Researcher Profile and Settings

Master

Affiliation (Master)

• Faculty of Pharmaceutical Sciences　Biopharmaceutical Sciences and Pharmacy　Biopharmaceutical Sciences and Pharmacy

Affiliation (Master)

• Faculty of Pharmaceutical Sciences　Biopharmaceutical Sciences and Pharmacy　Biopharmaceutical Sciences and Pharmacy

researchmap

Profile and Settings

Degree

• PhD（Hokkaido University）

Profile and Settings

• Name (Japanese)

  Nakamura

• Name (Kana)

  Takashi

• Name

  201301026023817381

Achievement

Research Interests

• 免疫細胞療法   がんワクチン   ナノ粒子   DDS   がん免疫療法   核酸   アジュバント   膀胱がん   がん免疫   ワクチン   ナノメディシン   リポソーム   ドラッグデリバリーシステム   

Research Areas

• Life sciences / Clinical pharmacy
• Life sciences / Biomedical engineering
• Life sciences / Biomaterials
• Life sciences / Tumor diagnostics and therapeutics
• Life sciences / Tumor biology

Research Experience

• 2023/07 - Today Hokkaido University Faculty of Pharmaceutical Sciences
• 2011/01 - 2023/06 Hokkaido University Faculty of Pharmaceutical Sciences
• 2012/11 - 2017/10 国立薬品食品衛生研究所 薬品部 協力研究員
• 2010/04 - 2010/12 Hokkaido University Faculty of Pharmaceutical Sciences
• 2007/04 - 2010/03 日本学術振興会 特別研究員（DC1）

Education

• 2007/04 - 2010/03  Hokkaido University  Graduate School of Life Science  Division of Life Sciences
• 2005/04 - 2007/03  Hokkaido University

Awards

• 2019/07 日本DDS学会 奨励賞
   

  受賞者: 中村 孝司
• 2018/03 日本薬学会 奨励賞
   

  受賞者: 中村 孝司
• 2016/05 日本薬剤学会 奨励賞
   

  受賞者: 中村孝司
• 2015/05 日本薬学会北海道支部 奨励賞
   

  受賞者: 中村孝司
• 2012/05 日本薬剤学会 日本薬剤学会第27年会 最優秀発表者賞
   

  受賞者: 中村孝司
• 2005/03 日本薬剤学会 日本薬剤学会創立20周年記念大会 最優秀発表者賞
   

  受賞者: 中村孝司

Published Papers

• Delivering mRNA to a human NK cell line, NK-92 cells, by lipid nanoparticles.

  Takashi Nakamura, Taisei Nakade, Yusuke Sato, Hideyoshi Harashima

  International journal of pharmaceutics 636 122810 - 122810 2023/04/05 

  In cancer immunotherapy, therapeutic methods targeting NK are highly expected. NK cell-based therapy using NK-92, a human NK cell line, has been clinically evaluated. Delivering mRNA into NK-92 cells is a potent strategy for enhancing its functions. However, the use of lipid nanoparticles (LNP) for this purpose has not yet been evaluated. We previously developed a LNP that was composed of CL1H6 (CL1H6-LNP) for the efficient delivery of siRNA to NK-92 cells, and the use of this material for delivering mRNA to NK-92 cells is reported in this study. Compared with a DLin-MC3-DMA based LNP, used as a benchmark, the CL1H6-LNP caused a high mRNA expression intensity and a cell transfection efficiency of 100%. The efficient mRNA delivery by this CL1H6-LNP is attributed to the high affinity for NK-92 cells and the intense, rapid fusion with the endosomal membrane. It therefore appears that the CL1H6-LNP could be a useful non-viral vector for modifying the NK-92 functions by mRNA. Our findings also provide some insights into the design and development of LNPs for delivering mRNA to NK-92 and NK cells.
• Interval- and cycle-dependent combined effect of STING agonist loaded lipid nanoparticles and a PD-1 antibody.

  Alaa M Khalifa, Takashi Nakamura, Yusuke Sato, Takanori Sato, Mamoru Hyodo, Yoshihiro Hayakawa, Hideyoshi Harashima

  International journal of pharmaceutics 624 122034 - 122034 2022/08/25 [Refereed][Not invited]
   

  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.
• Extrahepatic targeting of lipid nanoparticles in vivo with intracellular targeting for future nanomedicines.

  Takashi Nakamura, Yusuke Sato, Yuma Yamada, Mahmoud M Abd Elwakil, Seigo Kimura, Mahmoud A Younis, Hideyoshi Harashima

  Advanced drug delivery reviews 188 114417 - 114417 2022/07/03 [Refereed]
   

  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.
• Retrograde Axonal Transport of Liposomes from Peripheral Tissue to Spinal Cord and DRGs by Optimized Phospholipid and CTB Modification.

  Takafumi Fukui, Hironao Tateno, Takashi Nakamura, Yuma Yamada, Yusuke Sato, Norimasa Iwasaki, Hideyoshi Harashima, Ken Kadoya

  International journal of molecular sciences 23 (12) 2022/06/15 [Refereed]
   

  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.
• Innovative cancer nanomedicine based on immunology, gene editing, intracellular trafficking control.

  Yuma Yamada, Yusuke Sato, Takashi Nakamura, Hideyoshi Harashima

  Journal of controlled release : official journal of the Controlled Release Society 348 357 - 369 2022/06/09 [Refereed]
   

  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.
• Combined nano cancer immunotherapy based on immune status in a tumor microenvironment.

  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 2022/05 [Refereed]
   

  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.
• Novel antiangiogenic therapy targeting biglycan using tumor endothelial cell-specific liposomal siRNA delivery system.

  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 113 (5) 1855 - 1867 2022/03/09 [Refereed]
   

  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.
• Strategies for fighting pandemic virus infections: Integration of virology and drug delivery.

  Takashi Nakamura, Norikazu Isoda, Yoshihiro Sakoda, Hideyoshi Harashima

  Journal of controlled release : official journal of the Controlled Release Society 343 361 - 378 2022/02/03 [Refereed]
   

  Respiratory viruses have sometimes resulted in worldwide pandemics, with the influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) being major participants. Long-term efforts have made it possible to control the influenza virus, but seasonal influenza continues to take many lives each year, and a pandemic influenza virus sometimes emerges. Although vaccines for coronavirus disease 2019 (COVID-19) have been developed, we are not yet able to coexist with the SARS-CoV-2. To overcome such viruses, it is necessary to obtain knowledge about international surveillance systems, virology, ecology and to determine that immune responses are effective. The information must then be transferred to drugs. Delivery systems would be expected to contribute to the rational development of drugs. In this review, virologist and drug delivery system (DDS) researchers discuss drug delivery strategies, especially the use of lipid-based nanocarriers, for fighting to respiratory virus infections.
• The hydrophobic tail of a pH-sensitive cationic lipid influences siRNA transfection activity and toxicity in human NK cell lines

  Takashi Nakamura, Taisei Nakade, Koharu Yamada, Yusuke Sato, Hideyoshi Harashima

  International Journal of Pharmaceutics 609 121140 - 121140 0378-5173 2021/11 [Refereed]
   

  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.
• STING agonist loaded lipid nanoparticles overcome anti-PD-1 resistance in melanoma lung metastasis via NK cell activation

  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) e002852 - e002852 2021/07 [Refereed]
   

  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. 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. 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. 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.
• リポソームを応用した末梢組織から脊髄への低侵襲薬物輸送システムの開発

  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 (公社)日本整形外科学会 95 (2) S123 - S123 0021-5325 2021/03
• The nanomedicine rush: New strategies for unmet medical needs based on innovative nano DDS.

  Yusuke Sato, Takashi Nakamura, Yuma Yamada, Hideyoshi Harashima

  Journal of controlled release : official journal of the Controlled Release Society 330 305 - 316 2021/02/10 

  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.
• Dawn of lipid nanoparticles in lymph node targeting: Potential in cancer immunotherapy

  Takashi Nakamura, Hideyoshi Harashima

  Advanced Drug Delivery Reviews 167 78 - 88 0169-409X 2020/12 [Refereed][Invited]
   

  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.
• Evolution of drug delivery system from viewpoint of controlled intracellular trafficking and selective tissue targeting toward future nanomedicine.

  Yuma Yamada, Yusuke Sato, Takashi Nakamura, Hideyoshi Harashima

  Journal of controlled release : official journal of the Controlled Release Society 327 533 - 545 2020/11/10 

  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.
• 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 0546-0476 2020/10
• Lipid nanoparticles fuse with cell membranes of immune cells at low temperatures leading to the loss of transfection activity.

  Takashi Nakamura, Koharu Yamada, Yusuke Sato, Hideyoshi Harashima

  International journal of pharmaceutics 587 119652 - 119652 2020/07/17 [Refereed][Not invited]
   

  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.
• Distribution of BCG-CWS-Loaded Nanoparticles in the Spleen After Intravenous Injection Affects Cytotoxic T Lymphocyte Activity.

  Hideyuki Masuda, Takashi Nakamura, Hideyoshi Harashima

  Journal of pharmaceutical sciences 109 (6) 1943 - 1950 2020/06 [Refereed][Not invited]
   

  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.
• 【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 (株)ニュー・サイエンス社 52 (5) 245 - 248 1346-7557 2020/05 

  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)
• 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 (公社)日本薬剤学会 35年会 171 - 171 2020/05
• Vitamin E Scaffolds of pH-Responsive Lipid Nanoparticles as DNA Vaccines in Cancer and Protozoan Infection.

  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 2020/04/06 [Refereed][Not invited]
   

  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.
• 【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- 日本バイオマテリアル学会 38 (2) 92 - 99 1347-7080 2020/04 

  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)
• The Effect of Size and Charge of Lipid Nanoparticles Prepared by Microfluidic Mixing on Their Lymph Node Transitivity and Distribution.

  Takashi Nakamura, Minori Kawai, Yusuke Sato, Masatoshi Maeki, Manabu Tokeshi, Hideyoshi Harashima

  Molecular pharmaceutics 17 (3) 944 - 953 2020/03/02 [Refereed][Not invited]
   

  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.
• Innovative nanotechnologies for enhancing nucleic acids/gene therapy: Controlling intracellular trafficking to targeted biodistribution.

  Nakamura T, Yamada Y, Sato Y, Khalil IA, Harashima H

  Biomaterials 218 119329 - 119329 0142-9612 2019/10 [Refereed][Invited]
   

  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.
• The silencing of indoleamine 2,3-dioxygenase 1 (IDO1) in dendritic cells by siRNA-loaded lipid nanoparticles enhances cell-based cancer immunotherapy.

  Endo R, Nakamura T, Kawakami K, Sato Y, Harashima H

  Scientific reports 9 (1) 11335 - 11335 2019/08 [Refereed][Not invited]
   

  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.
• Application of BCG-CWS as a Systemic Adjuvant by Using Nanoparticulation Technology.

  Masuda H, Nakamura T, Noma Y, Harashima H

  Molecular pharmaceutics 15 (12) 5762 - 5771 1543-8384 2018/12 [Refereed][Not invited]
   

  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.
• DNA-loaded nano-adjuvant formed with a vitamin E-scaffold intracellular environmentally-responsive lipid-like material for cancer immunotherapy.

  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 1549-9634 2018/08 [Refereed][Not invited]
   

  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.
• Reducing the Cytotoxicity of Lipid Nanoparticles Associated with a Fusogenic Cationic Lipid in a Natural Killer Cell Line by Introducing a Polycation-Based siRNA Core

  Takashi Nakamura, Koharu Yamada, Yuki Fujiwara, Yusuke Sato, Hideyoshi Harashima

  Molecular Pharmaceutics 15 (6) 2142 - 2150 1543-8392 2018/06/04 [Refereed][Not invited]
   

  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.
• 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 (公社)日本薬剤学会 33年会 193 - 193 2018/05
• Liposome Microencapsulation for the Surface Modification and Improved Entrapment of Cytochrome c for Targeted Delivery

  Kazuaki Kajimoto, Tatsuhito Katsumi, Takashi Nakamura, Masatoshi Kataoka, Hideyoshi Harashima

  JAOCS, Journal of the American Oil Chemists' Society 95 (1) 101 - 109 0003-021X 2018/01/01 [Refereed][Not invited]
   

  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.
• [Development of a Nano DDS for Cancer Immunotherapy Based on Llipid Nanoparticles].

  Nakamura T

  Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan 138 (12) 1443 - 1449 0031-6903 2018 [Refereed][Invited]
  
• Integration of nano drug-delivery system with cancer immunotherapy

  Takashi Nakamura, Hideyoshi Harashima

  Therapeutic Delivery 8 (11) 987 - 1000 2041-6008 2017/11/01 [Refereed][Invited]
   

  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.
• 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 日本DDS学会 33回 175 - 175 2017/06
• Modifying Antigen-Encapsulating Liposomes with KALA Facilitates MHC Class I Antigen Presentation and Enhances Anti-tumor Effects

  Naoya Miura, Hidetaka Akita, Naho Tateshita, Takashi Nakamura, Hideyoshi Harashima

  MOLECULAR THERAPY 25 (4) 1003 - 1013 1525-0016 2017/04 [Refereed][Not invited]
   

  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.
• Modifying Cationic Liposomes with Cholesteryl-PEG Prevents Their Aggregation in Human Urine and Enhances Cellular Uptake by Bladder Cancer Cells

  Takashi Nakamura, Yosuke Noma, Yu Sakurai, Hideyoshi Harashima

  BIOLOGICAL & PHARMACEUTICAL BULLETIN 40 (2) 234 - 237 0918-6158 2017/02 [Refereed][Not invited]
   

  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.
• Innovative Technologies in Nanomedicines: From Passive Targeting to Active Targeting/From Controlled Pharmacokinetics to Controlled Intracellular Pharmacokinetics

  Yusuke Sato, Yu Sakurai, Kazuaki Kajimoto, Takashi Nakamura, Yuma Yamada, Hidetaka Akita, Hideyoshi Harashima

  MACROMOLECULAR BIOSCIENCE 17 (1) 1616-5187 2017/01 [Refereed][Invited]
   

  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.
• Development of a multifunctional envelope-type nano device and its application to nanomedicine

  Yusuke Sato, Takashi Nakamura, Yuma Yamada, Hideyoshi Harashima

  JOURNAL OF CONTROLLED RELEASE 244 (Pt B) 194 - 204 0168-3659 2016/12 [Refereed][Invited]
   

  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.
• Small-sized, stable lipid nanoparticle for the efficient delivery of siRNA to human immune cell lines

  Takashi Nakamura, Moeka Kuroi, Yuki Fujiwara, Shota Warashina, Yusuke Sato, Hideyoshi Harashima

  SCIENTIFIC REPORTS 6 37849  2045-2322 2016/11 [Refereed][Not invited]
   

  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.
• Development of a Drug Delivery System for Cancer Immunotherapy

  Takashi Nakamura

  YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 136 (11) 1477 - 1484 0031-6903 2016/11 [Refereed][Invited]
   

  Delivery systems are a powerful technology for enhancing the effect of cancer immunotherapy. We have been in the process of developing lipid-based delivery systems for controlling the physical properties and dynamics of immunofunctional molecules such as antigens and adjuvants. The lipid nanoparticulation of these molecules improves their physical properties, resulting in a good water dispensability, greater stability, and small size. The cell wall skeleton of bacille Calmette-Guerin (BCG-CWS) could be used to replace live BCG as a drug for treating bladder cancer, but problems associated with the physical properties of BCG-CWS have prevented its use. To overcome such problems, we developed a novel packaging method that permits BCG-CWS to be encapsulated into lipid nanoparticles, which induce antitumor responses against bladder cancer. Lipid nanoparticulation also improves the intracellular trafficking and biodistribution of immunofunctional molecules. Cyclic di-GMP (c-di-GMP) is an adjuvant that is recognized by the cytosolic sensor. However, c-di-GMP cannot pass through the cell membrane. We encapsulated c-di-GMP into lipid nanoparticles containing a pH-responsive lipid that was developed in our laboratory and achieved efficient cytosolic delivery and the induction of antitumor immunity. Furthermore, we are attempting to control the functions of immune cells by RNA interference. We have recently succeeded in the efficient delivery of small interfering RNA into mouse dendritic cells (DCs), which led to the enhancement of antitumor activity of DCs. In this review, our recent efforts regarding cancer immunotherapy using lipid-based nanoparticles are reviewed.
• Tri-membrane nanoparticles produced by combining liposome fusion and a novel patchwork of bicelles to overcome endosomal and nuclear membrane barriers to cargo delivery

  Asako Yamada, Asako Mitsueda, Mahadi Hasan, Miho Ueda, Susumu Hama, Shota Warashina, Takashi Nakamura, Hideyoshi Harashima, Kentaro Kogure

  Biomaterials Science 4 (3) 439 - 447 2047-4849 2016/03/01 [Refereed][Not invited]
   

  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.
• A lipid nanoparticle for the efficient delivery of siRNA to dendritic cells

  Shota Warashina, Takashi Nakamura, Yusuke Sato, Yuki Fujiwara, Mamoru Hyodo, Hiroto Hatakeyama, Hideyoshi Harashima

  JOURNAL OF CONTROLLED RELEASE 225 183 - 191 0168-3659 2016/03 [Refereed][Not invited]
   

  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.
• In Vivo Inverse Correlation in the Activation of Natural Killer T Cells Through Dual-Signal Stimulation via a Combination of alpha-Galactosylceramide-Loaded Liposomes and Interleukin-12

  Heba Abdelmegeed, Takashi Nakamura, Hideyoshi Harashima

  JOURNAL OF PHARMACEUTICAL SCIENCES 105 (1) 250 - 256 0022-3549 2016/01 [Refereed][Not invited]
   

  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.
• The intracellular pharmacodynamics of siRNA is responsible for the low gene silencing activity of siRNA-loaded nanoparticles in dendritic cells

  Takashi Nakamura, Yuki Fujiwara, Shota Warashina, Hideyoshi Harashima

  INTERNATIONAL JOURNAL OF PHARMACEUTICS 494 (1) 271 - 277 0378-5173 2015/10 [Refereed][Not invited]
   

  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.
• Liposomes loaded with a STING pathway ligand, cyclic di-GMP, enhance cancer immunotherapy against metastatic melanoma

  Takashi Nakamura, Hiroko Miyabe, Mamoru Hyodo, Yusuke Sato, Yoshihiro Hayakawa, Hideyoshi Harashima

  JOURNAL OF CONTROLLED RELEASE 216 149 - 157 0168-3659 2015/10 [Refereed][Not invited]
   

  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.
• Influence of Endosomal Escape and Degradation of alpha-Galactosylceramide Loaded Liposomes on CD1d Antigen Presentation

  Takashi Nakamura, Moeka Kuroi, Hideyoshi Harashima

  MOLECULAR PHARMACEUTICS 12 (8) 2791 - 2799 1543-8384 2015/08 [Refereed][Not invited]
   

  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.
• A KALA-modified lipid nanoparticle containing CpG-free plasmid DNA as a potential DNA vaccine carrier for antigen presentation and as an immune-stimulative adjuvant

  Naoya Miura, Sharif M. Shaheen, Hidetaka Akita, Takashi Nakamura, Hideyoshi Harashima

  NUCLEIC ACIDS RESEARCH 43 (3) 1317 - 1331 0305-1048 2015/02 [Refereed][Not invited]
   

  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.
• Mechanism responsible for the antitumor effect of BCG-CWS using the LEEL method in a mouse bladder cancer model

  Takashi Nakamura, Masafumi Fukiage, Yoshiteru Suzuki, Ikuya Yano, Jun Miyazaki, Hiroyuki Nishiyama, Hideyuki Akaza, Hideyoshi Harashima

  JOURNAL OF CONTROLLED RELEASE 196 161 - 167 0168-3659 2014/12 [Refereed][Not invited]
   

  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.
• Multifunctional envelope-type nano device for controlled intracellular trafficking and selective targeting in vivo

  Kazuaki Kajimoto, Yusuke Sato, Takashi Nakamura, Yuma Yamada, Hideyoshi Harashima

  JOURNAL OF CONTROLLED RELEASE 190 593 - 606 0168-3659 2014/09 [Refereed][Invited]
   

  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.
• Octaarginine-Modified Liposomes Enhance Cross-Presentation by Promoting the C-Terminal Trimming of Antigen Peptide

  Takashi Nakamura, Kouhei Ono, Yoshiteru Suzuki, Rumiko Moriguchi, Kentaro Kogure, Hideyoshi Harashima

  MOLECULAR PHARMACEUTICS 11 (8) 2787 - 2795 1543-8384 2014/08 [Refereed][Not invited]
   

  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.
• A new adjuvant delivery system 'cyclic di-GMP/YSK05 liposome' for cancer immunotherapy

  Hiroko Miyabe, Mamoru Hyodo, Takashi Nakamura, Yusuke Sato, Yoshihiro Hayakawa, Hideyoshi Harashima

  JOURNAL OF CONTROLLED RELEASE 184 20 - 27 0168-3659 2014/06 [Refereed][Not invited]
   

  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.
• Glycerol Monomycolate Is a Novel Ligand for the Human, but Not Mouse Macrophage Inducible C-type Lectin, Mincle

  Yuki Hattori, Daisuke Morita, Nagatoshi Fujiwara, Daiki Mori, Takashi Nakamura, Hideyoshi Harashima, Sho Yamasaki, Masahiko Sugita

  JOURNAL OF BIOLOGICAL CHEMISTRY 289 (22) 15405 - 15412 0021-9258 2014/05 [Refereed][Not invited]
   

  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.
• Nanoparticulation of BCG-CWS for application to bladder cancer therapy

  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 0168-3659 2014/02 [Refereed][Not invited]
   

  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.
• Multifunctional Enveloped Nanodevices (MENDs)

  Yusuke Sato, Takashi Nakamura, Yuma Yamada, Hidetaka Akita, Hideyoshi Harashima

  NONVIRAL VECTORS FOR GENE THERAPY LIPID- AND POLYMER-BASED GENE TRANSFER 88 139 - 204 0065-2660 2014 [Refereed][Not invited]
   

  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.
• Th1-skewed tissue responses to a mycolyl glycolipid in mycobacteria- infected rhesus macaques

  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 0006-291X 2013/11/08 [Refereed][Not invited]
   

  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-γ, 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. © 2013 Elsevier Inc. All rights reserved.
• A DNA microarray-based analysis of immune-stimulatory and transcriptional responses of dendritic cells to KALA-modified nanoparticles

  Hidetaka Akita, Soichiro Ishii, Naoya Miura, Sharif Mohammad Shaheen, Yasuhiro Hayashi, Takashi Nakamura, Noritada Kaji, Yoshinobu Baba, Hideyoshi Harashima

  BIOMATERIALS 34 (35) 8979 - 8990 0142-9612 2013/11 [Refereed][Not invited]
   

  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.
• Major T cell response to a mycolyl glycolipid is mediated by CD1c molecules in rhesus macaques

  Daisuke Morita, Yuki Hattori, Takashi Nakamura, Tatsuhiko Igarashi, Hideyoshi Harashima, Masahiko Sugita

  Infection and Immunity 81 (1) 311 - 316 0019-9567 2013/01 [Refereed][Not invited]
   

  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-γ) and tumor necrosis factor alpha (TNF-α), 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. © 2013, American Society for Microbiology.
• Incorporation of polyinosine-polycytidylic acid enhances cytotoxic T cell activity and antitumor effects by octaarginine-modified liposomes encapsulating antigen, but not by octaarginine-modified antigen complex

  Takashi Nakamura, Rumiko Moriguchi, Kentaro Kogure, Hideyoshi Harashima

  INTERNATIONAL JOURNAL OF PHARMACEUTICS 441 (1-2) 476 - 481 0378-5173 2013/01 [Refereed][Not invited]
   

  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.
• The nanoparticulation by octaarginine-modified liposome improves α-galactosylceramide-mediated antitumor therapy via systemic administration

  Takashi Nakamura, Daiki Yamazaki, Jun Yamauchi, Hideyoshi Harashima

  Journal of Controlled Release 171 (2) 216 - 224 1873-4995 2013 [Refereed][Not invited]
   

  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.
• Efficacy of DHMEQ, a NF-κB inhibitor, in islet transplantation: I. HMGB1 suppression by DHMEQ prevents early islet graft damage

  Daisuke Kuraya, Masaaki Watanabe, Yasuyuki Koshizuka, Masaomi Ogura, Tadashi Yoshida, Yoh Asahi, Hirofumi Kamachi, Takashi Nakamura, Hideyoshi Harashima, Michitaka Ozaki, Kazuo Umezawa, Michiaki Matsushita, Kenichiro Yamashita, Satoru Todo

  Transplantation 96 (5) 445 - 453 0041-1337 2013 [Refereed][Not invited]
   

  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.
• The liposome-incorporating cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guein can directly enhance the susceptibility of cancer cells to lymphokine-activated killer cells through up-regulation of natural-killer group 2, member D ligands

  Jun Miyazaki, Koji Kawai, Takahiro Kojima, Takehiro Oikawa, Akira Joraku, Toru Shimazui, Akihiro Nakaya, Ikuya Yano, Takashi Nakamura, Hideyoshi Harashima, Hideyuki Akaza

  BJU INTERNATIONAL 108 (9) 1520 - 1526 1464-4096 2011/11 [Refereed][Not invited]
   

  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.
• KALA-modified multi-layered nanoparticles as gene carriers for MHC class-I mediated antigen presentation for a DNA vaccine

  Sharif M. Shaheen, Hidetaka Akita, Takashi Nakamura, Shota Takayama, Shiroh Futaki, Atsushi Yamashita, Ryo Katoono, Nobuhiko Yui, Hideyoshi Harashima

  Biomaterials 32 (26) 6342 - 6350 0142-9612 2011/09 [Refereed][Not invited]
   

  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.
• A20 Silencing by Lipid Envelope-Type Nanoparticles Enhances the Efficiency of Lipopolysaccharide-Activated Dendritic Cells

  Shota Warashina, Takashi Nakamura, Hideyoshi Harashima

  BIOLOGICAL & PHARMACEUTICAL BULLETIN 34 (8) 1348 - 1351 0918-6158 2011/08 [Refereed][Not invited]
   

  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.
• Glycerol monomycolate, a latent tuberculosis-associated mycobacterial lipid, induces eosinophilic hypersensitivity responses in guinea pigs

  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 0006-291X 2011/06/03 [Refereed][Not invited]
   

  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. © 2011 Elsevier Inc.
• The Therapeutic Effects of R8-Liposome-BCG-CWS on BBN-Induced Rat Urinary Bladder Carcinoma

  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 0250-7005 2011/06 [Refereed][Not invited]
   

  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.
• A microbial glycolipid functions as a new class of target antigen for delayed-type hypersensitivity

  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 0021-9258 2011/05/13 [Refereed][Not invited]
   

  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 classicalDTHto protein antigens. Lymph nodeTcells from BCG-immunized guinea pigs specifically increased IFN-γ transcription in response to the GMM liposome, and this response was completely blocked by antibodies to CD1 lipid antigen-presenting molecules. Finally, whereas theTcells increased transcription of bothThelper (Th) 1-type (IFN-γ and TNF-α) 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. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.
• Noninvasive and efficient transdermal delivery of CpG-oligodeoxynucleotide for cancer immunotherapy

  Kaoru Kigasawa, Kazuaki Kajimoto, Takashi Nakamura, Susumu Hama, Kiyoshi Kanamura, Hideyoshi Harashima, Kentaro Kogure

  JOURNAL OF CONTROLLED RELEASE 150 (3) 256 - 265 0168-3659 2011/03 [Refereed][Not invited]
   

  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.
• Nanoparticles for ex vivo siRNA delivery to dendritic cells for cancer vaccines: Programmed endosomal escape and dissociation

  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 0168-3659 2010/05 [Refereed][Not invited]
   

  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.
• Enhanced antigen presentation and CTL activity by transduction of mature rather than immature dendritic cells with octaarginine-modified liposomes

  Atthachai Homhuan, Kentaro Kogure, Takashi Nakamura, Nilabh Shastri, Hideyoshi Harashima

  JOURNAL OF CONTROLLED RELEASE 136 (1) 79 - 85 0168-3659 2009/05 [Refereed][Not invited]
   

  To improve uptake and cross-presentation of exogenous antigens (Ag) by dendritic cells (DCs), octaarginine-modified liposomes (R8-Lip) were used as a novel strategy for protein-Ag transduction. Immature DCs endocytose macromolecules efficiently. While mature DCs lose their ability to capture Ag, but have an increased capacity for T-cell activation. Thus Ag-transduction has been performed mostly in immature DCs. In the present study, R8-Lip were efficiently taken up by both immature and mature DCs. DCs transduced after maturation were highly efficient at cross-presentation of Ag and induced higher cytotoxic T-lymphocytes (CTL) activity than were DCs transduced before maturation. The mechanism of Ag presentation involved the escape of R8-Lip from endosomes to cytosol, which require the acidic environment. The Ag released was then processed by a proteasome-dependent pathway. This novel transduction approach is clinically applicable, easy to perform, and has more practical advantages than current protein transduction methods. (C) 2009 Elsevier B.V. All rights reserved.
• Efficient MHC class I presentation by controlled intracellular trafficking of antigens in octaarginine-modified liposomes

  Takashi Nakamura, Rumiko Moriguchi, Kentaro Kogure, Nilabh Shastri, Hideyoshi Harashima

  MOLECULAR THERAPY 16 (8) 1507 - 1514 1525-0016 2008/08 [Refereed][Not invited]
   

  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.
• Delivery of condensed DNA by liposomal non-viral gene delivery system into nucleus of dendritic cells

  Takashi Nakamura, Rumiko Moriguchi, Kentaro Kogure, Arisa Minoura, Tomoya Masuda, Hidetaka Akita, Kazunori Kato, Hirofumi Hamada, Shiroh Futaki, Hideyoshi Harashima

  BIOLOGICAL & PHARMACEUTICAL BULLETIN 29 (6) 1290 - 1293 0918-6158 2006/06 [Refereed][Not invited]
   

  In this study, we developed novel double-membranous non-viral gene delivery system modified with SV-40 T antigen-derived nuclear localization signal (NLS-DMEND) for delivery of luciferase plasmid DNA to nucleus of non-dividing mouse bone marrow-derived dendritic cells (BMDC). Intracellular trafficking and gene expression of NLS-DMEND in the BMDC were evaluated. Condensed DNA was observed in the nucleus by confocal laser scanning microscopy, and the NLS-DMEND induced significant luciferase activity in the BMDC. It was suggested that the condensed DNA particle transferred into nucleus via energy dependent manner, since the nuclear transfer was inhibited by metabolic inhibitors. In conclusion, condensed plasmid DNA was delivered into the nucleus of non-dividing BMDC by NLS-DMEND.
• Unique features of a pH-sensitive fusogenic peptide that improves the transfection efficiency of cationic liposomes

  Shiroh Futaki, Yumi Masui, Ikuhiko Nakase, Yukio Sugiura, Takashi Nakamura, Kentaro Kogure, Hideyoshi Harashima

  Journal of Gene Medicine 7 (11) 1450 - 1458 1099-498X 2005/11 [Refereed][Not invited]
   

  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 μM 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 © 2005 John Wiley & Sons, Ltd.

MISC

• 難水溶性物質CoQ₁₀を搭載可能な脂質ナノ粒子設計の検討

  杉原崚太, 日比野光恵, 真栄城正寿, 渡慶次学, 中村孝司, 原島秀吉, 山田勇磨  生体膜と薬物の相互作用シンポジウム講演要旨集  44th-  2023
• 炎症性腸疾患におけるグルコシルセラミドの機能解析

  小室茉莉子, 永根大幹, 丹羽智瑛, 宮本貴祥, 遠藤力斗, 中村孝司, 原島秀吉, 相原尚之, 上家潤一, 山下匡  日本酸化ストレス学会学術集会プログラム・抄録集  74th (CD-ROM)-  2021
• 炎症性腸疾患におけるグルコシルセラミドの機能解析

  小室茉莉子, 永根大幹, 宮本貴祥, 丹羽智瑛, 遠藤力斗, 中村孝司, 原島秀吉, 相原尚之, 上家潤一, 山下匡  日本生化学会大会(Web)  94th-  2021
• Properties of lipid nanoparticles affecting their lymph node transitivity and distribution

  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
• T細胞のグルコシルセラミドは炎症性大腸炎を抑制する

  永根大幹, 山門みのり, 水野愛理, 遠藤力斗, 中村孝司, 原島秀吉, 相原尚之, 上家潤一, 山下匡  日本酸化ストレス学会学術集会プログラム・抄録集  73rd-  2020
• Size and charge effect of lipid nanoparticles on transitivity to lymph node and distribution in lymph node

  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
• Strategy for multiple cancer immunotherapy based on c-di-GMP loaded lipid nanoparticles

  中村 孝司, 原島 秀吉  細胞  48-  (13)  655  -659  2016/12
• A Lipid Nanoparticle for Efficient Delivery of siRNA to Dendritic Cells Towards Cell-based Cancer Therapy

  中村 孝司, 原島 秀吉  化學工業  67-  (7)  477  -482  2016/07
• 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  2015/03  [Not refereed][Not invited]
  
• Drug Delivery System Based on MEND

  秋田 英万, 佐藤 悠介, 中村 孝司  細胞工学  34-  (10)  956  -961  2015
• MENDで拓く遺伝子治療への道 (特集 Drug delivery system(DDS)の最新展望)

  佐藤 悠介, 中村 孝司, 山田 勇磨  医薬ジャーナル  50-  (7)  83  -87  2014/07
• 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  2014/03  [Not refereed][Not invited]
  
• Immunotherapy of bladder cancer with BCG cell wall skeleton encapsulated sub-200 nm nano particles

  T. Nakamura, M. Fukiage, M. Higuchi, A. Nakaya, I. Yano, J. Miyazaki, A. Joraku, H. Akaza, T. Ito, H. Hosokawa, T. Nakayama, H. Harashima  IMMUNOLOGY  137-  763  -763  2012/09  [Not refereed][Not invited]
  
• A Multifunctional Envelope-type Nanodevice for Use in Nanomedicine: Concept and Applications

  T. Nakamura, H. Akita, Y. Yamada, H. Hatakeyama, H. Harashima  ACCOUNTS OF CHEMICAL RESEARCH  45-  (7)  1113  -1121  2012/07  [Not refereed][Not invited]
   

  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
• SY11-3 BCG注入療法の新展開(11.ハイリスク筋層非浸潤性膀胱癌の再発・進展予防の新戦略,シンポジウム,第99回日本泌尿器科学会総会)

  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  2011/03/20  [Not refereed][Not invited]
  
• オクタアルギニン修飾リポソームによるMHCクラスI特異的抗原デリバリーメカニズムの解析

  小野 耕平, 森口 留美子, 中村 孝司, 小暮 健太朗, 原島 秀吉  薬剤学 = Journal of Pharmaceutical Science and Technology, Japan  67-  293  -293  2007/05/10

Association Memberships

• 日本核酸医薬学会   日本がん免疫学会   

Research Projects

• 炎症性腸疾患におけるスフィンゴ糖脂質による治療を目指した基礎的研究

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2023/04 -2026/03 

  Author : 山下 匡, 永根 大幹, 中村 孝司
• ナノDDSで挑む腫瘍血管内皮細胞を標的とした新機軸のがん治療法の開発

  日本学術振興会：科学研究費助成事業 挑戦的研究(萌芽)

  Date (from‐to) : 2022/06 -2025/03 

  Author : 中村 孝司
• PD-1抗体への獲得抵抗性を攻略する記憶NK細胞誘導型ナノがん免疫療法の開発

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  Date (from‐to) : 2020/04 -2023/03 

  Author : 中村 孝司

   

  本研究では、自然免疫記憶（trained innate immunity）を利用したnatural killer（NK）細胞のさらなる強化を実現するためのアジュバント搭載薬物送達システムを細胞内/体内動態の観点から構築し、獲得抵抗性を攻略するためのナノがん免疫療法を開発することを目的とする。本年度は、NK細胞の活性化やメモリー化を促進するためのアジュバントの選定をin vivoにて実施した。我々は、細胞内DNAセンサーであるstimulator of interferon genes（STING）経路のアゴニストを搭載した脂質ナノ粒子（STINGナノ粒子）の開発に成功しており、I型interferon（IFN）を強力に産生することでNK細胞を効果的に活性化できることを報告している。マウスに対しSTINGナノ粒子とアジュバントを同時に静脈内投与した結果、脾臓においてNK細胞のメモリー化に関与するフェノタイプへの分化が観察された。この結果は、異なる作用機序のアジュバントを組み合わせることで、NK細胞の活性化やメモリー化への影響を制御できることを示唆している。また、免疫チェックポイント阻害剤に対して獲得耐性を示す腫瘍モデルの構築についても実施した。免疫チェックポイント阻害剤が有効なMC38腫瘍マウスモデルにおいて、免疫チェックポイント阻害剤を投与した。大部分のマウスでは抗腫瘍活性が認められたが、一部のマウスでは腫瘍の再成長が観察された。再成長した腫瘍を回収し、獲得耐性を示す可能性のあるMC38細胞候補とした。
• Innovative gene/nucleic acid delivery system based on optimized intracellular trafficking steps

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2019/04 -2023/03 

  Author : 原島 秀吉, 佐藤 悠介, 中村 孝司, 山田 勇磨

   

  本研究は、ナノDDS技術 を駆使することでブラックボックスを解明する とともに新たな戦略を確立し、 最高水準のナノDDSを用いて、 がん免疫療法の進化へ貢献する。 さらに、これまで不可能であった疾患細胞のミトコンドリア (Mt) への遺伝子送達システムを 開発し、Mt遺伝子治療の臨床応 用へ展開する。 １ siRNA送達（佐藤）： siRNAへの核外輸送シグナルペプチドのコンジュゲートにより、脂質ナノ粒子をマウスに静脈内投与してから早い時間帯において肝臓中標的遺伝子のノックダウン効率が向上する傾向が認められた。コンジュゲートのリンカー部位の安定化による活性への影響は認められなかったことから、汎用的なリンカー構造で効果を発揮可能であることが示唆された。 ２ pDNA送達（原島）：肝臓選択的なキャリアと脾臓選択的なキャリアの体内分布・組織内分布を比較したところ、両者に大きな違いは見られず、pDNAの移行量だけでは遺伝子発現の差は説明できなかった。次に、核移行、転写、翻訳の各過程の効率を評価するため、各組織の核内pDNA量、mRNA発現量、タンパク質発現量を定量した。その結果、遺伝子発現の差には翻訳過程が大きく関与していることが示唆された。 ３ がん免疫ナノ療法（中村）：アジュバントと抗原を搭載したナノDDSを腫瘍内投与、静脈内投与した後、フローサイトメトリー解析により脾臓のT細胞フェノタイプを調べた結果、T細胞フェノタイプと抗腫瘍活性に相関を見出すことに成功した。 ４ ミトコンドリア (Mt)への送達(山田)：ナノDDSの神経細胞軸索輸送の検証を行うため、初代培養神経細胞を樹立し、共焦点レーザー走査型顕微鏡を用いたリアルタイム動態観察法を確立した。新型MITO-Porterが神経細胞軸索上を移動していることを確認した。
• Development of nano cancer immunotherapy based on immune status in tumor

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2018/06 -2021/03 

  Author : Nakamura Takashi

   

  In this study, we analyzed the mRNA level of cancer immunity-related genes in tumors of various mouse models, and identified the immune status reflecting antitumor activity by using our algorithm. We also found that the defined immune status can be useful for evaluating the antitumor activity induced by an adjuvant-loaded lipid nanoparticle (nano-DDS). Furthermore, we designed a new strategy based on the analysis of tumor, and succeeded in inducing antitumor effect against a refractory tumor having a resistance to the current cancer immunotherapy.
• 腫瘍リンパ節標的型クラスターナノDDSによる複合がん免疫療法

  文部科学省：科学研究費補助金(基盤研究(B)）

  Date (from‐to) : 2017/04 -2020/03 

  Author : 中村孝司
• Lipid immunity in rhesus monkeys and development of a new anti-tuberculosis vaccine

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2015/04 -2018/03 

  Author : SUGITA Masahiko

   

  Tuberculosis remains to be a global threat to humans. The only currently available anti-tuberculosis vaccine, BCG, has been used for decade, but development of alternative vaccines is definitely required. The principal investigator has attempted to develop a new type of lipid-based vaccines against tuberculosis. In this study, the vaccine candidate lipid was studied extensively in terms of is biosynthesis as well as its immunological properties. The findings obtained in this study provide an important basis for the development of anti-tuberculosis lipid vaccines.
• c-di-GMP搭載脂質ナノ粒子と抗PD-1抗体による複合的がん免疫療法

  文部科学省：科学研究費補助金(挑戦的萌芽研)

  Date (from‐to) : 2016/04 -2018/03 

  Author : 中村孝司
• Development of nano medicine for anti-bladder cancer based on human immunology

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2014/04 -2017/03 

  Author : NAKAMURA Takashi

   

  The purpose of this study is to develop a nanoparticle system achieving live BCG mediated antitumor effects against bladder cancer. We successfully found that the modification of PEGylated lipid into nanoparticles efficiently inhibited the aggregation of nanoparticle in human urine. We also developed the novel nanoparticle for efficient siRNA delivery to immune cells. The internalization of BCG cell wall skeleton in bladder cancer cells is essential for induction of antitumor effect. Moreover, we found key cytokines associated with antitumor effect of BCG by analyses with microarray and qRT-PCR. We currently progress selection of BCG components and development of Nano-BCG.
• The effect of intracellular trafficking of BCG-CWS loaded nanoparticles on anti-bladder cancer activity

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2014/04 -2016/03 

  Author : NAKAMURA Takashi

   

  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. Moreover, we investigated the effect of intracellular trafficking of lipid antigen loaded nanoparticles on CD1 antigen presentation. The CD1 antigen presentation is appeared to influence to anti-bladder cancer immune responses. Our study revealed that the accumulation of lipid antigen loaded nanoparticles in endosomes/lysosomes efficiently enhanced CD1 antigen presentation.
• Development of a new type of anti-tuberculosis vaccines by the use of lipid immunity

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2012/04 -2015/03 

  Author : SUGITA Masahiko, IGARASHI Tatuhiko, NAKAMURA Takashi, HARASHIMA Hideyoshi

   

  As about one third of the world population is infected with Mycobacterium tuberculosis, the infectious agent that causes human tuberculosis, the development of more effective vaccines than BCG, the only anti-tuberculosis vaccine currently available worldwide, is urgently needed. This principal investigator has contributed to advances in the frontier research of immune responses against tuberculosis-associated lipids. In this project, this research group has attempted to extend the basic achievements to the development of a new type of lipid-based vaccines against human tuberculosis. Using the guinea pig model of human tuberculosis, this group was successful in establishing a prototypic lipid vaccine. Furthermore, a new protocol capable of efficiently eliciting lipid-specific immune responses has been established for non-human primates, such as rhesus monkeys. These contributions provide the basis for the future development of anti-tuberculosis vaccines against human tuberculosis.
• MENDで拓く遺伝子治療への道：遺伝子の運び屋からナノマシンへ

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2010/05 -2014/03 

  Author : 原島 秀吉, 山田 勇磨, 中村 孝司, 兵藤 守, 馬場 嘉信, 篠原 康雄, 小暮 健太朗, 紙谷 浩之, 渡慶次 学, 松尾 保孝, 秋田 英万

   

  パッチワーク法による新しいMEND構築方法を確立し一枚膜でコア粒子をパッケージする新しい方法を開発し、平均粒子径が25nm程度でゼータ電位が-40mV程度の構造体を得ることに成功し、P-MENDと命名した。D-MEND法と組み合わせることでPD- MEND(3枚膜構造)を構築したところ、市販の試薬より高い活性を示した。マイクロ流体デバイスを開発し、最小粒径30nmのMENDを作製することに成功した。一方でマイクロ流路作製についての検討し、ナノインプリント手法によりガラスマイクロ流路の量産が可能であることをしめした。また、シースフローと電場を利用したマイクロフリーフロー電気泳動デバイスの構造・分離条件の最適化を行い、MEND精製に適用することでMENDの定性的かつ定量的評価法を確立した。KALAペプチドを導入した脂質膜封入型ナノ粒子にがん抗原等をコードした遺伝子を封入することで、抗腫瘍活性を得ることに成功した。また、本粒子は細胞に対し免疫活性化効果を有することを示し、その機構に細胞質内DNAセンサーが関与することを示唆する結果を得た。酵素耐性RNAアプタマーの単離に世界で始めて成功した。Mt移行性ペプチドを搭載したMTS-MITO-Porterを構築し、生細胞内での選択的Mt送達システムを開発し、MTS-MITO-Porterは従来型MITO-Porterと比較してミトコンドリア移行能が飛躍的に上昇した。GAL4とVP16の融合蛋白質を用いる自己活性化システムを構築し、reporterプラスミドDNAの単独投与に比べて数十倍にルシフェラーゼ活性が上昇した。アンチセンスRNA (ASO)をMITO-Porterを用いて細胞Mtに送達し、ミトコンドリアmRNAを40%抑制し、さらに標的mRNAがコードする内因性Mtタンパク質の発現量が低下した。
• Induction of antitumor immunity by nanoparticle incorporating alpha-galactocylceramide

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2012/04 -2014/03 

  Author : NAKAMURA Takashi

   

  Alpha-galactosylceramide (GC), a lipid antigen present on CD1d molecules, is predicted to have clinical applications as a new class of adjuvant, because GC strongly activates natural killer T (NKT) cells which produce large amounts of IFN-gamma. Here, we incorporated GC into stearylated octaarginine-modified liposomes (R8-Lip), our original delivery system developed for vaccines, and investigated the effect of nanoparticulation. The nanopaticulation of GC enhanced the GC presentation on CD1d of antigen presenting cells, NKT proliferation, IFN-gamma production, therapeutic effect against highly malignant B16 melanoma. These results strongly indicate that the nanopaticulation of GC by R8-Lip is promising technique for achieving systemic GC therapy.
• 細胞内生態解析に基づいた樹状細胞選択的DNAワクチンの開発

  日本学術振興会：科学研究費助成事業 特別研究員奨励費

  Date (from‐to) : 2007 -2009 

  Author : 中村 孝司

   

  前年度は、タンパク質抗原を用いた樹状細胞へのデリバリーを行い、抗原提示と抗腫瘍効果の評価を行った。本年度では、DNAワクチンの前段階としてsiRNAを樹状細胞に導入し、機能を示すキャリアを見出した。 R8/GALA-DMENDを用いた樹状細胞におけるSOCS1のノックダウン DNAワクチンの前段階としてsiRNAを樹状細胞に導入し、その効果を調べた。当研究室で開発されたR8/GALA-DMENDはエンドソーム脱出のための機能性素子であるGALAと膜枚数の制御により、培養細胞で高いRNAi効果を示すことが明らかになっている。本年度では、樹状細胞においてサイトカインシグナルの抑制因子であるSOCS1を標的とした R8/GALA-DMENDの機能評価を行った。siRNAを封入したR8/GALA-DMENDを樹状細胞に導入した結果、未処理の樹状細胞と比較して87.5%の発現抑制が認められた。またSOCS1をノックダウンした樹状細胞ではTNF-αやIL-6産生が増加した。さらにSOCS1をノックダウンした樹状細胞をマウスに投与した後、癌細胞をマウスに移植し、その増殖を観察した。その結果、コントロールsiRNAを用いた場合と比較して著しい腫瘍増殖抑制が認められた。以上のことから、R8/GALA-DMENDは樹状細胞においても十分なRNAi効果を誘導できるキャリアであることが明らかになった(Akita H et al.J.Control.Release)。 本年度の成果によりR8/GALA-DMENDが樹状細胞への核酸デリバリーシステムとして有用であることが示された。故にR8/GALA-DMENDはDNAワクチンのデリバリーシステムとしての応用が期待される。