Hideyoshi Harashima, Hiroyuki Kamiya
    DNA REPAIR 9 5 542 - 550 2010年05月 [査読有り][通常論文]
     
    8-Oxo-7,8-dihydroguanine (8-oxo-Gua, also known as 8-hydroxyguanine) is a major base lesion that is generated by reactive oxygen species in both the DNA and nucleotide pool. The role of DNA glycosylases, which initiate base excision repair, in the mutagenic processes of 8-oxo-Gua in DNA and 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP, also known as 8-hydroxy-2'-deoxyguanosine 5'-triphosphate) were investigated using supF shuttle plasmids propagated in human cells. The DNA glycosylases, OGG1, MUTYH, NTH1, and NEIL1, in 293T cells were individually knocked-down by siRNAs and plasmid DNAs containing an 8-oxo-Gua:C/8-oxo-Gua:A pair, and 8-oxo-dGTP plus unmodified plasmid DNA were then introduced into the knocked-down cells. The knock-down of OGG1, MUTYH, NTH1, and NEIL1 resulted in a significant increase in G:C -> T:A transversions caused by the 8-oxo-Gua:C pair in the shuttle plasmid. The knock-down of MUTYH resulted in a reduction in A:T C:G transversions induced by 8-oxo-dGTP and the 8-oxo-Gua:A pair, but the knockdown of OGG1, NTH1, and NEIL1 had no effect on mutagenesis. These results indicate that all of the above DNA glycosylases suppress mutations caused by 8-oxo-Gua:C in DNA. In contrast, it appears that MUTYH enhances A:T -> C:G mutations caused by 8-oxo-dGTP. (C) 2010 Elsevier B.V. All rights reserved.
  • Hidetaka Akita, Kentaro Kogure, Rumiko Moriguchi, Yoshio Nakamura, Tomoko Higashi, Takashi Nakamura, Satoshi Serada, Minoru Fujimoto, Tetsuji Naka, Shiroh Futaki, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 143 3 311 - 317 2010年05月 [査読有り][通常論文]
     
    We previously developed octaarginine (R8)-modified lipid envelope-type nanoparticles for siRNA delivery (R8-MEND). Herein, we report on their ex vivo siRNA delivery to primary mouse bone marrow-derived dendritic cells (BMDCs) for potential use as a cancer vaccine. Quantitative imaging analysis of the intracellular trafficking of siRNA revealed that the dissociation process, as well as the rate of endosomal escape limits the siRNA efficiency of the prototype R8-MEND, prepared by the hydration method (R8-MENDhydo). Successful endosomal escape was achieved by using a pH-dependent fusogenic peptide (GALA) modified on a lipid mixture that was optimized for endosomal fusion. Furthermore, a modified protocol for the preparation of nanoparticles, mixing the 5iRNA/STR-R8 complex and small unilamellar vesicles (R8/GALA-MENDSUV), results in a more homogenous, smaller particle size, and results in a more efficient intracellular dissociation. Gene knockdown of the suppressor of cytokine signaling 1 (SOCS1), a negative-feedback regulator of the immune response in BMDCs resulted in an enhanced phosphorylation of STAT1, and the production of proinflammatory cytokines. Moreover, SOCS1-silenced BMDCs were more potent in suppressing tumor growth. Collectively, these results show that siRNA loaded in R8/GALA-MENDSUV efficiently suppresses endogenous gene expression and consequently enhances dendritic cell-based vaccine potency in vivo. (C) 2010 Elsevier B.V. All rights reserved.
  • Hiroyuki Kamiya, Ayaka Yamaguchi, Tetsuya Suzuki, Hideyoshi Harashima
    MUTATION RESEARCH-FUNDAMENTAL AND MOLECULAR MECHANISMS OF MUTAGENESIS 686 1-2 90 - 95 2010年04月 [査読有り][通常論文]
     
    The formation of 8-hydroxyguanine (8-OH-Gua, 7,8-dihydro-8-oxoguanine) in DNA and in the nucleotide pool results in G:C --> T:A and A:T --> C:G substitution mutations, respectively, due to the ability of 8-OH-Gua to pair with both C and A. In this study, shuttle plasmid DNAs containing 8-OH-Gua paired with C and A in the supF gene were transfected into human 293T cells, in which specialized DNA polymerases were knocked-down. The DNAs replicated in the cells were recovered and then introduced into an indicator strain of Escherichia coli. Mutation analysis indicated that the knock-downs of DNA polymerases eta and zeta by siRNAs enhanced the G:C --> T:A mutations caused by 8-OH-Gua:C. The 8-OH-Gua:C-induced mutation frequency was not further increased by double knock-downs of DNA polymerases eta and zeta suggesting that the two DNA polymerases work in the same pathway. In addition, the reduction of DNA polymerase eta slightly decreased the A:T --> C:G substitutions caused by 8-OH-Gua:A. These results suggest that DNA polymerases eta and zeta are involved in the bypass of 8-OH-Gua in human cells. (C) 2010 Elsevier B.V. All rights reserved.
  • Mina Tamaru, Hidetaka Akita, Takahiro Fujiwara, Kazuaki Kajimoto, Hideyoshi Harashima
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 394 3 587 - 592 2010年04月 [査読有り][通常論文]
     
    Leptin is an appetite regulatory hormone that is secreted into the blood circulation by adipose tissue, and functions in the central nerve system (i.e hypothalamus) by crossing the blood brain barrier (BBB) In the present study, we investigated the function of a leptin-derived peptide (Lep(70-89)) as a ligand for mouse brain-derived endothelial cells (MBEC4) Lep(70-89)-modified liposomes, prepared with a polyethyleneglycol (PEG) spacer (Lep(70-89)-PEG-LPs) exhibited a significantly higher cellular uptake than peptide-unmodified liposomes (PEG-LPs) Furthermore, cellular uptake was inhibited by amiloride, while no significant inhibitory effect was observed by the presence of chlorpromazine and filipin III, suggesting that macropinocytosis largely contributed to the cellular uptake of Lep(70-89)-PEG-LPs. Imaging studies revealed that Lep(70-89)-PEG-LPs were not colocalized with endosome/lysosomes, whereas neutral dextran (70 kDa) was predominantly colocalized with these compartments This indicates that Lep(70-89)-PEG-LPs are taken up via macropinocytosis and are subject to non-classical intracellular trafficking, resulting in the circumvention of lysosomal degradation in endothelial cells (C) 2010 Elsevier Inc All rights reserved
  • Hiroyuki Kamiya, Masayuki Uchiyama, Jingshu Piao, Yoshimichi Nakatsu, Teruhisa Tsuzuki, Hideyoshi Harashima
    INTERNATIONAL JOURNAL OF PHARMACEUTICS 387 1-2 180 - 183 2010年03月 [査読無し][通常論文]
     
    Targeted sequence alteration would be an attractive method in gene therapy and biotechnology. To achieve in vivo targeted sequence alteration, a tailed duplex DNA consisting of annealed 35mer and 794mer single-stranded DNAs was delivered by means of hydrodynamic tail vein injection into liver of transgenic mouse harboring a reporter gene (the rpsL gene) in its genome. The tailed DNA was designed for a conversion of ATC to AGC at codon 80 of the rpsL transgene. The anticipated T -> G sequence alteration was induced in the transgene in the liver with an efficiency of similar to 0.1%. These results demonstrate the significant potential of this method for applications in gene therapy and biotechnology. (C) 2010 Elsevier B.V. All rights reserved.
  • Diky Mudhakir, Erdal Tan, Hidetaka Akita, Hideyoshi Harashima
    THIRD NANOSCIENCE AND NANOTECHNOLOGY SYMPOSIUM 2010 - NNSB2010 1284 199 - + 2010年 [査読有り][通常論文]
     
    In the present study, the enhancement of siRNA delivery into cytosol from the IRQ-modified multi-envelope type nano-device (IRQ-MEND) was investigated. It is known that IRQ-MEND efficiently enters cells by clathrin-mediated endocytosis and caveolar endocytosis pathways, resulting in gene silencing. In the present study, we controlled the number of layers of IRQ-MEND encapsulating siRNA by using double membranous nano-device strategy. This vector design overcomes intracellular barriers such as endosomal membrane, resulting drastically enhance transgene expression. In addition, the contribution of uptake mechanism of IRQ-modified nano-carrier to transgene expression was studied. The use of hypertonic treatment inhibited gene silencing, but it was not suppressed by the addition of filipin. These results indicate that low pH is important to induce the transgene expression. In conclusion, delivery of cytosolic siRNA can be enhanced by the use of double-membranous nano-carrier system which utilizes a clathrin-mediated endocytosis pathway to induce gene silencing.
  • Hiroto Hatakeyama, Hidetaka Akita, Kentaro Kogure, Hideyoshi Harashima
    NANO/MICRO BIOTECHNOLOGY 119 197 - + 2010年 [査読有り][通常論文]
     
    In this review we introduce a new concept for developing a nonviral gene delivery system which we call "Programmed Packaging." Based on this concept, we succeeded in developing a multifunctional envelope-type nano device (MEND), which exerts high transfection activities equivalent to those of an adenovirus in a dividing cell. The use of MEND has been extended to in vivo applications. PEG/peptide/DOPE ternary conjugate (PPD)-MEND, a new in vivo gene delivery system for the targeting of tumor cells that dissociates surface-modified PEG in tumor tissue by matrix metalloproteinase (MMP) and exerts significant transfection activities, was developed. In parallel with the development of MEND, a quantitative gene delivery system, Confocal Image-assisted 3-dimensionally integrated quantification (CIDIQ), also was developed. This method identified the rate-limiting step of the nonviral gene delivery system by comparing it with adenoviral-mediated gene delivery. The results of this analysis provide a new direction for the development of rational nonviral gene delivery systems.
  • Yuma Yamada, Taku Nomura, Hideyoshi Harashima, Atsushi Yamashita, Ryo Katoono, Nobuhiko Yui
    Biological and Pharmaceutical Bulletin 33 7 1218 - 1222 2010年 [査読有り][通常論文]
     
    It has been believed that nuclear gene delivery is the most important process for gene expression, and various non-viral vectors are currently being developed with this assumption. However, some of our earlier studies revealed a surprising difference in transfection activity between viral and non-viral vectors: this difference is largely due to the result of the intranuclear disposition of DNA rather than its delivery to the nucleus (Hama S. et al. (2006), Quantitative comparison of intracellular trafficking and nuclear transcription between adenoviral and lipoplex systems. Mol. Ther., 13, 786-794). Here, we report on some direct evidence that demonstrates the importance of the release of intranuclear DNA on transfection activity. The data show that transfection activity can be substantially enhanced by integrating a multifunctional envelope-type nano device (MEND) and a biocleavable polyrotaxane (DMAE-SS-PRX) as an artificial condenser. Our integration system showed significantly higher transfection activity compared to conventional gene delivery system. Moreover, this system provides a strong support for our hypothesis that intranuclear DNA disposition plays a critical role in gene expression for non-viral vectors. © 2010 Pharmaceutical Society of Japan.
  • Hiroyuki Kamiya, Kazuya Satou, Mika Hori, Ayaka Yamaguchi, Hideyoshi Harashima
    Nucleic acids symposium series (2004) 53 223 - 224 2009年12月01日 [査読無し][通常論文]
     
    To assess the involvement of specialized DNA polymerases in the mutagenesis induced by an oxidized form of dGTP, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP, 7,8-dihydro-8-oxo-2'-deoxyguanosine 5'-triphosphate), shuttle plasmid DNA containing the supF gene and 8-OH-dGTP were introduced into human 293T cells in which specialized DNA polymerases were knocked-down by siRNAs. The knock-downs of DNA polymerases eta and zeta, and REV1 reduced the A:T-->C:G substitution mutations, suggesting that these DNA polymerases are involved in the misincorporation of 8-OH-dGTP opposite A. In contrast, the knock-down of DNA polymerase tau did not affect the mutations induced by 8-OH-dGTP. The decrease in the induced mutation frequency was more evident by double knock-downs of DNA pols eta plus zeta and REV1 plus DNA pol zeta (but not by that of DNA pol eta plus REV1), suggesting that REV1-DNA pol eta and DNA pol zeta work in different steps. These results indicate that specialized DNA polymerases are involved in the mutagenesis induced by 8-OH-dGTP.
  • Hiroyuki Kamiya, Mika Hori, Takao Arimori, Mutsuo Sekiguchi, Yuriko Yamagata, Hideyoshi Harashima
    DNA REPAIR 8 10 1250 - 1254 2009年10月 [査読無し][通常論文]
     
    The human NUDT5 protein catalyzes the hydrolysis of 8-hydroxy-dGDP. To examine its substrate specificity, four oxidized deoxyribonucleotides (2-hydroxy-dADP, 8-hydroxy-dADP, 5-formyl-dUDP, and 5-hydroxy-dCDP) were incubated with the NUDT5 protein. Interestingly, all of the nucleotides, except for 5-hydroxy-dCDP, were hydrolyzed with various efficiencies. The kinetic parameters indicated that 8-hydroxy-dADP was hydrolyzed as efficiently as 8-hydroxy-dGDP. The hydrolyzing activities for their triphosphate counterparts were quite weak. These results suggest that the NUDT5 protein eliminates various oxidized deoxyribonucleoside diphosphates from the nucleotide pool and prevents their toxic effects. (C) 2009 Elsevier B.V. All rights reserved.
  • Hiroto Hatakeyama, Erika Ito, Hidetaka Akita, Motoi Oishi, Yukio Nagasaki, Shiroh Futaki, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 139 2 127 - 132 2009年10月 [査読有り][通常論文]
     
    Previously, we developed a multifunctional envelope-type nano device (MEND) for efficient delivery of both pDNA and siRNA. Modification of a MEND with polyuethylene glycol, i.e., PEGylation, is a potential strategy for in vivo delivery of MENDs to tumor tissue. However, PEGylation also inhibits both uptake and endosomal escape of MENDs. To overcome these limitations, we developed a PEG-peptide-DOPE (PPD) that can be cleaved in a matrix metalloproteinase (MMP)-rich environment. In this study, to further improve the silencing activity of encapsulated siRNA, we modified the PPD-MEND with a pH-sensitive fusogenic GALA peptide (GALA/PPD-MEND). First, we determined the GALA and PPD content that would optimize the synergistic functions of GALA and PPD. The most efficient gene silencing activity was achieved when GALA and either conventional PEG-lipid or PPD were used to modify the MEND at a molar ratio of 1:1. In this case, the silencing activity was comparable to that achieved when using a MEND that had not been modified with PEG (unmodified MEND). Furthermore, in vivo topical administration revealed that optimized PPD/GALA-MENDa resulted in more efficient gene silencing compared with unmodified MENDs. Collectively, data demonstrate that introduction of both of a pH-sensitive fusogenic GALA peptide and PPD into the MEND facilitates nanopartide endosomal escape, thereby enhancing the efficiency of siRNA delivery and gene silencing. (C) 2009 Elsevier B.V. All rights reserved.
  • Hiroyuki Kamiya, Hitomi Goto, Hideyoshi Harashima
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 108 1 18 - 21 2009年07月 [査読無し][通常論文]
     
    DNA conformation may be an important factor affecting gene transcription. In this study, we examined how DNA sequences with unusual conformations affect transgene expression. A(30) and (CG)(15) sequences that can adopt the B' and Z conformations, respectively, were introduced into a beta-actin promoter. Luciferase plasmids containing the manipulated promoter were transfected into NIH3T3 cells by electroporation and were delivered into mouse livers with a hydrodynamics-based injection. Expression from plasmid with the (CG)(15) sequence was multiple times higher than expression from control plasmid DNA. The A(30) sequence also tended to enhance expression. These results suggest that non-B DNA sequences could improve transgene expression in cells. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.
  • Hiroyuki Kamiya, Satoki Fukunaga, Takashi Ohyama, Hideyoshi Harashima
    INTERNATIONAL JOURNAL OF PHARMACEUTICS 376 1-2 99 - 103 2009年07月 [査読無し][通常論文]
     
    The intranuclear disposition of plasmid DNA is highly important for transgene expression. The effects of a left-handedly curved sequence with high histone affinity on transgene expression were examined in COS-7 cells with two kinds of carriers (Lipofectamine Plus and TransIT-LT1). Three plasmids containing the curved sequence at different positions were transfected. The transgene expression was affected by the position of the left-handedly curved sequence, and the sequence at appropriate locations enhanced the expression from plasmid DNAs. However, the position effects on the expression differed from those obtained by electroporation of the same plasmid DNAs in a naked form. In addition, the degree of expression enhancement seemed to depend on the carriers. These results suggest that the left-handedly curved sequence with high histone affinity could increase the transgene expression from a plasmid delivered with carriers. (C) 2009 Elsevier B.V. All rights reserved.
  • Dai Kurihara, Hidetaka Akita, Asako Kudo, Tomoya Masuda, Shiroh Futaki, Hideyoshi Harashima
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 32 7 1303 - 1306 2009年07月 [査読有り][通常論文]
     
    The nuclear delivery process is a crucial barrier to successful gene delivery, especially in non-dividing cells. We previously proposed a novel strategy for the nuclear delivery of plasmid DNA (pDNA), in which the pDNA is encapsulated in lipid bilayers that had been modified with nucleus-targeting signals, including nuclear localizing signals derived from SV40 (NLS) or sugar units. In the present study, we report on an investigation of the effect of the topology of the liposome-modified NLS on its ability to bind to the isolated nucleus. NLS was directly attached to a liposome (NLS-Lip) by incorporating stearylated NLS (STR-NSL), or by modification with a polyethyleneglycol (PEG) spacer (NLS-PEG-Lip). NLS-unmodified liposomes (PEG-Lip) were used as a control. The liposomes, after labeling with 7-nitrobenz-2-oxa-1,3-diazole (NBD), were incubated with a cell homogenate derived from JAWS 11 cells, followed by isolation of the nuclear fraction by centrifugation. The PEG-Lip preparation showed negligible binding to the nucleus. In contrast, the binding of NLS-Lips to the nucleus gradually increased in a STR-NLS density-dependent manner. Interestingly, the binding of NLS-PEG-Lips to the nucleus is highly effective even at low density, suggesting that the presence of the PEG spacer is an important factor in improving the binding activity of NLS-modified liposomes to the nucleus. This information will be useful for the design of nucleus-targeting carriers.
  • Kazuya Satou, Mika Hori, Kazuaki Kawai, Hiroshi Kasai, Hideyoshi Harashima, Hiroyuki Kamiya
    DNA REPAIR 8 5 637 - 642 2009年05月 [査読無し][通常論文]
     
    The mutagenicity of an oxidized form of dGTP, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP). was examined using human 293T cells. Shuttle plasmid DNA containing the supF gene was first transfected into the cells, and then 8-OH-dGTP was introduced by means of osmotic pressure. The DNAs replicated in the cells were recovered and then transfected into Escherichia coli. 8-OH-dGTP induced A:T -> C:G substitution mutations in the cells. The knock-downs of DNA polymerases eta and zeta, and REV1 by siRNAs reduced the A:T -> C:G substitution mutations, suggesting that these DNA polymerases are involved in the misincorporation of 8-OH-dGTP opposite A in human cells. In contrast. the knock-down of DNA polymerase iota did not affect the 8-OH-dGTP-induced mutations. The decrease in the induced mutation frequency was more evident by double knock-downs of DNA pols eta plus zeta and REV1 plus DNA pol zeta (but not by that of DNA pol eta plus REV1), suggesting that REV1-DNA pol eta and DNA pol zeta work in different steps. These results indicate that specialized DNA polymerases are involved in the mutagenesis induced by the oxidized dGTP. (C) 2008 Elsevier B,V. All rights reserved.
  • Sharif M. Shaheen, Hidetaka Akita, Atsushi Yamashita, Ryo Katoono, Nobuhiko Yui, Vasudevanpillai Biju, Mitsuru Ishikawa, Hideyoshi Harashima
    MOLECULAR THERAPY 17 S330 - S330 2009年05月 [査読有り][通常論文]
  • Hidetaka Akita, Asako Kudo, Arisa Minoura, Masaya Yamaguti, Ikrarny A. Khalil, Rumiko Moriguchi, Tomoya Masuda, Radostin Danev, Kuniaki Nagayama, Kentaro Kogure, Hideyoshi Harashima
    BIOMATERIALS 30 15 2940 - 2949 2009年05月 [査読有り][通常論文]
     
    Efficient targeting of DNA to the nucleus is a prerequisite for effective gene therapy. The gene-delivery vehicle must penetrate through the plasma membrane, and the DNA-impermeable double-membraned nuclear envelope, and deposit its DNA cargo in a form ready for transcription. Here we introduce a concept for overcoming intracellular membrane barriers that involves step-wise membrane fusion. To achieve this, a nanotechnology was developed that creates a multi-layered nanoparticle, which we refer to as a Tetra-lamellar Multi-functional Envelope-type Nano Device (T-MEND). The critical structural elements of the T-MEND are a DNA-polycation condensed core coated with two nuclear membrane-fusogenic inner envelopes and two enclosome-fusogenic outer envelopes, which are shed in stepwise fashion. A double-lamellar membrane structure is required for nuclear delivery via the stepwise fusion of double layered nuclear membrane structure. Intracellular membrane fusions to endosomes and nuclear membranes were verified by spectral imaging of fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores that had been dually labeled on the liposome surface. Coating the core with the minimum number of nucleus-fusogenic lipid envelopes (i.e., 2) is essential to facilitate transcription. As a result, the T-MEND achieves dramatic levels of transgene expression in non-dividing cells. (C) 2009 Elsevier Ltd. All rights reserved.
  • Yu Sakurai, Hiroto Hatakeyama, Hidetaka Akita, Motoi Oishi, Yukio Nagasaki, Shiro Futaki, Hideyoshi Harashima
    MOLECULAR THERAPY 17 Suppl. 1 S388 - S389 2009年05月 [査読有り][通常論文]
  • Hiroto Hatakeyama, Erika Itho, Hidetaka Akita, Motoi Oihi, Yukio Nagasaki, Shiroh Futaki, Hideyoshi Harashima
    MOLECULAR THERAPY 17 S388 - S388 2009年05月 [査読有り][通常論文]
  • Joraku A, Homhuan A, Kawai K, Yamamoto T, Miyazaki J, Kogure K, Yano I, Harashima H, Akaza H
    BJU international 103 5 686 - 693 2009年03月 [査読有り][通常論文]
  • Ayman El-Sayed, Shiroh Futaki, Hideyoshi Harashima
    AAPS JOURNAL 11 1 13 - 22 2009年03月 [査読有り][通常論文]
     
    Arginine-rich cell-penetrating peptides (AR-CPPs) are very promising tools for the delivery of therapeutic macromolecules such as peptides, proteins, and nucleic acids. These peptides allow efficient internalization of the linked cargos intracellularly through the endocytic pathway. However, when linked to bulky cargos, entrapment in the endocytic vesicles is a major limitation to the application of these peptides in cytosolic delivery. Attachment of a compatible endosomal escape device is, therefore, necessary to allow cytosolic delivery of the peptide-attached cargo. This review presents different endosomal escape devices currently in application in combination with AR-CPPs. Applications of fusogenic lipids, membrane-disruptive peptides, membrane-disruptive polymers, lysosomotropic agents, and photochemical internalization to enhance the cytosolic delivery of AR-CPPs-attached cargos are presented. The properties of each system and its mechanism of action for the enhancement of endosomal escape are discussed, together with its applications for the delivery of different macromolecules in vitro and, if applicable, in vivo.
  • Rumiko Moriguchi, Kentaro Kogure, Hideyoshi Harashima
    INTERNATIONAL JOURNAL OF PHARMACEUTICS 363 1-2 192 - 198 2008年11月 [査読有り][通常論文]
     
    Transfection efficiencies using LipofectAMINE varied by more than three orders of magnitude depending on the concentrations of lipid and plasmid DNA (pDNA) used to prepare the lipoplexes. When lipoplexes were formed at lower concentrations a striking positive but non-linear relationship was found between dose and transfection efficiency, while at higher (i.e., normally used) concentrations a linear relationship was maintained. To determine the contribution of intracellular pharmacokinetics (PK) and pharmacodynamics (PD) to the observed nonlinearity, we quantified pDNA in whole cells and nuclei by real-time PCR and compared the results with the transfection efficiencies. There was no significant difference in the efficiency of intracellular PK; however, a remarkable difference was observed in the efficiency of PD. Analysis of individual cells by confocal laser scanning microscopy (CLSM) revealed that the amount of nuclear-delivered pDNA was higher for lipoplexes prepared at the normal concentration (NCL) compared to those of lipoplexes prepared at low concentration (LCL). Moreover, the size of the NCL was larger than that of the LCL. Both the size of the lipoplex particle and the dose appear to contribute to the non-linear efficiency of PD. These results emphasize the need to control not only intracellular PK, but also PD for the rational development of non-viral gene delivery systems. (c) 2008 Elsevier B.V. All rights reserved.
  • Kenichi Niikura, Shota Sekiguchi, Takashi Nishio, Tomoya Masuda, Hidetaka Akita, Yasutaka Matsuo, Kentaro Kogure, Hideyoshi Harashima, Kuniharu Ijiro
    CHEMBIOCHEM 9 16 2623 - 2627 2008年11月 [査読有り][通常論文]
  • Atsushi Yamashita, Daizo Kanda, Ryo Katoono, Nobuhiko Yui, Tooru Ooya, Atsushi Maruyama, Hidetaka Akita, Kentaro Kogure, Hideyoshi Harashima
    J Control Release 131 2 137 - 144 2008年10月 [査読有り][通常論文]
     
    A novel strategy for gene delivery using biocleavable polyrotaxanes, in which dimethylaminoethyl-modified alpha-cyclodextrins (DMAE-alpha-CDs) are threaded onto a poly(ethylene glycol) (PEG) chain capped with benzyloxycarbonyl-L-tyrosine via disulfide linkages (DMAE-SS-PRX), involves the formation of a stable polyion complex (polyplex) against a counter polyanion and the intracellular plasmid DNA (pDNA) release from the polyplex accompanied by the supramolecular dissociation of DMAE-SS-PRXs. In this study, we prepared biocleavable polyrotaxanes with different numbers of threading alpha-CD and amino (DMAE) groups to enhance the transfection activity of DMAE-SS-PRXs. 29DMAE-alpha18-SS-PRX, in which the numbers of alpha-CD molecules and amino groups were 18 and 29 respectively, exhibited a high transfection activity compared with other PRXs. The transfection activity of DMAE-SS-PRXs seems to be related to the efficacy of pDNA release from those polyplexes, which was controlled by the number of alpha-CD and/or amino groups in the polyrotaxane carrier. Most of the DMAE-SS-PRX polyplexes released the pDNA only in the presence of both 10 mM DTT and of the counter-polyanion, as expected, e
  • Yuma Yamada, Hideyoshi Harashima
    ADVANCED DRUG DELIVERY REVIEWS 60 13-14 1439 - 1462 2008年10月 [査読有り][通常論文]
     
    Mitochondrial dysfunction has been implicated in a variety of human disorders-the so-called mitochondrial diseases. Therefore, the organelle is a promising therapeutic drug target. In this review, we describe the key role of mitochondria in living cells, a number of mitochondrial drug delivery systems and mitochondria-targeted therapeutic strategies. In particular, we discuss mitochondrial delivery of macro molecules, such as proteins and nucleic acids. The discussion of protein delivery is limited primarily to the mitochondrial import machinery. In the section on mitochondrial gene delivery and therapy, we discuss mitochondrial diseases caused by mutations in mitochondrial DNA, several gene delivery strategies and approaches to mitochondrial gene therapy. This review also summarizes our current efforts regarding liposome-based delivery system including use of a multifunctional envelope-type nano-device (MEND) and mitochondrial liposome-based delivery as anti-cancer therapies. Furthermore, we introduce the novel MITO-Porter-a liposome-based mitochondrial delivery system that functions using a membrane-fusion mechanism. (C) 2008 Elsevier B.V. All rights reserved.
  • Hiroyuki Kamiya, Masayuki Uchiyama, Yoshimichi Nakatsu, Teruhisa Tsuzuki, Hideyoshi Harashima
    JOURNAL OF BIOCHEMISTRY 144 4 431 - 436 2008年10月 [査読無し][通常論文]
     
    The correction of an inactivated hygromycin resistance and enhanced green fluorescent protein (Hyg-EGFP) fusion gene by a several hundred-base single-stranded (ss) DNA fragment has been reported. In this study, the effectiveness of this type of gene correction was examined for various positions in the rpsL gene. Sense and anti-sense ssDNA fragments were prepared, and the gene correction efficiencies were determined by co-introduction of the target plasmid containing the gene with the ssDNA fragments. The gene correction efficiency varied (0.8-9.3%), depending on target positions and sense/anti-sense strands. Sense ssDNA fragments corrected the target gene with equal or higher efficiencies as compared to their anti-sense counterparts. The target positions corrected with high efficiency by the sense fragments also tended to be corrected efficiently by the anti-sense fragments. These results suggest that the sense ssDNA fragments are useful for the correction of mutated genes. The variation in the correction efficiency may depend on the sequence of the target position in double-stranded DNA.
  • Atsushi Yamashita, Daizo Kanda, Ryo Katoono, Nobuhiko Yui, Tooru Ooya, Atsushi Maruyama, Hidetaka Akita, Kentaro Kogure, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 131 2 137 - 144 2008年10月 [査読有り][通常論文]
     
    A novel strategy for gene delivery using biocleavable polyrotaxanes, in which dimethylaminoethyl-modified alpha-cyclodextrins (DMAE-alpha-CDs) are threaded onto a poly(ethylene glycol) (PEG) chain capped with benzyloxycarbonyl-L-tyrosine via disulfide linkages (DMAE-SS-PRX), involves the formation of a stable polyion complex (polyplex) against a counter polyanion and the intracellular plasmid DNA (pDNA) release from the polyplex accompanied by the supramolecular dissociation of DMAE-SS-PRXs. In this study, we prepared biocleavable polyrotaxanes with different numbers of threading alpha-CD and amino (DMAE) groups to enhance the transfection activity of DMAE-SS-PRXs. 29DMAE-alpha 18-SS-PRX, in which the numbers of alpha-CD molecules and amino groups were 18 and 29 respectively, exhibited a high transfection activity compared with other PRXs. The transfection activity of DMAE-SS-PRXs seems to be related to the efficacy of pDNA release from those polyplexes, which was controlled by the number of alpha-CD and/or amino groups in the polyrotaxane carrier. Most of the DMAE-SS-PRX polyplexes released the pDNA only in the presence of both 10 mM DTT and of the counter-polyanion, as expected, except for 14DMAE-alpha 18-SS-PRX, which released pDNA in the absence of dextran sulfate once the DTT had been added to the polyplex solution. The transfection activity of 14DMAE-alpha 18-SS-PRX was significantly lower than that of 29DMAE-alpha 18-SS-PRX regardless of the above features. Confocal laser scanning microscopic (CLSM) observation suggested that the specific result for 14DMAE-alpha 18-SS-PRX might be due to a premature release of pDNA from the most dissociative 14DMAE-alpha 18-SS-PRX polyplex in the cytosol. Therefore, transfection activity seems to be related to an appropriate timing of pDNA release. (C) 2008 Elsevier B.V. All rights reserved.
  • Takashi Nakamura, Rumiko Moriguchi, Kentaro Kogure, Nilabh Shastri, Hideyoshi Harashima
    MOLECULAR THERAPY 16 8 1507 - 1514 2008年08月 [査読有り][通常論文]
     
    Recently, much attention has been paid to cell-penetrating peptides (CPPs) as an antigen-delivery tool for presentation through the major histocompatibility complex class I (MHC-I) pathway. However, escape of CPPs from the endosome is inefficient and therefore a bottleneck for antigen delivery. Previously, we showed the importance of topological control of octaarginine (R8) peptides on the liposome surface for regulating cellular uptake as well as intracellular trafficking, especially endosomal escape. In this study, we hypothesized that efficient MHC-I presentation could be achieved by controlled intracellular trafficking of antigen encapsulated in R8-modified liposomes (R8-Lip). The mechanism of uptake of both R8-Lip and cationic liposomes was shown to be by macropinocytosis in dendritic cells. However, confocal laser scanning microscopy (CLSM) revealed that R8-Lip are able to release significantly more antigen to the cytosol than are cationic liposomes. Processing of the antigens delivered by R8-Lip was shown to be proteasome-dependent, which is consistent with selective antigen presentation by R8-Lip via MHC-I. According to antigen-presentation analysis, R8-Lip can induce significantly higher MHC-I presentation at lower doses than either soluble ovalbumin ( OVA) or OVA in pH-sensitive or cationic liposomes. Moreover, R8-Lip showed an efficient antitumor effect in vivo. Therefore, R8-Lip is a promising new carrier for MHC-I-specific antigen presentation.
  • Hidetaka Akita, Hideyoshi Harashima
    EXPERT OPINION ON DRUG DELIVERY 5 8 847 - 859 2008年08月 [査読有り][通常論文]
     
    Background: Low transfection efficiency is an obstacle to the clinical use of non-viral gene vectors. Effective non-viral vectors require the ability to control intracellular trafficking of gene vectors for the delivery of exogenous DNA to the nucleus. Objective: To overcome multiple intracellular barriers, various types of devices must be integrated into one nano-particle so that each device performs its function at the appropriate location at the desired time. Such a strategy requires an understanding, based on quantitative information, of the rate-limiting processes that hinder intracellular trafficking. Methods: In this review, advancements in the development of multifunctional envelope-type nano-devices (MEND) are discussed. In particular, a novel method to quantitatively evaluate the rate-limiting steps in intracellular trafficking, based on a comparison of viral and non-viral gene-delivery systems, is described. Conclusion: MENDs are useful to integrate various kinds of devices to overcome intracellular barriers into one particle. Comparison of intracellular trafficking between adenoviruses and non-viral vectors indicates that a postnuclear delivery process is an important rate-limiting step for efficient transfection with non-viral vectors.
  • AbdulAziz Anas, Hidetaka Akita, Hideyoshi Harashima, Tamitake Itoh, Mitsuru Ishikawa, Vasudevanpillai Biju
    JOURNAL OF PHYSICAL CHEMISTRY B 112 32 10005 - 10011 2008年08月 [査読有り][通常論文]
     
    Strand breakages and nucleobase damages in plasmid DNA (pDNA) by CdSe-ZnS quantum dots (QDs) are investigated under different conditions of photoactivation. Here, streptavidin functionalized CdSe-ZnS QDs are conjugated to biotinylated pDNA, and photosensitized strand breakages and nucleobase damages in the conjugates are investigated using atomic force microscopy (AFM) imaging, gel electrophoreses analyses, and assay of reactive oxygen intermediates (ROI). Also, reactions of photoactivated pDNA-QD conjugates with base excision repair enzymes such as formamidopyrimidine glycosylase (Fpg) and endonuclease III (Endo 111) show damages of purine and pyrimidine bases. The base excision repair enzymes recognize and remove the damaged bases. The base excision reactions of photoactivated pDNA-QD conjugates resulted in pDNA strand breakages, which appeared as sheared bands in agarose gel images. On the basis of AFM imaging, reactions of Fpg and Endo III with damaged pDNA, ROI assay, and literature reports, we attribute the breakage and damage of pDNA to its reactions with ROI. The production of ROI by photoactivated QDs is confirmed by nitroblue tetrazolium (NBT) assay. The current work shows that photoactivation of QD-conjugated nucleic acids for an extended period of time is not favorable for their stability. On the other hand, photoinduced production of ROI by QDs is an emerging research area with potential applications in the photodynamic therapy of cancer. In this regard, photosensitized damage of pDNA observed in the current work shows possibilities of QDs in nucleus-targeted photodynamic therapy.
  • Hiroyuki Tsuchiya, Masayuki Uchiyarna, Kazuhiro Hara, Yoshimichi Nakatsu, Teruhisa Tsuzuki, Hideo Inoue, Hideyoshi Harashima, Hiroyuki Kamiya
    BIOCHEMISTRY 47 33 8754 - 8759 2008年08月 [査読無し][通常論文]
     
    A 606-base single-stranded (ss) DNA fragment, prepared by restriction enzyme digestion of ss phagemid DNA, corrects a hygromycin resistance and enhanced green fluorescent protein (Hyg-EGFP) fusion gene more efficiently than a PCR fragment, which is the conventional type of DNA fragment used in gene correction. Here, a tailed duplex, obtained by annealing an oligonucleotide to the ss DNA fragment, was used in the correction. The tailed duplex may be a good substrate for the RAD51 protein, an important enzyme in homologous recombination, which could be the gene correction pathway. The annealing of the oligonucleotides enhanced the correction efficiency of the Hyg-EGFP gene, especially when annealed in the 3'-region of the ss DNA fragment. Both the length and backbone structure of the oligonucleotides affected the gene correction efficiency. This type of gene correction device was also effective for another target gene, the rpsL gene. The results obtained in this study indicate that tailed duplex DNA fragments are effective nucleic acids for gene correction.
  • Hiroshi Kuramoto, Yeon-Su Park, Noritada Kaji, Manabu Tokeshi, Kentaro Kogure, Yasuo Shinohara, Hideyoshi Harashima, Yoshinobu Baba
    ANALYTICAL AND BIOANALYTICAL CHEMISTRY 391 8 2729 - 2733 2008年08月 [査読無し][通常論文]
     
    Microfluidic devices may be highly beneficial to the rapid fabrication of small quantities of various nonviral vectors with different functionalities, which is indispensable for effective order-made gene therapy. We adapted a microfluidic chip-based approach for fabricating small quantities of nonviral vectors in a short time in preparation for order-made gene therapy applications. This approach permitted us to fabricate multifunctional envelope-type nanodevices (MENDs), composed of a compacted (or condensed) DNA core and a lipid bilayer membrane shell, which are considered as promising nonviral vectors for gene therapy applications. The on-chip fabrication of the MEND was very simple, rapid, convenient, and cost-effective compared with conventional methods. The size of the MEND showed strong dependence on the concentration and flow rate of the reaction precursors and could be controlled to be much smaller than that achievable by conventional methods. This, together with abovementioned merits, makes our microfluidic chip-based approach very attractive for the fabrication of MENDs for effective application to order-made gene therapy.
  • Kentaro Sasaki, Kentaro Kogure, Shinji Chaki, Yoshio Nakamura, Rumiko Moriguchi, Hirofumi Hamada, Radostin Danev, Kuniaki Nagayama, Shiroh Futaki, Hideyoshi Harashima
    ANALYTICAL AND BIOANALYTICAL CHEMISTRY 391 8 2717 - 2727 2008年08月 [査読有り][通常論文]
     
    We previously reported that transferrin (Tf)-modified liposomes (Tf-L) additionally modified with a cholesterylated pH-sensitive fusogenic peptide (Chol-GALA) can release an encapsulated aqueous phase marker to cytosol via endosomal membrane fusion. However, further obstacles need to be overcome to bring the Tf-L to the level of a viral-like gene delivery system. In this study, we developed a novel packaging method to encapsulate condensed plasmid DNA into PEgylated Tf-L (Tf-PEG-L) to form a core-shell-type nanoparticle. The most difficult challenge was to provide a mechanism of escape for the condensed core from endosome to cytosol in the presence of polyethylene glycol (PEG). We hypothesized that a membrane-introduced Chol-GALA and a PEgylated GALA would interact synergistically to induce membrane fusion between liposome and endosome. By simultaneously incorporating Chol-GALA into the membrane of Tf-PEG-L and GALA at tips of PEG chains, a condensed core was released into cytosol, and transfection acitivty increased 100-fold. We concluded that topological control was responsible for the synergistic effect of GALA derivatives introduced on Tf-PEG-L.
  • Ayman El-Sayed, Ikramy A. Khalil, Kentaro Kogure, Shiroh Futaki, Hideyoshi Harashima
    JOURNAL OF BIOLOGICAL CHEMISTRY 283 34 23450 - 23461 2008年08月 [査読有り][通常論文]
     
    The present study examines the role of surface modification with an octaarginine peptide (R8) in liposomal escape from endocytic vesicles, using octalysine (K8) as a control cationic peptide; the mechanism of endosomal escape of liposomes was also investigated. Gene expression of condensed plasmid DNA encapsulated in R8-modified nanoparticles was more than 1 order of magnitude higher than that of K8-modified nanoparticles, and 2 orders of magnitude higher than gene expression using unmodified nanoparticles. The difference in gene expression could not be attributed to differences in uptake, as R8- and K8-modified liposomes were taken up primarily via macropinocytosis with comparable efficiency. The extent of R8-nanoparticle escape to the cytosol was double that of K8-nanoparticles. Suppression of endosome acidification inhibited R8-nanoparticle endosomal escape, but enhanced that of K8-nanoparticles. Using spectral imaging in live cells, we showed that R8- and K8-liposomes escaped from endocytic vesicles via fusion between the liposomes and the endosomal membrane. R8- liposomes fused efficiently at both acidic and neutral pH, whereas K8- liposomes fused only at neutral pH. Similar behavior was observed during in vitro lipid mixing and calcein-release experiments. Co-incubation of cells with distinctly labeled K8- and R8-modified nanoparticles confirmed a common uptake pathway and different rates of endosomal escape particularly at longer time intervals. Therefore, it was concluded that R8 on the liposome surface stimulates efficient escape from endocytic vesicles via a fusion mechanism that works at both neutral and acidic pH; in contrast, K8 mediates escape mainly at neutral pH.
  • Atthachai Homhuan, Hideyoshi Harashima, Ikuya Yano
    SCIENCEASIA 34 2 179 - 185 2008年06月 [査読有り][通常論文]
     
    The cell wall derived from Mycobacterium bovis bacillus Calmette-Guerin (BCG) is a potent immunopotentiator and has recently been suggested as an alterative treatment for in situ bladder carcinoma. In contrast to the live BCG, the loss of infectivity and the negatively charged nature of BCG cell wall physically inhibit its attachment and subsequent internalization to the urothelial bladder cells. As part of our research involving the delivery of macromolecules to target cells, we developed cationic liposomes that anchor arginine octamers on the liposome surface. In this study, we used cationic liposomes as a delivery tool to facilitate the attachment and internalization of the BCG cell wall. Using confocal scanning microscopy, we verified that cationic liposomes incorporated with BCG cell wall could attach to the cellular membrane of murine bladder tumour (MBT-2) cells and become internalized. Cationic liposomes containing BCG cell wall were taken up by MBT-2 cells mainly via clathrin-mediated endocytosis. These results would be useful to understand the mechanism of action of BCG cell wall against bladder tumour cells as well as to develop an immunotherapeutic agent for clinical use.
  • Krarny A. Khalil, Kentaro Kogure, Shiroh Futaki, Hideyoshi Harashima
    INTERNATIONAL JOURNAL OF PHARMACEUTICS 354 1-2 39 - 48 2008年04月 [査読有り][通常論文]
     
    Gene therapy is a promising new approach for treating a variety of genetic and acquired diseases. While viral vectors are highly efficient for gene therapy, their use is associated with high toxicity and immunogenicity. Synthetic or nonviral vectors are attractive alternatives to viral vectors because of their low immunogenicity and low acute toxicity. The main disadvantage of the nonviral vectors is the low transfection efficiency compared to viral vectors. Novel functional devices to enhance the transfection activities of nonviral vectors are needed. In this review, we discuss the modification of liposomal drug carriers with a novel functional device, the octaarginine (R8) peptide, for drug and gene delivery. Decoration of liposomes with R8 enhanced their cellular uptake. In addition, by optimizing the density of the peptide as well as its topology, the liposomes could be internalized via clathrin-independent pathways, which improved the intracellular trafficking through avoiding lysosomal degradation. A special emphasis is given to the need for optimizing the conditions of using the peptide to not only enhance the cellular uptake but also to improve the intracellular trafficking of its cargos. In addition, the use of R8-modified liposomes and nano-particles in gene delivery is discussed. (C) 2007 Elsevier B.V. All rights reserved.
  • Kentaro Kogure, Hidetaka Akita, Yuma Yamada, Hideyoshi Harashima
    ADVANCED DRUG DELIVERY REVIEWS 60 4-5 559 - 571 2008年03月 [査読有り][通常論文]
     
    In this review, we describe a key role of octaarginine (R8) in developing our new concept of "Programmed Packaging", by which we succeeded in creating a multifunctional envelope-type nano device (MEND) as a non-viral gene-delivery system. This concept can be applied not only to nuclear targeting of plasmid DNA (pDNA) but also to cytosolic delivery of functional nucleic acids such as oligonucleotides or siRNA, This concept has been extended to other organelles such as mitochondria as a foundation for innovative nanomedicine. Finally, we discuss the rate-limiting step in gene delivery by comparing non-viral and viral gene delivery systems, which clearly indicates the importance of nuclear disposition of pDNA for efficient transfection. (C) 2007 Elsevier B.V. All rights reserved.
  • Katsuhiko Hidaka, Masami Yamada, Hiroyuki Kamiya, Chikahide Masutani, Hideyoshi Harashima, Fumio Hanaoka, Takehiko Nohmi
    DNA repair 7 3 497 - 506 2008年03月01日 [査読有り][通常論文]
     
    Aberrant oxidation is a property of many tumor cells. Oxidation of DNA precursors, i.e., deoxynucleotide triphosphates (dNTPs), as well as DNA is a major cause of genome instability. Here, we report that human DNA polymerase eta (h Poleta) incorporates oxidized dNTPs, i.e., 2-hydroxy-2'-deoxyadenosine 5'-triphosphate (2-OH-dATP) and 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP), into DNA in an erroneous and efficient manner, thereby inducing various types of mutations during in vitro gap-filling DNA synthesis. When 2-OH-dATP was present at a concentration equal to those of the four normal dNTPs in the reaction mixture, DNA synthesis by h Poleta enhanced the frequency of G-to-T transversions eight-fold higher than that of the transversions in control where only the normal dNTPs were present. When 8-OH-dGTP was present at an equimolar concentration to the normal dNTPs, it enhanced the frequency of A-to-C transversions 17-fold higher than the control. It also increased the frequency of C-to-A transversions about two-fold. These results suggest that h Poleta incorporates 2-OH-dATP opposite template G and incorporates 8-OH-dGTP opposite template A and slightly opposite template C during DNA synthesis. Besides base substitutions, h Poleta enhanced the frequency of single-base frameshifts and deletions with the size of more than 100 base pairs when 8-OH-dGTP was present in the reaction mixture. Since h Poleta is present in replication foci even without exogenous DNA damage, we suggest that h Poleta may be involved in induction of various types of mutations through the erroneous and efficient incorporation of oxidized dNTPs into DNA in human cells.
  • Kentaro Kogure, Hidetaka Akita, Hiroyuki Kamiya, Hideyoshi Harashima
    Modern Biopharmaceuticals: Design, Development and Optimization 4 1521 - 1536 2008年02月05日 [査読無し][通常論文]
     
    Dramatic advances have occurred in the field of drug delivery systems (DDS) during the past 10 years. Tumor targeting with long-circulating liposomes that contain antitumor agents (passive targeting) has been successfully demonstrated in clinical trials. Active targeting with ligands specific to cell surface receptors has also been developed. In this chapter, we will focus on the next generation of DDS "Programmed Packaging", in which intracellular trafficking and the disposition of DNA for gene therapy are controlled. © 2005 WILEY-VCH Verlag GmbH & Co. KGaA.
  • Yuma Yamada, Hidetaka Akita, Hiroyuki Kamiya, Kentaro Kogure, Takenori Yamamoto, Yasuo Shinohara, Kikuji Yamashita, Hideo Kobayashi, Hiroshi Kikuchi, Hideyoshi Harashima
    Biochimica et biophysica acta 1778 2 423 - 32 2008年02月 
    Mitochondria are the principal producers of energy in higher cells. Mitochondrial dysfunction is implicated in a variety of human diseases, including cancer and neurodegenerative disorders. Effective medical therapies for such diseases will ultimately require targeted delivery of therapeutic proteins or nucleic acids to the mitochondria, which will be achieved through innovations in the nanotechnology of intracellular trafficking. Here we describe a liposome-based carrier that delivers its macromolecular cargo to the mitochondrial interior via membrane fusion. These liposome particles, which we call MITO-Porters, carry octaarginine surface modifications to stimulate their entry into cells as intact vesicles (via macropinocytosis). We identified lipid compositions for the MITO-Porter which promote both its fusion with the mitochondrial membrane and the release of its cargo to the intra-mitochondrial compartment in living cells. Thus, the MITO-Porter holds promise as an efficacious system for the delivery of both large and small therapeutic molecules into mitochondria.
  • Tomoya Masuda, Hidetaka Akita, Takashi Nishio, Kenichi Niikura, Kentaro Kogure, Kuniharu Ijiro, Hideyoshi Harashima
    BIOMATERIALS 29 6 709 - 723 2008年02月 [査読有り][通常論文]
     
    Efficient nuclear gene delivery is essential for successful gene therapy. This study developed a novel system that mimics the mechanism of nuclear entry of adenovirus (Ad) by means of a Multifunctional Envelope-type Nano Device (MEND). In this system, plasmid DNA (pDNA) was condensed with polycation, followed by encapsulation in a lipid membrane. To target MEND to the nuclear pore complex (NPC), sugar served as a NPC-mediated nuclear targeting device was modified on the surface of the lipid envelope. This was accomplished via synthesis of a sugar-cholesterol conjugate. After binding of the MEND to the NPC, the pDNA core was transferred into the nucleus in conjunction with a breakdown of the lipid envelope. Sugar-modified MEND showed higher transfection efficiency compared with unmodified MEND, in non-dividing and dividing cells. Confocal microscopy confirmed that nuclear transfer of pDNA was improved by sugar modification of MEND. Furthermore, destabilization of the lipid envelope significantly enhanced transfection activity: therefore, nuclear-delivery efficiency was closely related to lipid envelope stability. Moreover, quantitative evaluation of cellular uptake and nuclear transfer processes by real-time PCR confirmed that the surface sugars affected nuclear transfer, but not cellular uptake. In summary, a novel system for the nuclear delivery of pDNA was successfully developed by using a sugar-modified MEND and by optimizing the lipid envelope stability. (c) 2007 Elsevier Ltd. All rights reserved.
  • Hidetaka Akita, Hideyoshi Harashima
    GENE THERAPY FOR RENAL DISEASES AND TRANSPLANTATION 159 13 - 29 2008年 [査読有り][通常論文]
     
    Gene and RNA interference therapies are promising cures for intractable renal failure. However, low delivery efficiency of the therapeutic nucleic acid into the nucleus of the target cell is a significant obstacle in the clinical application of nonviral gene therapy. Various mechanical techniques (hydrodynamic injection, electroporation and ultrasound-microbubble) and topically applied preparations (HVJ liposome and cationic liposome/polymer), which introduce transgenes into specific renal compartments depending on the administration route, have been reported. Additional improvements in renal application of nonviral gene vectors must address the important issue of how to control intracellular trafficking. Therefore, novel vectors based on the 'programmed packaging' concept are desirable in which all functional devices are integrated into a single system so that each function occurs at the appropriate time and correct place. In parallel with development of the carrier, quantitative evaluation of intracellular trafficking is essential to determine the efficacy of the modified devices in the cellular environment. In particular, comparison of the intracellular trafficking of the engineered devices with that of viruses (i.e. adenovirus) is useful in identifying the rate-limiting intracellular processes of the vectors during development. Copyright (c) 2008 S. Karger AG, Basel.
  • Ayman El-Sayed, Ikramy A. Khalil, Kentaro Kogure, Shiroh Futaki, Hideyoshi Harashima
    YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 128 111 - 112 2008年 [査読有り][通常論文]
  • Yamashita A, Kanda D, Katoono R, Yui N, Ooya T, Maruyama A, Akita H, Kogure K, Harashima H
    8th World Biomaterials Congress 2008 4 2008年 [査読有り][通常論文]
  • Tetsuya Suzuki, Kazuo Yamamoto, Hideyoshi Harashima, Hiroyuki Kamiya
    Nucleic acids symposium series (2004) 51 - 52 2007年12月01日 [査読無し][通常論文]
     
    To examine whether base excision repair suppresses mutations induced by oxidized deoxyribonucleotide 5'-triphosphates in the nucleotide pool, 8-hydroxy-dGTP (8-OH-dGTP) was introduced into Escherichia coli strains deficient in endonucleases III (Nth) and VIII (Nei), and MutY, and mutations in the chromosomal rpoB gene were analyzed. The spontaneous rpoB mutant frequency was also examined in mutT/nth and mutT/nei strains, to assess the influence on the mutations induced by the endogenous 8-OH-dGTP accumulated in the mutT mutant. Exogenous 8-OH-dGTP increased the rpoB mutant frequency more efficiently in the nth strain than that in the wild-type strain. The spontaneous mutant frequency in the mutT/nth strain was 1.8-fold higher than that in the mutT strain. These results suggest that E. coli endonuclease III also acts as a defense against the mutations caused by 8-OH-dGTP in the nucleotide pool.
  • Kentaro Kogure, Hidetaka Akita, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 122 3 246 - 251 2007年10月 [査読有り][通常論文]
     
    In this review, a new concept called "Programmed Packaging" is proposed for developing non-viral gene delivery systems. A Multifunctional Envelope-type Nano Device (MEND) was developed based on this concept, which can enter into cells via macropinocytosis and facilitate transfection activities as efficiently as an adenovirus. Intracellular trafficking of MEND was optimized in order to prevent lysosomal degradation and to enhance nuclear translocation. A quantitative analytical method (CIDIQ) was also established to evaluate each step in intracellular trafficking of genes as well as carriers. A comparative study between Lipofectamin and an adenovirus showed that intra-nuclear disposition, which includes the transcriptional process, translational process, etc., can be the limiting step for Lipofectamin PLUS. These studies indicated the importance of controlling intra-nuclear disposition in the development of an efficient non-viral gene delivery system. (c) 2007 Elsevier B.V. All rights reserved.
  • Hiroto Hatakeyama, Hidetaka Akita, Emi Ishida, Koichi Hashimoto, Hideo Kobayashi, Takanori Aoki, Junko Yasuda, Kenichi Obata, Hiroshi Kikuchi, Tatsuhiro Ishida, Hiroshi Kiwada, Hideyoshi Harashima
    INTERNATIONAL JOURNAL OF PHARMACEUTICS 342 1-2 194 - 200 2007年09月 [査読有り][通常論文]
     
    Immunoliposomes are potent carriers for targeting of therapeutic drugs to specific cells. Membrane type-1 matrix metalloproteinase(MT1-MMP), which plays an important role in angiogenesis, is expressed on angiogenic endothelium cells as well as tumor cells. Then, the MT1-MMP might be useful as a target molecule for tumor and neovascularity. In the present study, we addressed a utility of antibodies against the MT1-MMP as a targeting ligand of liposomal anticancer drug. Fab' fragments of antibody against the MT1-MMP were modified at distal end of polyethylene glycol (PEG) of doxorubicin (DXR)-encapsulating liposomes, DXR-sterically stabilized immunoliposomes (DXR-SIL[anti-MT1-MMP(Fab ')]). Modification with the antibody significantly enhanced cellular uptake of DXR-SIL[anti-MT1-MMP(Fab ')] into the HT1080 cells, which highly express MT1-MMP, compared with the non-targeted liposomes (DXR-stealthliposomes (DXR-SL)), suggesting that MT1-MMP antibody (Fab') is a potent targeting ligand for the MT1-MMP expressed cells. In vivo systemic administration of DXR-SIL[anti-MT1-MMP(Fab ')] into the tumor-bearing mice showed significant suppression of tumor growth compared to DXR-SL. This is presumably due to the active targeting of immunoliposomes for tumor and neovascularity. However, tumor accumulation of DXR-SIL[anti-MT1-MMP(Fab ')] and DXR-SL were comparable, suggesting that both liposomal formulations accumulated in tumor via enhanced permeation and retention (EPR) effect, but not via targeting to the MT1-MMP expressed on both the endothelial and tumor cells. It appears that the enhanced antitumor activity of DXR-SIL[anti-MT1-MMP(Fab ')] resulted from acceleration of cellular uptake of lioposomes owing to the incorporated antibody after extravasation from capillaries in tumor. (c) 2007 Elsevier B.V. All rights reserved.
  • Atthachal Homhuan, Kentaro Kogure, Hideyuki Akaza, Shiroh Futaki, Takashi Naka, Yukiko Fujita, Ikuya Yano, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 120 1-2 60 - 69 2007年07月 [査読有り][通常論文]
     
    Despite the potential of mycobacterial cell wall (CW) components to serve as immunotherapeutic agents, this application is hampered by the molecules' unfavorable physicochemical properties, such as its high molecular weight, poor solubility and negatively charged nature. Here we describe a new mycobacterial CW delivery system that uses an efficient and simple packaging method. This is achieved by incorporating mycobacterial CW into liposomes and attaching arginine octamers (R8) to the liposome surface. R8-modified liposomes improve the uptake of mycobacterial CW by dendritic cells (DC) and enhance its immunostimulatory activity. High R8 surface density promoted high levels of mycobacterial CW uptake by DC compared to low density R8-modified liposomes. Maturation markers (CD80, CD86, MHC Class II molecules) showed significantly enhanced expression on DC pulsed with high density R8-modified liposomes containing mycobacterial CW. Moreover, R8-modified liposomes with mycobacterial CW incorporated induced production of IL-12 p40 by DC, at levels similar to those produced by lipopolysaccharide-pulsed DC. We assert that R8-modified liposomes with mycobacterial CW incorporated should have tremendous potential as immune-potentiating agents. (C) 2007 Elsevier B.V. All rights reserved.
  • Kazuya Satou, Hiroshi Kasai, Chikahide Masutani, Fumio Hanaoka, Hideyoshi Harashima, Hiroyuki Kamiya
    Biochemistry 46 22 6639 - 46 2007年06月05日 [査読有り][通常論文]
     
    The coexistence effects of multiple kinds of oxidized deoxyribonucleotides were examined using an SV40 origin-dependent in vitro replication system with a HeLa extract. Oxidized dGTP and dATP, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP) and 2-hydroxy-2'-deoxyadenosine 5'-triphosphate (2-OH-dATP), were used in this study. The mutation frequency synergistically increased when the two oxidized deoxyribonucleotides were together in the reaction. 2-OH-dATP enhanced the mutagenicity of 8-OH-dGTP, since the induced mutations were A.T --> C.G transversions. The contribution of the highly error-prone DNA polymerase eta was unlikely, since similar results were observed with an XP-V cell extract. The possible involvement of 2-hydroxyadenine in the complementary (template) strand was excluded on the basis of experiments using plasmids containing 2-hydroxyadenine as templates in the reactions with 8-OH-dGTP. 2-OH-dATP suppressed hydrolysis of 8-OH-dGTP, suggesting that the inhibition of the MTH1 protein played the major role in the enhancement. These results highlight the importance of specific hydrolysis of 8-OH-dGTP for the suppression of its induced mutation.
  • Yoshio Nakamura, Kentaro Kogure, Shiroh Futaki, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 119 3 360 - 367 2007年06月 [査読有り][通常論文]
     
    The multifunctional envelope-type nano device (MEND) is a novel non-viral gene delivery system for plasmid DNA (pDNA) and oligodeoxynucleotides (ODN). We showed previously that octaarginine-modified MEND (R8-MEND) produces a high transfection activity without cytotoxicity via macropinocytosis and efficient release of a condensed DNA core to the cytosol. In the present study, we succeeded in developing an efficient method for packaging siRNA into the R8-MEND, and its silencing effect was compared with that of transfection reagent TransIT-TKO. A polycation able to condense siRNA was screened for by measuring the size and zeta-potential of complexes formed between siRNA and the polycations poly-l-lysine (PLL), stearyl octaarginine (STR-R8) and protamine. Only STR-R8 was able to condense siRNA to form nano particles (<100 nm), whereas all three polycations were able to condense pDNA or ODN. The siRNA packaged in R8-MEND inhibited luciferase activity by more than 80% in HeLa cells stably expressing luciferase. Much amount of siRNA was internalized into the cells as R8-MEND, and siRNA was effectively released from lipid envelope of MEND to cytoplasm near the nucleus. Consequently, R8-MEND can deliver condensed siRNA into cells to produce an efficient and persistent silencing effect with minimum cytotoxicity. (c) 2007 Published by Elsevier B.V.
  • Masatomi Shimizu, Petr Gruz, Hiroyuki Kamiya, Chikahide Masutani, Yan Xu, Yukio Usui, Hiroshi Sugiyama, Hideyoshi Harashima, Fumio Hanaoka, Takehiko Nohmi
    Biochemistry 46 18 5515 - 22 2007年05月08日 [査読有り][通常論文]
     
    Altered oxidative metabolism is a property of many tumor cells. Oxidation of DNA precursors, i.e., dNTP pool, as well as DNA is a major source of mutagenesis and carcinogenesis. Here, we report the remarkable nature of human DNA polymerase eta that incorporates oxidized dNTPs into a nascent DNA strand in an efficient and erroneous manner. The polymerase almost exclusively incorporated 8-hydroxy-dGTP (8-OH-dGTP) opposite template adenine (A) at 60% efficiency of normal dTTP incorporation, and incorporated 2-hydroxy-dATP (2-OH-dATP) opposite template thymine (T), guanine (G), or cytosine (C) at substantial rates. The synthetic primers having 8-hydroxy-G paired with template A or 2-hydroxy-A paired with template T, G, or C at the termini were efficiently extended. In contrast, human DNA polymerase iota incorporated 8-OH-dGTP opposite template A with much lower efficiency and did not incorporate 2-OH-dATP opposite any of the template bases. It did not extend the primers having the oxidized bases at the termini either. We propose that human DNA polymerase eta may participate in oxidative mutagenesis through the efficient and erroneous incorporation of oxidized dNTPs during DNA synthesis.
  • Hiroyuki Kamiya, Satoki Fukunaga, Takashi Ohyama, Hideyoshi Harashima
    ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 461 1 7 - 12 2007年05月 [査読無し][通常論文]
     
    The intranuclear disposition of a plasmid is extremely important for transgene expression. The effects of a left-handedly curved sequence with high histone affinity on plasmid expression were examined in vivo. A naked luciferase-plasmid was delivered into mouse liver by a hydrodynamics-based injection, and the luciferase activities were quantitated at various time points. The location of the left-handedly curved sequence determined the transgene expression, without affecting the amount of intranuclear exogenous DNA. The plasmid containing the curved sequence at the location that results in the exposure of the TATA box out of the nucleosome core showed the highest expression. These results suggest that sequences with high histone affinity could control transgene expression from plasmids in vivo. (c) 2007 Elsevier Inc. All rights reserved.
  • I. A. Khalil, K. Kogure, S. Futaki, S. Hama, H. Akita, M. Ueno, H. Kishida, M. Kudoh, Y. Mishina, K. Kataoka, M. Yamada, H. Harashima
    GENE THERAPY 14 8 682 - 689 2007年04月 [査読有り][通常論文]
     
    This study describes a multifunctional envelope-type nano device (MEND) that mimics an envelope-type virus based on a novel packaging strategy. MEND particles contain a DNA core packaged into a lipid envelope modified with an octaarginine peptide. The peptide mediates internalization via macropinocytosis, which avoids lysosomal degradation. MEND-mediated transfection of a luciferase expression plasmid achieved comparable efficiency to adenovirus-mediated transfection, with lower associated cytotoxicity. Furthermore, topical application of MEND particles containing constitutively active bone morphogenetic protein (BMP) type IA receptor (caBmpr1a) gene had a significant impact on hair growth in vivo. These data demonstrate that MEND is a promising non-viral gene delivery system that may provide superior results to existing non-viral gene delivery technologies.
  • Kenichi Niikura, Takashi Nishio, Hidetaka Akita, Yasutaka Matsuo, Ryosuke Kamitani, Kentaro Kogure, Hideyoshi Harashima, Kuniharu Ijiro
    CHEMBIOCHEM 8 4 379 - 384 2007年03月 [査読有り][通常論文]
  • Tomotaka Kobayashi, Tatsuhiro Ishida, Yurie Okada, Saori Ise, Hideyoshi Harashima, Hiroshi Kiwada
    INTERNATIONAL JOURNAL OF PHARMACEUTICS 329 1-2 94 - 102 2007年02月 [査読有り][通常論文]
     
    The over-expression of P-glycoprotein (P-gp) has been associated with the development of multidrug resistance (MDR) in cancer cells. In this study, we examined whether transferrin receptor (Tf-R) targeted liposomes can efficiently deliver encapsulated doxorubicin (DXR) into MDR cells (SBC-3/ADM) via Tf-R-mediated endocytosis thus overcoming MDR by by-passing P-gp-mediated drug efflux. We prepared four types of liposome, i.e. untargeted and Tf-R-targeted, made of either egg-PC/cholesterol or hydrogenated egg PC/cholesterol. Only with the targeted EPC-liposome we achieved significant delivery of encapsulated DXR and increased cytotoxicity of encapsulated DXR on the MDR cells (3.5-fold higher than free DXR). Confocal microscopy and an intracellular drug-accumulation assay indicated that the targeted liposomes efficiently delivered DXR into cells where it readily accumulated in the nucleus, in both drug-sensitive and MDR cells. These findings suggest that the targeted liposomes are rapidly internalized via Tf-R-mediated endocytosis followed by release of their contents into the cytoplasm. The rapid internalization and content release, most likely facilitated by the higher fluidity of the EPC-based liposomes, may explain why only targeted EPC-liposomes were able to prevent drug efflux by P-gp and to consequently circumvent MDR. Our results indicate that in order to achieve MDR circumvention by means of liposomal encapsulation of DXR the liposomes not only need to be targeted, but also to have the proper physicochernical properties for adequate release of the drug. Furthermore, these in vitro results suggest that Tf-R targeted EPC-liposomes are a potentially useful drug delivery system to circumvent P-gp-mediated MDR of tumors. (c) 2006 Elsevier B.V. All rights reserved.
  • Hiroshi Kuramoto, Noritada Kaji, Kentarou Kogure, Manabu Tokeshi, Yasuo Shinohara, Hideyoshi Harashima, Yoshinobu Baba
    Proceedings of the 11th International Conference on Miniaturized Systems for Chemistry and Life Sciences, uTAS 2007 781 - 783 2007年 
    A multifunctional envelope-type nano device (MEND) is an extremely efficient technology for a non-viral gene delivery system. However, the construction processes of the MEND by the existing method are very complicated. Therefore we develop a new method to construct it quickly and easily in the previous work. In this work, we report a new on-chip construction method of the MEND which is much more suitable for the gene therapy.
  • Hiroyuki Kamiya, Emiko Iida, Hideyoshi Harashima
    Genes and Environment 29 2 63 - 66 2007年 [査読無し][通常論文]
     
    The Escherichia coli Orf 135 (NudG) protein, a MutT-type enzyme, catalyzes the hydrolysis of 2-hydroxy-dATP and 8-hydroxy-dGTP, and its deficiency causes an increase in the mutation frequency. In this study, Orf135 proteins with substitutions at the Gly-36, Gly-37, Lys-38, Glu-43, Arg-51, Glu-52, Leu-53, Glu-55, and Glu-56 residues, which are conserved in three MutT-type proteins (Orf135, MutT, and MTH1), were each expressed in the orf135- strain, and the rpoB mutant frequency upon H2O2 treatment was examined. The in vivo mutation suppression abilities and the invitro enzymatic activities obtained in a previous study were compared. The expression of the enzymatically active Orf135 mutants in the orf135- strain tended to reduce the rpoB mutant frequency induced by H2O2. This result suggests the importance of the phosphohydrolase activity in the suppression of mutations by the Orf135 protein. Key words: Orf135, phosphohydrolase activity, mutation suppression, nucleotide pool sanitization. © 2007, The Japanese Environmental Mutagen Society. All rights reserved.
  • H. Hatakeyama, H. Akita, K. Kogure, M. Oishi, Y. Nagasaki, Y. Kihira, M. Ueno, H. Kobayashi, H. Kikuchi, H. Harashima
    GENE THERAPY 14 1 68 - 77 2007年01月 [査読有り][通常論文]
     
    For successful cancer gene therapy via intravenous (i.v.) administration, it is essential to optimize the stability of carriers in the systemic circulation and the cellular association after the accumulation of the carrier in tumor tissue. However, a dilemma exists regarding the use of poly(ethylene glycol) (PEG), which is useful for conferring stability in the systemic circulation, but is undesirable for the cellular uptake and the following processes. We report the development of a PEG-peptide-lipid ternary conjugate (PEG-Peptide-DOPE conjugate (PPD)). In this strategy, the PEG is removed from the carriers via cleavage by a matrix metalloproteinase (MMP), which is specifically expressed in tumor tissues. An in vitro study revealed that the PPD-modified gene carrier (Multifunctional Envelope-type Nano Device: MEND) exhibited pDNA expression activity that was dependent on the MMP expression level in the host cells. In vivo studies further revealed that the PPD was potent in stabilizing MEND in the systemic circulation and facilitating tumor accumulation. Moreover, the i.v. administration of PPD or PEG/PPD dually-modified MEND resulted in the stimulation of pDNA expression in tumor tissue, as compared with a conventional PEG-modified MEND. Thus, MEND modified with PPD is a promising device, which has the potential to make in vivo cancer gene therapy achievable.
  • Atsushi Yamashita, Nobuhiko Yui, Tooru Ooya, Arihiro Kano, Atsushi Maruyama, Hidetaka Akita, Kentaro Kogure, Hideyoshi Harashima
    Nat Protoc 1 6 2861 - 2869 2006年12月 [査読有り][通常論文]
     
    This protocol provides a method for synthesizing a biocleavable polyrotaxane/plasmid DNA (pDNA) polyplex and for using it to deliver pDNA into cell nuclei. The biocleavable polyrotaxane is synthesized in four steps: (i) introduction of disulfide linkages at both terminals of PEG, (ii) preparation of an inclusion complex between disulfide-containing PEG and alpha-cyclodextrins (alpha-CDs), (iii) synthesis of polyrotaxane and (iv) modification of alpha-CDs in the polyrotaxane with dimethylethylenediamine. A polyplex of pDNA with the polyrotaxane is formed when the two compounds are dissolved together in a phosphate buffer. Subcellular localization of rhodamine-labeled pDNA in fluorescently labeled organelles is quantified by Z-series of confocal images captured by confocal laser scanning microscopy. Significant amounts of pDNA delivered to the nucleus can be expected as well as high transfection activity of the polyplex. This protocol can be completed in 23-32 d.
  • Hiroyuki Kamiya, Akihiro Suzuki, Kazuaki Kawai, Hiroshi Kasai, Hideyoshi Harashima
    Nucleic acids symposium series (2004) 99 - 100 2006年12月01日 [査読無し][通常論文]
     
    Oxidation of RNA precursors may disturb genetic information by mispair formations. In this study, effects of oxidized ribonucleoside triphosphates on in vitro transcription catalyzed by T7 RNA polymerase were examined. 8-Hydroxyguanosine 5'-triphosphate (8-OH-GTP) and 2-hydroxyadenosine 5'-triphosphate (2-OH-ATP) reduced amount of RNA. In addition, mRNA was converted to cDNA by reverse transcriptase, and the cDNA was then amplified by PCR. The PCR product was subsequently cloned into plasmid DNA and sequence of DNA was analyzed for each bacterial colony. The two oxidized ribonucleotides induced mutations in cDNA, suggesting disturbance of genetic information during transcription and/or reverse transcription. 8-OH-GTP induced T-->G plus T-->C mutations and 2-OH-ATP caused T-->C mutations. These results indicate that formation of these oxidized RNA precursors in cells affects transcription quantitatively and qualitatively. In addition, they are potential antiviral drugs that cause mutations in genomic RNA.
  • Tianzhi Yang, Alamdar Hussain, Shuhua Bai, Ikramy A. Khalil, Hideyoshi Harashima, Fakhrul Ahsan
    JOURNAL OF CONTROLLED RELEASE 115 3 289 - 297 2006年10月 [査読有り][通常論文]
     
    This study tests the hypothesis that positively charged polyethylenimines (PEIs) enhance nasal absorption of low molecular weight heparin (LMWE) by reducing the negative surface charge of the drug molecule. Physical interactions between PEIs and LMVM were studied by Fourier transform infrared (FTIR) spectroscopy, particle size analysis, conductivity measurements, zeta potential analysis, and azure A assay. The efficacy of PEIs in enhancing nasal absorption of LMWH was studied by administering LMWH formulated with PEI into the nose of anesthetized rats and monitoring drug absorption by measuring plasma anti-factor Xa activity. The metabolic stability of LMWH was evaluated by incubating the drug in rat nasal mucosal homogenates. FUR spectra of the LMWH-PEI formulation showed a shift in peak position compared to LMWH or PEI alone. Decreases in conductivity, zeta potential and the amount of free LMWH in the PEI-LMWH formulation, as revealed by azure A assay, suggest that PEIs possibly neutralize the negative surface charge of LMWH. The efficacy of PEI in enhancing the bioavailability of nasally administered LMWH can be ranked as PEI-1000 kDa >= PEI-750 kDa > PEI-25 kDa. When PEI-1000 kDa was used at a concentration of 0.25%, there was a 4-fold increase in both the absolute and relative bioavailabilities of LMWH compared to the control formulation. Overall, these results indicate that polyethylenimines can be used as potential carriers for nasally administered LMWHs. (c) 2006 Elsevier B.V. All rights reserved.
  • Takahiro Fujiwara, Hidetaka Akita, Katsuko Furukawa, Takashi Ushida, Hiroyuki Mizuguchi, Hideyoshi Harashima
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 29 7 1511 - 1515 2006年07月 [査読有り][通常論文]
     
    An in vitro cell culture model that mimics in vivo extracellular environment would be useful in developing in vivo gene delivery system. In the present study, a parallel flow model was applied to investigate the impact of convective flow on cellular uptake and transfection activity in endothelial cells. LipofectAMINE PLUS and adenovirus were used as model vectors, which bind cells via electrostatic- and ligand-receptor interactions, respectively. Whereas a convective flow increased the total amount of vector passing through the flow chamber by 3 orders of magnitude, uptake was increased by less than 10-fold, suggesting that the flow severely inhibited cellular uptake by reducing the retention time in the chamber and/or by diminishing the affinity between the cell and vector. Moreover, the uptake of both vectors was increased in a shear stress-dependent manner to a comparable extent, suggesting that the effect of flow on the cellular uptake was not significant. In contrast, transfection efficiency (TE), expressed as the transfection activity normalized by the cellular uptake of vectors was dramatically stimulated by shear stress, only when LipofectAMINE PLUS was used. Since the activities of the CMV promoter were unaffected by a shear stress, it is possible that altered intracellular trafficking may responsible for the improvement in lipoplex-mediated TE, presumably related to the cellular uptake pathway.
  • M Yamada, T Nunoshiba, M Shimizu, P Gruz, H Kamiya, H Harashima, T Nohmi
    JOURNAL OF BACTERIOLOGY 188 13 4992 - 4995 2006年07月 [査読無し][通常論文]
     
    Escherichia coli DNA polymerase IV incorporated 2-hydroxy-dATP opposite template guanine or thymine and 8-hydroxy-dGTP exclusively opposite adenine in vitro. Mutator phenotypes in sod/fur strains were substantially diminished by deletion of dinB and/or umuDC. DNA polymerases IV and V may be involved in mutagenesis caused by incorporation of the oxidized deoxynucleoside triphosphates.
  • Akitada Iwasa, Hidetaka Akita, Ikramy Khalil, Kentaro Kogure, Shiroh Futaki, Hideyoshi Harashima
    Biochimica et biophysica acta 1758 6 713 - 20 2006年06月 
    Intracellular trafficking is a determining factor in the transgene expression efficiency of gene vectors. In the present study, the mechanism of the cellular uptake of octaarginine (R8)-modified liposomes, when introduced at 37 degrees C and 4 degrees C, was investigated in living cells. Compared with 37 degrees C, the uptake of R8-liposomes was only slightly reduced at 4 degrees C. Dual imaging of liposomes and plasma membranes revealed that R8-liposomes were internalized by vesicular transport, and partially escaped to the cytosol at the perinuclear region at 37 degrees C. When introduced at 4 degrees C, intracellular liposomes were observed within a specific region close to the plasma membrane, and internalization of the plasma membrane was completely inhibited. Therefore, at 4 degrees C, R8-liposomes appear to enter cells via unique pathway, which is separate and distinct from energy-dependent vesicular transport. The subsequent nuclear delivery of encapsulated pDNA, when introduced at 4 degrees C, was less prominent compared with those introduced at 37 degrees C. Collectively, these findings demonstrate that a vesicular transport-independent pathway is responsible for the cellular uptake of liposomes. In addition, the uptake route is closely related to the subsequent nuclear delivery process; the operation of an endogenous vesicular sorting system is advantageous for the nuclear delivery of pDNA.
  • Hiroshi Ochiai, Hideyoshi Harashima, Hiroyuki Kamiya
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 29 6 1294 - 1296 2006年06月 [査読無し][通常論文]
     
    The intranuclear disposition of exogenous DNA is highly important for the therapeutic effects of the administrated DNA. Naked luciferase-plasmid DNA was transfected into cultured cells including HeLa by electroporation, and the amounts of intranuclear plasmid DNA and luciferase were quantitated at various time points. Decrease in expression efficiency from one copy of the exogenous DNA over time occurred as the case of mouse liver, and its degrees varied between cell lines. These results suggest that exogenous DNA is 'silenced' in the cultured cells as well as in mouse hepatocytes.
  • Akitada Iwasa, Hidetaka Akita, Ikramy Khalil, Kentaro Kogure, Shiroh Futaki, Hideyoshi Harashima
    BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 1758 6 713 - 720 2006年06月 [査読有り][通常論文]
     
    Intracellular trafficking is a determining factor in the transgene expression efficiency of gene vectors. In the present study, the mechanism of the cellular uptake of octaarginine (R8)-modified liposomes, when introduced at 37 degrees C and 4 degrees C, was investigated in living cells. Compared with 37 degrees C, the uptake of R8-liposomes was only slightly reduced at 4 degrees C. Dual imaging of liposomes and plasma membranes revealed that R8-liposomes were internalized by vesicular transport, and partially escaped to the cytosol at the perinuclear region at 37 degrees C. When introduced at 4 degrees C, intracellular liposomes were observed within a specific region close to the plasma membrane, and internalization of the plasma membrane was completely inhibited. Therefore, at 4 degrees C, R8-liposomes appear to enter cells via unique pathway, which is separate and distinct from energy-dependent vesicular transport. The subsequent nuclear delivery of encapsulated pDNA, when introduced at 4 degrees C, was less prominent compared with those introduced at 37 degrees C. Collectively, these findings demonstrate that a vesicular transport-independent pathway is responsible for the cellular uptake of liposomes. In addition, the uptake route is closely related to the subsequent nuclear delivery process; the operation of an endogenous vesicular sorting system is advantageous for the nuclear delivery of pDNA. (c) 2006 Elsevier B.V All rights reserved.
  • Kentaro Kogure, Yoshio Nakamura, Ikramy A. Khalil, Shiroh Futaki, Hideyoshi Harashima
    MOLECULAR THERAPY 13 S202 - S203 2006年05月 [査読有り][通常論文]
  • Akitada Iwasa, Hidetaka Akita, Ikramy Khalil, Kentaro Kogure, Shiroh Futaki, Hideyoshi Harashima
    MOLECULAR THERAPY 13 S129 - S129 2006年05月 [査読有り][通常論文]
  • Hideki Abe, Mitsuhiro Fujihara, Hiroshi Azuma, Hisami Ikeda, Kenji Ikebuchi, Shinji Takeoka, Eishun Tsuchida, Hideyoshi Harashima
    Artificial Cells, Blood Substitutes, and Biotechnology 34 1 1 - 10 2006年04月01日 [査読有り][通常論文]
     
    Hemoglobin vesicles (HbVs), cellular-type artificial oxygen carriers containing human hemoglobin, were assessed for their biocompatibility by mixing with human plasma in vitro. Among three kinds of HbVs (PEG-DPEA-HbV, PEG-DPPG-HbV and DPPG-HbV), PEG-DPEA-HbV did not affect the extrinsic or intrinsic coagulation activities of the plasma, while PEG-DPPG-HbV and DPPG-HbV tended to shorten the intrinsic coagulation time. The kallikrein-kinin cascade of the plasma was slightly activated by PEG-DPPG-HbV and DPPG-HbV, but not by PEG-DPEA-HbV. The complement consumption of the plasma was observed by incubation with DPPG-HbV, but not with PEG-DPEA-HbV or PEG-DPPG-HbV. These results indicate that PEG-DPEA-HbV has a higher biocompatibility with human plasma. Copyright © Taylor & Francis Group, LLC.
  • S Hama, H Akita, R Ito, H Mizuguchi, T Hayakawa, H Harashima
    MOLECULAR THERAPY 13 4 786 - 794 2006年04月 [査読有り][通常論文]
     
    To develop nonviral gene vectors that are sufficient for clinical application, it is necessary to understand why and to what extent nonviral vectors are inferior to viral vectors, which in general show a more efficient transfection activity. This study describes a systematic and quantitative comparison of the cellular uptake and subsequent intracellular distribution (e.g., endosome/lysosome, cytosol, and nucleus) of exogenous DNA transfected by viral and nonviral vectors in living cells, using a combination of TaqMan PCR and a recently developed confocal image-assisted three-dimensionally integrated quantification method. As a model, adenovirus (Ad) and Lipofectamine Plus (LFN) were used for comparison since they are highly potent and widely used viral and nonviral vectors, respectively. The findings indicate that the efficiency of cellular uptake for LFN is significantly higher than that for Ad. Once taken up by a cell, Ad exhibited comparable endosomal escape and slightly higher nuclear transfer efficiency compared with LFN. In contrast, LFN requires 3 orders of magnitude more intranuclear gene copies to exhibit a transgene expression comparable to that of the Ad, suggesting that the difference in transfection efficiency principally arises from differences in nuclear transcription efficiency and not from a difference in intracellular trafficking between Ad and LFN.
  • IA Khalil, K Kogure, H Akita, H Harashima
    PHARMACOLOGICAL REVIEWS 58 1 32 - 45 2006年03月 [査読有り][通常論文]
     
    The successful delivery of therapeutic genes to the designated target cells and their availability at the intracellular site of action are crucial requirements for successful gene therapy. Nonviral gene delivery is currently a subject of increasing attention because of its relative safety and simplicity of use; however, its use is still far from being ideal because of its comparatively low efficiency. Most of the currently available nonviral gene vectors rely on two main components, cationic lipids and cationic polymers, and a variety of functional devices can be added to further optimize the systems. The design of these functional devices depends mainly on our understanding of the mechanisms involved in the cellular uptake and intracellular disposition of the therapeutic genes as well as their carriers. Macromolecules are internalized into cells by a variety of mechanisms, and their intracellular fate is usually linked to the entry mechanism. Therefore, the successful design of a nonviral gene delivery system requires a deep understanding of gene/carrier interactions as well as the mechanisms involved in the interaction of the systems with the target cells. In this article, we review the different uptake pathways that are involved in nonviral gene delivery from a gene delivery point of view. In addition, available knowledge concerning cellular entry and the intracellular trafficking of cationic lipid-DNA complexes ( lipoplexes) and cationic polymer-DNA complexes ( polyplexes) is summarized.
  • T Ooya, HS Choi, A Yamashita, N Yui, Y Sugaya, A Kano, A Maruyama, H Akita, R Ito, K Kogure, H Harashima
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 128 12 3852 - 3853 2006年03月 [査読有り][通常論文]
  • I.A. Khalil, K. Kogure, S. Futaki, H. Harashima
    Journal of Biological Chemistry 281 6 3544 - 3551 2006年02月10日 [査読有り][通常論文]
  • Kogure K, Akita H, Harashima H
    Nihon rinsho. Japanese journal of clinical medicine 64 2 258 - 262 2006年02月 [査読有り][通常論文]
     
    We introduced a new concept "Programmed Packaging" for the rational development of non viral gene delivery system. There are many non-viral gene delivery system, however, their utility is limited in the in vitro experiments. The major reason is considered that it is difficult to package many functional devices into a nanoparticle. The new concept which we introduce here is named as Programmed Packaging, which is composed of the strategy to targeting, molecular design of functional devices to overcome barriers, and assembly technique to package them into a nanoparticle. In this article, we explain this concept and provide newly developed gene delivery systems based on this concept.
  • H Ochiai, H Harashima, H Kamiya
    FEBS LETTERS 580 3 918 - 922 2006年02月 [査読無し][通常論文]
     
    The intranuclear disposition of exogenous DNA is highly important for the therapeutic effects of the administrated DNA. Naked luciferase-plasmid DNA was delivered into mouse liver by a hydrodynamics-based injection, and the amounts of intranuclear plasmid DNA, luciferase, and its mRNA were quantitated at various time points. Methylation of the promoter of the luciferase gene was also analyzed. Expression efficiency from one copy of the exogenous DNA dramatically decreased over time, and the DNA was methylated and degraded into fragments. Unexpectedly, methylation of the intact plasmid DNA was low and did not increase over time. Rather, the fragmented DNA was methylated more frequently than the intact plasmid. These results suggest that the CpG methylation and the degradation of exogenous DNA, and its 'silencing', occurred in parallel in the nucleus. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  • Yamashita A, Ooya T, Kanda D, Choi H.S, Yui N, Maruyama A, Akita H, Kogure K, Harashima H
    Polymer Preprints, Japan 55 1 2006年 [査読有り][通常論文]
  • Hidetaka Akita, Susumu Hama, Hiroyuki Mizuguchi, Hideyoshi Harashima
    Progress on Post-Genome Technologies 521 - 525 2006年 [査読有り][通常論文]
     
    For developing of non-viral gene vectors sufficient for clinical application, it is necessary to understand why and to what extent non-viral vectors are inferior to the viral vectors, which in general exhibit more efficient transfection activity. This study describes a systematic and quantitative comparison of the cellular uptake and subsequent intracellular distribution (e. g. endosome/lysosome, cytosol and nucleus) of exogenous DNA transfected by viral and non-viral vectors in living cells, using a combination of TaqMan PCR and a recently developed confocal image-assisted 3-dimensionally integrated quantification (CIDIQ) method. As a model, adenovirus and LipofectAMINE PLUS (LFN) was used for comparison since they are highly potent and widely used viral and non-viral vectors, respectively. The findings indicate that the efficiency of cellular uptake for LFN is significantly higher than that for adenovirus. Once taken up by a cell, adenovirus exhibited comparable endosomal escape and slightly more higher nuclear transfer efficiency compared with LFN. In contrast, LFN requires three orders of magnitude more intra-nuclear gene copies to exhibit a trans-gene expression comparable to the adenovirus, suggesting that difference of transfection efficiency principally arises from differences in nuclear transcription efficiency, and not from a difference in intracellular trafficking between adenovirus and LFN.
  • Hiroyuki Kamiya, Hiroshi Ochiai, Hideyoshi Harashima, Mana Ito, Akira Matsuda
    NUCLEOSIDES NUCLEOTIDES & NUCLEIC ACIDS 25 4-6 553 - 560 2006年 [査読無し][通常論文]
     
    The Drosophila melanogaster deoxynucleoside kinase gene was introduced into HeLa cells with cationic lipids to allow its transient expression, and cytotoxic effects of several nucleoside analogs in the transfected cells were examined. Of the analogs tested, cytotoxicities of 1-beta-D-arabinofuranosylcytosine (araC), 5-fluorodeoxyuridine (FUdR), and 1-(2-deoxy-2-methylene-beta-D-erythro-pentofuranosyl)cytosine (DMDC) were increased by the deoxynucleoside kinase gene. These results suggest that the combination of the transient expression of the Drosophila deoxynucleoside kinase gene and these nucleoside analogs is a candidate for the suicide gene therapy.
  • H. Akita, M. Tanimoto, T. Masuda, K. Kogure, S. Hama, K. Ninomiya, S. Futaki, H. Harashima
    Journal of Gene Medicine 8 2 198 - 206 2006年 [査読有り][通常論文]
     
    BACKGROUND: The efficient nuclear delivery of plasmid DNA (pDNA) is essential for the development of a promising non-viral gene vector. In an attempt to achieve nuclear delivery, NLS-mu, a novel pDNA condenser, was prepared. This consists of mu, a highly potent polypeptide for condensing the pDNA, and a SV40 T antigen-derived nuclear localization signal (NLS(SV40)). METHODS: The utility of NLS-mu was assessed in terms of green fluorescent protein (GFP) expression after cytoplasmic and nuclear microinjection of GFP-encoding pDNA along with the transfection, and compared with mu and poly-L-lysine (PLL). Trans-gene expression after cytoplasmic microinjection was affected by the efficiencies of nuclear transfer and following intra-nuclear transcription. To evaluate the nuclear transfer process separately, we introduced a parameter, a nuclear transfer score (NT score), which was calculated as the trans-gene expression after cytoplasmic microinjection divided by that after nuclear microinjection. RESULTS: As expected, the rank order of trans-gene expression after the transfection and cytoplasmic microinjection was NLS-mu > mu > PLL. However, the calculated NT scores were unexpectedly ranked as mu = NLS-mu > PLL, suggesting that mu, and not NLS(SV40), is responsible for the nuclear delivery of pDNA. In addition, confocal images of rhodamine-labeled pDNA indicated that pDNA condensed with mu and NLS-mu was delivered as a condensed form. In comparing the nuclear transcription, the rank order of trans-gene expression after nuclear microinjection was PLL = NLS-mu > mu, suggesting that intra-nuclear transcription is inhibited by efficient condensation by mu, and is avoided by the attachment of NLS(SV40). CONCLUSIONS: Collectively, NLS-mu, which consists of chimeric functions, is an excellent DNA condenser, and the process is based on mu-derived nuclear transfer and NLS(SV40)-derived efficient intra-nuclear transcription.
  • B Ji, A Maeda, M Higuchi, K Inoue, H Akita, H Harashima, T Suhara
    LIFE SCIENCES 78 8 851 - 855 2006年01月 [査読有り][通常論文]
     
    Lactoferrin (Lf) is an iron-binding glycoprotein belonging to the transferrin (Tf) family. Lf was reported to cross the blood brain barrier (BBB) via receptor-mediated transcytosis in an in vitro model of the BBB. In the present study, we compared the in vivo brain uptake of Lf with that of OX26, an anti-Tf receptor antibody, and Tf. These three proteins were radiolabeled with 125 1 and administered to rats by i.v. injection. We found that Lf was more rapidly eliminated from the blood compared with OX26 and Tf (The half-life of Lf was approximately 8 and 6 times shorter than that of OX26 and Tf, respectively; the area under the blood concentration- time curve of Lf was approximately 15 and 17 times smaller than that of OX26 and Tf, respectively), and mainly accumulated in the liver, spleen, and kidney. Markedly high brain uptake was observed for Lf relative to Tf and OX26. Lf might be useful as a ligand for facilitating drug delivery into the brain. (c) 2005 Elsevier Inc. All rights reserved.
  • Kanda D, Yamashita A, Katoono R, Ooya T, Yui N, Maruyama A, Akita H, Kogure K, Harashima H
    Polymer Preprints, Japan 55 2 5367 - 5368 2006年 [査読有り][通常論文]
  • Atsushi Yamashita, Nobuhiko Yui, Tooru Ooya, Arihiro Kano, Atsushi Maruyama, Hidetaka Akita, Kentaro Kogure, Hideyoshi Harashima
    NATURE PROTOCOLS 1 6 2861 - 2869 2006年 [査読有り][通常論文]
     
    This protocol provides a method for synthesizing a biocleavable polyrotaxane/plasmid DNA (pDNA) polyplex and for using it to deliver pDNA into cell nuclei. The biocleavable polyrotaxane is synthesized in four steps: (i) introduction of disulfide linkages at both terminals of PEG, (ii) preparation of an inclusion complex between disulfide-containing PEG and alpha-cyclodextrins (alpha-CDs), (iii) synthesis of polyrotaxane and (iv) modification of alpha-CDs in the polyrotaxane with dimethylethylenediamine. A polyplex of pDNA with the polyrotaxane is formed when the two compounds are dissolved together in a phosphate buffer. Subcellular localization of rhodamine-labeled pDNA in fluorescently labeled organelles is quantified by Z-series of confocal images captured by confocal laser scanning microscopy. Significant amounts of pDNA delivered to the nucleus can be expected as well as high transfection activity of the polyplex. This protocol can be completed in 23-32 d.
  • Hiroyuki Tsuchiya, Tomoko Sawamura, Masayuki Uchiyama, Hideyoshi Harashima, Hiroyuki Kamiya
    Nucleic acids symposium series (2004) 93 - 94 2005年12月01日 [査読無し][通常論文]
     
    The correction of a mutated gene is a highly attractive approach for gene therapy. This in vivo mutagenesis method will also be an effective tool in biotechnology. However, the current small fragment homologous replacement (SFHR) method with a heat-denatured double-stranded PCR fragment yielded the low correction efficiency. Single-stranded DNA fragments were prepared from single-stranded phagemid DNAs and tested in a gene correction assay with a Hyg-EGFP fusion gene inactivated by a substitution mutation, as a model target. A 606-nt sense, single-stranded DNA fragment dramatically (12-fold) improved the gene correction efficiency, although the antisense strand showed only minimal correction efficiency. On the other hand, correction of frameshift mutations with the sense single-stranded DNA fragment were 2-3-fold as efficient as that with the PCR fragment. These results suggest that the use of a sense, single-stranded DNA fragment is useful in the SFHR method for the correction of mutated genes.
  • H Tsuchiya, H Harashima, H Kamiya
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 336 4 1194 - 1200 2005年11月 [査読無し][通常論文]
     
    A 606-nt single-stranded (ss) DNA fragment, prepared by restriction enzyme digestion of ss phagemid DNA, improves the gene correction efficiency by 12-fold as compared with a PCR fragment, which is the conventional type of fragment used in the small fragment homologous replacement method [H. Tsuchiya, H. Harashima, H. Kamiya, Increased SFHR gene correction efficiency with sense single-stranded DNA, J. Gene Med. 7 (2005) 486-493]. To reveal the characteristic features of this gene correction with the ss DNA fragment, the effects on the gene correction in CHO-K1 cells of the chain length, 5'-phosphate, adenine methylation, and transcription were studied. Moreover, the possibility that the ss DNA fragment is integrated into the target DNA was examined with a radioactively labeled ss DNA fragment. The presence of methylated adenine, but not the 5'-phosphate, enhanced the gene correction efficiency, and the optimal length of the ss DNA fragment (similar to 600 nt) was determined. Transcription of the target gene did not affect the gene correction efficiency. In addition, the target DNA recovered from the transfected CHO-K1 cells was radioactive. The results obtained in this study indicate that length and adenine methylation were important factors affecting the gene correction efficiency, and that the ss DNA fragment was integrated into the double-stranded target DNA. (c) 2005 Elsevier Inc. All rights reserved.
  • H. S. Choi, A. Yamashita, OOYA Tooru, N. Yui, H. Akita, K. Kogure, R. Ito, H. Harashima
    ChemPhysChem Vol. 6, pp. 1986-1990 11 1986 - 90 2005年11月 [査読有り][通常論文]
  • S Futaki, Y Masui, Nakase, I, Y Sugiura, T Nakamura, K Kogure, H Harashima
    JOURNAL OF GENE MEDICINE 7 11 1450 - 1458 2005年11月 [査読有り][通常論文]
     
    Background One of the critical steps in intracellular gene delivery using cationic liposomes is the endosomal escape of the plasmid/liposome complexes to the cytosol. The addition of GALA, a pH-sensitive fusogenic peptide, is a promising method to accelerate this step in order to enhance the expression of the desired proteins. Detailed studies on the methods of enhancement would broaden the horizon of its application. Methods Using representative commercially available cationic liposomes (Lipofectin, Lipofectamine, and Lipofectamine 2000), the effects of GALA on transfection. efficiency were studied by luciferase assay and confocal microscopic observations. Results A concentration-dependent increase in the transfection efficiency was observed for GALA. Addition of 0.1 mu M GALA to the plasmid/liposome complex significantly increased the transfection efficiency, especially in the case of Lipofectin, but higher concentration of GALA decreased transfection efficiency. Successful reduction in the liposomal dosage was attained by employing GALA while maintaining a high transfection efficiency. Interestingly, although the transfection efficiency was higher in the presence of GALA, a lower amount of the plasmid DNA was taken up by the cells. Confocal microscopic observations of the rhodamine-labeled plasmid did not show a significant difference in the cellular localization among cells incubated in the presence or absence of GALA, suggesting that a slight increase in GALA-induced release of the plasmid to the cytosol may cause a significant change in the transfection efficiency. Conclusion The unique features of GALA to mediate improved transfection efficiencies were identified. Copyright (C) 2005 John Wiley & Sons, Ltd.
  • H Ochiai, H Harashima, H Kamiya
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 28 10 2019 - 2022 2005年10月 [査読無し][通常論文]
     
    Cationic lipid-mediated transfer of DNA is promising in gene therapy. However, one disadvantage with this approach is the induction of an inflammatory response, which may decrease transgene expression. Recently, we found that plasmid DNA containing N-6-methyladenine (N-6-MeA), a bacterium-specific modified base, induced cytokine twice as efficiently as plasmid DNA without N6-MeA, when complexed with cationic lipids. Thus, plasmid DNA without N-6-MeA might express a transgene more efficiently than that containing N-6-MeA in vivo. To evaluate the effects of adenine methylation on transgene expression in vivo, we injected luciferase-encoding plasmid DNA, complexed with cationic lipids or a cationic polymer, intravenously into mice. When the plasmid DNA-cationic lipid complexes were injected, the luciferase expression from the methylated and unmethylated plasmids was similar, although cytokine was more efficiently elicited by the methylated DNA than the unmethylated DNA. Hydrodynamics-based injections of plasmid DNA-cationic polymer complexes did not induce cytokine, and the luciferase expression from the unmethylated plasmid was slightly lower than that from the methylated plasmid DNA. These results suggest that the presence of N-6-MeA did not reduce transgene expression in vivo.
  • R. Moriguchi, K. Kogure, H. Akita, S. Futaki, M. Miyagishi, K. Taira, H. Harashima
    International Journal of Pharmaceutics 301 1-2 277 - 285 2005年09月14日 [査読有り][通常論文]
     
    A multifunctional envelope-type nano device (MEND) for use in the delivery of siRNA expression plasmids is described. The plasmid DNA encoding anti-luciferase short interfering RNA (siRNA) was condensed by poly-L-lysine (PLL) and packaged into the MEND. The silencing effect of the MEND(PLL) showed a 96% inhibition of luciferase activities in a co-transfection study. The silencing effect was maintained at more than 60%, even under the 100-fold diluted conditions. In the luciferase transformed cells, however, the MEND(PLL) showed no significant silencing effect (10%), indicating heterogeneity in transfection by the MEND(PLL). To solve this problem, the DNA condensing agents were optimized by comparing PLL, stearyl octaarginine (STR-R8) and protamine (Prot). No difference in silencing effect (95-97%) was found among these MENDs in a co-transfection study. However, the MEND(Prot) showed a 70% silencing effect in the transformed cells. These results suggest that the MEND(Prot) has less heterogeneity in transfection, while the MEND(PLL) and the MEND(STR-R8) have large heterogeneities. These results demonstrate that MEND(Prot) is a promising gene delivery system for siRNA expression plasmids with less heterogeneity associated with the transfection.
  • K Satou, M Yamada, T Nohmi, H Harashima, H Kamiya
    CHEMICAL RESEARCH IN TOXICOLOGY 18 8 1271 - 1278 2005年08月 [査読無し][通常論文]
     
    To reveal the roles of Y family DNA polymerases in the mutagenesis induced by oxidatively damaged DNA precursors, 2-hydroxy-dATP (2-OH-dATP) and 8-hydroxy-dGTP (8-OH-dGTP) were introduced into Escherichia coli strains deficient in the Y family polymerases, DNA polymerase IV (pol IV, encoded by the dinB gene) and DNA polymerase V (pol V, encoded by the umuDC locus). The mutation induced by 2-OH-dATP, but not that induced by 8-OH-dGTP, occurred less frequently in the dinB(-) strain than in the wild-type (wt) strain, suggesting the involvement of pol IV in the mutagenesis by 2-OH-dATP. Expression of pol IV from plasmid enhanced the mutagenesis by 2-OH-dATP in the dinB- strain. This enhancement depends on the polymerase activity since the expression of a mutant pol IV lacking the polymerase activity did not increase the mutations induced by 2-OH-dATP. In contrast, both 2-OH-dATP and 8-OH-dGTP caused mutations more efficiently in the umuDC(-) strain than in the wt strain, suggesting that the umuDC gene products suppressed the mutagenesis by these oxidized DNA precursors. The DNA polymerase activity was not required for the suppressive effects because expression of the umuDC gene products lacking the polymerase activity also suppressed the mutagenesis. These results suggest that the E. coli pol IV was involved in mutagenesis by 2-OH-dATP and that the umuDC gene products play suppressive role(s) in the mutagenesis by damaged nucleotides.
  • Y Yumada, H Kamiya, H Harashima
    INTERNATIONAL JOURNAL OF PHARMACEUTICS 299 1-2 34 - 40 2005年08月 [査読無し][通常論文]
     
    The production of an exogenous protein by the transfection of a plasmid DNA encoding the protein was kinetically analyzed, to determine the efficiency of the transfection. Cultured NIH3T3 or HeLa cells, and the luciferase protein were used as a model system in this experiment. The findings indicate that at least a 8 x 10(4)- and 4 x 10(3)-fold molar amounts of luciferase protein was produced from one copy of the plasmid DNA molecule in NIH3T3 and HeLa cells, respectively. The rate of elimination of luciferase activity upon DNA transfection was smaller than that for the luciferase protein itself (k(el) for DNA transfection < k(cl) for the luciferase protein), suggesting that a decrease in intranuclear active DNA was the main determinant of the elimination rate in this case. A preliminary pharmacokinetic model is proposed, based on the results obtained. (c) 2005 Elsevier B.V. All rights reserved.
  • K Sasaki, K Kogure, S Chaki, Y Kihira, M Ueno, H Harashima
    INTERNATIONAL JOURNAL OF PHARMACEUTICS 296 1-2 142 - 150 2005年05月 [査読有り][通常論文]
     
    A novel assembly method "SUV*-fusion method" was developed for the construction of a small and homogenous multifunctional envelope-type nano device (MEND) by utilizing a detergent-rich small unilamellar vesicle (SUV*). The method consists of three steps: (1) DNA condensation with a polycation, (2) electrostatic interaction of the SUV* with the DNA/polycation complex (DPC) and (3) lipid coating of DPC by SUV* fusion via removal of the detergent. We confirmed the construction of the MEND by sucrose density gradient centrifugation, and isolated the MEND only from the boundary between 25% and 40% sucrose. The isolated MEND had a small diameter (155 nm), was negatively charged (-24 mV), and encapsulated 30% of the total DNA. The MEND was formed by only SUV*, not by a lipid/detergent micelle. This confirms that a small and homogenous MEND can be constructed by the SUV*-fusion method. Furthermore, we confirmed that a transferrin-modified MEND could deliver a gene into a cell through receptor-mediated endocytosis. Consequently, we report on the successful construction of a small and homogenous MEND by a novel SUV*-fusion method. (C) 2005 Elsevier B.V. All rights reserved.
  • H Tsuchiya, H Harashima, H Kamiya
    JOURNAL OF GENE MEDICINE 7 4 486 - 493 2005年04月 [査読無し][通常論文]
     
    Background The correction of a mutated gene by the small fragment homologous replacement (SFHR) method is a highly attractive approach for gene therapy. However, the current SFHR method with a heat-denatured double-stranded PCR fragment yielded a low correction efficiency. Methods Single-stranded (ss) DNA fragments were prepared from ss phagemid DNA and tested in a gene correction assay with an inactivated Hyg-EGFP fusion gene, as a model target. Results A 606-nt sense, ss DNA fragment dramatically (12-fold) improved the gene correction efficiency, although the antisense strand showed only minimal correction efficiency. Conclusions These results suggest that the use of a sense, single-stranded DNA fragment is useful in the SFHR method for the correction of mutated genes. Copyright (c) 2004 John Wiley C Sons, Ltd.
  • E Iida, K Satou, M Mishima, C Kojima, H Harashima, H Kamiya
    BIOCHEMISTRY 44 15 5683 - 5689 2005年04月 [査読無し][通常論文]
     
    The Escherichia coli Orf135 protein, a MutT-type enzyme, hydrolyzes mutagenic 2-hydroxy-dATP (2-OH-dATP) and 8-hydroxy-dGTP, in addition to dCTP and 5-methyl-dCTP, and its deficiency causes increases in both the spontaneous and H2O2-induced mutation frequencies. To identify the amino acid residues that interact with these nucleotides, the Glu-33, Arg-72, Arg-77, and Asp-118 residues of Orf135, which are candidates for residues interacting with the base, were substituted, and the enzymatic activities of these mutant proteins were examined. The mutant proteins with a substitution at the 33rd, 72nd, and 118th amino acid residues displayed activities affected to various degrees for each substrate, suggesting the involvement of these residues in substrate binding. On the other hand, the mutant protein with a substitution at the 77th Arg residue had activitiy similar to that of the wild-type protein, excluding the possibility that this Arg side chain is involved in base recognition. In addition, the expression of some Orf135 mutants in orf135(-) E. coli reduced the level of formation of rpoB mutants elicited by H2O2. These results reveal the residues involved in the substrate binding of the E. coli Orf135 protein.
  • A. Yamashita, H. S. Choi, OOYA Tooru, N. Yui, H. Akita, K. Kogure, R. Ito, H. Harashima
    ChemPhysChem Vol. 7, pp. 297-302 297 - 302 2005年02月 [査読有り][通常論文]
  • Hidetaka Akita, Ikramy A. Khalil, Kentaro Kogure, Hideyoshi Harashima
    Non-viral Gene Therapy: Gene Design and Delivery 135 - 154 2005年 [査読有り][通常論文]
     
    Since the first report, in the 1980s, of gene delivery with a cationic liposome, numerous attempts have been made to improve non-viral gene vectors. It has gradually become clear that transgene expression can be interrupted by several intracellular barriers (Fig. 1). The cellular association of naked DNA molecules is very poor, since negative charges on both the cell surface and the DNA molecules interrupt contact with each other via electrostatic interactions. Thus, in order to enhance cellular association, DNA was condensed with cationic polymers (Oupicky et al. 2000 Brown et al. 2001) and cationic liposomes (Li et al. 1999 Tranchant et al. 2004) that neutralize the effect of the negative charge. A cationic vector enhances cell-surface binding through interactions with the negative constituents of the cell surface (e.g. heparan sulfate proteoglycans) or through selective binding to specific receptors, resulting in a strong transgene expression. This method of condensation also enables targeting of the cells by modulating different ligands to the surfaces of the gene vectors (Table 1).
  • IA Khalil, K Kogure, S Futaki, H Harashima
    YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 125 94 - 95 2005年 [査読有り][通常論文]
  • Hiroyuki Kamiya, Hiroyuki Tsuchiya, Hideyoshi Harashima
    Non-viral Gene Therapy: Gene Design and Delivery 315 - 322 2005年 [査読無し][通常論文]
     
    Gene correction therapy entails the conversion of a mutated gene to one with the normal (or desired) sequence (Richardson et al. 2002). The corrected genes should be properly expressed under the control of the original promoter, and respond appropriately to the intracellular and extracellular environments.When corrected, the "therapeutic effects" are expected to be life-long. Gain-of-function or predominant mutations, such as activated oncogenes, could be suitable subjects for gene correction, making it a highly attractive therapeutic strategy.
  • M Hori, K Fujikawa, H Kasai, H Harashima, H Kamiya
    DNA REPAIR 4 1 33 - 39 2005年01月 [査読無し][通常論文]
     
    To determine whether the Orf17 (NtpA) protein of Escherichia coli, a MutT-type enzyme, functions as a hydrolyzing enzyme for a damaged deoxyribonucleotide, we purified the recombinant Orf17 protein and incubated it with oxidized deoxyribonucleotides. Of the deoxyribonucleoside 5'-triphosphates tested, 8-hydroxy-2'-deoxyadenosine 5'-triphosphate was hydrolyzed by this protein. Unexpectedly, the Orf17 protein degraded 8-hydroxy-2'-deoxyadenosine 5-diphosphate 2.3-fold more efficiently than the corresponding triphosphate. Thus, this protein is the first MutT-type enzyme that hydrolyzes both the triphosphate and diphosphate derivatives of a deoxyribonucleoside, with similar efficiencies. These results suggest that the Orf17 protein may be involved in the hydrolysis of oxidized dATP and dADP. (C) 2004 Elsevier B.V. All rights reserved.
  • H Tsuchiya, T Matsuda, H Harashima, H Kamiya
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 326 4 777 - 781 2005年01月 [査読有り][通常論文]
     
    Unmethylated CpG dinucleotides in DNA contribute to a rapid inflammatory response in mammals. Here we show that N-6-methyladenine (N-6-MeA), a bacterium-specific modified base, also causes cytokine production. An oligodeoxyribonucleotide (ODN) containing N-6-MeA induced cytokines when injected into mice. Co-injection of N-6-MeA and CpG ODNs enhanced cytokines 2- to 3-fold, as compared with the injection of a CpG ODN alone. Plasmid DNA containing N-6-MeA, complexed with cationic lipids, induced IL-12. These results indicate that the bacterium-specific base, in addition to the unmethylated CpG motif, triggers the mammalian immune response, and suggest that N-6-MeA-containing DNA could be useful for cellular immunotherapy and DNA vaccine. (C) 2004 Elsevier Inc. All rights reserved.
  • D. Mudhakir, H. Akita, I.A. Khalil, S. Futaki, H. Harashima
    Drug metabolism and pharmacokinetics. 20 4 275 - 281 2005年 [査読有り][通常論文]
     
    We recently found that octaarginine modified liposomes (R8-Lip) can be efficiently internalized by cultured cells. The purpose of the present study was to quantitatively determine the effect of R8-density on the tissue distribution of R8-Lip in mice, using their clearance as an index. R8 was introduced in the form of stearylated R8 (STR-R8). The liposomes were composed of cholesterol and egg phosphatidylcholine and were labeled with [(3)H]cholesteryl hexadecyl ether. Various densities of R8 (3%, 10% and 30%) containing liposomes were prepared with a diameter of approximately 70-80 nm. The tissue distribution of R8-Lip was determined after their i.v. administration into mice and the effect of R8-density on tissue distribution was compared with uptake clearance, the calculated tissue distribution divided by the area under the blood concentration-time course. As results, R8-Lip were more rapidly eliminated from circulating blood and distributed to many tissues, especially liver depending on the R8-density. However, the tissue uptake clearance represented similar value to that of positively charge liposomes. Based on these results, we conclude that the R8-dependent increase in R8-Lip in various tissues tested indicates that positive charge, but not PTD function derived from R8 predominantly responsible for the enhancement of tissue distribution. Therefore, it is suggested that topology control of R8 is important to exhibit the PTD function.
  • Hiroyuki Kamiya, Kazuya Satou, Mika Hori, Emiko Iida, Chieko Ishiguro, Hideyoshi Harashima
    Nucleic acids symposium series (2004) 48 271 - 272 2004年12月01日 [査読無し][通常論文]
     
    Oxidized deoxyribonucleotides, 2-hydroxydeoxyadenosine 5'-triphosphate (2-OH-dATP) and 8-hydroxydeoxyguanosine 5'-triphosphate (8-OH-dGTP), were introduced into Escherichia coli strains deficient in DNA polymerase IV (a Y-family DNA polymerase encoded in the dinB gene), and the MutT and Orfl35 proteins to examine their in vivo roles in mutagenesis elicited by 2-OH-dATP and 8-OH-dGTP. 2-OH-dATP elicited mutations less efficiently in the dinB- strain than in the wild type strain, suggesting involvement of DNA polymerase IV in 2-OH-dATP-induced mutations. 8-OH-dGTP and 2-OH-dATP elicited mutations more efficiently in mutT- and orfl35- strains, respectively, than those in their isogenic mutT+ and orfl35+ strains. These results indicate that these proteins play important roles in mutagenesis induced by 2-OH-dATP and 8-OH-dGTP in vivo.
  • H Kamiya, E Iida, H Harashima
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 323 3 1063 - 1068 2004年10月 [査読無し][通常論文]
     
    The Escherichia coli Orf135 protein, a Muff-type enzyme, hydrolyzes 2-hydroxy-dATP and 8-hydroxy-dGTP, in addition to dCTP and 5-methyl-dCTP, and its deficiency causes increases in both the spontaneous and H2O2-induced mutation frequencies. In this study, the Gly-36 Gly-37, Lys-38,. Glu-43, Arg-51, Glu-52, Leu-53, Glu-55, and Glu-56 residues of Orf135, which are conserved in the three MutT-type proteins (Orf135, Muff, and MTH1), were substituted, and the enzymatic activity of these mutant proteins was examined. The mutant proteins with a substitution at the 36th, 37th, 52nd, and 56th amino acid residues completely lost their activity. On the other hand, the mutant proteins with a substitution at the 38th, 43rd, 51st, 53rd, and 55th residues could hydrolyze 5-methyl-dCTP. Some mutants with detectable activity for 5-methyl-dCTP did not hydrolyze dCTP. Activities for known substrates (5-methyl-dCTP, dCTP, 2-hydroxy-dATP, and 8-hydroxy-dGTP) were examined in detail with the four mutants, K38R, E43A, L53A, and E55Q. These results indicate the essential residues for the activity of the Orf135 protein. (C) 2004 Elsevier Inc. All rights reserved.
  • KOGURE K
    J Control Release 98 2 317 - 323 2004年08月11日 [査読有り][通常論文]
  • H Hatakeyama, H Akita, K Maruyama, T Suhara, H Harashima
    INTERNATIONAL JOURNAL OF PHARMACEUTICS 281 1-2 25 - 33 2004年08月 [査読有り][通常論文]
     
    Liposomes, coated with transferrin (Tf)-coupled polyethylene glycol are considered to be potent carriers for drug delivery to various organs via receptor-mediated endocytosis. Since Tf receptors were ubiquitously expressed in various organs, additional perturbation of the liposomes such as regulation of the size may be required to exhibit the tissue selectivity. In the present study, the effect of size on the uptake of transferrin-coupled polyethylene glycol liposomes (Tf-PEG-L) to various organs was investigated. In liver and brain, Tf-dependent uptake was found to be dependent on the size of the liposomes used. In small liposomes with a diameter of 60-80 nm, Tf-PEG-L was taken up to these organs more efficiently than PEG-L. This Tf-dependent uptake for small liposomes decreased by the high dose administration, suggested that Tf-PEG-L is taken Lip via Tf receptor-mediated endocytosis even under the physiological condition, in which plasma concentration of endogenous Tf remains high. On the other hand, Tf receptor-mediated uptake was also observed in the heart, but size-dependency was not observed in this case. Collectively, these results indicate that size dependency in the uptake of Tf-PEG-L is tissue-dependent and therefore, controlling the size of Tf-PEG-L may be useful for the Success of tissue targeting. (C) 2004 Elsevier B.V. All rights reserved.
  • David I. Fisher, Jared L. Cartwright, Jared L. Cartwright, Hideyoshi Harashima, Hiroyuki Kamiya, Alexander G. McLennan
    BMC Biochemistry 5 1 - 24 2004年05月17日 [査読無し][通常論文]
     
    Background: Nudix hydrolases form a protein family whose function is to hydrolyse intracellular nucleotides and so regulate their levels and eliminate potentially toxic derivatives. The genome of the radioresistant bacterium Deinococcus radiodurans encodes 25 nudix hydrolases, an unexpectedly large number. These may contribute to radioresistance by removing mutagenic oxidised and otherwise damaged nucleotides. Characterisation of these hydrolases is necessary to understand the reason for their presence. Here, we report the cloning and characterisation of the DR0975 gene product, a nudix hydrolase that appears to be unique to this organism. Results: The DR0975 gene was cloned and expressed as a 20 kDa histidine-tagged recombinant product in Escherichia coli. Substrate analysis of the purified enzyme showed it to act primarily as a phosphatase with a marked preference for (deoxy)nucleoside 5′ -diphosphatase (dGDP > ADP > dADP > GDP > dTDP > UDP > dCDP > CDP). Kmfor dGDP was 110 μM (and kcatwas 0.18 s-1under optimal assay conditions (pH 9.4, 7.5 mM Mg2+). 8-Hydroxy-2′-(deoxyguanosine 5′-diphosphate (8-OH-dGDP) was also a substrate with a Kmof 170 μM and kcatof 0.13 s-1. Thus, DR0975 showed no preference for 8-OH-dGDP over dGDP. Limited pyrophosphatase activity was also observed with NADH and some (di)adenosine polyphosphates but no other substrates. Expression of the DR0975 gene was undetectable in logarithmic phase cells but was induced at least 30-fold in stationary phase. Superoxide, but not peroxide, stress and slow, but not rapid, dehydration both caused a slight induction of the DR0975 gene. Conclusion: Nucleotide substrates for nudix hydrolases conform to the structure NDP-X, where X can be one of several moieties. Thus, a preference for (d)NDPs themselves is most unusual. The lack of preference for 8-OH-dGDP over dGDP as a substrate combined with the induction in stationary phase, but not by peroxide or superoxide, suggests that the function of DR09075 may be to assist in the recycling of nucleotides under the very different metabolic requirements of stationary phase. Thus, if DR0975 does contribute to radiation resistance, this contribution may be indirect.
  • T Kakudo, S Chaki, S Futaki, Nakase, I, K Akaji, T Kawakami, K Maruyama, H Kamiya, H Harashima
    BIOCHEMISTRY 43 19 5618 - 5628 2004年05月 [査読有り][通常論文]
     
    dLiposomes are one of the most promising systems for selective cellular targeting via introduction of specific ligands for cell-surface receptors. After being taken up by the cells, these liposomes usually follow intracellular pathways of receptor-mediated endocytosis. Control of intracelluar trafficking is required for optimized drug delivery. In this study, we elucidated the intracellular fate of transferrin-modified liposomes and succeeded in altering it by introducing the pH-sensitive fusogenic peptide, GALA (WEAALAEALAEALAEHLAEALAEALEALAA). Transferrins that are chemically attached to a liposomal surface (Tf-L) were internalized via receptor-mediated endocytosis more slowly than unmodified transferrins. In contrast to the recyclable nature of transferrin, liposome-attached transferrins together with encapsulated rhodamines were retained in vesicular compartments. When GALA was introduced into liposomal membranes using a cholesteryl moiety for anchoring (Choi-GALA), rhodamines were efficiently released and diffused into the cytosol. The addition of GALA to the Tf-L-containing medium or the encapsulation of GALA in Tf-L did not induce similar effects. These results clearly indicate that GALA must be present on the surface of liposomes to exert its function. In vitro energy transfer and dynamic light scattering experiments suggested that the endosomal escape of the encapsulates in Tf-L equipped with Chol-GALA can be attributed to pH-dependent membrane fusion. With GALA present on the surface, intracellular trafficking of liposomes after receptor-mediated endocytosis could be successfully controlled.
  • IA Khalil, S Futaki, M Niwa, Y Baba, N Kaji, H Kamiya, H Harashima
    GENE THERAPY 11 7 636 - 644 2004年04月 [査読有り][通常論文]
     
    The internalization mechanisms associated with octaarginine and stearyl-octaarginine were investigated using confocal laser microscopy and flow cytometric analysis. Octaarginine is able to translocate through cell membranes in a manner that does not exactly involve the classical endocytic pathways of internalization. However, when a stearyl moiety is attached to the N-terminus of octaarginine, the internalization shifts mainly to an endocytosis-dependent pathway. The transfection efficiency of stearyl-octaarginine was significantly higher than that of octaarginin. To understand the mechanism of the improved gene transfer by the N-terminal stearylation of octaarginine, the gene transfer processes mediated by octaarginine or stearyl-octaarginine were compared. Both octaarginine and stearyl-octaarginine are able to carry plasmid DNA into cells. The amount of plasmid DNA internalized as well as that delivered to the nucleus was higher in the case of stearyl-octaarginine. Even though the internalization mechanisms of octaarginine and stearyl-octaarginine were different, their complexes with plasmid DNA were internalized via the same pathway, presumably, the clathrin-mediated pathway of endocytosis. The results of the atomic force microscopy revealed that stearyl-octaarginine, but not octaarginine, can completely condense the DNA into stable complexes that can be highly adsorbed to the cell surface and subsequently highly internalized. Therefore, using stearylated-octaarginine provided higher internalization of plasmid DNA into cells, due to enhanced cellular association, as well as higher nuclear delivery. The results presented in this study provide a better understanding of the mechanisms of improved transfection using stearylated-octaarginine. The concept of using stearylated peptides may aid in the development of more efficient nonviral gene vectors.
  • H. Akita, R. Ito, I.A. Khalil, S. Futaki, H. Harashima
    Molecular Therapy 9 3 443 - 451 2004年03月 [査読有り][通常論文]
  • Hiroyuki Kamiya, Hiroyuki Yakushiji, Laurence Dugué, Mitsuhide Tanimoto, Sylvie Pochet, Yusaku Nakabeppu, Hideyoshi Harashima
    Journal of molecular biology 336 4 843 - 50 2004年02月27日 [査読有り][通常論文]
     
    To examine the substrate recognition mechanism of the human MTH1 protein, which hydrolyzes 2-hydroxy-dATP, 8-hydroxy-dATP, and 8-hydroxy-dGTP, ten nucleotide analogs (8-bromo-dATP, 8-bromo-dGTP, deoxyisoinosine triphosphate, 8-hydroxy-dITP, 2-aminopurine-deoxyriboside triphosphate, 2-amino-dATP, deoxyxanthosine triphosphate, deoxyoxanosine triphosphate, dITP, and dUTP) were incubated with the MTH1 protein. Of these, the former five nucleotides were hydrolyzed with various efficiencies. The fact that the syn-oriented brominated nucleotides were hydrolyzed suggests that the MTH1 protein binds to deoxynucleotides adopting the syn-conformation. However, 8-hydroxy-dITP, which lacks the 2-amino group of 8-hydroxy-dGTP, was degraded with tenfold less efficiency as compared with 8-hydroxy-dGTP. In addition, deoxyisoinosine triphosphate, lacking the 6-amino group of 2-hydroxy-dATP, was hydrolyzed as efficiently as 8-hydroxy-dGTP, but less efficiently than 2-hydroxy-dATP. These results clarify the effects of the anti/syn conformation and the functional groups on the 2 and 6 positions of the purine ring on the recognition by the human MTH1 protein.
  • R Tachibana, N Ide, Y Shinohara, H Harashima, CA Hunt, H Kiwada
    INTERNATIONAL JOURNAL OF PHARMACEUTICS 270 1-2 315 - 321 2004年02月 [査読有り][通常論文]
     
    To design better delivery systems that enhance transfection efficiency of nonviral vectors, we need to improve our understanding of the mechanisms governing both the amounts of plasmid delivered to the nucleus and gene expression. What is needed is a measure of transcriptional availability (TA): the average level of gene expression per plasmid delivered to the nucleus over the course of an experiment. We describe a method to measure TA and demonstrate its application. The chloramphenicol acetyltransferase reporter gene was transfected into NIH/3T3 cells using either cationic liposomes (TFL-3; O,O'-ditetradecanoyl-N-(alpha-trimethylammonioacetyl) diethanolamine chloride (DC-6-14), dioleoylphosphatidylethanotamine (DOPE) and cholesterol, molar ratio 1/0.75/0.75) or cationic polymer (PEI; polyethylenimine). The time courses of both nuclear delivery of plasmids and reporter gene expression were measured for 4 h thereafter. For the conditions used, time courses of gene expression and plasmid nuclear delivery for the two vectors were different. To understand the origins of those differences, we applied a simple pharmacokinetic model, used the data to estimate the values of the model parameters, and interpret differences in estimated parameter values. The rate constant of delivery of plasmids into the nucleus for the TFL-3 vector was twice that of the PEI vector, whereas rate constant of elimination of plasmids in the nucleus for the PEI vector was four times that for the TFL-3 vector. The gene expression rate constant for the TFL-3 vector was estimated to be seven times larger than that of the PEI vector for the conditions used. The pharmacokinetically determined average exposure of a nucleus to plasmid was about 17 times larger for the TFL-3 vector, relative to the PEI vector. That greater exposure resulted in increased relative gene expression. Overall, the TA from the TFL-3 vector was about 13 times greater than from the PEI vector. The experimental design combined with the adoption of pharmacokinetic concepts and principles provide a method to measure TA along with detailed insights into the mechanisms governing gene delivery and expression. (C) 2003 Elsevier B.V. All rights reserved.
  • LA Khalil, K Kogure, S Futaki, H Harashima
    YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 124 113 - 116 2004年 [査読有り][通常論文]
     
    This study was conducted to prepare efficient and safe non-viral gene vectors using the unique properties of the octaarginine (R8) peptide. We have investigated the factors influencing the uptake mechanism of R8-modified liposomes (R8-Lip). Liposomes with high density of R8 on the surface were taken up through pathways different from the classical clathrin-dependent endocytosis while those modified with low amount of R8 were taken up through clathrin-mediated endocytosis. Liposomes were internalized as vesicles where they slowly migrated towards the nucleus and the presence of heparin dramatically inhibited their uptake. R8-Lip could transfect cells with plasmid DNA encapsulated into their cores. However, the inclusion of fusogenic lipids in the envelope further increased the transfection activity. We found that fusogenic lipids did not significantly alter the uptake pathway, however, they increased the endosomal escape and nuclear delivery of plasmid DNA. Complexes formed between the R8 peptide and plasmid DNA were internalized through classical endocytosis, indicating the importance of peptide incorporation in a lipid envelope to target different entrance pathways.
  • Hiroyuki Tsuchiya, Keizou Kuwata, Sekio Nagayama, Kazumasa Yamashita, Hiroyuki Kamiya, Hideyoshi Harashima
    Drug metabolism and pharmacokinetics 19 206 - 215 2004年01月01日 [査読無し][通常論文]
     
    TAS-102, a new oral drug, is composed of an antitumor drug, alpha,alpha,alpha-trifluorothymidine (FTD), and its metabolic inhibitor, 5-chloro-6-(2-iminopyrrolidine-1-yl)methyl-2,4(1H,3H)-pyrimidinedione hydrochloride (TPI). It has been reported that the oral administration of TAS-102 increases the AUC of FTD in rodents and monkeys in different manners. In this study, a pharmacokinetic model was developed, in an attempt to evaluate the bioavailability of FTD in these animals after the co-administration of TPI. Since TPI inhibits FTD metabolism competitively, a time-dependent as well as concentration-dependent model for the hepatic intrinsic clearance of FTD was developed including the time courses of both FTD and TPI. Based on this modeling, we were able to quantitatively explain the TPI dose-dependent enhancement of AUC of FTD in monkeys, while little increase was observed in rats. These results are consistent to observations that thymidine phosphorylase (TPase) is predominantly expressed in monkeys; while uridine phosphorylase (UPase) is superior to TPase in rats. Since TPase is also predominantly expressed in humans, the pharmacokinetic model developed in this study can be used to explain the bioavailability of TAS-102 in humans.
  • KAMIYA H, IIDA E, MURATA(KAMIYA) N, YAMAMOTO Y, MIKI T, HARASHIMA H
    Genes to Cells 8 12 941 - 950 2003年12月 [査読無し][通常論文]
     
    H. Kamiya, E. Iida, N. Murata-Kamiya, Y. Yamamoto, T. Miki, and H. Harashima: Suppression of spontaneous and hydrogen peroxide-induced mutations by a MutT-type nucleotide pool sanitization enzyme, the Escherichia coli Orf135 protein. Genes Cells 8, 941-950 (2003)*
  • H Kamiya, H Akita, H Harashima
    DRUG DISCOVERY TODAY 8 21 990 - 996 2003年11月 [査読無し][通常論文]
     
    During gene therapy the concentration of plasmid DNA or oligonucleotides in the plasma can be quite different from their concentrations in the nucleus or cytosol where they exert their actions. For a better understanding of the apparent discrepancies between pharmacokinetics (PK) and pharmacodynamics (PD), a new concept for intracellular PK with an emphasis on the final efficacy of gene transcription is needed. Here, the conventional PK and intracellular PK and PD of non-viral gene delivery systems are discussed, together with a new concept, referred to as controlled intracellular disposition, which integrates these factors to gain a better understanding of gene expression in the nucleus. The importance of optimizing the system from a transcriptional point of view in the nucleus is also discussed. These new concepts must be integrated to develop an optimized non-viral gene delivery system.
  • M Tanimoto, H Kamiya, N Minakawa, A Matsuda, H Harashima
    BIOCONJUGATE CHEMISTRY 14 6 1197 - 1202 2003年11月 [査読無し][通常論文]
     
    Efficient nuclear entry of exogenous DNA is one of the key factors toward gene therapy success with nonviral vectors. To re-address the effects of a nuclear localization signal (NLS) peptide attached directly to DNA, we prepared three dumbbell-shaped, green fluorescent protein (GFP)-encoding DNAs containing one or two NLS peptides. The peptide was conjugated to the loop-forming oligodeoxyribo-nucleotides by cross-linking reactions between the peptide and a modified uracil base with a dioxaoctylamino linker, and the oligonucleotides were then ligated to the DNA molecules. The NLS-conjugated DNA dumbbells were microinjected into the cytosols and nuclei of simian COS-7 cells. In addition, unconjugated DNA dumbbells, with or without a modified uracil base, were also examined for comparison. The GFP gene was expressed with efficiencies in the order of the unmodified DNA greater than or equal to the NLS-conjugated DNA > the unconjugated DNA with the base modification, with both cytosolic and intranuclear microinjections. Thus, we concluded that (i) one or two NLS peptide(s) did not dramatically improve the nuclear entry of DNA and that (ii) chemical modification of DNA reduced the transcription efficiency or stability in the nucleus.
  • H Matsuo, K Funato, H Harashima, H Kiwada
    JOURNAL OF DRUG TARGETING 11 5 273 - 277 2003年06月 [査読有り][通常論文]
     
    In the elimination of injected liposomes in vivo , it is considered that several serum components play an important role on hepatic uptake of them. This study was conducted to clarify the hepatic uptake mechanism of cetylmannoside (Man)-modified multilamellar vesicles (Man-MLV) using perfused rat liver. In the presence of serum, Man-MLV was taken up by the liver-depending on the serum concentration, and it showed an approximately two-fold higher accumulation than MLV without any surface modifications (PC-MLV). These heptic uptakes of liposomes were obviously inhibited by preheating the serum at 56degreesC for 30 min or by the treatment with anti-rat C3 antiserum. Further, SDS-PAGE followed by immunoblot analysis showed the deposition of iC3b on the opsonized Man-MLV. These results obtained in the present study suggested that hepatic uptake of Man-MLV was mainly mediated by complement receptor rather than mannose receptor on Kupffer cells- in vivo .
  • K Satou, H Harashima, H Kamiya
    NUCLEIC ACIDS RESEARCH 31 10 2570 - 2575 2003年05月 [査読無し][通常論文]
     
    The mutagenicity of an oxidized form of dATP, 2-hydroxydeoxyadenosine 5'-triphosphate (2-OH-dATP), was examined using an SV40 origin-dependent in vitro replication system with a HeLa extract. 2-OH-dATP induced mutations in a dose-dependent manner and elicited substitution and deletion mutations. Of the substitutions, a G.C-->A.T transition including a tandem (CC-->TT) mutation was mainly observed. This result agrees with our previous observation that mammalian DNA polymerase alpha misincorporates the oxidized nucleotide opposite C, but is in contrast to the finding that 2-OH-dATP elicits G.C-->T.A transversions in Escherichia coli. This type of mutation was also elicited, but to a lesser extent. Interestingly, the mutagenicity of 2-OH-dATP was enhanced in the presence of 2-hydroxydeoxyadenosine 5'-diphosphate, an inhibitor of the MTH1 protein, suggesting that this protein functions in the hydrolysis of 2-OH-dATP in the replication reaction mixture, and probably in living cells. These results indicate that 2-OH-dATP is mutagenic and that its mutagenicity is suppressed by the MTH1 protein in mammalian cells.
  • Masatomi Shimizu, Petr Gruz, Hiroyuki Kamiya, Su-Ryang Kim, Francesca M Pisani, Chikahide Masutani, Yusuke Kanke, Hideyoshi Harashima, Fumio Hanaoka, Takehiko Nohmi
    EMBO reports 4 3 269 - 73 2003年03月 [査読有り][通常論文]
     
    Deranged oxidative metabolism is a property of many tumour cells. Oxidation of the deoxynucleotide triphosphate (dNTP) pool, as well as DNA, is a major cause of genome instability. Here, we report that two Y-family DNA polymerases of the archaeon Sulfolobus solfataricus strains P1 and P2 incorporate oxidized dNTPs into nascent DNA in an erroneous manner: the polymerases exclusively incorporate 8-OH-dGTP opposite adenine in the template, and incorporate 2-OH-dATP opposite guanine more efficiently than opposite thymine. The rate of extension of the nascent DNA chain following on from these incorporated analogues is only slightly reduced. These DNA polymerases have been shown to bypass a variety of DNA lesions. Thus, our results suggest that the Y-family DNA polymerases promote mutagenesis through the erroneous incorporation of oxidized dNTPs during DNA synthesis, in addition to facilitating translesion DNA synthesis. We also report that human DNA polymerase eta, a human Y-family DNA polymerase, incorporates the oxidized dNTPs in a similar erroneous manner.
  • Mohamad Radwan Almofti, Hideyoshi Harashima, Yasuo Shinohara, Ammar Almofti, Yoshinobu Baba, Hiroshi Kiwada
    Archives of biochemistry and biophysics 410 2 246 - 53 2003年02月15日 [査読有り][通常論文]
     
    To identify factors affecting cationic liposome-mediated gene delivery efficiency, we studied the relationship between the biophysical characteristics of liposome/DNA complexes (lipoplexes) at different (+/-) charge ratios, their structures as monitored by atomic force microscopy (AFM), and their mechanism(s) of internalization into the cells. Significant changes were observed in the particle size and zeta potential of liposomes and their structures assessed by AFM upon addition of DNA, which depended on (+/-) charge ratios. AFM images showed that lipoplexes were formed from extensively fused and apparently homogeneous lipid particles encapsulating DNA. Lipoplexes were found to internalize the cells through the endocytosis pathway. Lipoplex-cell fusion was found to occur mainly at the plasma membrane level; however, this lipoplex-cell membrane fusion was found to be essential for the uptake of the large particles. A new perspective for the internalization of large lipoplex particles into cytoplasm is discussed.
  • MR Almofti, H Harashima, Y Shinohara, A Almofti, WH Li, H Kiwada
    MOLECULAR MEMBRANE BIOLOGY 20 1 35 - 43 2003年01月 [査読有り][通常論文]
     
    In order to identify factors affecting cationic liposome-mediated gene transfer, the relationships were examined among cationic liposome/DNA complex (lipoplex)-cell interactions, lipoplex size and lipoplex-mediated transfection (lipofection) efficiency. It was found that lipofection efficiency was determined mainly by lipoplex size, but not by the extent of lipoplex-cell interactions including binding, uptake or fusion. In addition, it was found that serum affected mainly lipoplex size, but not lipoplex-cell interactions, which effect was the major reason behind the inhibitory effect of serum on lipofection efficiency. It was concluded that, in the presence or absence of serum, lipoplex size is a major factor determining lipofection efficiency. Moreover, in the presence or absence of serum, lipoplex size was found to affect lipofection efficiency by controlling the size of the intracellular vesicles containing lipoplexes after internalization, but not by affecting lipoplex-cell interactions. In addition, large lipoplex particles showed, in general, higher lipofection efficiency than small particles. These results imply that, by controlling lipoplex size, an efficient lipid delivery system may be achieved for in vitro and in vivo gene therapy.
  • Immunoliposomes having high membrane fluidity delivery a large amount of liposomal doxorubicin into not only doxorubicin sensitive SBC-3 but also doxorubicin resistant SBC-3/ADM cell lines
    Kobayashi, T, Okada, Y, Ishida, T, Sone, S, Harashima, H, Maruyama, K, Kiwada, H
    J. Liposome Res. 13 67  2003年 [査読有り][通常論文]
  • Naomi Nishimoto, Hiroyuki Kamiya, Hideyoshi Harashima, Shunji Izuta
    Nucleic acids research. Supplement (2001) 3 299 - 300 2003年01月01日 [査読無し][通常論文]
     
    To elucidate the behavior of DNA polymerase eta against the oxidized purine nucleotides, we determined the utilization efficiency of 2-hydroxy-dATP and 8-hydroxy-dGTP by the recombinant yeast DNA polymerase eta using the primer extension assay with the synthetic oligonucleotide template-primers, and compared those by DNA polymerase alpha. Results indicate that DNA polymerase eta incorporates 2-hydroxy-dATP opposite template G in addition to template T and 8-hydroxy-dGTP opposite A in addition to C, respectively. Kinetic analysis revealed that the rate of mutation caused by 2-OH-dATP and 8-OH-dGTP with DNA polymerase eta should be much higher than those with DNA polymerase alpha.
  • Kazuya Satou, Hideyoshi Harashima, Hiroyuki Kamiya
    Nucleic acids research. Supplement (2001) 325 - 326 2003年01月01日 [査読無し][通常論文]
     
    The mutagenicity of oxidized dATP, 2-hydroxydeoxyadenosine 5'-triphosphate (2-OH-dATP), was examined using an SV40 origin-dependent in vitro replication system with a HeLa extract. 2-OH-dATP mainly elicited a G x C to A x T transition, and a G x C to T x A transversion to a lesser extent. Interestingly, the mutagenicity of 2-OH-dATP was enhanced in the presence of 2-hydroxydeoxyadenosine 5'-diphosphate, an inhibitor of the MTH1 protein, suggesting that this protein functions in the hydrolysis of 2-OH-dATP in the replication reaction mixture. These results indicate that 2-OH-dATP is mutagenic and that its mutagenicity is suppressed by the MTH1 protein in mammalian cells.
  • R Tachibana, S Futaki, H Harashima, H Kiwada
    LIPOSOMES, PT B 372 349 - 361 2003年 [査読有り][通常論文]
  • H. Kamiya, J. Yamazaki, H. Harashima
    Gene Therapy 9 1500 - 1507 2002年11月01日 [査読無し][通常論文]
     
    We prepared a series of linearized DNA duplexes of various lengths in order to examine the effects of topology and the size of exogenous, plasmid-derived DNAs on transgene expression. These linearized DNA duplexes were capped at each end with a highly stable loop (5'-GCGAAGC-3') to produce a dumbbell-shaped construct that is refractory to exonuclease digestion in comparison to the analogous uncapped DNA duplexes. Intranuclear microinjection of the DNA dumbbells into simian COS-7 cells allowed the expression of the green fluorescent protein (GFP) gene on the linearized molecules, which was expressed five- to 10-fold more than that on the circular DNA of the same size. In addition, the expression by the dumbbell DNA was higher than that by the circular plasmid for at least 14 days. Interestingly, the size of the dumbbell DNA affected the transgene expression upon their microinjection into cell nuclei. The GFP expression efficiency increased with decreasing DNA size below a DNA size of 5.7 kb. The effects of topology and size on the expression of DNAs transfected with cationic lipids are similar to those of DNAs microinjected into cell nuclei. In contrast, microinjection into the cytosol showed the inverse size dependency over a range of 2.3 to 9.4 kb. Thus, transcription of a transgene in the nucleus, but not endocytosis or nuclear entry, was influenced by the exogenous DNA structure, and this was the primary determinant of transgene expression upon transfection under our conditions. These results indicate that small, linearized DNA duplexes that are end-capped with a highly stable loop (dumbbell-shaped DNA) would be very useful for nonviral gene therapy.
  • H Kamiya, Y Fujimura, Matsuoka, I, H Harashima
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 298 4 591 - 597 2002年11月 [査読有り][通常論文]
     
    To visualize the intracellular trafficking of exogenous DNAs delivered by cationic liposomes, rhodamine-labeled DNAs were transfected into NIH3T3 cells and observed by confocal laser microscopy. After 0.5- to 1-h incubations, the DNAs reached the nucleus with a much higher frequency than that expected from the cell division rate. This result suggests that DNAs can enter the nucleus in the presence of the nuclear membrane. Interestingly, some DNAs appeared to extend through the nuclear membrane in the aggregated form which were much larger than the nuclear pore complex. The DNAs which have passed through the nuclear membrane were stained with SYTO 24, a DNA labeling reagent. The stained part may be "naked" DNA that is free of lipids or proteins. This observation indicates that a complex containing DNA fuses with the nuclear membrane and then naked DNA is released into the nucleus. (C) 2002 Elsevier Science (USA). All rights reserved.
  • R Tachibana, H Harashima, N Ide, S Ukitsu, Y Ohta, N Suzuki, H Kikuchi, Y Shinohara, H Kiwada
    PHARMACEUTICAL RESEARCH 19 4 377 - 381 2002年04月 [査読有り][通常論文]
     
    Purpose. A quantitative understanding of the intracellular trafficking of plasmids delivered by nonviral vectors is essential for optimizing vector functions to increase their transfection efficiency. In this study, quantitative methods were developed to measure plasmids delivered to the nucleus, and the relationship between transfection activity and the number of plasmids in the nucleus were analyzed. Methods. AH130 cells were transfected with plasmids in cationic liposomes at various doses. The nuclear fraction was isolated after NP-40 lysis, and the unincorporated plasmids were enzymatically degraded and washed away. Intranuclear plasmids were amplified by quantitative PCR, and the number of plasmids was determined. Plasmid amounts in the nucleus were also measured by Southern analysis to confirm the quantification. Results. Both methods led to similar results in measuring the nuclear plasmids within the same order of magnitude. A remarkable saturation was found for transfection activity vs. number of plasmids in the nucleus, whereas no saturation was observed in nuclear-delivered plasmids vs. dose. Conclusions. These results clearly demonstrate the importance of the quantitative measurement of intracellular trafficking of plasmids after transfection. The findings herein described suggest that efficient transgene expression as well as enhanced nuclear delivery is required in order to achieve the maximal transfection activity of nonviral vectors.
  • Akihito Abe, Yoshimitsu Kiriyama, Masako Hirano, Toshiaki Miura, Hiroyuki Kamiya, Hideyoshi Harashima, Yukiko Tokumitsu, Yukiko Tokumitsu, Yukiko Tokumitsu
    European Journal of Pharmacology 436 1-2 7 - 13 2002年02月 [査読無し][通常論文]
     
    We examined the effects of troglitazone, one of thiazolidinedione derivatives on human basophilic leukemia cell line KU812. Troglitazone caused the suppression of cell growth, which was suggested to result from the decrease in cyclin E and the hyperphosphorylated form of retinoblastoma tumor suppressor gene product (pRb). In addition, troglitazone caused a decrease in histamine secretion due to the reduced expression of histidine decarboxylase mRNA. Peroxisome proliferator-activated receptor (PPAR)-γ mRNA was undetectable by reverse transcription-polymerase chain reaction (RT-PCR) in KU812 cells. These findings suggested that troglitazone suppressed cell growth and histamine synthesis independently of PPARγ. © 2002 Elsevier Science B.V.
  • Ishida, T, Harashima, H, Kiwada, H
    Biosci. Rep. 22 197 - 224 2002年 [査読無し][通常論文]
  • H Harashima, T Ishida, H Kamiya, H Kiwada
    CRITICAL REVIEWS IN THERAPEUTIC DRUG CARRIER SYSTEMS 19 3 235 - 275 2002年 [査読無し][通常論文]
     
    The optimization of drug disposition in the body leads to an increase in its therapeutic effect and to a decrease in adverse effects. Liposomes can serve as a potential drug carrier for achieving this. However, the behavior of a drug carrier system under in vivo conditions is complex. Therefore, a more complete understanding of the pharmacokinetics of liposomes themselves, as well as that of the encapsulated drug, is required. The optimization of the pharmacokinetics of liposomes can be performed by linking a pharmacodynamic model of the free drugs that are encapsulated into liposomes. Sensitivity analysis was applied to optimize the delivery system to maximize the antitumor effect of liposomal doxorubicin (DOX). Advanced technology for ligand-mediated selective targeting and intracellular. targeting is also introduced for antitumor agents and for gene delivery systems.
  • Hiroyuki Kamiya, Laurence Dugué, Hiroyuki Yakushiji, Sylvie Pochet, Yusaku Nakabeppu, Hideyoshi Harashima
    Nucleic acids research. Supplement (2001) 2 85 - 6 2002年 [査読有り][通常論文]
     
    A nucleotide pool sanitizing enzyme, the human MTH1 protein, hydrolyzes 2-hydroxy-dATP, 8-hydroxy-dATP, and 8-hydroxyd-GTP. To examine the substrate recognition mechanism of the MTH1 protein, ten nucleotide analogs (8-bromo-dATP, 8-bromod-GTP, deoxyisoinosine triphosphate, 8-hydroxy-dITP, 2-aminopurine-deoxyriboside triphosphate, 2-amino-dATP, deoxyxanthosine triphosphate, deoxyoxanosine triphosphate, dITP, and dUTP) were incubated with the protein. Of these, the former five nucleotides were hydrolyzed with various efficiencies. This results suggests the importance of the anti/syn-conformation and the functional groups on the 2 and 6-positions of the purine ring.
  • H Kamiya, H Tsuchiya, J Yamazaki, H Harashima
    ADVANCED DRUG DELIVERY REVIEWS 52 3 153 - 164 2001年11月 [査読無し][通常論文]
     
    The objective of this review is to summarize the critical steps in intracellular trafficking and the principal factors involved in transgene expression by a comparison of viral and non-viral gene vectors. Intracellular trafficking of viral and non-viral gene vectors are reviewed kinetically as well as from the mechanistic point of view. The peptide-dependent specific mechanism of endosomal escape by viral vectors is compared with the non-specific mechanism of non-viral vectors. Regarding the nuclear transport of DNA, a number of recently developed strategies in non-viral vectors such as the application of nuclear localization signals or cell specific transcription factors are summarized in comparison with viral nuclear gene delivery. The molecular mechanisms of transcription and the translation of delivered genes to nucleus are also summarized in view of drug delivery systems. This information is intended to serve as a basis for developing a new gene delivery system for both viral and non-viral gene vectors. Optimizing the gene delivery system by integrating this intracellular trafficking as well as transgene expression will be required in order to develop an efficient and an safe gene delivery system. (C) 2001 Elsevier Science B.V. All rights reserved.
  • H Kamiya, N Murata-Kamiya, E Iida, H Harashima
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 288 3 499 - 502 2001年11月 [査読無し][通常論文]
     
    To examine the possibility that the Orf135 protein of Escherichia coli functions as a hydrolyzing enzyme for a damaged DNA precursor (deoxyribonucleoside 5'-triphosphate), we purified the recombinant Orf135 protein and incubated it with oxidized deoxynucleotides. Of the nucleotides tested, 2-hydroxydeoxyadenosine 5'-triphosphate, and somewhat less efficiently, 8-hydroxydeoxyguanosine 5'-triphosphate, were hydrolyzed by this protein. These damaged deoxynucleotides elicit transversion mutations in E. coli (Inoue, M, Kamiya, H., Fujikawa, K., Ootsuyama, Y., Murata-Kamiya, N., Osaki, T., Yasumoto, K., Kasai, IL (1998) J. Biol. Chem. 273, 11069-11074). These results suggest that this protein may be involved in the prevention of mutations induced by these oxidized deoxynucleotides. (C) 2001 Academic Press.
  • S. Futaki, W. Ohashi, T. Suzuki, M. Niwa, S. Tanaka, K. Ueda, H. Harashima, Y. Sugiura
    Bioconjugate Chemistry 12 6 1005 - 1011 2001年11月 [査読有り][通常論文]
  • T Ishida, K Yasukawa, H Kojima, H Harashima, H Kiwada
    INTERNATIONAL JOURNAL OF PHARMACEUTICS 224 1-2 69 - 79 2001年08月 [査読有り][通常論文]
     
    Liposomes composed of hydrogenated egg phosphatidylcholine (HEPC) and cholesterol (CHOL) were found to activate the rat complement (C) system in a CHOL content-dependent manner. Liposomes containing 22 or 33 mol% CHOL activated the C system in a Ca2+-dependent manner, suggesting that C activation occurred via the classical pathway. Liposomes containing 44 mol% CHOL activated the C system in a Ca2+ independent manner, suggesting that C activation occurred via the alternative pathway. The CHOL content appeared to dictate the pathway by which the C system was activated. This C activation was inhibited by removal of serum component(s), which adsorb to the liposomes. Activation of the alternative pathway, induced by the liposomes, was reduced by the depletion of IgG and I-M, whereas the classical pathway activation was reduced by the depletion of IgG, but not IgM. In addition, the removal of adsorbed serum component(s) by treatment with 44 mol% CHOL-containing liposomes decreased serum I.-G and IgM levels that adsorb to the same liposomes, whereas the removal of adsorbed serum component(s) by treatment with 22 mol% CHOL-containing liposomes only slightly decreased serum IgG levels, which adsorbs to the same liposomes. Collectively, both IgG and IgM, which are specifically adsorbed to the liposomes in a CHOL-content dependent manner, were responsible for C activation via the alternative pathway induced by the 44 mol% CHOL containing liposomes. I.-G alone would be partially responsible for C activation via the classical pathway induced by 22 or 33 mol% CHOL-containing liposomes. The discovery of this unique C-activating property of liposomes will be of value in attempts to decipher the underlying mechanism of C activation by providing a useful model membrane system. (C) 2001 Elsevier Science B.V. All rights reserved.
  • TM Huong, T Ishida, H Harashima, H Kiwada
    INTERNATIONAL JOURNAL OF PHARMACEUTICS 215 1-2 197 - 205 2001年03月 [査読有り][通常論文]
     
    In this study, we investigated the contribution of the complement system to the biodistribution of phosphatidylserine (PS)-containing liposomes in rat and guinea pig. It appeared that the inclusion of PS in the liposome formulation accelerates the rate of liposome uptake by liver, resulting in rapid elimination of the liposomes from blood circulation. Pretreatment with K76COOH (K76), an anti-complement agent, decreased the rapid uptake of PS-containing liposomes by guinea pig liver, resulting in increasing blood concentration of the liposomes. Significant complement-dependent liposome destabilization was observed in vitro in both animals, whereas the complement-dependent destabilization in vivo was likely only a part of the process of the clearance of the PS-containing liposomes. This discrepancy suggests that the rate of complement-dependent liposome uptake by liver is much faster than the rate of complement-dependent liposome destabilization in vivo. Pretreatment of K76 dramatically inhibited the binding of C3 fragments, one of dominant opsonins, to PS-containing liposomes in guinea pig under both in vivo and in vitro conditions. This finding suggests that the C3 fragments in the system are responsible for the clearance of the PS-containing liposomes in guinea pig. In rat, in contrast to guinea pig, in vivo binding of C3 fragments was not inhibited by K76-pretreatment, while in vitro binding was inhibited. This discrepancy may be due to different experimental conditions between in vitro and in vivo assay. Nevertheless, based on the observations in this study, the complement components are most likely involved in the clearance of the PS-containing liposomes in rat. Taken together, the activity of PS in enhancing the liposome clearance appears to be mediated by the complement components, presumably C3 fragments, in both guinea pig and rat. This is a first report showing the mechanism on the hepatic uptake of the PS-containing liposomes in guinea pig. (C) 2001 Elsevier Science B.V. All rights reserved.
  • Ishida, T, Harashima, H, Kiwada, H
    Current Drug Metabolism 2 397 - 409 2001年 [査読無し][通常論文]
  • Yoshinobu Baba, Mitsuhiko Shionoya, Hideyoshi Harashima
    European Journal of Pharmaceutical Sciences 13 1 1  2001年 [査読有り][通常論文]
  • Ishida, T, Kojima, H, Harashima, H, Kiwada, H
    Int. J. Pharm. 205 No.1-2 183 - 193 2000年09月15日 [査読有り][通常論文]
     
    The biodistribution of liposomes with two different kind phospholipids (hydrogenated egg phosphatidylcholine and egg phosphatidylcholine) plus cholesterol (CHOL) were investigated after intravenous administration to rats. Elimination of liposomes from blood circulation was affected by the lipid composition. It appeared that the inclusion of CHOL in liposomes accelerates the rate of liposome uptake by liver, resulting in rapid elimination of liposomes. The amount of C3 fragments bound to liposomes was quantitatively determined to assess the contribution of the complement system to liposome accumulation into organs and liposome destabilization in vivo and in vitro. The amount of bound C3 fragments was directly proportional to CHOL content, and the amount was also proportional to the CLh, CLs as well as CLrel. This relationship suggests that the complement system is responsible for the elimination of liposomes from blood circulation, presumably as a consequence of opsonization by C3 fragments and assembly of membrane attack complex (MAC) onto liposomes. In addition, substitution of cholesteryl methyl ether into the liposome formulation for CHOL significantly diminished not only the binding of C3 fragments but also the CLh, CLs and CLrel, resulting in increased mean resident time (MRT) of the liposomes. This result suggests that the hydroxyl-group on CHOL is a binding site for C3 fragments on the liposomes and that CHOL in a liposome formulation promotes the accumulation of liposomes into the liver and spleen, probably due to their uptake by phagocytic cells, and impairs the stability of the liposomes in blood circulation, via a mechanism involving the complement system.
  • T Ishida, H Kojima, H Harashima, H Kiwada
    INTERNATIONAL JOURNAL OF PHARMACEUTICS 205 1-2 183 - 193 2000年09月 [査読有り][通常論文]
     
    The biodistribution of liposomes with two different kind phospholipids (hydrogenated egg phosphatidylcholine and egg phosphatidylcholine) plus cholesterol (CHOL) were investigated after intravenous administration to rats. Elimination of liposomes from blood circulation was affected by the lipid composition. It appeared that the inclusion of CHOL in liposomes accelerates the rate of liposome uptake by liver, resulting in rapid elimination of liposomes. The amount of C3 fragments bound to liposomes was quantitatively determined to assess the contribution of the complement system to liposome accumulation into organs and liposome destabilization in vivo and in vitro, The amount of bound C3 fragments was directly proportional to CHOL content, and the amount was also proportional to the CLh. CLs as well as CLrel. This relationship suggests that the complement system is responsible for the elimination of liposomes from blood circulation, presumably as a consequence of opsonization by C3 fragments and assembly of membrane attack complex (MAC) onto liposomes. In addition, substitution of cholesteryl methyl ether into the liposome formulation for CHOL significantly diminished not only the binding of C3 fragments but also the CLh, CLs and CLI el, resulting in increased mean resident time (MRT) of the liposomes. This result suggests that the hydroxyl-group on CHOL is a binding site for C3 fragments on the liposomes and that CHOL in a liposome formulation promotes the accumulation of liposomes into the liver and spleen, probably due to their uptake by phagocytic cells, and impairs the stability of the liposomes in blood circulation, via a mechanism involving the complement system. (C) 2000 Elsevier Science B.V. All rights reserved.
  • Masamichi Kuwajima, Masahisa Horiuchi, Hideyoshi Harashima, Kang-Mo Lu, Miyuki Hayashi, Masako Sei, Kiyokazu Ozaki, Tomoko Kudo, Hiroshi Kamido, Akira Ono, Takeyori Saheki, Kenji Shima
    FEBS Letters 443 3 261 - 266 1999年01月29日 [査読有り][通常論文]
     
    The long-term administration of L-carnitine was very effective in preventing cardiomegaly in juvenile visceral steatosis (JVS) mice, which was confirmed by heart weight as well as the lipid contents in heart tissue. After i.p. injection of L-carnitine, the concentration of free carnitine in heart remained constant, although serum free carnitine level increased up to 80-fold. On the other hand, a significant increase in short-chain acyl-carnitine level in heart was observed. These results suggest that increased levels of short-chain acyl-carnitine, not free carnitine, might be a key compound in the protective effect of L-carnitine administration in JVS mice. Copyright (C) 1999 Federation of European Biochemical Societies.
  • S Futaki, R Tachibana, M Shono, M Azumano, M Niwa, H Kiwada, H Harashima
    PEPTIDE SCIENCE - PRESENT AND FUTURE 699 - 700 1999年 [査読有り][通常論文]
  • R Tachibana, H Harashima, M Shono, M Azumano, M Niwa, S Futaki, H Kiwada
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 251 2 538 - 544 1998年10月 [査読有り][通常論文]
     
    The objective of this study is to present a rational strategy to target macromolecules to the nucleus via the endocytic pathway. The two major barriers in this route to the nucleus are known as endosomal escape and nuclear transport. pH-sensitive liposomes were used in order to achieve endosomal escape under the conditions of low pH in endosomes. Bovine serum albumin (alb) served as a model compound to be delivered to nucleus and was encapsulated into the pH-sensitive Liposomes. The liposomes are composed of dioleoyl phosphatidyl ethanolamine: cholesterylhemisuccinate. They were taken up by rat peritoneal macrophages via endocytosis and subsequently underwent degradation, principally by lysosomal enzymes. By using pH-sensitive liposomes, intracellular degradation was reduced by a significant extent, as expected, via endosomal escape. Cytosolic delivery of FITC-labelled alb was also detected by confocal microscopy. Selective targeting to the nucleus was performed by adding the nuclear localization signal (NLS) of the SV-40 large T antigen to the FITC-alb, which were then encapsulated into the pH-sensitive liposomes, Confocal microscopy revealed that FITC-alb, in the presence of NLS was successfully delivered into nucleus, while no transport was observed in the absence of NLS. These results provide a useful strategy for the nuclear targeting of macromolecules using pH-sensitive Liposomes in conjunction with NLS, (C) 1998 Academic Press.
  • TM Huong, H Harashima, H Kiwada
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 21 9 969 - 973 1998年09月 [査読有り][通常論文]
     
    The uptake mechanisms of liposomes by rat peritoneal macrophages (PMs) were investigated. Incubation of liposomes with fresh rat serum enhanced the uptake of liposomes depending on the liposome size and cholesterol (CH) content. The binding of liposomes was also enhanced by serum, and this increase depended on the size and CH content as in the case of liposome uptake, which suggested that the binding of opsonized liposomes with PMs govern the extent in liposome uptake. The rate constant for the internalization (k(int)) was calculated by measuring both uptake and binding. The k(int) cannot explain the variation of liposome uptake for different sizes and CH contents. The kint values for liposomes with high (44%) and medium (33%) CH contents were constant (2.5 h(-1)), while those for liposomes with low (22%) CH content were significantly elevated (5-9 h(-1)). These results indicate the presence of at least two kinds of uptake mechanisms of liposomes, Treatment of serum with anti-C3 antibody completely inhibited the enhanced uptake of CH-high, large liposomes, which suggested that complement receptor-mediated phagocytosis may be an uptake mechanism for CH-high and -medium liposomes. In addition, complement-independent enhanced uptake was suggested for CH-low liposomes, since no inhibition was observed for CH-low liposomes by anti-C3 antibody and these liposomes were disintegrated in serum via compliment-independent pathway. These results provided evidence that PMs take up liposomes via complement-dependent and independent mechanisms depending on the CH content of the liposomes.
  • M Yamada, H Harashima, H Kiwada
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 21 9 964 - 968 1998年09月 [査読有り][通常論文]
     
    The size of liposomes is considered to be an important factor in determining the liposome-complement interaction. In this study, the release of carboxyfluorescein (CF) from liposomes was measured continuously for three different diameters (800, 400 and 200 nm) by changing the liposome concentration from 1 to 1000 nmol/ml. At a low liposome concentration range (1-10 nmol/ml), small liposomes (200 nm) released CF to a similar extent (approximately 35%) as in the medium (400 nm) and large (800 nm) liposomes. The affinity (K-m) and capacity (L-max) of a complement system to release liposomally encapsulated CF were estimated by kinetic analysis of the liposome-complement interaction. Surprisingly, there was no remarkable size dependency in the K-m and L-max in terms of liposome number, although these parameters depended on the size of liposomes in terms of lipid concentration. These results indicated the possibility that the complement system does not discriminate according to liposome size.
  • N Mizuno, Y Kato, K Shirota, Y Izumi, T Irimura, H Harashima, H Kiwada, N Motoji, A Shigematsu, Y Sugiyama
    JOURNAL OF HEPATOLOGY 28 5 878 - 885 1998年05月 [査読有り][通常論文]
     
    Background/Aims/Methods: The distribution characteristics of a human colon carcinoma cell line, KM12-HX cells, were examined. After intraportal vein (i.p.v.) or intravenous (i.v.) injection into rats, almost all the injected tumor cells are distributed to liver or lung, respectively, both after 30 s and 30 min. Our previous kinetic analysis of the fate of tumor cells revealed that the cumulative amount of tumor cells distributed in the liver is a factor determining the degree of metastasis, Thus, we examined the mechanism of initial efficient trapping of tumor cells by the liver in more detail. Results: Thirty minutes after tumor cells were injected into the left ventricle of the heart, the distribution of tumor cells was more restricted in several tissues (kidney, small intestine, large intestine and spleen), as compared with the distribution of microspheres undergoing 100% extraction, indicating that the first-pass extraction of KM12-HX cells is incomplete in these organs. The hepatic first-pass distribution of these tumor cells was unaffected by pretreatment of liposomes, such that the preinjected amount was sufficient to saturate the phagocytotic function of macrophages, Thus, the mechanism of initial distribution of the tumor cells to the liver is different from the mechanism of liposome uptake by macrophages. Considering that the diameter of microvessels in sinusoid and KM12-HX cells is approximately 7 and 12 mu m, respectively, it is possible that these turner cells are trapped physically in hepatic microvessels. In fact, after i.p.v. injection of microspheres 5 mu m in diameter, only 20% of the dose was distributed to liver and the rest to other tissues. In contrast, almost 100% of microspheres 10 mu m in diameter were distributed to the liver. Conclusions: These results support the hypothesis that the initial organ distribution of blood-borne tumor cells is determined by mechanical and physical properties of the cells.
  • Takafumi Komori, Kazuki Shimoishi, Hideyoshi Harashima, Masaki Otagiri
    Biological and Pharmaceutical Bulletin 21 12 1376 - 1378 1998年 [査読有り][通常論文]
     
    The goal of the present work was to determine the effect of clarithromycin (CAM) administration on the pharmacokinetic properties of pindolol in rats. The binding of pindolol to serum components increases proportionally with increasing α1-acid glycoprotein (AGP) concentration, indicating that AGP might play a major role in the binding of pindolol. After intravenous administration of pindolol to rats, the CAM-treated group showed a decrease in the volume of distribution, an increase in AUC and no change in the half-life as compared to the control group. Treatment with CAM increased the AGP concentration only. The serum concentration of albumin and creatinine, as well as the metabolic activity of hepatic microsomes towards pindolol, were not altered. Good correlation was observed between the AUC of pindolol in rats and the AGP concentration in serum. Moreover, at 5 min after the administration of an intravenous bolus dose of pindolol to CAM-treated rats, the free concentration of pindolol was lower but the total concentration was higher, compared with the control rats. These results suggested that the influence of CAM on the pharmacokinetic properties of pindolol in CAM-treated rats can be explained by protein binding which, in turn, may be associated with variations in AGP concentration.
  • D. Russell Wada, Sven Björkman, William F. Ebling, Hideyoshi Harashima, Sandra R. Harapat, Donald R. Stanski
    Anesthesiology 87 4 884 - 899 1997年10月 [査読有り][通常論文]
     
    Background: Understanding the influence of physiological variables on thiopental pharmacokinetics would enhance the scientific basis for the clinical usage of this anesthetic. Methods: A physiological pharmacokinetic model for thiopental previously developed in rats was scaled to humans by substituting human values for tissue blood flows, tissue masses, and elimination clearance in place of respective rat values. The model was validated with published serum concentration data from 64 subjects. The model was simulated after intravenous thiopental administration, 250 mg, over 1 min, to predict arterial plasma concentrations under conditions of different cardiac outputs, degrees of obesity, gender, or age. Results: The human pharmacokinetic model is characterized by a steady state volume of distribution of 2.2 1/kg, an elimination clearance of 0.22 l/min, and a terminal half-life of 9 h. Measured thiopental concentrations are predicted with an accuracy of 6 ± 37% (SD). Greater peak arterial concentrations are predicted in subjects with a low versus a high cardiac output (3.1 and 9.4 l/min), and in subjects who are lean versus obese (56 and 135 kg). Acutely, obesity influences concentrations because it affects cardiac output. Prolonged changes are due to differences in fat mass. Changes with gender and age are relatively minor. Conclusions: The physiological pharmacokinetic model developed in rats predicts thiopental pharmacokinetics in humans. Differences in basal cardiac output may explain much of the variability in early thiopental disposition between subjects.
  • Hideyoshi Harashima, William F. Ebling, D. Russell Wada, Donald R. Stanski
    Experimental Gerontology 32 3 315 - 324 1997年05月 [査読有り][通常論文]
     
    With increasing human age (20-80 years), the electroencephalogram (EEG) dose requirement for the intravenous anesthetic thiopental decreases approximately 10% per decade of life. The goal of this study was to compare the dose required to attain isoelectric EEG in young (4-5-month) vs. aged (24-25-month) Fischer 344 rats. One second isoelectricity was found to be an endpoint where minimal cardiorespiratory depression occurred. The effects of age, infusion rate, and repeated administration were examined in nine young and nine old rodents. Thiopental dose requirement increased with increasing infusion rates. Repeated administration at two-day intervals did not demonstrate tolerance to thiopental. No difference in thiopental dose requirement was detected in the young vs. elderly rats. In a separate group of five young and five old rats, thiopental plasma, brain, heart, and CSF concentrations were measured when five seconds of EEG isoelectricity was achieved no consistent differences were noted. The rat may not be an appropriate model to investigate acute age-related anesthetic effects in humans, because cardiovascular changes with age are dissimilar between species.
  • H Harashima, TM Huong, T Ishida, Y Manabe, H Matsuo, H Kiwada
    PHARMACEUTICAL RESEARCH 13 11 1704 - 1709 1996年11月 [査読有り][通常論文]
     
    Purpose. The effect of liposome sim and cholesterol (CH) content on the pharmacokinetics of liposomes was investigated in rats. Methods. The pharmacokinetics of liposomes was examined using 5(6)-carboxyfluorescein (CF) as an aqueous phase marker. The extent of complement activation (EGA) was also measured by the release of CF from liposomes in serum. Results. Both the size and the CH content influenced the mean residence time, total body clearance, and the hepatic uptake clearance (CLh) of liposomes. The increase of the size of liposomes increased the CLh at each CH content. There was no CH dependency of CLh in small liposomes (200 nm in diameter), although the CLh increased with the increase in the CH content in large (800 nm) and medium (400 nm) liposomes. A significant interaction effect was observed between liposome size and the CH content on CLh according to the analysis of variance. The good correlation between CLh and ECA indicated the role of complements as opsonins in enhancing the hepatic uptake of liposomes. The interaction effect between the size and CH content on CLh was explained principally by the product of the size and CH content. Conclusions. A synergistic effect was observed between the size and the CH content on CLh An underlying hypothesis of the synergistic effect was postulated based on the size dependent recognition of liposomes by complement system.
  • H Harashima, S Komatsu, S Kojima, C Yanagi, Y Morioka, M Naito, H Kiwada
    PHARMACEUTICAL RESEARCH 13 7 1049 - 1054 1996年07月 [査読有り][通常論文]
     
    Purpose. The species difference in the pharmacokinetics of liposomes was investigated in mice, rats and rabbits. Methods. Liposomes were intravenously injected at doses of 1, 10 and 100 (nmol/g body weight), and the time courses of liposomes in blood, liver and spleen were measured. Pharmacokinetic parameters were regressed as a function of body weight (BW) and dose of liposomes (D). The uptake mechanism of liposomes was also examined with the isolated perfused liver between rats and mice. Results. Mean residence time increased with the increase of BW and D of liposomes. This increase of mean residence time resulted from the decreased total body clearance, which was principally explained by the species difference in the hepatic uptake clearance (CLh) of liposomes. The parameter CLh was regressed well by a multiple regression as a function of BW and D. In this analysis, an exponent for BW was around 0.5, which clearly indicates that smaller animals have higher uptake clearance per unit BW. Immunohistochemical analysis revealed that there was no significant difference in the density of Kupffer cells among these species. This suggest that the species difference in CLh resulted not from the density of Kupffer cells but from the uptake ability of Kupffer cells amoung species. In the isolated perfused liver, the hepatic uptake of liposomes was mainly explained by opsonin dependent uptake in rats, while opsonin independent uptake in mice. Conclusions. These quantitative and qualitative information on the species difference of liposome disposition will provide an useful information for constructing a drug delivery system using liposomes.
  • Masamichi Kuwajima, Kang-Mo Lu, Hideyoshi Harashima, Akira Ono, Izumi Sato, Akira Mizuno, Takashi Murakami, Hiromu Nakajima, Jun-Ichiro Miyagawa, Mitsuyoshi Namba, Toshiaki Hanafusa, Jun-Ichiro Hayakawa, Yuji Matsuzawa, Kenji Shima
    Biochemical and Biophysical Research Communications 223 2 283 - 287 1996年06月14日 [査読有り][通常論文]
     
    Juvenile visceral steatosis (JVS) mice are associated with systemic carnitine deficiency. In order to investigate the cause of this deficiency, we compared fibroblast carnitine transport activities in normal mice and JVS mice. The kinetic analysis showed that in normal fibroblasts, the Km and Vmax values for saturable uptake was 15.6 μM and 2.56 pmol/min/mg protein, respectively. In JVS fibroblasts, however, saturable uptake was not observed. There was no great difference in the linear component of uptake between normal and JVS fibroblasts. At the physiological concentration (50 μM) of carnitine, the fibroblast carnitine transport activity in JVS mice was decreased to 18% of that in the normal mice. Thus there is hardly any carnitine transport activity in the fibroblasts of JVS mice, indicating that the JVS mouse can be regarded as an animal model of primary carnitine deficiency.
  • D. Russell Wada, Hideyoshi Harashima, William F. Ebling, Eileen W. Osaki, Donald R. Stanski
    Anesthesiology 84 3 596 - 604 1996年03月 [査読有り][通常論文]
     
    Background: The goal of this investigation was to characterize the effects of thiopental on cardiac output and regional blood flows in the rat. Blood flows influence thiopental pharmacokinetics. Acquisition of these data may ultimately permit evaluation of the contribution of thiopental-induced alterations in regional blood flows to the disposition and hypnotic effect of this drug. Methods: Chronically instrumented unrestrained Wistar rats (n = 20) aged 3-4 months received either a dose of thiopental sufficient to induce a brief period of unconsciousness (20 mg · kg2+) or a larger dose achieving electroencephalographic burst suppression (45 mg · kg-1). Cardiac output and blood flows to 14 tissues were determined at 4 times in each rat for a period of 420 min using injections of radioactive microspheres (expressed as mean ± SD). Mean arterial pressure, heart rate, and blood gas tensions were determined at all measurement times. Arterial plasma concentrations were sampled at postinfusion times. Results: No important changes in systemic cardiovascular measurements were detected after the smaller dose of thiopental. One minute after the larger dose, cardiac output decreased from baseline (123 ± 14 to 84 ± 11 ml · min-1, P < 0.01), flow to muscle and fat decreased, and muscle and fat resistance increased. At 5 min, compared to baseline, no difference in cardiac output was detected (123 ± 14 vs. 119 ± 11 ml · min-1), intestinal flows increased, and intestinal resistances decreased. Cardiac output was again depressed at 30, 90, and 180 min. Brain blood flow decreased 25 ± 19% (P < 0.01) from baseline for the duration of the study. Conclusions: Thiopental acutely decreases cardiac output, and blood flows to muscle and fat tissue. The temporary return of cardiac output to baseline may be related to intestinal vasodilation. These blood flow alterations may influence the pharmacokinetics of thiopental.
  • AJ Ferdous, T Ishida, M Shinohara, H Harashima, H Kiwada
    BIOPHARMACEUTICS & DRUG DISPOSITION 17 2 145 - 154 1996年03月 [査読無し][通常論文]
     
    The interaction of liposomes with human plasma was investigated using 6(5)-carboxyfluorescein (CF) as an aqueous phase marker of cetylmannoside-modified multilamellar vesicles (Man-MLVs) of various sizes. The release of CF decreased with increasing liposome concentration. The time courses of the CF release from Man-MLVs were monitored continuously and were analysed kinetically. The curves were characterized by two phases, the first-order release process and the maximum release, which represent the rate and the extent of CF release, respectively. The increase of liposome size increased the rate of release by 42% and the extent of release by 121%, respectively. These effects of liposome size on the release processes were suggested to result from the size-dependent affinities of liposomes to the human complement system. The assay system of liposomally bound fragments of complement component 3 (C3), such as C3b and/or iC3b, was developed by applying a sandwich enzyme-linked immunospecific assay. The percentage of C3 fragments to total proteins bound to liposomes increased with the size of liposomes and there was a good correlation between the extent of CF release and the percentage of C3 fragments bound. These results indicated that Man-MLVs released entrapped CF via activating the human complement system and the affinity of Man-MLV to complement increased with the size of Man-MLVs in human plasma. These in vitro results suggest the role of complement as an opsonin in the disposition of Man-MLVs in humans.
  • Hideyoshi Harashima, Hiroshi Kiwada
    Journal of Liposome Research 6 1 61 - 75 1996年 [査読有り][通常論文]
     
    A useful system was developed for studies on the mechanism of uptake of liposomes by isolated perfused liver. The uptake of multilamellar vesicles (MLV) composed of hydrogenated egg phosphatidylcholine (HEPC), cholesterol and dicetylphosphate (ratio, 5:4:1) by perfused rat liver was enhanced by preincubation with serum. This opsonic activity decreased with increase in the liposome concentration, suggesting the depletion of opsonins. Saturation of hepatic uptake without depletion of opsonins on increasing the liposome concentration was also demonstrated. Liposomes of this composition were shown to activate the complement system in an in vitro release study using 6(5)- carboxyfluorescein (CF), so their uptake via complement receptor was examined after pretreatment of the serum with heat (56°C, 30 min) or antiC3- antiserum. The opsonic activity was completely inhibited by these treatments, suggesting complement receptor-mediated phagocytosis. K76COOH, an inhibitor of C5a formation, also inhibited the opsonic activity, indicating activation of Kupffer cells by formation of C5a. Activation of the complement system by liposomes was shown to depend on the liposome size, those of less than 200 nm showing little release of entrapped 6CF and thus its enhanced hepatic uptake. These results indicate uptake pathways, one opsonin-dependent and the other opsonin-independent. The opsonin-dependent pathway involves complement receptor-mediated phagocytosis, shows saturation and depends on liposome size. The opsonin-independent pathway shows little saturation and independent of liposome size. Cetylmannoside modified MLV, like HEPC-MLV, was found to be taken up by the liver via complement receptor mediated phagocytosis, not through the mannose receptor. We have further examined the species difference in the hepatic uptake of liposomes by comparing the results described above with those in perfused mouse liver. In mice, there was no release of entrapped CF into the serum or enhancement of hepatic uptake by serum, although the uptake clearance per g of liver is much higher than in rats. These results suggest species-dependent differences in the mechanisms of liposome uptake by the liver.
  • S Iida, Y Urakami, H Harashima, H Kiwada
    23RD INTERNATIONAL SYMPOSIUM ON CONTROLLED RELEASE OF BIOACTIVE MATERIALS, 1996 PROCEEDINGS 653 - 654 1996年 [査読有り][通常論文]
  • K Yoshimura, Y Morioka, H Harashima, H Kiwada
    23RD INTERNATIONAL SYMPOSIUM ON CONTROLLED RELEASE OF BIOACTIVE MATERIALS, 1996 PROCEEDINGS 683 - 684 1996年 [査読有り][通常論文]
  • Effect between size and cholesterol content in the enhanced hepatic uptake clearance of liposomes through complement activation in rats
    Harashima, H, Huong, T.M, Ishida, T, Manabe, Y, Matsuo, H, Kiwada, H
    Progress in Drug Delivery System V 93 - 96 1996年 [査読無し][通常論文]
  • K UCHIYAMA, A NAGAYASU, Y YAMAGIWA, T NISHIDA, H HARASHIMA, H KIWADA
    INTERNATIONAL JOURNAL OF PHARMACEUTICS 121 2 195 - 203 1995年07月 [査読有り][通常論文]
     
    The distribution of liposomes with different membrane fluidity and vesicle size in tumors after intravenous injection was investigated in Yoshida sarcoma-bearing rats. Liposomes composed of egg phosphatidylcholine (EPC) or hydrogenated egg phosphatidylcholine (HEPC), dicetyl phosphate and cholesterol in a molar ratio of 5:1:4 were prepared. Their size was adjusted so that they had various mean diameters, ranging from 40 to 400 nm. In EPC liposomes, whose membranes were more fluid than those of HEPC liposomes, tumor accumulation increased with increasing area under the blood concentration-time curve (AUG). The size of liposomes which showed the greatest tumor accumulation and AUC was around 100 nm in diameter. In HEPC liposomes, the less fluid type, the size dependence of tumor accumulation and AUC differed. The greatest tumor accumulation or AUC were found in liposomes with a diameter of about 100 or 40 nm, respectively. This discrepancy indicates that the tumor accumulation of liposomes is not always correlated with their circulation time in the blood. To clarify the process by which these liposomes accumulate from the vascular space into the tumor, we calculated tumor uptake clearance (CL(tu)), which can separate the contribution of the blood concentration from the accumulation in tumor. The CL(tu) values for EPC and HEPC liposomes agreed at all sizes, liposomes with a diameter of 100 nm showing the highest values. These findings indicate that the accumulation of liposomes from the vascular space into the tumor is primarily governed by their size and not by their membrane fluidity or blood circulation time. When tumor blood flow was selectively enhanced by the infusion of angiotensin II, the CL(tu) of 100-nm liposomes decreased to the level of that in 40-nm liposomes, suggesting that some histological factor(s) in the tumor may be responsible for the localization of 100-nm liposomes in tumor. In an in vitro experiment using cultured Yoshida sarcoma cells, 59-nm HEPC liposomes were directly taken up by the tumor cells to an extent at least 2.5-times greater than larger liposomes (greater than or equal to 100 nm). We conclude that 100-nm liposomes may predominantly localize in the interstitial space, whereas some liposomes of smaller size may be taken up by tumor cells.
  • H HARASHIMA, N HIRAI, H KIWADA
    BIOPHARMACEUTICS & DRUG DISPOSITION 16 2 113 - 123 1995年03月 [査読有り][通常論文]
     
    The objective of this study was to quantify and model the degradation process of liposomes in peritoneal macrophages (PMs). Iodinated albumin (I-125-alb) was chosen to be the marker of liposome degradation. The time course of the degradation of free I-125- alb after pinocytosis by PMs followed first-order kinetics with a half-life of 23 min. The degradation of liposomally encapsulated I-125-alb was also quantified. Kinetic modelling of liposome degradation indicated the existence of two kinetically different processes, one with a half-life of 13 min and the other with a half-life of 7.5 h. Comparing the degradation of liposomal and free I-125-alb suggested that I-125-alb was delivered to lysosomes much faster through phagocytosis than pinocytosis. These results indicate that the intracellular degradation kinetics of pinosomes and phagosomes is different. This method can quantify the rate and extent of liposomal degradation in macrophages and provide kinetic information on the intracellular destiny of liposomally encapsulated compounds.
  • H Harashima, T Hiraiwa, Y Ochi, H Kiwada
    JOURNAL OF DRUG TARGETING 3 4 253 - 261 1995年 [査読有り][通常論文]
     
    The degradation of liposomes in blood circulation is important in regulating the releasing rate of encapsulated compounds. In this study, the effect of liposome size-one of the principal determining factors in liposome disposition-on their degradation in serum/blood was evaluated quantitatively both in vitro and in vivo. In the in vitro study, the time courses of the degradation of liposomes in fresh rat serum were measured continuously using 5(6)-carboxyfluorescein (CF) as an aqueous phase marker and were described by the kinetic model with the lag time (tau), first order degradation rate constant (k), and the maximum degradation (alpha). Both k and alpha increased with the increase of liposome size, which indicated a higher affinity of larger liposomes for complement activation. In the in vivo study, the degradation of liposomes was evaluated sensitively by a first order degradation rate constant (kd) in blood circulation. The kd was obtained by kinetically modeling the liposome degradation in vivo using H-3-inulin as an aqueous phase marker. The size dependent kd correlated well with the hepatic uptake clearance, which suggests an underlying complement activation mechanism common to both degradation and hepatic uptake of liposomes. There was a good correlation in the degradation rate constant between in vitro and in vivo trials. These kinetic analyses validate the quantitative evaluation of liposome degradation in blood circulation and provide a useful way to predict the degradation of liposomes in vivo from in vitro experiments.
  • Size dependent characterization of cetylmannoside-modified liposomes (Man-MLVs) in human plasma
    Ishida, T, Ferdous, A.J, Shinohara, M, Harashima, H, Kiwada, H
    J.Liposome Res. 6 252  1995年 [査読無し][通常論文]
  • H HARASHIMA, Y OCHI, H KIWADA
    BIOPHARMACEUTICS & DRUG DISPOSITION 15 3 217 - 225 1994年04月 [査読有り][通常論文]
     
    The purpose of this study is to propose a new method for quantitative evaluation of liposome degradation in serum. The time course of liposome degradation in rat serum was monitored continuously, using 6(5)-carboxyfluorescein as an aqueous phase marker. The degradation curves exhibited three characteristic phases: lag time, degradation, and plateau. This curve was described by a kinetic model with three parameters: lag time (tau), first-order degradation rate constant (k), and maximum degradation (alpha). The rate and extent of the degradation of liposomes were evaluated separately in terms of k and alpha, respectively. The effects of size and concentration of liposomes on their degradation kinetics were examined using this method. Both k and alpha increased with increasing liposomal size. The increased affinity of larger liposomes for complement was suggested to increase both k and alpha. On the other hand, alpha decreased with increasing liposomal concentration without altering k. The decreased extent of degradation was considered to result from the depletion of complement components. There was no significant effect of size and concentration of liposomes on tau. Quantitative evaluation of the rate and extent of degradation of liposomes will provide deeper insights into the interaction between liposomes and serum components, and basic information on liposomes as potential drug carriers.
  • H HARASHIMA, K SAKATA, K FUNATO, H KIWADA
    PHARMACEUTICAL RESEARCH 11 3 402 - 406 1994年03月 [査読有り][通常論文]
     
    The objective of this study was to differentiate the roles of opsonins and phagocytic cells in the size-dependent hepatic uptake of liposomes in the submicron region. The extent of opsonization decreased with the decrease in size of liposomes (from 800 to 200 nm in diameter) and no enhancement of uptake was observed at 200 nm. There was no effect of liposome size on the uptake of unopsonized liposomes. Serum was pretreated with empty liposomes of each size and its opsonic activity was measured in the perfused liver. The small liposomes could not consume the opsonic activity, while the larger ones did so substantially. These results suggest that opsonins bind to liposomes depending on the size of liposomes and phagocytic cells take up liposomes in proportion to the extent of opsonization. Size-dependent liposome degradation in serum was also found, which was consistent with the size-dependent complement activation, because liposomes with this composition have been shown to be degraded by complement. The mechanism of opsonization was examined by treating serum at 56 degrees C for 30 min or with anti-C3 antiserum. Since both treatments inhibited the opsonic activity, the hepatic uptake of liposomes is considered to occur via complement receptor. In conclusion, the size of liposomes affected complement recognition, and the liposomes were taken up by the liver depending on the extent of opsonization.
  • 松尾 浩民, 船戸 耕一, 山下 親正, 原島 秀吉, 際田 弘志
    薬物動態 9 118 - 121 The Japanese Society for the Study of Xenobiotics 1994年 
    In the elimination of injected liposomes in vivo, it is considered that several serum components play an important role on hepatic uptake of them. This study was conducted to clarify the hepatic uptake mechanism of cetylmannoside (Man) -modified multilamellar vesicles (Man-MLV) using perfused rat liver. In the presence of serum, Man-MLV was taken up by the liver depending on the serum concentration, and it showed an approximately 2-fold higher accumulation than non-modified MLV (PC-MLV). These hepatic uptake of liposomes were obviously inhibited by preheating the serum at 56 °C for 30 min or by the treatment with anti-rat C3 antiserum. Further, SDS-PAGE followed by immunoblot analysis showed the deposition of iC3b on the opsonized Man-MLV. These results obtained in the present study suggested that hepatic uptake of Man-MLV was mainly mediated by complement receptor rather than mannose receptor on Kupffer cells in vivo.
  • H MATSUO, K FUNATO, H HARASHIMA, H KIWADA
    JOURNAL OF DRUG TARGETING 2 2 141 - 146 1994年 [査読有り][通常論文]
     
    In the elimination of injected liposomes in vivo, it is considered that several serum components play an important role on hepatic uptake of them. This study was conducted to clarify the hepatic uptake mechanism of cetylmannoside (Man)-modified multilamellar vesicles (Man-MLV) using perfused rat liver. In the presence of serum, Man-MLV was taken up by the liver depending on the serum concentration, and it showed an approximately two-fold higher accumulation than MLV without any surface modifications (PC-MLV). These heptic uptakes of liposomes were obviously inhibited by preheating the serum at 56-degrees-C for thirty minutes or by the treatment with anti-rat C3 antiserum. Further, SDS-PAGE followed by immunoblot analysis showed the deposition of iC3b on the opsonized Man-MLV. These results obtained in the present study suggested that hepatic uptake of Man-MLV was mainly mediated by complement receptor rather than mannose receptor on Kupffer cells in vivo.
  • H HARASHIMA, Y MIDORI, S OHSHIMA, K YACHI, H KIKUCHI, H KIWADA
    BIOPHARMACEUTICS & DRUG DISPOSITION 14 7 595 - 608 1993年10月 [査読有り][通常論文]
     
    The objective of this study is to perform kinetic modelling of the tissue distribution of doxorubicin encapsulated into liposomes (L-DXR), especially to the heart and liver. The release process of doxorubicin (DXR) from liposomes in blood was quantified by a release clearance. This parameter defines a release rate of DXR based on the concentration of L-DXR in blood and was estimated from kinetic modelling of DXR distribution to the heart after L-DXR administration. The distribution of free DXR to the heart was modelled separately. The experimental data for this modelling were reported previously (Harashima et al., Biopharm. Drug. Disposit., 13, 155-170 (1992)). This analysis provided a free DXR concentration profile as well as a release clearance of DXR after L-DXR administration. There was a remarkable difference in the free DXR concentration in blood between free and liposomal administration. The area under the DXR curve in the heart was reduced by approximately one third from that for the first two hours after DXR administration by liposomal encapsulation, which could be the reason for reduced cardiac toxicity. In our previous report, the distribution of L-DXR to the liver was shown to be explained by a sequentially linked two-compartment model with efflux process. The validity of this efflux model was examined in this study by a repeated dose study. The apparent uptake clearance decreased with time and showed a second peak after the repeated dose, which justified the efflux model. These kinetic analyses give quantitative understanding of the effect of liposomal encapsulation on the tissue distribution of DXR.
  • Sven Björkman, Donald R. Stanski, Hideyoshi Harashima, Robert Dowrie, Sandra R. Harapat, D.Russell Wada, William F. Ebling
    Journal of Pharmacokinetics and Biopharmaceutics 21 3 255 - 279 1993年06月 [査読有り][通常論文]
     
    Traditionally, physiological pharmacokinetic models assume that arterial blood flow to tissue is the rate-limiting step in the transfer of drug into tissue parenchyma. When this assumption is made the tissue can be described as a well-stirred single compartment. This study presents the tissue washout concentration curves of the two opioid analgesics fentanyl and alfentanil after simultaneous 1-min iv infusions in the rat and explores the feasibility of characterizing their tissue pharmacokinetics, modeling each of the 12 tissues separately, by means of either a one-compartment model or a unit disposition function. The tissue and blood concentrations of the two opioids were measured by gas-liquid chromatography. The well-stirred one-compartment tissue model could reasonably predict the concentration-time course of fentanyl in the heart, pancreas, testes, muscle, and fat, and of alfentanil in the brain and heart only. In most other tissues, the initial uptake of the opioids was considerably lower than predicted by this model. The unit disposition functions of the opioids in each tissue could be estimated by nonparametric numerical deconvolution, using the arterial concentration times tissue blood flow as the input and measured tissue concentrations as the response function. The observed zero-time intercepts of the unit disposition functions were below the theoretical value of one, and were invariably lower for alfentanil than for fentanyl. These findings can be explained by the existence of diffusion barriers within the tissues and they also indicate that alfentanil is less efficiently extracted by the tissue parenchyma than the more lipophilic compound fentanyl. The individual unit disposition functions obtained for fentanyl and alfentanil in 12 rat tissues provide a starting point for the development of models of intratissue kinetics of these opioids. These submodels can then be assembled into full physiological models of drug disposition. © 1993 Plenum Publishing Corporation.
  • H HARASHIMA, C YAMANE, Y KUME, H KIWADA
    JOURNAL OF PHARMACOKINETICS AND BIOPHARMACEUTICS 21 3 299 - 308 1993年06月 [査読有り][通常論文]
     
    The objective of this study was to examine the AUC dependency of saturable hepatic clearance (CL(h)) of liposomes and to postulate a mathematical model to describe the characteristics. The AUC dependency of saturable CL(h) was examined wider intravenous rapid administration at various doses. The CL(h) increased with increasing blood concentration but decreased with the increase of AUC at each dose. In addition, the relationship between AUC and CLA(h) was consistent with that observed in previously reported infusion studies. These experimental data confirm the AUC dependency of saturable CL(h) of liposomes. A mathematical model was developed for this, AUC dependency. The decrease of CL(h) was described by the uptake amount (X) as follows: CL(h) = CL(m)(1 - X/X(m)), where CL(m) and X(m) represent the maximum uptake clearance and the maximum uptake amount, respectively. The rate equation for uptake was analytically solved as CL(h) = X/AUC = X(m)/AUC(1 - exp(CL(m)/X(m)AUC)). Uptake clearance can be described by CL(m), X(m), and AUC, and so uptake clearance is constant if AUC is constant. These experimental analyses and theoretical considerations show the validity of the AUC-dependent saturable CL(h) of liposomes.
  • H HARASHIMA, Y KUME, C YAMANE, H KIWADA
    BIOPHARMACEUTICS & DRUG DISPOSITION 14 3 265 - 270 1993年04月 [査読有り][通常論文]
     
    The aim of this study is to develop a kinetic model for the quantitative evaluation of, and to examine dose dependency in liposome degradation in blood circulation in vivo. Multilamellar liposomes labeled with H-3-inulin were administered intravenously into rats and the time courses of blood concentration and urinary excretion of H-3-inulin were measured. The dosages of liposomes were fixed at 1, 5, and 100 mumolPCkg-1. Remarkable saturation was found in the time courses of both blood concentration and urinary excretion. Then a kinetic model for the degradation of liposomes in blood was developed, assuming that the degradation follows the first order rate process for each dose. The model fitted the observed time courses of excreted H-3-inulin well, and dose dependency could be observed in the rate constants for liposome degradation, which are more sensitive than urinary excretion of H-3-inulin. The degradation rate constant correlated well with the uptake rate constant, which suggests the same underlying mechanism for both uptake and degradation. These results indicate the usefulness of kinetic modeling in the quantitative evaluation of liposome degradation in blood circulation in vivo.
  • H HARASHIMA, K SAKATA, H KIWADA
    PHARMACEUTICAL RESEARCH 10 4 606 - 610 1993年04月 [査読有り][通常論文]
     
    Opsonins play a role in the hepatic uptake of particles such as bacteria, lipid emulsion, and liposomes. The objective of this study was to distinguish between opsonin depletion and uptake saturation in the dose-dependent hepatic uptake of liposomes. The uptake of opsonized and unopsonized liposomes was determined in the isolated perfused liver. Serum (2.9 mL) was required to opsonize 1 mumol liposomes fully, indicating that a rat (250 g with 10 mL of serum) can opsonize 3.5 mumol liposomes. Next the dose effect on hepatic uptake of opsonized and unopsonized liposomes was examined. Saturation of uptake was found only for the opsonized liposomes. On the other hand, the hepatic uptake clearance decreased dose dependently from 4.31 to 0.79 (mL/min), with increasing doses from 0.075 to 17 mumol/250 g, respectively, after i.v. administration. Thus, the decrease in the hepatic uptake clearance at the medium dose was due to the saturation of uptake alone, and at the high dose it was due to opsonin depletion as well. These results show that the saturation of liposomal uptake in the liver and the depletion of opsonins occurred at different liposome dosage levels.
  • 原島 秀吉, 越智 良仁, 平岩 智子, 杉野 香織, 際田 弘志
    薬物動態 8 751 - 754 The Japanese Society for the Study of Xenobiotics 1993年 
    Kinetic analysis of the hepatic uptake of liposomes was performed in comparison to the degradation of liposomes in blood and liver. The degradation clearance of liposomes in blood was shown to depend on the size of liposomes. In vitro kinetic study on the degradation of liposomes suggested the mechanism of this size dependency due to the Increased affinity of liposomes for complement. There was two kinds of hepatic uptake components of liposomes, one is a size dependent opsonized pathway, and the other is a size Independent unopsonized pathway. The size dependent opsonization of liposomes was shown to result from the size dependent complement activation and this component explained the size dependended hepatic uptake clearance of liposomes in vivo. The kinetic analysis of the hepatic degradation of liposomes was also performed by labeling liposomes with both 125I-albumin and 3H-cholesterylhexadecylether. Although the mechanism of hepatic uptake are considered to be different depending on the size of liposomes, the kinetics of hepatic degradation of liposomes was same for each size of liposomes and lay in the order of hour.
  • H HARASHIMA, S OHSHIMA, Y MIDORI, K YACHI, H KIKUCHI, H KIWADA
    BIOPHARMACEUTICS & DRUG DISPOSITION 13 3 155 - 170 1992年04月 [査読有り][通常論文]
     
    The purpose of this study was to perform a kinetic analysis of the tissue distribution of doxorubicin (DXR) and liposomes separately after intravenous administration of DXR entrapped in liposomes in rats. Liposomes were double labeled with 14C-DXR (L-DXR) and 3H-inulin (L-INU). Blood and tissues were sampled at specified times until 120 min. Blood clearance of L-DXR was similar to that of L-INU. Distribution of both L-DXR and L-INU into the liver was parallel and extensive, while in the heart, the pattern of distribution differed between L-DXR and L-INU after peak concentration. Time courses of tissue concentration were explained well by dividing tissue into a shallow compartment with efflux and a deep compartment without efflux. In the liver, pharmacokinetic parameters of L-DXR and L-INU were similar, and the two kinetically different compartments may correspond to different uptake processes in hepatic endocytosis. In the heart, the shallow compartment was considered to correspond to the cardiac vascular space, and the intercompartmental rate constant (k3) for L-DXR was much larger than that for L-INU. The estimated half-life for this process was 20 min. The half-life for the degradation of liposomes in blood circulation was also estimated at 20 min from data on the urinary excretion of released 3H-inulin. These results suggest that the release of DXR from liposomes may be the rate-limiting process in the tissue distribution of DXR to the heart.
  • Hideyoshi Harashima, Michio Mamiya, Masayo Yamazaki, Yasufumi Sawada, Tatsuji Iga, Manabu Hanano, Yuichi Sugiyama
    Pharmaceutical Research: An Official Journal of the American Association of Pharmaceutical Scientists 9 12 1607 - 1611 1992年 [査読有り][通常論文]
     
    The significance of the binding to Na,K-ATPase in the tissue distribution of ouabain was previously documented (Harashima et al., Pharm. Res. 9:474-479, 1992). The purpose of this study was to obtain a kinetic model of ouabain tissue distribution. In most tissues, the ouabain concentration continued to rise after the termination of infusion (5 min), with the peak tissue concentration at approximately 20 min. This delay could not be explained by the rapid equilibrium model (RE model), nor could the kinetics of ouabain be explained by an RE model modified for saturable binding. Since ouabain binding to Na,K-ATPase is slow, the association and dissociation processes were incorporated into a model that can accurately fit the observed time courses of ouabain. The obtained binding parameters corresponded well with the observed values in the in vitro binding experiments, except for muscle. These results quantitatively support the role of the slow and saturable binding of ouabain to Na,K-ATPase in its tissue distribution. © 1992, Plenum Publishing Corporation. All rights reserved.
  • Hideyoshi Harashima, Michio Mamiya, Masayo Yamazaki, Yuichi Sugiyama, Yasufumi Sawada, Tatsuji Iga, Manabu Hanano
    Pharmaceutical Research: An Official Journal of the American Association of Pharmaceutical Scientists 9 4 474 - 479 1992年 [査読有り][通常論文]
     
    Ouabain binds specifically to Na,K-ATPase on the plasma membrane and therefore serves to measure the tissue concentration of Na,K-ATPase. We examined the role of ouabain binding to Na,K-ATPase in its overall tissue distribution. The tissue-to-plasma concentration ratio (Kp,vivo) was defined in each tissue after intravenous administration of 3H-ouabain in guinea pigs, and specific binding of ouabain to Na,K-ATPase was measured in tissue homogenate to obtain the dissociation constant and binding capacity in each tissue. A predicted tissue-to-plasma concentration ratio (Kp,vitro) was calculated using the obtained binding parameters and the volume of extracellular space in each tissue. The absolute values of Kp,vitro were comparable to those of Kpvivo, except in brain. Regression analysis showed that the specific binding capacity of Na,K-ATPase in each tissue is the main factor in the tissue variation of Kp,vivo. Therefore, the binding of ouabain to Na,K-ATPase plays a significant role in the tissue distribution of ouabain. © 1992, Plenum Publishing Corporation. All rights reserved.
  • Y KUME, F MAEDA, H HARASHIMA, H KIWADA
    JOURNAL OF PHARMACY AND PHARMACOLOGY 43 3 162 - 166 1991年03月 [査読有り][通常論文]
     
    Multilamellar vesicles (300-350 nm) were infused into the rat femoral vein at the rate of 4, 40 and 400 nmol phosphatidycholine min-1 for 6 h using [H-3]inulin as an aqueous marker. The time courses of blood concentration of vesicles, normalized for infusion rate, were not superimposable, showing the non-linearity of liposome disposition in the blood circulation. These time courses of blood concentration were well fitted by a single Michaelis-Menten equation. On the other hand, the time courses of tissue content could not be so accommodated. Additionally, the observed relationship between the uptake of liposomes by the liver and their clearance from it and other organs differed essentially from a simulation based on Michaelis-Menten type saturable kinetics. Therefore, it is suggested that there is a time-dependent non-Michaelis-Menten type process in the phagocytosis of macrophages in the reticuloendothelial system.
  • Youn Bok Chung, Seiji Miyauchi, Yuichi Sugiyama, Hideyoshi Harashima, Tatsuji Iga, Manabu Hanano
    Journal of Pharmacokinetics and Biopharmaceutics 18 4 313 - 333 1990年08月 [査読有り][通常論文]
     
    The dose dependency in the hepatic transport of an anionic fluorescent dye, 1-anilino-8-naphthalene sulfonate (ANS), was investigated by measuring the plasma disappearance and biliary excretion in rats. Bulk of the administered ANS distributed into the liver at 10 min after iv bolus injection. The plasma disappearance curves of ANS were then kinetically analyzed based on a two-compartment model, in which the ligand is eliminated only from the peripheral compartment (liver compartment). The total body clearance (CLtot) decreased with increasing dose of ANS. That is, the values of CLtot were 4.06 and 1.98 ml/min/per kg at the doses of 3 and 100 Μmol/kg, respectively. The clearances of the uptake and sequestration processes (CLup and CLseq, respectively) for a total ligand were constant irrespective of dose, while the efflux clearance (CLeff) for a total ligand was increased by twofold with increasing dose. A mechanism for the increase in the CLeff value might be explained by a saturation of the ANS binding to the intracellular proteins. The hepatocellular distribution and the binding of ANS to cytosolic proteins were then determined. ANS mainly distributed to the cytosol fraction, and the unbound fraction in the cytosol increased from approximately 0.04 to 0.09 when the cytosolic concentrations of ANS increased from 40 to 900 ΜM, respectively. In,spite of such increase in the unbound fraction in the cytosol, the CLseq values remained unchanged with increasing dose, suggesting that the saturation of sequestration clearance for unbound ANS might occur. Furthermore, the plasma disappearance curves of ANS at various doses were simultaneously analyzed based on three nonlinear kinetic models: Model I is a model incorporating both saturable intracellular binding and saturable sequestration Model II is a model incorporating only saturable intracellular binding Model III is the model incorporating only saturable sequestration. Goodness- of- fit evaluated by AIC value was best for Model I. Taken together, the nonlinearity in the plasma clearance of ANS was confirmed to be attributed to saturation of both its binding to cytosolic proteins and sequestration process. © 1990 Plenum Publishing Corporation.
  • Youn Bok Chung, Seiji Miyauchi, Yuichi Sugiyama, Hideyoshi Harashima, Tatsuji Iga, Manabu Hanano
    Journal of Hepatology 11 2 240 - 251 1990年 [査読有り][通常論文]
     
    The effects of various organic anions on the hepatic transport of an anionic fluorescent dye, 1-anilino-8-naphthalene sulfonate (ANS) were investigated by measuring the plasma disappearance-time profiles in rats. Ten min after the i.v. administration of ANS (3 μmol/kg), various organic anions (60 μmol/kg) were injected in a bolus. Sulfobromophthalein (BSP),bromophenol blue (BPB) and rose bengal (RB) induced a transient increase in the plasma concentration of ANS (the so-called 'counter-transport' phenomena). The effect of rose bengal was somewhat different. After the administration of rose bengal, the plasma concentration of ANS decreased rapidly followed by a gradual increase. On the other hand, after the administration of bilirubin and taurocholate, the transient increases in plasma ANS concentrations were minimal. No effect was observed after the administration of phenolsulfophthalein (PSP) or oleate. The effects of these organic anions on the binding of ANS to rat liver cytosols were examined by equilibrium dialysis. Sulfobromophthalein, bromophenol blue and rose bengal, which yielded an in vivo 'counter-transport' phenomena, markedly inhibited ANS binding to cytosolic proteins. On the other hand, the other organic anions examined had very small, if any, inhibitory effect. The ANS binders in the cytosol were then identified by gel filtration. ANS bound mainly to X and Y (ligandin) fractions in the cytosol. Sulfobromophthalein, which is one of the organic anions exhibiting the in vivo 'counter-transport' phenomenon, remarkably inhibited ANS binding to ligandin fraction. It was thus suggested that the in vivo 'counter-transport' phenomena may be also explained by the enhancement of back diffusion due to the displacement of intracellular binding. In conclusion, one should be more cautious in interpreting data obtained from so-called in vivo 'counter-transport' experiments. © 1990.
  • H HARASHIMA, Y SUGIYAMA, T IGA, M HANANO
    DRUG METABOLISM AND DISPOSITION 16 4 645 - 649 1988年07月 [査読有り][通常論文]
  • H HARASHIMA, Y SUGIYAMA, Y SAWADA, K SHIGENOBU, Y KASUYA, T IGA, M HANANO
    CHEMICAL & PHARMACEUTICAL BULLETIN 35 7 2923 - 2927 1987年07月 [査読有り][通常論文]
  • H HARASHIMA, Y SAWADA, Y SUGIYAMA, T IGA, M HANANO
    JOURNAL OF PHARMACOKINETICS AND BIOPHARMACEUTICS 13 4 425 - 440 1985年08月 [査読有り][通常論文]
  • Y SAWADA, M HANANO, Y SUGIYAMA, H HARASHIMA, T IGA
    JOURNAL OF PHARMACOKINETICS AND BIOPHARMACEUTICS 12 6 587 - 596 1984年 [査読有り][通常論文]

書籍

  • THE創薬 小資源国家にっぽんの生きる道
    (担当:分担執筆範囲:薬物送達システムから革新的医薬品の創出へ)
    日本薬学会編、薬事日報社 2021年03月
  • DDSキャリア設計入門
    原島秀吉 (担当:編者(編著者)範囲:編集と総論の執筆)
    丸善出版株式会社 2019年01月
  • Mitochondrial Biology and Experimental Therapeutics
    山田勇磨, 原島秀吉 (担当:分担執筆範囲:Targeting the Mitochondrial Genome Through a Nanocarrier and the Regulation of Mitochondrial Gene Expression)
    Springer International Publishing Switzerland 2018年
  • 製剤学(改訂第7版)
    原島秀吉 (担当:共著範囲:薬物速度論)
    南江堂 2017年
  • パートナー薬剤学(改訂第3版)
    原島秀吉, 伊藤智夫, 寺田勝英 (担当:共編者(共編著者)範囲:編集、序章薬剤学総論と「分布」の執筆)
    南江堂 2017年
  • 医療・診断・創薬の化学 医療分野に挑む革新的な化学技術
    原島秀吉 (担当:共著範囲:新しい遺伝子キャリア:多機能性エンベロープ型ナノ構造体の創製)
    日本化学会編 2017年
  • スタンダード薬学シリーズII
    原島秀吉 (担当:共著範囲:VI.薬の生体内運命 第6章 薬物速度論)
    東京化学同人 2016年
  • Pharmacology of Mitochondria. Handbook of Experimental Pharmacology 240
    原島秀吉 (担当:分担執筆範囲:MITO-Porter for Mitochondrial Delivery and Mitochondrial Functional Analysis)
    Springer International Publishing Switzerland 2016年
  • 生物薬剤学(改訂第3版)
    原島秀吉 (担当:共著範囲:生理学的モデル)
    南江堂 2015年
  • 公益社団法人日本薬剤学会薬剤学概史 私はこう見る120人による俯瞰図
    原島秀吉 (担当:共訳範囲:薬剤学発展に影響をお呼びした会議、セミナーと国立大学 北海道大学)
    公益社団法人日本薬剤学会 2015年
  • Mitochondrial Medicine, Volume II, Manipulating Mitochondrial Function. (Methods in Molecular Biology 1265 Springer Protocols)
    山田勇磨, 原島秀吉 (担当:分担執筆範囲:Targeting the mitochondrial genome via a dual function MITO-oter: Evaluation of mtDNA levels and mitochondrial function)
    Humana Press 2015年
  • 遺伝子治療・診断の最先端技術と新しい医薬品・診断薬の開発
    秋田英万, 原島秀吉 (担当:分担執筆範囲:非ウイルスベクターでの適切なプロトコール、トラブル対策)
    技術情報協会 2014年
  • 薬剤学実験法必携マニュアル~Pharmaceutical Scientistのために~ II 生物薬剤学
    秋田英万, 原島秀吉 (担当:分担執筆範囲:7.1 核酸製剤)
    南江堂 2014年
  • 非経口投与製剤の開発と応用~次世代型医薬品の新規投与携帯の開拓を目指して~
    中村孝司, 梶本和昭, 原島秀吉 (担当:分担執筆範囲:遺伝子の体内動態・細胞内動態制御システムの構築とその最適化)
    株式会社シーエムシー出版 2013年
  • 応用が拡がるDDS
    秋田英万, 山田勇磨, 畠山浩人, 林泰弘, 原島秀吉 (担当:分担執筆範囲:第6章 遺伝子治療におけるDDS. 1. 非ウイルスベクター)
    株式会社エヌ・ティー・エス 2013年
  • 毛髪再生の最前線
    小暮健太朗, 原島秀吉 (担当:分担執筆範囲:人工遺伝子デリバリーシステムMENDによる毛孔内へのBMPR1A遺伝子の送達)
    株式会社シーエムシー出版 2013年
  • ファインケミカルシリーズ、ペプチド医薬の最前線
    梶本和昭, 原島秀吉 (範囲:第3章 ペプチド創薬に向けてー製剤化・安定化・投与法ー、8 脂肪血管指向性ペプチドを搭載したナノデバイスによる肥満治療の新戦略)
    シーエムシー出版 2012年
  • 先端バイオマテリアルハンドブック
    小暮健太朗, 林泰弘, 原島秀吉 (担当:分担執筆範囲:第3章 DDS用バイオマテリアル 第5節多機能性エンベロープ型ナノ構造体による遺伝子デリバリー)
    株式会社エヌ・ティー・エス 2012年
  • ドラッグデリバリーシステム(高分子学会 編集)
    中村孝司, 原島秀吉 (担当:分担執筆範囲:遺伝子治療DDS」最先端材料システムone pointシリーズ(9巻))
    共立出版 2012年
  • 原島 秀吉 (担当:編者(編著者))
    南江堂 2011年12月 (ISBN: 4524402861) 458
  • 原島 秀吉 
    南江堂 2011年 (ISBN: 9784524402861)
  • 居城 邦治, 有賀 寛万, 五十嵐 靖之, 伊藤 悦朗, 上出 利光, 長田 義仁, 小野江 和則, 小林 淳一, 志田 壽利, 高田 賢三, 田村 守, 西村 伸一郎, 野口 昌幸, 畠山 昌則, 原島 秀吉, 松田 彰, 研究成果編集委員会 (担当:共著)
    北海道大学図書刊行会 2007年03月 (ISBN: 4832981781) 355
  • 高畑 雅一, 原島 秀吉, 田中 勲, 辻井 薫, 出村 誠, 永井 健治, 中村 貴義, 山口 淳二, 守内 哲也, 金城 政孝, 芳賀 永, 長田 義仁, 稲垣 冬彦, 山下 正兼, 田中 一馬, 笹木 敬司, 下村 政嗣, 鈴木 範男, 土田 義和, 研究成果編集委員会 (担当:共著)
    北海道大学図書刊行会 2007年03月 (ISBN: 4832981773) 363
  • 原島 秀吉, 田畑 泰彦, 原島 秀吉, 田畑 泰彦 (担当:共編者(共編著者))
    メディカルドゥ 2006年04月 (ISBN: 4944157355) 268

講演・口頭発表等

  • Multifunctional Envelope-type Nano Device for Gene Delivery: Application for Nanomedicine  [招待講演]
    原島秀吉
    大塚創薬化学シンポジウム2018、徳島 2018年11月 口頭発表(招待・特別)
  • 多機能性エンベロープ型ナノ構造体の創製と核酸ナノ医療への展開  [招待講演]
    原島秀吉
    日本薬剤学会経口吸収FG第8回合宿討論会、小樽 2018年10月 口頭発表(招待・特別)
  • Multifunctional Envelope-type Nano Device for Gene Delivery: Concept and Application for Nanomedicine  [招待講演]
    原島秀吉
    13th France-Japan Drug Delivery System Symposium、Mie, Japan 2018年10月 口頭発表(招待・特別)
  • 最新のナノDDS戦略  [招待講演]
    原島秀吉
    第16回社内勉強会(ナノキャリア) 2018年09月 口頭発表(招待・特別)
  • Multifunctional Envelope-type Nano Device for Gene Delivery: Concept and Clinical Application for Nanomedicine  [招待講演]
    原島秀吉
    The 19th International Pharmaceutical Technology Symposium, Antalya, Turkey 2018年09月 口頭発表(招待・特別)
  • Multifunctional Envelope-type Nano Device for Nanomedicine  [招待講演]
    原島秀吉
    The 5th International Biomaterials Symposium, Chanchun, China 2018年08月 口頭発表(招待・特別)
  • Controlled Intracellular Trafficking and Selective Tissue Targeting by Multifunctional Envelope-type Nano Device for Nucleic Acid Nanomedicine  [招待講演]
    原島秀吉
    The 45th CRS Annual Meeting & Exposition, New York, USA 2018年07月 シンポジウム・ワークショップパネル(指名)
  • リポソームからの薬物放出と殺細胞効果の関係について  [招待講演]
    原島秀吉
    第6回事例報告会、製剤種差検討会、京都 2018年07月 口頭発表(招待・特別)
  • 多機能性エンベロープ型ナノ構造体の開発とナノ医療への展開:ナノ医療の現状と今後の展開  [招待講演]
    原島秀吉
    日本薬剤学会第33回年会、学術シンポジウム:微粒子製剤設計と医薬品開発~現状と将来展望~、静岡 2018年05月 シンポジウム・ワークショップパネル(指名)
  • 多機能性エンベロープ型ナノ構造体の開発と核酸ナノ医薬の展望  [招待講演]
    原島秀吉
    核酸分析化学特論、広島大学大学院医歯薬保健学研究科 2018年05月 公開講演,セミナー,チュートリアル,講習,講義等
  • DDSの現状と核酸医薬への展望  [招待講演]
    原島秀吉
    徳島大学特別講演会、徳島 2018年02月 口頭発表(招待・特別)
  • リポソーム研究の現状と産学官の連携で目指すDDS  [招待講演]
    原島秀吉
    講演会(小野薬品工業株式会社、水無瀬研究所) 2017年12月 口頭発表(招待・特別)
  • Multifunctional Envelope-type Nano Device for Gene Delivery: Concept and Application for Nanomedicine  [招待講演]
    原島秀吉
    3rd International Conference on Biomaterials Science in Tokyo 2017年11月 口頭発表(招待・特別)
  • 多機能性エンベロープ型ナノ構造体の創製とナノ医療への展開  [招待講演]
    原島秀吉
    講演会(秋吉ERATO)、京都 2017年11月 口頭発表(招待・特別)
  • EPR効果はヒトでも有効か?:DoxilとDoxorubicinのメタ解析から  [招待講演]
    原島秀吉
    製剤種差検討会、第3回事例報告会、東京 2017年06月 口頭発表(招待・特別)
  • 多機能性エンベロープ型ナノ構造体の創製とナノ医療への展開  [招待講演]
    原島秀吉
    日本薬剤学会第32年会、ラウンドテーブル2、核酸・遺伝子医薬の臨床応用に立ちはだかる障壁について考える、大宮 2017年05月 口頭発表(招待・特別)
  • 多機能性エンベロープ型ナノ構造体の創製とナノ医療への応用  [招待講演]
    原島秀吉
    社内講演会(アンジェス)、大阪 2017年04月 口頭発表(招待・特別)
  • Multifunctional Envelope-type Nano Device for Nanomedicine  [招待講演]
    原島秀吉
    AsiaTIDES: Oligonucleotide & Peptide Therapeutics, Kyoto 2017年02月 口頭発表(招待・特別)
  • 多機能性エンベロープ型ナノ構造体の開発とナノ医療への展開  [招待講演]
    原島秀吉
    薬物動態懇話会 第39年会、薬物動態研究と繋がるサイエンス~DDSと毒性バイオマーカーを薬物動態から俯瞰する~、浜松 2016年11月 口頭発表(招待・特別)
  • Multifunctional Envelope-type Nano Device for Gene Delivery: Concept and Application for Nanomedicine  [招待講演]
    原島秀吉
    12th France-JapanDrug Delivery System Symposium, Paris 2016年10月 口頭発表(招待・特別)
  • Multifunctional Envelope-type Nano Device for Gene Delivery: Concept and Application for Nanomedicine  [招待講演]
    原島秀吉
    18th International Pharmaceutical Technology Symposium, Antalya, Turkey 2016年09月 口頭発表(招待・特別)
  • 多機能性エンベロープ型ナノ構造体の創製とナノ医療への展開  [招待講演]
    原島秀吉
    ポスト片岡CRESTミニシンポジウム、川崎 2016年07月 口頭発表(招待・特別)
  • Multifunctional Envelope-type Nano Device for Gene Delivery: Concept and Application for Nanomedicine  [招待講演]
    原島秀吉
    30th Anniversary Symposium of The Nagai Foundation Tokyo、“Link to the Past and Bridge to the Future” 2016年07月 口頭発表(招待・特別)
  • 多機能性エンベローブ型ナノ構造体の創製とナノ医療への展開  [招待講演]
    原島秀吉
    第31回日本薬剤学会、薬剤学会賞受賞講演、岐阜 2016年05月 口頭発表(招待・特別)
  • Multifunctional Envelope-type Nano Device for Gene Delivery: Concept and Application for Nanomedicine  [招待講演]
    原島秀吉
    14th European Symposium on Controlled Drug Delivery, Egmond, Netherland 2016年04月 口頭発表(招待・特別)
  • 多機能性エンベロープ型ナノ構造体の開発とナノ医療への展開  [招待講演]
    原島秀吉
    講演会(理研杉山雄一研究室)、横浜 2016年01月 口頭発表(招待・特別)
  • 多機能性エンベロープ型ナノ構造体の開発とナノ医療への展開  [招待講演]
    原島秀吉
    第1回日本核酸医薬学会年会 シンポジウム 2015年12月 シンポジウム・ワークショップパネル(指名)
  • 大学発ナノ医薬品の創出拠点の形成と早期実用化  [招待講演]
    原島秀吉
    日本薬物動態学会第30年会 シンポジウム-8、船堀 2015年11月 シンポジウム・ワークショップパネル(指名)
  • 多機能性エンベロープ型ナノ構造体の創製とナノ医療への展開  [招待講演]
    原島秀吉
    講演会、徳島大学、徳島 2015年10月 口頭発表(招待・特別)
  • Multifunctional lipid-based systems for drug and gene delivery  [招待講演]
    原島秀吉
    20th International Symposium on Microencapsulation, Boston, USA 2015年10月 口頭発表(招待・特別)
  • Activities of Education and Researches at Faculty of Pharmaceutical Sciences in Hokkaido University  [招待講演]
    原島秀吉
    The UP-HU Joint Workshop for Academic Exchange, university of Pretoria, South Africa 2015年09月 公開講演,セミナー,チュートリアル,講習,講義等
  • Multifunctional envelope-type nano device for gene delivery: Concept and application for Nanomedicine  [招待講演]
    原島秀吉
    International Seminar on Pharmaceutics 2015, Bandung, Indonesia 2015年08月 口頭発表(招待・特別)
  • Multifunctional envelope-type nano device for gene delivery: Concept and application for Nanomedicine  [招待講演]
    原島秀吉
    Biomaterials International 2015, Kenting, Taiwan 2015年06月 口頭発表(招待・特別)
  • アカデミア発DDSによる医療イノベーション  [招待講演]
    原島秀吉
    日本薬剤学会第30年会、学術シンポジウム-3:アカデミア発DDS製剤シーズによる医療イノベーションを考える、長崎 2015年05月 シンポジウム・ワークショップパネル(指名)
  • Multifunctional envelope-type nano device for gene delivery: Concept and application for Nanomedicine  [招待講演]
    原島秀吉
    The 5th Asian Biomaterials Congress, Taipei, Taiwan 2015年05月 口頭発表(招待・特別)
  • 日本発革新的ナノ医薬品の創出を目指して  [招待講演]
    原島秀吉, 秋田英万, 加藤くみ子, 松村保広, 片岡一則
    日本薬学会第135年会 シンポジウム:健康・医療戦略推進法の与えるインパクト~先端的研究開発の実現に向けて~、神戸学院大学 2015年03月 シンポジウム・ワークショップパネル(指名)
  • 多機能性エンベロープ型ナノ構造体の創製とナノ医療への展開  [招待講演]
    原島秀吉
    第114回 医工学フォーラム、京都大学再生医科学研究所 2014年12月 口頭発表(招待・特別)
  • 多機能性エンベロープ型ナノ構造体による遺伝子デリバリーとナノ医療への展開  [招待講演]
    原島秀吉
    日本認知症学会、シンポジウム6:核酸創薬とドラッグデリバリーシステムの革新による認知症の遺伝子治療への道、横浜 2014年11月 口頭発表(招待・特別)
  • 11) Hideyoshi Harashima, Multifunctional Envelope-type Nano Device for Nanomedicine  [招待講演]
    原島秀吉
    Workshop on Global Future Healthcare, on Materials Science and Engineering, Jointly Organized by Nanyang Technological University (NTU) and Hokkaido University 2014年10月 公開講演,セミナー,チュートリアル,講習,講義等
  • Multifunctional Envelope-type Nano Device for gene delivery: Concept and application for Nanomedicine  [招待講演]
    原島秀吉
    The 11th France - Japan DDS Symposium, Awaji, Tokushima 2014年10月 口頭発表(招待・特別)
  • 多機能性エンベロープ型ナノ構造体の創製とナノメディシンへの応用  [招待講演]
    原島秀吉
    化学とマイクロ・ナノシステム学会、第30回研究会、北海道大学 2014年10月 口頭発表(招待・特別)
  • 多機能性エンベロープ型ナノ構造体の開発とナノメディシンへの展開  [招待講演]
    原島秀吉
    第3回創薬イノベーション懇話会in札幌 2014年09月 口頭発表(招待・特別)
  • 遺伝子・核酸デリバリーシステムの開発とナノメディシンへの展開  [招待講演]
    原島秀吉
    平成26年度 未踏科学サマー道場、次世代バイオマテリアルとその応用、湘南 2014年08月 口頭発表(招待・特別)
  • Multifunctional envelope-type nano device for gene delivery: Concept and application for Nanomedicine  [招待講演]
    原島秀吉
    Current and Future Aspect of Drug Delivery, National Yang-Ming University, Taiwan 2014年08月 口頭発表(招待・特別)
  • 「血管を標的とする革新的医薬品分子送達方の基盤技術の確立」のまとめと「血管を標的とするナノ医療の実用化に向けた拠点形成」へ向けて  [招待講演]
    原島秀吉
    公開シンポジウム「血管を標的とするナノ医療の実用化に向けた拠点形成、北海道大学 2014年06月 シンポジウム・ワークショップパネル(指名)
  • MITO-Porter: Membrane-type nano device for mitochondrial drug delivery via membrane fusions  [招待講演]
    原島秀吉
    Euromit 2014, Tampere, Finland 2014年06月 口頭発表(招待・特別)
  • 多機能性エンベロープ型ナノ構造体の創製とナノ医療への展開  [招待講演]
    原島秀吉
    Biotech2014、BIO-3:ナノテクノロジーがもたらす革新的医薬品開 2014年05月 口頭発表(招待・特別)
  • Breakthrough technology based on controlled intracellular trafficking of functional nucleic acids with MEND  [招待講演]
    原島秀吉
    5th FIP Pharmaceutical Sciences World Congress (PSWC), APSA Symposium, Intracellular drug trafficking and targeting, Melbourne, Australia 2014年04月 口頭発表(招待・特別)
  • 多機能性エンベロープ型ナノ構造体の創製とナノ医療への展開  [招待講演]
    原島秀吉
    講演会、日本化薬 2014年01月 口頭発表(招待・特別)
  • 多機能性エンベロープ型ナノ構造体の創製とナノ医療への展開  [招待講演]
    原島秀吉
    第10回レギュラトリーサイエンス学会シンポジウム、~ナノメディシンの実現に向けて~、東京 2014年01月 口頭発表(招待・特別)

その他活動・業績

  • 山田勇磨, 山田勇磨, 日比野光恵, 伊藤百, 荒井愛永, 佐々木大輔, 真栄城正寿, 渡慶次学, 渡慶次学, 太田善浩, 原島秀吉, 原島秀吉 日本薬剤学会年会講演要旨集(CD-ROM) 36th 2021年
  • 中村孝司, 河合美典, 佐藤悠介, 真栄城正寿, 渡慶次学, 原島秀吉 日本薬剤学会年会講演要旨集(CD-ROM) 36th 2021年
  • 多機能性エンベロープ型ナノ構造体の開発とナノ医療への展開
    原島 秀吉 日本小児臨床薬理学会雑誌 33 (1) 88 -88 2020年12月 [招待有り]
  • ミトコンドリアを標的とする遺伝子治療用RNAナノカプセルの創製
    山田 勇磨, 原島 秀吉 遺伝子医学 10 (4) 74 -79 2020年10月 [招待有り]
     
    多彩な機能を有するミトコンドリアの機能破綻は様々な疾患を誘発する。これらの疾患の一部は,ミトコンドリアDNA(mtDNA)の変異・欠失が原因であることが報告されている。そのため,ミトコンドリアを標的とした遺伝子治療研究は,革新的医薬品の創製につながると期待されている。本稿では,われわれが創製したミトコンドリア標的型drug delivery system(DDS)"MITO-Porter"を基盤とした「ミトコンドリアを標的とする遺伝子治療用RNAナノカプセル」に関する研究を中心に紹介する。(著者抄録)
  • ミトコンドリア強化幹細胞(MITO Cell)の製造および細胞移植療法への展開
    山田 勇磨, 阿部 二郎, 佐々木 大輔, 原島 秀吉 BIO Clinica 35 (5) 448 -450 2020年05月 [招待有り]
     
    心筋幹細胞(Cardiac Progenitor Cell[CP Cell])移植は、心筋症に対する治療法として大規模臨床試験において、その有用性が認められている。一方で、持続的な移植効果維持が困難とされており、移植細胞のミトコンドリア機能不全などが原因として報告されている。我々はこれまでに、酸化ストレス発生源であり細胞内ATP産生を担うミトコンドリアを強化した心筋幹細胞(MITO Cell)の製造を行い、細胞移植療法の治療有効性を報告している。MITO Cellの構築は、MITO-Porter(ミトコンドリア標的型ナノカプセル)を用いてCP Cellのミトコンドリアに機能性分子を送達し構築した。本稿では、ミトコンドリア毒性を有するドキソルビシン心筋症モデルマウスを用いた。MITO Cellの細胞移植療法の心筋症治療の成績向上に関する我々の研究成果を紹介する。(著者抄録)
  • 【核酸医薬の最近の話題】核酸DDS技術を用いたがん免疫療法
    中村 孝司, 原島 秀吉 細胞 52 (5) 245 -248 2020年05月 [招待有り]
     
    免疫チェックポイント阻害療法の臨床での成功は、2018年のノーベル生理学・医学賞へと繋がった。さらに、遺伝子改変T細胞療法は血液がんに対する顕著な奏効を実現する。これらの治療法はがん治療に革命をもたらし、がん免疫療法は今や最も注目されている領域となっている。しかしながら、免疫チェックポイント阻害剤単剤での有効率は全体として20-30%であり、全てのがん患者に対して有効ではない。また、遺伝子改変T細胞療法は固形がんに対する有効性、煩雑性、高コストなどが課題となっている。そのような中、Drug Delivery System(DDS)は現在のがん免疫療法が抱える課題を解決することが可能な技術として注目されている。本稿では、核酸を搭載したDDS技術ががん免疫療法に与えるインパクトを我々の成果を実例として紹介する。(著者抄録)
  • 【核酸創薬に貢献するバイオマテリアル】バイオマテリアルに基づいた核酸ナノ医療の創製
    山田 勇磨, 中村 孝司, 佐藤 悠介, 原島 秀吉 バイオマテリアル-生体材料- 38 (2) 92 -99 2020年04月 [招待有り]
     
    核酸ナノ医療の時代が始まり、医薬品業界ではゴールドラッシュが巻き起こっている。一方で、ゾルゲンスマに代表される超高額医療費の経済的問題は、国家予算の破綻も招きかねず、新たなジレンマに直面することが予想される。本稿では、前半ではCAR-T療法、パティシラン、ゾルゲンスマについて紹介し、革新的核酸医薬・遺伝子治療の最先端技術について考察する。後半では、我々の研究成果に基づいて、細胞内動態制御の重要性とポストEPRの時代のDDS戦略について紹介し、バイオマテリアルによってこのジレンマをいかにして突破するべきか、議論したい。(著者抄録)
  • 中村孝司, 河合美典, 佐藤悠介, 真栄城正寿, 渡慶次学, 原島秀吉 日本薬学会年会要旨集(CD-ROM) 140th (Web) 2020年
  • 佐藤悠介, 鈴木裕一, 小沼はるの, 佐藤里咲, 真栄城正寿, 渡慶次学, 原島秀吉 日本DDS学会学術集会プログラム予稿集 36th 2020年
  • 日比野光恵, 山田勇磨, 真栄城正寿, 渡慶次学, 石塚洋一, 原島秀吉 日本DDS学会学術集会プログラム予稿集 36th 2020年
  • 真栄城正寿, 真栄城正寿, 岡田悠斗, 佐藤悠介, 石田晃彦, 谷博文, 原島秀吉, 渡慶次学 日本DDS学会学術集会プログラム予稿集 36th 2020年
  • 岡田悠斗, 真栄城正寿, 真栄城正寿, 佐藤悠介, 石田晃彦, 谷博文, 原島秀吉, 渡慶次学 化学とマイクロ・ナノシステム学会研究会講演要旨集 42nd (Web) 2020年
  • 原島 秀吉, 畠山 浩人, 金沢 貴憲 薬剤学: 生命とくすり 80 (1) 13 -23 2020年01月 [査読無し][招待有り]
  • 山田 勇磨, 原島 秀吉 実験医学 37 (12) 2067 -2073 2019年08月 [査読無し][招待有り]
     
    多彩な機能を有するミトコンドリアへ目的分子を送達するdrug delivery system(DDS)は、医療・ライフサイエンス分野の発展に大きく貢献すると期待されている。本稿では、DDS研究者の立場から、「ミトコンドリアDDS研究の魅力を伝えること」を目標とし、さらに、「ミトコンドリアを標的とする未来医療の可能性」も提示したい。ここでは、ミトコンドリアを標的とするナノDDSについて概説するとともに、われわれが創製したミトコンドリア標的型ナノDDS"MITO-Porter"の開発研究に関して紹介する。(著者抄録)
  • 心不全病態に対する多面的アプローチ 基礎から治療へ 虚血再灌流モデルマウスを用いた心不全に対するミトコンドリア活性化心筋幹細胞を用いた細胞移植療法の治療効果の検討
    佐々木 大輔, 阿部 二郎, 武田 充人, 原島 秀吉, 山田 勇磨 日本小児循環器学会雑誌 35 (Suppl.1) s1 -125 2019年06月
  • 山田 勇磨, 原島 秀吉 小児科診療 82 (4) 523 -528 2019年04月 [査読無し][招待有り]
     
    ●ミトコンドリアの機能異常は種々の疾患(進行性変性疾患、虚血性疾患、糖尿病、ミトコンドリア遺伝病、など)を誘発することが報告されている。●そのため、ミトコンドリアを標的とした医薬品の開発研究は、新たな疾患治療法を提供する次世代医薬品の創製につながると期待される。●本稿では、筆者らが創製したミトコンドリア標的型drug delivery system(DDS)"MITO-Porter"の開発研究に関して、特に「ミトコンドリアを標的とする遺伝子治療戦略」および「ミトコンドリア強化幹細胞を用いた移植療法」に焦点をあて紹介する。(著者抄録)
  • 佐藤 悠介, 原島 秀吉 医薬ジャーナル 55 (2) 615 -619 2019年02月 [招待有り]
     
    <文献概要>Short interfering RNA(siRNA)は,RNA干渉により任意の遺伝子発現が抑制可能な機能性核酸である。多くの難治性疾患に対する新規治療モダリティとして魅力的であるが,単独では組織移行能を示さないことから,医薬としての応用には目的細胞へ選択的かつ効率的にsiRNAを送達する技術(drug delivery system:DDS)の開発が不可欠である。これまでに脂質,ポリマーやリガンド分子コンジュゲート等の多様な分子設計・マテリアル開発がなされてきており,複数の製剤が臨床試験に進んでいる。2018年8月には世界初のsiRNA医薬品が認可され,まさにsiRNA医薬時代の幕開けとなった。本稿では,近年のsiRNAのDDS開発状況を概説するとともに,今後の課題や展望について述べる。
  • 木村笑, 真栄城正寿, 岡部奈々, 佐藤悠介, 石田晃彦, 谷博文, 原島秀吉, 渡慶次学 化学とマイクロ・ナノシステム学会研究会講演要旨集 39th 2019年
  • ミトコンドリアへの分子送達技術
    山田 勇磨, 原島 秀吉 BIO Clinica 33 (7) 637 -640 2018年07月 [招待有り]
     
    オートファジーに関連するミトコンドリアの品質管理システム・マイトファジーが注目を集めている。人工的にマイトファジーを誘導するシステムは、進行性神経変性疾患などの疾患解明・治療に大きく貢献する事が期待される。本戦略を実現するためには、マイトファジー誘導分子をミトコンドリアに送達するDrug Delivery Systemが必要不可欠である。本稿では、ミトコンドリアを標的としたDDSを概説する。また、ミトコンドリア標的型ナノカプセル"MITO-Porter"に関する我々の最新の研究成果も紹介する。(著者抄録)
  • 櫻井 遊, 原島 秀吉 Drug Delivery System 33 (2) 98 -104 2018年03月 [招待有り]
     
    EPR効果の発見により、ナノ粒子を用いて抗がん剤を送達する戦略を基盤としたナノ粒子製剤の開発が大きな進展を見せた。しかしながら、近年のドキソルビシン封入リポソームの複数の臨床試験のメタ解析の結果から、ヒトにおいて統計学的に有意な治療効果が認められないことが報告された。今回はこの報告について紹介する。また、ナノ粒子への腫瘍送達に与える腫瘍微小環境の影響の評価と送達効率の向上のために、筆者らは最近、血管へのsiRNA送達による微小環境リモデリングに基づくナノ粒子送達戦略を提唱している。本稿では、これまでに得られた血管正常化とナノ粒子送達効率の関係性について概説したい。(著者抄録)
  • 木村笑, 真栄城正寿, 岡部奈々, 佐藤悠介, 石田晃彦, 谷博文, 原島秀吉, 渡慶次学, 渡慶次学, 渡慶次学, 渡慶次学 化学とマイクロ・ナノシステム学会研究会講演要旨集 37th 2018年
  • 真栄城正寿, 木村笑, 佐藤悠介, 石田晃彦, 谷博文, 原島秀吉, 渡慶次学 化学工学会秋季大会研究発表講演要旨集(CD-ROM) 49th 2017年
  • pH感受性脂質を基盤とした脂質ナノ粒子の開発と核酸ナノメディシンへの応用
    佐藤悠介, 原島秀吉 化学工業 67 (11) 21 -27 2016年11月 [査読無し][招待有り]
  • 畠山 浩人, 原島 秀吉 Drug Delivery System 31 (4) 293 -299 2016年09月 [招待有り]
     
    核酸医薬のがん治療への応用にはDDSが必要不可欠である。血中投与後のがんへの送達には、DDSへのPEG修飾による血中滞留性向上とEPR効果が重要であるが、これは同時に標的細胞における著しい活性の低下を引き起こす。この体内動態と細胞内動態に対する相反するPEGの性質を「PEGのジレンマ」として提唱し、この問題の解決が、効率的なDDS開発にとって最も重要な課題であると考えている。本稿ではこのPEGのジレンマの解決にむけた筆者らの戦略や取り組みについて紹介したい。(著者抄録)
  • マイクロ流体デバイスによる脂質ナノ粒子作製とDDSへの応用
    真栄城正寿, 佐藤悠介, 原島秀吉, 渡慶次学 機能材料 36 15 -21 2016年07月 [査読無し][招待有り]
  • 加藤 くみ子, 石原 比呂之, 友田 寬, 松田 嘉弘, 永井 尚美, 花田 博幸, 久田 茂, 小野寺 博志, 西山 伸宏, 原島 秀吉, 松村 保広, 片岡 一則, 合田 幸広, 奥田 晴宏, 川西 徹 医薬品医療機器レギュラトリーサイエンス = Pharmaceutical and medical device regulatory science 47 (5) 333 -341 2016年05月 [査読無し][通常論文]
     
    リポソーム製剤は有効成分をリポソームの脂質二分子膜または内相に封入することにより作製される。今回、ナノメートルサイズのリポソーム製剤で、有効成分の生体内安定性、組織移行性プロファイルをはじめとする薬物動態、細胞内動態等の向上を目的として設計された製剤について、以下の項目に分けて述べた。1)リポソーム製剤に特有の品質特性と製法変更時の同等性/同質性評価、2)非臨床薬物動態、3)非臨床毒性、として述べた。
  • 紙谷浩之, 紙谷浩之, 紙谷浩之, 西原実香, 西原実香, 神田元紀, 神田元紀, 鈴木哲矢, 山門振一郎, 原島秀吉 日本DDS学会学術集会プログラム予稿集 32nd 2016年
  • 血管を標的とする薬物送達システムに基づいた新しい治療法
    櫻井遊, 梶本和昭, 原島秀吉 Dementia Japan 30 (1) 30 -40 2016年 [査読無し][招待有り]
  • BCG-CWS搭載ナノ粒子を用いた膀胱がん免疫療法剤の開発
    中村 孝司, 吹上 雅文, 鈴木 嘉晃, 矢野 郁也, 宮崎 淳, 西山 博之, 赤座 英之, 中山 俊憲, 原島 秀吉 日本薬学会年会要旨集 135年会 (4) 84 -84 2015年03月 [査読無し][通常論文]
  • 多機能性エンベロープ型ナノ構造体の創世とナノ医療への展開
    佐藤悠介, 山田勇磨, 梶本和昭, 原島秀吉 化学とマイクロ・ナノシステム 14 (1) 15 -23 2015年 [査読無し][招待有り]
  • Yu Sakurai, Kazuaki Kajimoto, Hideyoshi Harashima BIOMATERIALS SCIENCE 3 (9) 1253 -1265 2015年 [査読有り][通常論文]
     
    Sophisticated drug delivery systems (DDS) are required for delivering drugs, especially macromolecules such as nucleic acids or proteins, to their sites of action. Therefore it is a prerequisite that future DDS are designed to selectively target a tissue. In this review, we focus on systems that actively target the vasculature in tumors or adipose tissues. For targeting tumor vasculatur, a new strategy referred to as dual-targeting is proposed that uses a combination of a receptor specific ligand and a cell penetrating peptide, which can induce the synergistic enhancement of tissue selectivity under in vivo conditions. A novel pH-sensitive cationic lipid was designed to enhance the endosomal release of encapsulated compounds such as siRNA as well as to improve the stability in blood circulation after intravenous administration. A cyclic RGD peptide is used as an active targeting ligand. For targeting adipose vasculature, prohibitin, which is expressed on the surface of adipose endothelial cells, was targeted with KGGRAKD peptides on the surface of PEGylated nanoparticles. Prohibitin targeted nanoparticles (PTNP) encapsulating Cytochrome c (CytC) can selectively target adipose vasculature by optimizing the lengths of the PEG linkers and can deliver CytC to adipose endothelial cells. PTNP can successfully induce anti-obese effects as well as apoptosis by delivering CytC to the cytosol in endothelial cells. Unexpectedly, the EPR (enhanced permeability and retention) effect, which is usually observed in tumor tissue, was also observed in the adipose vasculature, especially in obese mice, where PEGylated nanoparticles can pass through the endothelial barriers in adipose tissue. We believe that these achievements in active targeting will allow a greatly expanded use of DDS for nanomedicines.
  • M. Maeki, T. Saito, Y. Node, Y. Sato, T. Yasui, N. Kaji, A. Ishida, H. Tani, Y. Baba, H. Harashima, M. Tokeshi MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences 838 -840 2015年 [査読有り][通常論文]
     
    © 15CBMS-0001. This paper described a simple preparation method for small-size and monodispersed lipid nanoparticles (LNPs) by using microfluidic devices. The fundamental role and importance of chaotic micromixer in the microfluidic device was demonstrated. The suitable cycle number of chaotic micromixer was confirmed for precise controlling LNPs size with narrow distribution under the any flow rate conditions. In addition, LNPs containing siRNA was synthesized for evaluation of penetration efficiency via in vivo experiment. The PEGylated LNPs containing siRNA with a diameter of 30 nm could penetrate to the mouse parenchymal liver cells rather than the LNPs with a diameter of 50 nm.
  • 肝臓集積性DDSおよび肝類洞内皮細胞選択性DDSを用いた肝炎治療効果の検討
    田野井 智倫, 田村 孝史, 中山 健, 佐野 直樹, 村田 聡一郎, 鳥谷部 尚之, 櫻井 遊, 原島 秀吉, 大河内 信弘 日本DDS学会学術集会プログラム予稿集 30回 178 -178 2014年07月 [査読無し][通常論文]
  • Hideyoshi Harashima JOURNAL OF GENE MEDICINE 16 (7-8) 204 -205 2014年07月 [査読無し][通常論文]
  • Yukari Yasuzaki, Yuma Yamada, Tsutomu Kanefuji, Dexi Liu, Hideyoshi Harashima JOURNAL OF GENE MEDICINE 16 (7-8) 275 -275 2014年07月 [査読無し][通常論文]
  • 【先端的医薬品等におけるレギュラトリーサイエンス研究の現状・課題】 ナノテクノロジーを基盤とした医薬品のレギュラトリーサイエンス研究への取り組み
    原島 秀吉, 秋田 英万, 加藤 くみ子, 石井 武彦, 松村 保広, 片岡 一則 Drug Delivery System 29 (3) 217 -225 2014年07月 [査読無し][通常論文]
     
    ナノメートルサイズの物質は、バルク物質が有する特性とは、化学的、物理的、または生物学的に区別しうる特性をしばしば有し、このような特異な性質は、医薬品開発にも応用されつつある。ナノテクノロジーを基盤とした医薬品(ナノ医薬品)は、従来の製剤とは体内での挙動や生体との相互作用などさまざまな点で異なると考えられるため、ナノ医薬品の特性に配慮した評価が必要とされている。筆者らは、主として有効成分の生体内安定性や生体内分布等の体内動態の制御による薬効の向上と毒性低減等を目的とした脂質ナノ粒子並びに高分子ミセルの製剤化研究や非臨床、臨床試験を行っている。本稿では、筆者らが取り組んでいるナノ医薬品のレギュラトリーサイエンス研究について紹介する。(著者抄録)
  • 山田 勇磨, 原島 秀吉 化學工業 65 (6) 418 -423 2014年06月
  • 肝類洞内皮細胞を標的としたDrug Delivery Systemによる新規肝炎治療法の開発
    田野井 智倫, 田村 孝史, 村田 聡一郎, 鳥谷部 尚之, 櫻井 遊, 原島 秀吉, 大河内 信弘 肝臓 55 (Suppl.1) A329 -A329 2014年04月 [査読無し][通常論文]
  • BCG-CWS搭載ナノ粒子を基盤とした膀胱癌治療剤の開発
    中村 孝司, 吹上 雅文, 中谷 彰洋, 矢野 郁也, 宮崎 淳, 西山 博之, 赤座 英之, 伊藤 俊宏, 細川 裕之, 中山 俊憲, 原島 秀吉 泌尿器外科 27 (3) 338 -339 2014年03月 [査読無し][通常論文]
  • 西原実香, 神田元紀, 神田元紀, 山門振一郎, 原島秀吉, 原島秀吉, 紙谷浩之, 紙谷浩之, 紙谷浩之 日本DDS学会学術集会プログラム予稿集 30th 2014年
  • 西原実香, 神田元紀, 神田元紀, 山門振一郎, 原島秀吉, 原島秀吉, 紙谷浩之, 紙谷浩之, 紙谷浩之, 紙谷浩之 日本分子生物学会年会プログラム・要旨集(Web) 37th 2014年
  • 高岡晃教, 佐藤精一, LI Kai, 亀山武志, 林隆也, 原島秀吉, 秋田英万, 櫻井遊 B型肝炎ウイルスの持続感染を再現する効率的な培養細胞評価系の開発に関する研究 平成25年度 総括・分担研究報告書 55‐59 2014年 [査読無し][通常論文]
  • 加藤 くみ子, 中西 健, 小崎 雅人, 松田 嘉弘, 平野 舞, 花田 博幸, 久田 茂, 小野寺 博志, 西山 伸宏, 原島 秀吉, 松村 保広, 片岡 一則, 奥田 晴宏, 川西 徹 医薬品医療機器レギュラトリーサイエンス 44 (12) 968 -975 2013年12月 [査読無し][通常論文]
  • 櫻木 誠, 安西 智宏, 木村 廣道, 原島 秀吉 薬剤学 = The archives of practical pharmacy 73 (5) 312 -320 2013年09月01日
  • Nano Packagingが拓く次世代核酸医療
    松尾保隆, 加地範匡, 畠山浩人, 渡慶次学, 小暮健太朗, 馬場嘉信, 原島秀吉 表面 51 227-240 2013年05月 [査読有り][通常論文]
  • DNAマイクロアレイとin vivo siRNA送達システムの融合による新規2型糖尿病治療薬創出
    末光 永理奈, 林 泰弘, 梶本 和昭, 佐藤 悠介, Akhter Afsana, 櫻井 遊, 畠山 浩人, 兵藤 守, 加地 範匡, 馬場 嘉信, 原島 秀吉 日本薬剤学会年会講演要旨集 28年会 237 -237 2013年04月 [査読無し][通常論文]
  • 網羅的遺伝子発現情報に基づいた遺伝子キャリアの免疫応答メカニズムの解析
    畠山 浩人, 山本 桃子, 林 泰弘, 梶本 和昭, 加地 範匡, 馬場 嘉信, 原島 秀吉 日本薬学会年会要旨集 133年会 (4) 83 -83 2013年03月 [査読無し][通常論文]
  • 革新的医薬品の創出に向けたレギュラトリーサイエンス(RS)の推進 ナノテクノロジーを基盤とした革新的医薬品に関する評価方法
    原島 秀吉, 松村 保広, 片岡 一則 日本薬学会年会要旨集 133年会 (1) 132 -132 2013年03月 [査読無し][通常論文]
  • 神田元紀, 神田元紀, 神田元紀, 小林三和子, 松岡一郎, 原島秀吉, 原島秀吉, 紙谷浩之, 紙谷浩之, 紙谷浩之 日本分子生物学会年会プログラム・要旨集(Web) 36th 2013年
  • 重中大輔, 鵜川真美, 加地範匡, 渡慶次学, 秋田英万, 原島秀吉, 馬場嘉信 日本化学会講演予稿集 93rd (2) 2013年
  • 原島 秀吉 薬剤学 = The archives of practical pharmacy 73 (1) 1 -1 2013年01月01日
  • Noritada Kaji, Daisuke Shigenaka, Masami Ukawa, Manabu Tokeshi, Hidetaka Akita, Hideyoshi Harashima, Yoshinobu Baba 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013 2 1170 -1172 2013年 [査読有り][通常論文]
     
    We developed microfluidic continuous purification devices based on free-flow electrophoresis for gene-delivery multifunctional envelope-type nanodevices (MEND) which consists of DNA core and phospholipid bilayer envelope. Various impurities, such as DNA, DNA-peptide complex, and liposomes, produced during the fabrication process were removed in an efficient manner and over 75% collection of the input plasmid DNA which corresponds to purified MEND was achieved.
  • 山田勇磨, 野村政壽, 原島秀吉 人工臓器(日本人工臓器学会) 41 (3) 212 -214 2012年12月15日 [査読無し][通常論文]
  • S. Shaheen, H. Akita, I. Souichirou, N. Miura, H. Harashima CANCER RESEARCH 72 2012年12月 [査読無し][通常論文]
  • Yasuhiro Hayashi, Ryoichi Mizuno, Khalil A. Ikramy, Hidetaka Akita, Hideyoshi Harashima NUCLEIC ACID THERAPEUTICS 22 (5) 360 -363 2012年10月 [査読無し][通常論文]
     
    We previously reported that an octaarginine- and pH-sensitive fusogenic peptide-modified multifunctional envelope-type nano device (R8-GALA-MEND) produces a high level of gene expression in the liver. In this study, we report on an examination of whether this gene delivery system exerts potent hepatoprotective effects against lipopolysaccharide/D-galactosamine (LPS/D-GalN)-induced acute liver injury. In vivo-jetPEI (TM)-Gal, a commercially available in vivo transfection reagent, was used as a reference. The systemic administration of the R8-GALA-MEND or in vivo-jetPEI (TM)-Gal showed that the latter was more toxic than the R8-GALA-MEND, indicating that R8-GALA-MEND is a safer system than in vivo-jetPEI (TM)-Gal. Pretreatment with R8-GALA-MEND or in vivo-jetPEI (TM)-Gal loaded with hepatocyte growth factor (HGF) pDNA inhibited serum GPT and GOT levels from becoming elevated. However, the survival rate of the mice was significantly enhanced in the case of R8-GALA-MEND, but not for the in vivo-jetPEI (TM)-Gal treatment. These results demonstrate that R8-GALA-MEND has the potential for use in the pretreatment of an acute liver injury.
  • 秋田 英万, 原島 秀吉 高分子 61 (8) 533 -535 2012年08月01日
  • Yuma Yamada, Eriko Kawamura, Hideyoshi Harashima JOURNAL OF NANOPARTICLE RESEARCH 14 (8) 1 -15 2012年08月 [査読無し][通常論文]
     
    Mitochondrial gene therapy has the potential for curing a variety of diseases that are associated with mitochondrial DNA mutations and/or defects. To achieve this, it will be necessary to deliver therapeutic agents into the mitochondria in diseased cells. A number of mitochondrial drug delivery systems have been reported to date. However, reports of mitochondrial-targeted DNA delivery are limited. To achieve this, the therapeutic agent must be taken up by the cell (1), after which, the multi-processes associated with intracellular trafficking must be sophisticatedly regulated so as to release the agent from the endosome and deliver it to the cytosol (2) and to pass through the mitochondrial membrane (3). We report herein on the mitochondrial delivery of oligo DNA as a model therapeutic using a Dual Function (DF)-MITO-Porter, an innovative nano carrier designed for mitochondrial delivery. The critical structural elements of the DF-MITO-Porter include mitochondria-fusogenic inner envelopes and endosome-fusogenic outer envelopes, modified with octaarginine which greatly assists in cellular uptake. Inside the cell, the carrier passes through the endosomal and mitochondrial membranes via step-wise membrane fusion. When the oligo DNA was packaged in the DF-MITO-Porter, cellular uptake efficiency was strongly enhanced. Intracellular observation using confocal laser scanning microscopy showed that the DF-MITO-Porter was effectively released from endosomes. Moreover, the findings confirmed that the mitochondrial targeting activity of the DF-MITO-Porter was significantly higher than that of a carrier without outer endosome-fusogenic envelopes. These results support the conclusion that mitochondrial-targeted DNA delivery using a DF-MITO-Porter can be achieved when intracellular trafficking is optimally regulated.
  • Kazuhiro Takara, Hiroto Hatakeyama, Golam Kibria, Noritaka Ohga, Kyoko Hida, Hideyoshi Harashima JOURNAL OF CONTROLLED RELEASE 162 (1) 225 -232 2012年08月 [査読無し][通常論文]
     
    Anti-angiogenic therapy is a potential chemotherapeutic strategy for the treatment of drug resistant cancers. However, a method for delivering such drugs to tumor endothelial cells remains to be a major impediment to the success of anti-angiogenesis therapy. We designed liposomes (LPs) with controlled diameter of around 300 nm, and modified them with a specific ligand and a cell penetrating peptide (CPP) (a dual-ligand LP) for targeting CD13-expressing neovasculature in a renal cell carcinoma (RCC). We modified the LPs with an NGR motif peptide on the top of poly(ethylene glycol) and tetra-arginine (R4) on the surface of the liposome membrane as a specific and CPP ligand, respectively. The large size prevented extravasation of the dual-ligand LP, which allowed it to associate with target vasculature. While a single modification with either the specific or CPP ligand showed no increase in targetability, the dual-ligand enhanced the amount of delivered liposomes after systemic administration to OS-RC-2 xenograft mice. The anti-tumor activity of a dual-ligand LP encapsulating doxorubicin was evaluated and the results were compared with Doxil (R), which is clinically used to target tumor cells. Even though Doxil showed no anti-tumor activity, the dual-ligand LP suppressed tumor growth because the disruption of tumor vessels was efficiently induced. The comparison showed that tumor endothelial cells (TECs) were more sensitive to doxorubicin by 2 orders than RCC tumor cells, and the disruption of tumor vessels was efficiently induced. Collectively, the dual-ligand LP is promising carrier for the treatment of drug resistant RCC via the disruption of TECs. (C) 2012 Elsevier B. V. All rights reserved.
  • 原島 秀吉, 秋田 英万 Drug delivery system : DDS : official Journal of the Japan Society of Drug Delivery System 27 (3) 218 -220 2012年07月
  • T. Nakamura, H. Akita, Y. Yamada, H. Hatakeyama, H. Harashima ACCOUNTS OF CHEMICAL RESEARCH 45 (7) 1113 -1121 2012年07月 [査読無し][通常論文]
     
    In the 21st century, drug development has shifted toward larger molecules such as proteins and nucleic adds, which require the use of new chemical strategies. In this process, the drug delivery system plays a central role and intracellular targeting using nanotechnology has become a key technology for the development of successful new medicines. We have developed a new delivery system, a multifunctional envelope-type nanodevice (MEND) based on "Programmed Packaging." In this new concept of packaging, multifunctional nanodevices are integrated into a nanocarrier system according to a program designed to overcome all barriers during the course of biodistribution and intracellular trafficking. In this Account, we introduce our method for delivering nucleic adds or proteins to intracellular sites of action such as the cytosol, nucleus, and mitochondria and for targeting selective tissues in vivo via systemic administration of the nanodevices. First, we introduce an octaarginine-modified MEND (R8-MEND) as an efficient intracellular delivery system, designed especially for vaccinations and transgene expression. Many types of cells can internalize the R8-MEND, mainly by inducing macropinocytosis, and the MEND escapes from macropinosomes via membrane fusion, which leads to efficient antigen presentation via the major histocompatibility complex I pathway in antigen-presenting cells. In addition, the transfection activities of the R8-MEND in dividing cells, such as Hela or A549 cells, are as high as those for adenovirus. However, because the R8-MEND cannot induce sufficient transgene activity in primary cultured dendritic cells, which are critical regulators of the immune response, we converted the R8-MEND into a tetralamellar MEND (T-MEND). The T-MEND uses a new packaging method and delivers condensed pDNA into the nucleus via fusion between the envelopes and the nuclear membrane. To achieve efficient transfection activity, we also optimized the decondensation of nucleic adds within the nucleus. To optimize mitochondrial drug delivery, we introduced the MITOPorter. Many types of materials can be packaged into this liposome-based nanocarrier and then delivered to mitochondria via membrane fusion mechanisms. Finally, we describe an integrated strategy for in vivo tumor delivery and optimization of intracellular trafficking. Successful tumor delivery typically requires coating the surfaces of nanoparticles with PEG, but PEG can also limit uptake by the reticuloendothelial system and reduce the efficiency of intracellular trafficking Here we integrate the optimum biodistribution and intracellular trafficking of the MEND with an Innovative strategy such as enzymatically cleavable PEG and a short membrane peptide, GALA. Some of these strategies will soon be tested in the clinic
  • Mayumi Takahashi, Chisato Nagai, Hiroto Hatakeyama, Noriaki Minakawa, Hideyoshi Harashima, Akira Matsuda NUCLEIC ACIDS RESEARCH 40 (12) 5787 -5793 2012年07月 [査読無し][通常論文]
     
    Chemically modified siRNAs are expected to have resistance toward nuclease degradation and good thermal stability in duplex formation for in vivo applications. We have recently found that 2'-OMe-4'-thioRNA, a hybrid chemical modification based on 2'-OMeRNA and 4'-thioRNA, has high hybridization affinity for complementary RNA and significant resistance toward degradation in human plasma. These results prompted us to develop chemically modified siRNAs using 2'-OMe-4'-thioribonucleosides for therapeutic application. Effective modification patterns were screened with a luciferase reporter assay. The best modification pattern of siRNA, which conferred duration of the gene-silencing effect without loss of RNAi activity, was identified. Quantification of the remaining siRNA in HeLa-luc cells using a Heat-in-Triton (HIT) qRT-PCR revealed that the intracellular stability of the siRNA modified with 2'-OMe-4'-thioribonucleosides contributed significantly to the duration of its RNAi activity.
  • 古川亮, 山田勇磨, 原島秀吉 日本DDS学会学術集会プログラム予稿集 28th 134 2012年06月05日 [査読無し][通常論文]
  • 安崎友香理, 山田勇磨, 兼藤努, LIU Dexi, 原島秀吉 日本DDS学会学術集会プログラム予稿集 28th 119 2012年06月05日 [査読無し][通常論文]
  • 肺血管内皮細胞標的型siRNAキャリアの標的メカニズムとその抗腫瘍効果
    楠本 憲司, 秋田 英万, 松本 有, 野本 貴大, 片岡 一則, 原島 秀吉 日本DDS学会学術集会プログラム予稿集 28回 119 -119 2012年06月 [査読無し][通常論文]
  • マイクロ流体チップによる多機能性エンベロープ型ナノ構造体の作製
    加地 範匡, 北添 雄眞, 重中 大輔, 小暮 健太朗, 秋田 英万, 原島 秀吉, 渡慶次 学, 馬場 嘉信 日本DDS学会学術集会プログラム予稿集 28回 182 -182 2012年06月 [査読無し][通常論文]
  • Mst Naznin Ara, Mamoru Hyodo, Noritaka Ohga, Kyoko Hida, Hideyoshi Harashima CANCER RESEARCH 72 2012年04月 [査読無し][通常論文]
  • 古川亮, 山田勇磨, 原島秀吉 日本薬学会年会要旨集 132nd (1) 246 2012年03月05日 [査読無し][通常論文]
  • 中村宏平, 山田勇磨, 古川亮, 河村恵理子, 奥田勝博, 新藤充, 原島秀吉 日本薬学会年会要旨集 132nd (4) 209 2012年03月05日 [査読無し][通常論文]
  • 紙谷浩之, 紙谷浩之, 紙谷浩之, 紙谷浩之, 宮本紫甫, 後藤仁美, 神田元紀, 神田元紀, 小林三和子, 松岡一郎, 原島秀吉, 原島秀吉 日本分子生物学会年会プログラム・要旨集(Web) 35th 2012年
  • 紙谷浩之, 紙谷浩之, 紙谷浩之, 紙谷浩之, 宮本紫甫, 後藤仁美, 神田元紀, 神田元紀, 小林三和子, 松岡一郎, 原島秀吉, 原島秀吉 日本DDS学会学術集会プログラム予稿集 28th 2012年
  • 重中大輔, 鵜川真美, 加地範匡, 岡本行広, 渡慶次学, 秋田英万, 原島秀吉, 馬場嘉信 日本化学会講演予稿集 92nd (2) 2012年
  • DDS技術による遺伝子・核酸医薬の将来
    梶本和昭, 兵藤守, 小暮健太朗, 原島秀吉 月刊ファインケミカル 特集 先端医療実用化のための最新DDS技術の動向 41 (4) 31 -36 2012年 [査読無し][招待有り]
  • 多機能性エンベロープ型ナノ構造体の創薬への応用
    林泰弘, 梶本和昭, 原島秀吉 細胞. 特集 疾患治療とドラッグデリバリーシステム 44 8 -11 2012年 [査読無し][招待有り]
  • ドラッグデリバリーシステムの新展開
    原島 秀吉, 松村 保広, 木村 廣道, 片岡 一則 HUMAN SCIENCE 23 (1) 4 -12 2012年01月 [査読無し][通常論文]
  • 紙谷 浩之, 山崎 大樹, 原島 秀吉 日本環境変異原学会大会プログラム・要旨集 0 (40) 138 -138 2011年10月20日 [査読無し][通常論文]
  • H. Hatakeyama, M. Yamamoto, Y. Hayashi, K. Kajimoto, H. Akita, N. Kaji, Y. Baba, H. Harashima NUCLEIC ACID THERAPEUTICS 21 (5) A34 -A34 2011年10月 [査読無し][通常論文]
  • Yusuke Sato, Hiroto Hatakeyama, Yu Sakurai, Mamoru Hyodo, Hidetaka Akita, Hideyoshi Harashima NUCLEIC ACID THERAPEUTICS 21 (5) A41 -A42 2011年10月 [査読無し][通常論文]
  • Yu Sakurai, Hiroto Hatakeyama, Yusuke Sato, Hidetaka Akita, Hideyoshi Harashima NUCLEIC ACID THERAPEUTICS 21 (5) A41 -A41 2011年10月 [査読無し][通常論文]
  • Yasuhiro Hayashi, Jun Yamauchi, Ikramy A. Khalil, Kazuaki Kajimoto, Hidetaka Akita, Hideyoshi Harashima INTERNATIONAL JOURNAL OF PHARMACEUTICS 419 (1-2) 308 -313 2011年10月 [査読無し][通常論文]
     
    The cell-penetrating peptide (CPP) is one of the most attractive tools for efficiently delivering biomolecules to a target organelle. Here, we describe the use of octaarginine (R8)-modified lipid nanoparticles for the efficient and targeted in vivo delivery of siRNA to the liver. In this study, SR-BI (a scavenger receptor class B, member 1) was targeted by this nanoparticle. Our results demonstrate that R8-modified lipid nanoparticles can be used for the efficient and targeted delivery of liver siRNA to induce the specific knock-down of an endogenous gene with minimum liver toxicity and immune response, and that this CPP based technology holds considerable promise for further in vivo biological applications of siRNA. (C) 2011 Elsevier B.V. All rights reserved.
  • 原島 秀吉 バイオイメージング 20 (2) 49 -50 2011年08月10日 [査読無し][通常論文]
  • Yuma Yamada, Ryo Furukawa, Yukari Yasuzaki, Hideyoshi Harashima MOLECULAR THERAPY 19 (8) 1449 -1456 2011年08月 [査読無し][通常論文]
     
    Mitochondrial dysfunction is associated with a variety of human diseases including inherited mitochondrial diseases, neurodegenerative disorders, diabetes mellitus, and cancer. Effective medical therapies for mitochondrial diseases will ultimately require an optimal drug delivery system, which will likely be achieved through innovations in the nanotechnology of intracellular trafficking. To achieve efficient mitochondrial drug delivery, two independent processes, i.e., "cytoplasmic delivery through the cell membrane" and "mitochondrial delivery through the mitochondrial membrane" are required. In previous studies, we developed an octaarginine (R8) modified nano carrier for efficient cytoplasmic delivery, showing that R8-modified liposomes were internalized into cells efficiently. On the other hand, we also constructed MITO-Porter for the mitochondrial delivery of macromolecules, a liposome-based carrier that delivers cargos to mitochondria via membrane fusion. Here, we report the development of a dual function MITO-Porter (DF-MITO-Porter), based on the concept of integrating both R8-modified liposomes and MITO-Porter. We show that the DF-MITO-Porter effectively delivers exogenous macro-biomolecules into the mitochondrial matrix, and provide a demonstration of its potential use in therapies aimed at mitochondrial DNA.
  • Hidetaka Akita, Yuko Umetsu, Dai Kurihara, Hideyoshi Harashima INTERNATIONAL JOURNAL OF PHARMACEUTICS 415 (1-2) 218 -220 2011年08月 [査読無し][通常論文]
     
    Heterogeneity of transgene expression is a severe disadvantage in the use of cationic lipid-mediated gene vectors. We previously demonstrated that heterogeneity of the post-nuclear delivery process, as well as intracellular trafficking (i.e. nuclear delivery) is a major determinant in the overall heterogeneity in gene expression, when plasmid DNA (pDNA) is transfected to HeLa cells using a lipoplex. In this study, we explored the mechanism underlying this heterogeneity in a post-nuclear transport process by the dual imaging of mRNA and its encoded protein (histone H2B-tagged mTFP1: mTFP1-H2B) in a single cell. To establish a highly sensitive imaging system for mRNA, we used fluorescence in situ hybridization (FISH) combined with tyramide signal amplification (TSA) and a semiconductor quantum dot (QD) probe. The mRNA expression and protein production were quantified by counting the total pixel intensity in the region of interest (r.o.i.) surrounding single cells. As a result, the correlation was poor in a scattered plot of mRNA expression versus protein production in individual cells. These findings demonstrate that cell-to-cell differences in the translation process are also a key factor in heterogeneous gene expression. (C) 2011 Elsevier B.V. All rights reserved.
  • Taichi Ishitsuka, Hidetaka Akita, Hideyoshi Harashima JOURNAL OF CONTROLLED RELEASE 154 (1) 77 -83 2011年08月 [査読無し][通常論文]
     
    The targeted delivery of genes to endothelial cells is a potential strategy for curing certain types of disorders including cancer, inflammation and obesity. We previously reported that a liposome (IRQ-LP) modified with the IRQ peptide (IRQRRRR) was taken up by cells via a unique pathway, namely caveolar endocytosis, a cellular uptake pathway that is involved in the blood-to-tissue uptake of macromolecules in vascular endothelial cells. In the present study, we initally investigated the effect of IRQ peptide-modification on the biodistribution of poly(ethyleneglycol) (PEG)-coated liposomes (PEG-LP) after i.v. administration. The IRQ peptide-modified PEG-LP (IRQ-PEG-LP), as well as the PEG-LP were found to be mainly accumulated in the liver. Nevertheless, the fold increase in the lung accumulation of IRQ-PEG-LP, compared to the PEG-LP (approximately 20-folds) was substantially higher than other tissues (<5-fold). Thus, IRQ could function as a target ligand for lungs. We then used the IRQ peptide as a model for a ligand for targeting normal tissue endothelial cells, and then applied it to a gene delivery system. We previously developed a multifunctional envelope-type nano device (MEND), in which plasmid DNA is condensed using a polycation to form a core particle that is encapsulated in a lipid envelope. We modified the IRQ-modified PEG to the MEND (IRQ-PEG-MEND) and marker gene expression was evaluated after i.v. administration. However the transgene expression of the IRQ-PEG-MEND in lungs was low. This is most likely due to the inhibitory effect of the PEG spacer on intracellular trafficking (especially endosomal escape) of the IRQ-PEG-MEND. To overcome the dilemma associated with PEGylation, we improved the MEND system from the point of view of PEG length, lipid chain of the PEG derivative, the polycation and cationic lipid. As a result, transgene expression in lungs was enhanced in stepwise manner, and was finally improved by 5 orders of magnitude compared with the original IRQ-PEG-MEND. Overcoming the dilemma of PEGylation is critical issue for in vivo applications of gene delivery targeting endothelial cells. (c) 2011 Elsevier B.V. All rights reserved.
  • 楠本 憲司, 畠山 浩人, 原島 秀吉 ファルマシア 47 (8) 719 -723 2011年08月01日 [査読無し][通常論文]
  • Hiroto Hatakeyama, Erika Ito, Momoko Yamamoto, Hidetaka Akita, Yasuhiro Hayashi, Kazuaki Kajimoto, Noritada Kaji, Yoshinobu Baba, Hideyoshi Harashima MOLECULAR THERAPY 19 (8) 1487 -1498 2011年08月 [査読無し][通常論文]
     
    The purpose of this study was to investigate the host response to systemically administered lipid nanoparticles (NPs) encapsulating plasmid DNA (pDNA) in the spleen using a DNA microarray. As a model for NPs, we used a multifunctional envelope-type nano device (MEND). Microarray analysis revealed that 1,581 of the differentially expressed genes could be identified by polyethylene glycol (PEG)-unmodified NP using a threefold change relative to the control. As the result of PEGylation, the NP treatment resulted in the reduction in the expression of most of the genes. However, the expression of type I interferon (IFN) was specifically increased by PEGylation. Based on the microarray and a pathway analysis, we hypothesize that PEGylation inhibited the endosomal escape of NP, and extended the interaction of toll-like receptor-9 (TLR9) with CpG-DNA accompanied by the production of type I IFN. This hypothesis was tested by introducing a pH-sensitive fusogenic peptide, GALA, which enhances the endosomal escape of PEGylated NP. As expected, type I IFN was reduced and interleukin-6 (IL-6) remained at the baseline. These findings indicate that a carrier design based on microarray analysis and the manipulation of intracellular trafficking constitutes a rational strategy for reducing the host immune response to NPs.
  • Golam Kibria, Hiroto Hatakeyama, Noritaka Ohga, Kyoko Hida, Hideyoshi Harashima JOURNAL OF CONTROLLED RELEASE 153 (2) 141 -148 2011年07月 [査読無し][通常論文]
     
    The objective of this study was to develop an efficient dual-ligand based PEGylated liposomal delivery system that had target specificity as well as properties that would enhance cellular uptake. PEGylated liposomes (PEG-LP) were prepared by the lipid film hydration method by adding distearoyl phosphoethanolamine-polyethylene-glycol-2000 conjugate (DSPE-PEG2000) to a lipid mixture. The cyclic RGD (Arg-Gly-Asp) peptide, a specific ligand with affinity for Integrin alpha(v)beta(3) was coupled to the distal end of the PEG on the PEG-LP (RGD-PEG-LP). Stearylated octaarginine (STR-R8) was incorporated on the surface of the RGD-PEG-LP as dual-ligand (R8/RGD-PEG-LP) that functions as a cell penetrating peptide (CPP). RGD-PEG-LP and R8/RGD-PEG-LP were preferentially taken up by caveolae-mediated and clathrin-mediated endocytosis pathways, respectively. Compared to PEG-LP, R8/RGD-PEG-LP showed an enhanced cellular uptake as well as a higher transfection efficiency in Integrin alpha(v)beta(3) expressing cells. However, the amount of cellular uptake or gene expression by the single ligand versions was negligible, even in Integrin alpha(v)beta(3) expressing cells. No remarkable difference in cellular uptake or gene expression was observed for cells in which the expression of targeted receptors was absent. It can be concluded that dual-ligand modified PEG-LP possesses a strong capability for the efficient internalization of PEG-LP and consequently would be an effective tool for the targeted delivery of macromolecules or chemotherapeutics through accelerated cellular uptake. (C) 2011 Elsevier B.V. All rights reserved.
  • Golam Kibria, Hiroto Hatakeyama, Hideyoshi Harashima INTERNATIONAL JOURNAL OF PHARMACEUTICS 412 (1-2) 106 -114 2011年06月 [査読無し][通常論文]
     
    Protective antigen (PA) is a nontoxic protein present in anthrax toxin. Domain 4 of PA (PA-D4) acts as a receptor binding site for tumor endothelial marker 8 (TEM8). In this study, KYND motif from PA-D4 was utilized as a ligand against TEM8. The efficiency of KYND motif on cellular association was assessed by evaluating the cellular uptake of PEGylated liposomes (PEG-LPs) in TEM8 positive and negative cells. The peptide was attached on the top of the PEG of PEG-LP. Compared to PEG-LP, KYND modified PEG-LP (KYND-PEG-LP) enhanced the cellular uptake to a greater extent in all cell lines. Based on the inhibition assay, no receptor involvement was observed in the cellular association of KYND-PEG-LP, suggesting that KYND motif functions as a cell penetrating peptide (CPP) which facilitated the internalization of PEG-LP via clathrin mediated endocytosis pathway. Further enhancement of cellular uptake was observed when KYND-PEG-LP was combined with octaarginine (R8) on the surface of lipid membrane as dual-CPP ligand formulation, however, when PEG-LP combined with only R8, only negligible enhancement was observed. These findings suggest that two CPP ligands act in a synergistic fashion; therefore the dual-CPP ligand based liposomal formulation can be assumed to be an effective delivery system. (C) 2011 Elsevier B.V. All rights reserved.
  • 古川亮, 山田勇磨, 松島雄一, 後藤雄一, 原島秀吉 Drug Deliv Syst 26 (3) 317 2011年05月28日 [査読無し][通常論文]
  • 山田勇磨, 原島秀吉 Drug Deliv Syst 26 (3) 285 2011年05月28日 [査読無し][通常論文]
  • 山田勇磨, 河村恵理子, 安崎友香理, 古川亮, 原島秀吉 日本薬剤学会年会講演要旨集 26th 102 2011年05月01日 [査読無し][通常論文]
  • 原島 秀吉 バイオマテリアル : 生体材料 : journal of Japanese Society for Biomaterials 29 (2) 77 -78 2011年04月28日 [査読無し][通常論文]
  • 常樂 晃, 宮崎 淳, 中谷 彰洋, 中村 孝司, 河合 弘二, 矢野 郁也, 原島 秀吉, 赤座 英之 日本泌尿器科學會雜誌 102 (2) 148 -148 2011年03月20日 [査読無し][通常論文]
  • 山田勇磨, 鈴木亮佑, 原島秀吉 日本薬学会年会要旨集 131st (4) 159 2011年03月05日 [査読無し][通常論文]
  • 河村恵理子, 山田勇磨, 原島秀吉 日本薬学会年会要旨集 131st (4) 159 2011年03月05日 [査読無し][通常論文]
  • 神田元紀, 神田元紀, 神田元紀, 福永賢輝, 棚瀬潤一, 原島秀吉, 原島秀吉, 大山隆, 紙谷浩之, 紙谷浩之, 紙谷浩之 日本分子生物学会年会プログラム・要旨集(Web) 34th 2011年
  • KAWAMURA Eriko, YAMADA Yuma, HARASHIMA Hideyoshi 日本ミトコンドリア学会年会要旨集 11th 178 2011年 [査読無し][通常論文]
  • YASUZAKI Yukari, YAMADA Yuma, KANEFUJI Tsutomu, LIU Dexi, HARASHIMA Hideyoshi 日本ミトコンドリア学会年会要旨集 11th 170 2011年 [査読無し][通常論文]
  • FURUKAWA Ryo, YAMADA Yuma, MATSUSHIMA Yuichi, GOTO Yu‐ichi, HARASHIMA Hideyoshi 日本ミトコンドリア学会年会要旨集 11th 179 2011年 [査読無し][通常論文]
  • YAMADA Yuma, HARASHIMA Hideyoshi 日本ミトコンドリア学会年会要旨集 11th 149 2011年 [査読無し][通常論文]
  • Tetsuya Suzuki, Hideyoshi Harashima, Hiroyuki Kamiya Genes and Environment 33 (3) 103 -108 2011年 [査読無し][通常論文]
     
    The loss of tumor suppressor proteins, p53 and retinoblastoma (Rb), causes genomic instability. 8-Oxo-7,8-dihydroguanine (G O, 8-hydroxyguanine) is a major oxidatively damaged base that induces G:C←T:A transversions in cells. In this study, the effects of p53 and Rb reductions on the mutagenicity of GO in DNA were investigated, using a supF shuttle plasmid propagated in human U2OS and HT1080 cells. The p53 and Rb proteins were individually knocked-down by siRNAs, and then the plasmid DNA containing G O was introduced into the knocked-down cells. The knock-downs of p53 and Rb had quite weak effects on mutagenesis by G O in the shuttle plasmid. These results suggested that p53 and Rb minimally affect the G O-induced mutagenic processes in living cells. © The Japanese Environmental Mutagen Society.
  • 紙谷 浩之, 鈴木 哲矢, 原島 秀吉 日本環境変異原学会大会プログラム・要旨集 0 (39) 98 -98 2010年10月29日 [査読無し][通常論文]
  • Md. Nazir Hossen, Kazuaki Kajimoto, Hidetaka Akita, Mamoru Hyodo, Taichi Ishitsuka, Hideyoshi Harashima JOURNAL OF CONTROLLED RELEASE 147 (2) 261 -268 2010年10月 [査読無し][通常論文]
     
    Ligand-based targeted delivery is an emerging platform in nanomedicine. We report herein on a peptide modified nanocarrier for a ligand-based targeted delivery system. The liposomal surface of the carrier was first modified with a linear peptide, followed by an adipose tissue-specific circular peptide (KGGRAKD) via a polyethylene glycol (PEG) spacer. To evaluate the specificity of the carrier, we purified primary cells from the endothelium of adipose tissue. The liposomes bound only to isolated primary cultured endothelial cells derived from inguinal adipose tissue (pcEC-IWAT) and not to other endothelial cell lines, such as MBEC-4 and MFLM-4. Cellular uptake was confirmed both qualitatively and quantitatively by confocal laser scanning microscopy (CLSM) and flow cytometry. The mechanism for the intracellular uptake of tPep-PEG-LPs into pcEC-IWAT, as evidenced by three independent experiments, involves saturation of receptor binding sites by excess free peptide, the blocking of receptors by an anti-prohibitin antibody and low temperature (4 C) experiments, resulting in the inhibition of uptake of tPep-PEG-LPs into pcEC-IWAT, suggesting that receptor mediated endocytosis largely contributed to the observed cellular uptake. A co-localization study using double labeled modified liposomes (lipid membrane: NBD-DOPE and aqueous phase: rhodamine) indicated that a predominant part of tPep-PEG-LPs was found without co-localization with lysosomes and retained their intactness. The selective delivery of tPep-PEG-LPs to endothelial cells in adipose tissue represents a potential approach for the design of diverse nanocarrier-based targeted delivery systems for targeting the vasculature in adipose tissue. (C) 2010 Elsevier B.V. All rights reserved.
  • Kazuhiro Takara, Hiroto Hatakeyama, Noritaka Ohga, Kyoko Hida, Hideyoshi Harashima INTERNATIONAL JOURNAL OF PHARMACEUTICS 396 (1-2) 143 -148 2010年08月 [査読無し][通常論文]
     
    In this study, a dual-ligand liposomal system comprised of a specific ligand and a cell penetrating peptide (CPP) is described to enhance selectivity and cellular uptake. Dual-ligand PEGylated liposomes were prepared by modifying the end of the PEG with an NGR motif peptide, followed by a surface coating of the liposomes with stearylated oligoarginine (STR-RX) The NGR motif recognizes CD13. a marker protein located on tumor endothelial cells A suitable number of RX units was determined to be R4, since it can be masked by the PEG aqueous layer Although no enhanced cellular uptake was observed when a single modification of PEGylated liposomes with either NGR- or STR-R4 was used, the dual-modification with NGR and STR-R4 stimulated uptake of PEGylated liposomes by CD13 positive cells, and this uptake was superior to that obtained by PEG-unmodified liposomes modified with STR-R4 The dual-ligand system shows a synergistic effect on cellular uptake Collectively, the dual-ligand system promises to be useful in the development efficient and specific drug delivery systems. (C) 2010 Elsevier B V All rights reserved.
  • Mana Ito, Yusuke Suda, Hideyoshi Harashima, Hiroyuki Kamiya BIOLOGICAL & PHARMACEUTICAL BULLETIN 33 (7) 1223 -1227 2010年07月 [査読無し][通常論文]
     
    To enhance the levels of transgene expression from plasmid-based nonviral vectors, replicating plasmids containing the SV40 origin and the SV40 large T antigen gene, as a model replicating unit, were constructed. The replicating luciferase plasmid DNA produced the luciferase protein more efficiently than the non-replicating luciferase plasmid DNA, as expected. Surprisingly, the introduction of the replicating plasmid DNA containing the Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) gene was highly cytotoxic and caused cell death without nucleoside analogs. Our results confirm that transgenes on a replicating plasmid represent an excellent tool for effective