Researcher Profile and Settings

Affiliation

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

Job Title

• Professor

Degree

• PhD（Hokkaido Univ.）

URL

http://kaken.nii.ac.jp/d/r/60451431.ja.html

J-Global ID

• 201301001298698134

Research Interests

• ミトコンドリア   薬学   イメージング   ナノテクノロジー   バイオテクノロジー   癌   遺伝子治療   細胞内動態制御   ナノマシン   タンパク質送達   遺伝子送達   共焦点レーザースキャン顕微鏡   細胞・組織   共焦点顕微鏡   細胞・動物   オクタアルギニン   多機能性エンベロープ型ナノ構造体   抗腫瘍効果   ナノパッケージング   遺伝子デリバリー   トランスサイトーシス   MHCクラス-I   ミトコンドリア送達   細胞透過性ペプチド   pH-感受性カチオニック脂質   遺伝子導入   ハイドロダイナミクス法   がん治療   MEND   siRNA   抗原提示   ワクチン   MMP   PPD   アジュバント   

Research Areas

• Life sciences / Pharmaceuticals - chemistry and drug development
• Life sciences / Biomaterials
• Life sciences / Biomedical engineering
• Life sciences / Clinical pharmacy

Educational Organization

•  Bachelor's degree program　School of Pharmaceutical Sciences and Pharmacy
•  Master's degree program　Graduate School of Life Science
•  Doctoral (PhD) degree program　Graduate School of Life Science

Academic & Professional Experience

• 2024 - Today 北海道大学産業創出講座 [細胞・オルガネラ製剤学分野]
• 2023/04 - Today Hokkaido University Faculty of Pharmaceutical Sciences Professor
• 2021/04 - Today Fusion Oriented REsearch for disruptive Science and Technology (FOREST) FOREST program researcher
• 2020/04 - Today 北海道大学産業創出講座 [バイオDDS実用化分野]
• 2019/04 - Today ルカサイエンス株式会社 科学顧問（兼務）
• 2017/04 - Today Hokkaido University
• 2009/04 - Today Hokkaido University Hokkaido University Hospital
• 2008/10 - 2016/03 Hokkaido University
• 2007/01 - 2008/09 Hokkaido University Faculty of Pharmaceutical Sciences
• 2006/04 - 2006/12 日本学術振興会 特別研究員 (DC2)

Education

• 2008/09 - 2008/09  Hokkaido University
• 2005/04 - 2006/12  Hokkaido University
• 2003/04 - 2005/03  Hokkaido University
• 1999/04 - 2003/03  Hokkaido University  Faculty of Pharmaceutical Sciences

Association Memberships

• THE JAPANESE SOCIETY FOR BIOMATERIALS   札幌学校薬剤師会   日本病院薬剤師会   日本核酸医薬学会   THE JAPANESE CANCER ASSOCIATION   SOCIETY FOR FREE RADICAL RESEARCH JAPAN   Mitochondrial Research Society   THE JAPANESE SOCIETY OF MITOCHONDRIAL RESEARCH AND MEDICINE   THE ACADEMY OF PHARMACEUTICAL SCIENCE AND TECHNOLOGY, JAPAN   日本DDS学会   THE PHARMACEUTICAL SOCIETY OF JAPAN   

Research Activities

Published Papers

• Investigation of the Nanoparticulation Method and Cell-Killing Effect following the Mitochondrial Delivery of Hydrophobic Porphyrin-Based Photosensitizers

  Rina Naganawa, Hanjun Zhao, Yuta Takano, Masatoshi Maeki, Manabu Tokeshi, Hideyoshi Harashima, Yuma Yamada

  International Journal of Molecular Sciences 2024/04/12 [Refereed]
  
• Activation of Mitochondrial Oxygen Consumption Rate by Delivering Coenzyme Q10 to Mitochondria of Rat Skeletal Muscle Cell (L6)

  Itsumi Sato, Mitsue Hibino, Atsuhito Takeda, Hideyoshi Harashima, Yuma Yamada

  Journal of Pharmaceutical Sciences 0022-3549 2024/02
• Human cardiosphere-derived cells with activated mitochondria for better myocardial regenerative therapy.

  Masahiro Shiraishi, Daisuke Sasaki, Mitsue Hibino, Atsuhito Takeda, Hideyoshi Harashima, Yuma Yamada

  Journal of controlled release : official journal of the Controlled Release Society 2024/01/29 

  Cell transplantation is a promising therapeutic strategy for myocardial regeneration therapy. To improve therapeutic effects, we developed a culture medium additive that enhances the mitochondrial function of cardiomyocytes for transplantation. A mitochondrial targeting drug delivery system (MITO-Porter system) was used to deliver mitochondrial activation molecules to mouse-derived cardiac progenitor cells. In this study, we investigated whether the mitochondrial function of human-derived myocardial precursor cells could be enhanced using MITO-Porter. Human cardiosphere-derived cells (CDCs) were isolated from myocardium which was excised during surgery for congenital heart disease. MITO-Porter was added to the cell culture medium to generate mitochondrial activated CDCs (human MITO cells). The human MITO cells were transplanted into myocardial ischemia-reperfusion rat model rat, and the effect was investigated. The transplanted human MITO cells improved the cardiac function and suppressed myocardial fibrosis compared to conventional cell transplantation methods. These effects were observed not only with myocardial administration but also by intravenous administration of human MITO cells. This study is the first study that assessed whether the mitochondrial delivery of functional compounds improved the outcome of human-derived myocardial cell transplantation therapy.
• Development of Liposomes That Target Axon Terminals Encapsulating Berberine in Cultured Primary Neurons

  Ikuma Hori, Hideyoshi Harashima, Yuma Yamada

  Pharmaceutics 16 (1) 49 - 49 2023/12/28 

  Most of the energy in neurons is produced in mitochondria. Mitochondria generate the ATP that is essential for neuronal growth, function, and regeneration. Mitochondrial axonal transport plays a crucial role in maintaining neuronal homeostasis and biological activity. Decreased mitochondrial axonal transport at axon terminals, where the metabolism of substances is likely to be delayed, may contribute to neurological dysfunction. Therefore, regulation of mitochondrial dynamics at axon terminals has attracted considerable interest as a strategy to modulate neuronal function. Nanoparticles may be useful in controlling local mitochondrial dynamics. Nevertheless, there are few reports on the influence of drug delivery that nanoparticles impart on the mitochondrial dynamics in neurons. This paper reports the results of a study using liposomes (LPs) to examine local drug delivery and pharmacological actions on neurons. We tested berberine (BBR), which is an activator of AMP-activated protein kinase (AMPK), to examine the utility of this drug as a cellular energy sensor. Axon terminals targeting LPs were prepared. The amount of axon terminals targeting LPs was increased compared with treatment using cationic LPs. Moreover, axon terminal-targeting LPs increased anterograde transport by about 40% compared with that of either naked BBR or cationic LPs and suppressed axonal retraction. Our findings suggest that local drug delivery to neurons is important for enhancing pharmacological activity in axon terminals.
• π-extended porphyrin-based near-infrared photosensitizers for mitochondria-targeted photodynamic therapy

  Hanjun Zhao, Rina Naganawa, Yuma Yamada, Yasuko Osakada, Mamoru Fujitsuka, Hideyuki Mitomo, Yukiko Miyatake, Hideyoshi Harashima, Vasudevanpillai Biju, Yuta Takano

  Journal of Photochemistry and Photobiology A: Chemistry 115397 - 115397 1010-6030 2023/12
• Development of light-induced disruptive liposomes (LiDL) as a photoswitchable carrier for intracellular substance delivery.

  Taichi Tsuneishi, Keiichi Kojima, Fumika Kubota, Hideyoshi Harashima, Yuma Yamada, Yuki Sudo

  Chemical communications (Cambridge, England) 59 (49) 7591 - 7594 2023/06/15 

  Light-driven inward proton pump rhodopsin RmXeR was embedded in pH-sensitive liposomes. Substance release from the proteoliposomes was observed following light illumination both in vitro and in cells, indicating the successful production of light-induced disruptive liposomes (LiDL). Thus, LiDL is a photoswitchable carrier utilized for intracellular substance delivery.
• Differences in the Intracellular Localization of Methylated β-Cyclodextrins-Threaded Polyrotaxanes Lead to Different Cellular States

  Yuma Yamada, Shinnosuke Daikuhara, Atsushi Tamura, Kei Nishida, Nobuhiko Yui, Hideyoshi Harashima

  Biomolecules 13 (6) 903 - 903 2023/05/29 

  Activation of autophagy represents a potential therapeutic strategy for the treatment of diseases that are caused by the accumulation of defective proteins and the formation of abnormal organelles. Methylated β-cyclodextrins-threaded polyrotaxane (Me-PRX), a supramolecular structured polymer, induces autophagy by interacting with the endoplasmic reticulum. We previously reported on the successful activation of mitochondria-targeted autophagy by delivering Me-RRX to mitochondria using a MITO-Porter, a mitochondria-targeted nanocarrier. The same level of autophagy induction was achieved at one-twentieth the dosage for the MITO-Porter (Me-PRX) compared to the naked Me-PRX. We report herein on the quantitative evaluation of the intracellular organelle localization of both naked Me-PRX and the MITO-Porter (Me-PRX). Mitochondria, endoplasmic reticulum and lysosomes were selected as target organelles because they would be involved in autophagy induction. In addition, organelle injury and cell viability assays were performed. The results showed that the naked Me-PRX and the MITO-Porter (Me-PRX) were localized in different intracellular organelles, and organelle injury was different, depending on the route of administration, indicating that different organelles contribute to autophagy induction. These findings indicate that the organelle to which the autophagy-inducing molecules are delivered plays an important role in the level of induction of autophagy.
• A system that delivers an antioxidant to mitochondria for the treatment of drug-induced liver injury.

  Mitsue Hibino, Masatoshi Maeki, Manabu Tokeshi, Yoichi Ishitsuka, Hideyoshi Harashima, Yuma Yamada

  Scientific reports 13 (1) 6961 - 6961 2023/05/10 

  Mitochondria, a major source of reactive oxygen species (ROS), are intimately involved in the response to oxidative stress in the body. The production of excessive ROS affects the balance between oxidative responses and antioxidant defense mechanisms thus perturbing mitochondrial function eventually leading to tissue injury. Therefore, antioxidant therapies that target mitochondria can be used to treat such diseases and improve general health. This study reports on an attempt to establish a system for delivering an antioxidant molecule coenzyme Q10 (CoQ10) to mitochondria and the validation of its therapeutic efficacy in a model of acetaminophen (APAP) liver injury caused by oxidative stress in mitochondria. A CoQ10-MITO-Porter, a mitochondrial targeting lipid nanoparticle (LNP) containing encapsulated CoQ10, was prepared using a microfluidic device. It was essential to include polyethylene glycol (PEG) in the lipid composition of this LNP to ensure stability of the CoQ10, since it is relatively insoluble in water. Based on transmission electron microscope (TEM) observations and small angle X-ray scattering (SAXS) measurements, the CoQ10-MITO-Porter was estimated to be a 50 nm spherical particle without a regular layer structure. The use of the CoQ10-MITO-Porter improved liver function and reduced tissue injury, suggesting that it exerted a therapeutic effect on APAP liver injury.
• Development of a Mitochondrial Targeting Lipid Nanoparticle Encapsulating Berberine

  Ikuma Hori, Hideyoshi Harashima, Yuma Yamada

  International Journal of Molecular Sciences 24 (2) 903 - 903 2023/01/04 

  Delivering drugs to mitochondria, the main source of energy in neurons, can be a useful therapeutic strategy for the treatment of neurodegenerative diseases. Berberine (BBR), an isoquinoline alkaloid, acts on mitochondria and is involved in mechanisms associated with the normalization and regulation of intracellular metabolism. Therefore, BBR has attracted considerable interest as a possible therapeutic drug for neurodegenerative diseases. While BBR has been reported to act on mitochondria, there are few reports on the efficient delivery of BBR into mitochondria. This paper reports on the mitochondrial delivery of BBR using a lipid nanoparticle (LNP), a “MITO-Porter” that targets mitochondria, and its pharmacological action in Neuro2a cells, a model neuroblastoma. A MITO-Porter containing encapsulated BBR (MITO-Porter (BBR)) was prepared. Treatment with MITO-Porter (BBR) increased the amount of BBR that accumulated in mitochondria compared with a treatment with naked BBR. Treatment with MITO-Porter (BBR) resulted in increased ATP production in Neuro2a cells, which are important for maintaining life phenomena, compared with treatment with naked BBR. Treatment with MITO-Porter (BBR) also increased the level of expression of mitochondrial ubiquitin ligase (MITOL), which is involved in mitochondrial quality control. Our findings indicate that increasing the accumulation of BBR into mitochondria is important for inducing enhanced pharmacological actions. The use of this system has the potential for being important in terms of the regulation of the metabolic mechanism of mitochondria in nerve cells.
• Fine-tuning the encapsulation of a photosensitizer in nanoparticles reveals the relationship between internal structure and phototherapeutic effects.

  Fumika Kubota, Satrialdi, Yuta Takano, Masatoshi Maeki, Manabu Tokeshi, Hideyoshi Harashima, Yuma Yamada

  Journal of biophotonics 16 (3) e202200119  2022/09/01 

  Photodynamic therapy (PDT) is a cancer therapy that uses a photosensitizer (PS) in the presence of oxygen molecules. Since singlet oxygen is highly reactive, it is important to deliver it to the target site. Thus, an efficient drug delivery system (DDS) is essential for enhancing the efficacy of such a treatment and protecting against the side effects of PDT. Here, we report on attempts to increase the therapeutic effect of PDT by using a DDS, a lipid nanoparticle (LNP). We prepared a porphyrin analog, rTPA (PS) that was encapsulated in LNPs using a microfluidic device. The findings indicated that the internal structure of the prepared particles changed depending on the amount of rTPA in LNPs. The photoactivity and cell-killing effect of PS in LNPs also changed when the amount of the cargo increased. These results suggest that the internal structure of LNPs is important factors that affect drug efficacy. This article is protected by copyright. All rights reserved.
• 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 

  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 

  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 

  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.
• Validation of a therapeutic strategy involving the mitochondrial delivery of thiamine pyrophosphate using brain damage induced mouse model

  Yuma Yamada, Takuya Ishimaru, Hideyoshi Harashima

  Clinical and Translational Discovery 2 (2) 2768-0622 2022/06
• Recent advances in delivering RNA-based therapeutics to mitochondria.

  Yuma Yamada, Sen Ishizuka, Manae Arai, Minako Maruyama, Hideyoshi Harashima

  Expert opinion on biological therapy 1 - 11 2022/05/11 

  INTRODUCTION: After the emergence of lipid nanoparticles (LNP) containing therapeutic mRNA as vaccines for use against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the clinical usefulness of nucleic acid-encapsulated LNPs is now a fact. In addition to the nucleus and cytoplasm, mitochondria, which have their own genome, are a site where nucleic acids function in the cell. Gene therapies targeting mitochondria are expected to pave the way for the next generation of therapies. AREAS COVERED: Methods for delivering nucleic acids to mitochondria are needed in order to realize such innovative therapies. However, only a few reports on delivery systems targeting mitochondria have appeared. In this review, we summarize the current state of research on RNA-based therapeutics targeted to mitochondria, with emphasis on mitochondrial RNA delivery therapies and on therapies that involve the use of mitochondrial genome editing devices. EXPERT OPINION: We hope that this review article will focus our attention to this area of research, stimulate more interest in this field of research, and lead to the development of mitochondria-targeted nucleic acid medicine. It has the potential to become a major weapon against urgent and unknown diseases, including SARS-CoV-2 infections.
• Transplantation of MITO cells, mitochondria activated cardiac progenitor cells, to the ischemic myocardium of mouse enhances the therapeutic effect.

  Daisuke Sasaki, Jiro Abe, Atsuhito Takeda, Hideyoshi Harashima, Yuma Yamada

  Scientific reports 12 (1) 4344 - 4344 2022/03/22 

  Given the potential for myocardial stem cell transplantation as a promising treatment for heart failure, numerous clinical trials have been conducted and its usefulness has been clearly confirmed. However, the low rate of engraftment of transplanted cells has become a clinical problem, and this needs to be improved in the case of transplanting cells to the heart. To address this issue, we report on attempts to prepare mitochondria-activated stem cells (MITO cells) for use in transplantation. MITO cells, which is cardiac progenitor cells (CPCs) activated by the mitochondrial delivery of resveratrol with an anti-oxidant and mitochondrial activation effects were successfully prepared using a mitochondrial targeting nanocarrier (MITO-Porter). The purpose of this study was to validate the therapeutic effect of cell transplantation by the MITO cells using a mouse model of myocardial ischemia-reperfusion. Mouse CPCs were used as transplanted cells. The transplantation of CPCs and MITO cells were conducted after myocardial ischemia-reperfusion, and the therapeutic effect was determined. The MITO cells transplanted group showed increase in postoperative weight gain, improve cardiac function and inhibition of fibrosis compared to the non-transplanted group and the CPC group. The transplantation of MITO cells to the ischemic myocardium showed a stronger transplantation effect compared to conventional CPC transplantation.
• [Development of an Online Role-play-based Medical Interview Training Method for Fourth-year Pharmacy Students].

  Ayako Mori, Izumi Kato, Hitoshi Kashiwagi, Shungo Imai, Katsuya Narumi, Yuki Sato, Ayako Furugen, Yuma Yamada, Masaki Kobayashi

  Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan 142 (6) 661 - 674 2022 

  With the coronavirus disease 2019 pandemic, businesses are rapidly expanding their online practices, and the online medical care system has been established and is growing. The field of pharmacy education is also looking for ways to conduct practical online training. Hence, we developed an online role-play-based medical interview training method for fourth-year pharmacy students. The purpose of this study was to describe in detail this method and to clarify the effect of online on medical interviewing practice. The training sessions were conducted using video teleconferencing software. Two settings were used for the role-play scenarios: the pharmacy and hospital. To evaluate the effectiveness of the sessions, a questionnaire was sent to the students, and the results were analyzed using text mining. The most important requirement for successfully conducting the interviews was a stable voice connection, and we reduced audio interruptions and delays by connecting the host personal computer to a wired local area network. We also solved the problem of howling when multiple terminals were installed in the same room by muting all devices in the room. Results of the analysis of the questionnaires suggested that students were more tense online. We also found that students perceived a difference between online and face-to-face interviews in terms of eye contact and the presentation of documents. In this way, we succeeded in conducting smooth online role-playing sessions while taking countermeasures against infection. In the future, it will be necessary to devise nonverbal communication methods and digital methods of presenting the training material.
• Resveratrol-Encapsulated Mitochondria-Targeting Liposome Enhances Mitochondrial Respiratory Capacity in Myocardial Cells.

  Takao Tsujioka, Daisuke Sasaki, Atsuhito Takeda, Hideyoshi Harashima, Yuma Yamada

  International journal of molecular sciences 23 (1) 2021/12/22 

  The development of drug delivery systems for use in the treatment of cardiovascular diseases is an area of great interest. We report herein on an evaluation of the therapeutic potential of a myocardial mitochondria-targeting liposome, a multifunctional envelope-type nano device for targeting pancreatic β cells (β-MEND) that was previously developed in our laboratory. Resveratrol (RES), a natural polyphenol compound that has a cardioprotective effect, was encapsulated in the β-MEND (β-MEND (RES)), and its efficacy was evaluated using rat myocardioblasts (H9c2 cells). The β-MEND (RES) was readily taken up by H9c2 cells, as verified by fluorescence-activated cell sorter data, and was observed to be colocalized with intracellular mitochondria by confocal laser scanning microscopy. Myocardial mitochondrial function was evaluated by a Seahorse XF Analyzer and the results showed that the β-MEND (RES) significantly activated cellular maximal respiratory capacity. In addition, the β-MEND (RES) showed no cellular toxicity for H9c2 cells as evidenced by Premix WST-1 assays. This is the first report of the use of a myocardial mitochondria-targeting liposome encapsulating RES for activating mitochondrial function, which was clearly confirmed based on analyses using a Seahorse XF Analyzer.
• An effective in vivo mitochondria-targeting nanocarrier combined with a pi-extended porphyrin-type photosensitizer

  Satrialdi, Yuta Takano, Eri Hirata, Natsumi Ushijima, Hideyoshi Harashima, Yuma Yamada

  NANOSCALE ADVANCES 3 (20) 5919 - 5927 2516-0230 2021/10 

  A photochemical reaction mediated by light-activated molecules (photosensitizers) in photodynamic therapy (PDT) causes molecular oxygen to be converted into highly reactive oxygen species (ROS) that are beneficial for cancer therapy. As the active oxygen consumer and the primary regulator of apoptosis, mitochondria are known as an important target for optimizing PDT outcomes. However, most of the clinically used photosensitizers exhibited a poor tumor accumulation profile as well as lack of mitochondria targeting ability. Therefore, by applying a nanocarrier platform, mitochondria-specific delivery of photosensitizers can be materialized. The present research develops an effective mitochondria-targeting liposome-based nanocarrier system (MITO-Porter) encapsulating a pi-extended porphyrin-type photosensitizer (rTPA), which results in a significant in vivo antitumor activity. A single PDT treatment of the rTPA-MITO-Porter resulted in a dramatic tumor inhibition against both human and murine tumors that had been xenografted in a mouse model. Furthermore, depolarization of the mitochondrial membrane was observed, implying the damage of the mitochondrial membrane due to the photochemical reaction that occurred specifically in the mitochondria of tumor cells. The findings presented herein serve to verify the significance of the mitochondria-targeted nanocarrier system for advancing the in vivo PDT effectivity in cancer therapy regardless of tumor type.
• Validation of the mitochondrial delivery of vitamin B1 to enhance ATP production using SH-SY5Y cells, a model neuroblast.

  Yuma Yamada, Takuya Ishimaru, Kohei Ikeda, Hideyoshi Harashima

  Journal of pharmaceutical sciences 111 (2) 432 - 439 2021/08/31 

  Large amounts of ATP are produced in mitochondria especially in the brain and heart, where energy consumption is high compared with other organs. Thus, a decrease in ATP production in such organs could be a cause of many diseases such as neurodegenerative diseases and heart disease. Based on thus assumption, increasing intracellular ATP production in such organs could be a therapeutic strategy. In this study, we report on the delivery of vitamin B1, a coenzyme that activates the tricarboxylic acid (TCA) cycle, to the inside of mitochondria. Since the TCA cycle is responsible for ATP production, we hypothesized delivering vitamin B1 to mitochondria would enhance ATP production. To accomplish this, we used a mitochondrial targeted liposome a "MITO-Porter" as the carrier. Using SH-SY5Y cells, a model neuroblast, cellular uptake and intracellular localization were analyzed using flow cytometry and confocal laser scanning microscopy. The optimized MITO-Porter containing encapsulated vitamin B1 (MITO-Porter (VB1)) was efficiently accumulated in mitochondria of SH-SY5Y cells. Further studies confirmed that the level of ATP production after the MITO-Porter (VB1) treatment was significantly increased as compared to a control group that was treated with naked vitamin B1. This study provides the potential for an innovative therapeutic strategy in which the TCA cycle is activated, thus enhancing ATP production. Relative ATP ratio (%) = IS/IU × 100, where IS and IU represent the intracellular ATP amounts for the treated and untreated cells with samples, respectively.
• Targeting the Mitochondrial Genome Via a MITO-Porter : Evaluation of mtDNA and mtRNA Levels and Mitochondrial Function.

  Yuma Yamada, Hideyoshi Harashima

  Methods in molecular biology (Clifton, N.J.) 2275 227 - 245 2021 

  Genetic mutations and defects in mitochondrial DNA (mtDNA) are associated with certain types of mitochondrial dysfunctions, ultimately resulting in the emergence of a variety of human diseases. To achieve an effective mitochondrial gene therapy, it will be necessary to deliver therapeutic agents to the innermost mitochondrial space (the mitochondrial matrix), which contains the mtDNA pool. We recently developed a MITO-Porter, a liposome-based nanocarrier that delivers cargo to mitochondria via a membrane-fusion mechanism. In this chapter, we discuss the methodology used to deliver bioactive molecules to the mitochondrial matrix using a Dual Function (DF)-MITO-Porter, a liposome-based nanocarrier that delivers it cargo by means of a stepwise process, and an evaluation of mtDNA levels and mitochondrial activities in living cells. We also discuss mitochondrial gene silencing by the mitochondrial delivery of antisense RNA oligonucleotide (ASO) targeting mtDNA-encoded mRNA using the MITO-Porter system.
• 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 2020/12/20 

  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.
• 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.
• The use of a MITO-Porter to deliver exogenous therapeutic RNA to a mitochondrial disease's cell with a A1555G mutation in the mitochondrial 12S rRNA gene results in an increase in mitochondrial respiratory activity.

  Yuma Yamada, Minako Maruyama, Tomoko Kita, Shin-Ichi Usami, Shin-Ichiro Kitajiri, Hideyoshi Harashima

  Mitochondrion 55 134 - 144 2020/11 

  We report on validating a mitochondrial gene therapeutic strategy using fibroblasts derived from patients with an A1555G point mutation in mitochondrial DNA coding 12S ribosomal RNA (rRNA (12S)). Wild-type rRNA (12S) as a therapeutic RNA was encapsulated in a mitochondrial targeting liposome, a MITO-Porter (rRNA-MITO-Porter), and an attempt was made to deliver the MITO-Porter to mitochondria of the diseased cells. It was confirmed that the rRNA-MITO-Porter treatment significantly decreased the ratio of the mutant rRNA content. Moreover, it was shown that the mitochondrial respiratory activities of the diseased cells were improved as the result of the mitochondrial transfection of the rRNA-MITO-Porter.
• Challenges in Promoting Mitochondrial Transplantation Therapy.

  Yuma Yamada, Momo Ito, Manae Arai, Mitsue Hibino, Takao Tsujioka, Hideyoshi Harashima

  International journal of molecular sciences 21 (17) 2020/09/02 

  Mitochondrial transplantation therapy is an innovative strategy for the treatment of mitochondrial dysfunction. The approach has been reported to be useful in the treatment of cardiac ischemic reperfusion injuries in human clinical trials and has also been shown to be useful in animal studies as a method for treating mitochondrial dysfunction in various tissues, including the heart, liver, lungs, and brain. On the other hand, there is no methodology for using preserved mitochondria. Research into the pharmaceutical formulation of mitochondria to promote mitochondrial transplantation therapy as the next step in treating many patients is urgently needed. In this review, we overview previous studies on the therapeutic effects of mitochondrial transplantation. We also discuss studies related to immune responses that occur during mitochondrial transplantation and methods for preserving mitochondria, which are key to their stability as medicines. Finally, we describe research related to mitochondrial targeting drug delivery systems (DDS) and discuss future perspectives of mitochondrial transplantation.
• Mitochondrial Delivery of an Anticancer Drug Via Systemic Administration Using a Mitochondrial Delivery System That Inhibits the Growth of Drug-Resistant Cancer Engrafted on Mice.

  Yuma Yamada, Reina Munechika, Satrialdi, Fumika Kubota, Yusuke Sato, Yu Sakurai, Hideyoshi Harashima

  Journal of pharmaceutical sciences 109 (8) 2493 - 2500 2020/08 

  Mitochondrial delivery of an anticancer drug targeting cancer cells would eventually result in cell death. To achieve this, a drug delivery system targeting mitochondria is needed. We recently developed a MITO-Porter, a liposome that delivers its cargo to mitochondria. We reported that such a MITO-Porter could deliver doxorubicin (DOX), an anticancer drug, to mitochondria in OS-RC-2 cells, a drug resistant cancer cell, resulting in inhibiting the cell growth, based in in vitro experiments. Herein, we report on validating the benefit of such a therapeutic strategy for treating drug resistant cancers by the in vivo targeting of mitochondria. We prepared a DOX-MITO-Porter, in which DOX was encapsulated in the MITO-Porter and optimized its retention in blood circulation. When the DOX-MITO-Porter was administered to mice bearing OS-RC-2 cells via tail vein injection, tumor size was significantly decreased, compared to DOX itself and to the DOX-encapsulated polyethylene glycol-modified liposome (DOX-PEG-LP). Intracellular observation confirmed that the DOX-MITO-Porter had accumulated in tumor mitochondria. It was also found a relationship between anti-tumor effect and the mitochondrial function, as indicated by the depolarization of mitochondrial membrane potential. This study provides support for the utility of an in vivo mitochondrial delivery system in drug resistant cancer therapies.
• Validation of Gene Therapy for Mutant Mitochondria by Delivering Mitochondrial RNA Using a MITO-Porter.

  Eriko Kawamura, Minako Maruyama, Jiro Abe, Akira Sudo, Atsuhito Takeda, Shingo Takada, Takashi Yokota, Shintaro Kinugawa, Hideyoshi Harashima, Yuma Yamada

  Molecular therapy. Nucleic acids 20 687 - 698 2020/06/05 [Refereed][Not invited]
   

  Here, we report on validating a mitochondrial gene therapy by delivering nucleic acids to mitochondria of diseased cells by a MITO-Porter, a liposome-based carrier for mitochondrial delivery. We used cells derived from a patient with a mitochondrial disease with a G625A heteroplasmic mutation in the tRNAPhe of the mitochondrial DNA (mtDNA). It has been reported that some mitochondrial gene diseases are caused by heteroplasmic mutations, in which both mutated and wild-type (WT) genes are present, and the accumulation of pathological mutations leads to serious, intractable, multi-organ diseases. Therefore, the decrease of the mutated gene rate is considered to be a useful gene therapy strategy. To accomplish this, wild-type mitochondrial pre-tRNAPhe (pre-WT-tRNAPhe), prepared by in vitro transcription, was encapsulated in the MITO-Porter. The pre-WT-tRNAPhe encapsulated in the MITO-Porter was transfected into diseased mitochondrial cells, and the resulting mutant levels were examined by an amplification refractory mutation system (ARMS)-quantitative PCR. The mutation rate of tRNAPhe was decreased, and this therapeutic effect was sustained even on the 8th day after transfection. Furthermore, mitochondrial respiratory activity of the disease cells was increased after the transfection of therapeutic pre-WT-tRNAPhe. These results support the conclusion that the mitochondrial delivery of therapeutic nucleic acids represents a viable strategy for mitochondrial gene therapy.
• Validation of a mitochondrial RNA therapeutic strategy using fibroblasts from a Leigh syndrome patient with a mutation in the mitochondrial ND3 gene.

  Yuma Yamada, Kana Somiya, Akihiko Miyauchi, Hitoshi Osaka, Hideyoshi Harashima

  Scientific reports 10 (1) 7511 - 7511 2020/05/05 

  We report on the validation of a mitochondrial gene therapeutic strategy using fibroblasts from a Leigh syndrome patient by the mitochondrial delivery of therapeutic mRNA. The treatment involves delivering normal ND3 protein-encoding mRNA as a therapeutic RNA to mitochondria of the fibroblasts from a patient with a T10158C mutation in the mtDNA coding the ND3 protein, a component of the mitochondrial respiratory chain complex I. The treatment involved the use of a liposome-based carrier (a MITO-Porter) for delivering therapeutic RNA to mitochondria via membrane fusion. The results confirmed that the mitochondrial transfection of therapeutic RNA by the MITO-Porter system resulted in a decrease in the levels of mutant RNA in mitochondria of diseased cells based on reverse transcription quantitative PCR. An evaluation of mitochondrial respiratory activity by respirometry also showed that transfection using the MITO-Porter resulted in an increase in maximal mitochondrial respiratory activity in the diseased cells.
• Development of a nanoparticle that releases nucleic acids in response to a mitochondrial environment.

  Yuma Yamada, Yutaka Fukuda, Daisuke Sasaki, Minako Maruyama, Hideyoshi Harashima

  Mitochondrion 52 67 - 74 2020/02/22 [Refereed][Not invited]
   

  The delivery of nucleic acids targeting mutant mtDNA represent a potential strategy for addressing a variety of mitochondria-related diseases. We previously developed a MITO-Porter, a nano carrier that is capable of delivering nanoparticles of nucleic acids to mitochondria of human cells. Here, we report on an investigation of a series of nanoparticles formed with various poly cationic peptides that can release nucleic acids in response to a mitochondrial environment. A significant relationship was found between the number of and the location of arginine and histidine residues in the peptide sequence and the release of nucleic acids in a mitochondrial environment.
• The optimization of cancer photodynamic therapy by utilization of a pi-extended porphyrin-type photosensitizer in combination with MITO-Porter.

  Satrialdi, Reina Munechika, Vasudevanpillai Biju, Yuta Takano, Hideyoshi Harashima, Yuma Yamada

  Chemical communications (Cambridge, England) 56 (7) 1145 - 1148 2020/01/23 [Refereed][Not invited]
   

  The uncontrolled production of reactive oxygen species during photodynamic therapy (PDT) induces oxidative stress. The full potential of PDT is accomplished by delivery of a pi-extended porphyrin-type photosensitizer into mitochondria of tumor cells using a MITO-Porter, a mitochondrial targeting nanodevice. This strategy can be implemented for innovative cancer therapy.
• Therapeutic Strategies for Regulating Mitochondrial Oxidative Stress.

  Yuma Yamada, Yuta Takano, Satrialdi, Jiro Abe, Mitsue Hibino, Hideyoshi Harashima

  Biomolecules 10 (1) 2020/01/05 [Refereed][Not invited]
   

  There have been many reports on the relationship between mitochondrial oxidative stress and various types of diseases. This review covers mitochondrial targeting photodynamic therapy and photothermal therapy as a therapeutic strategy for inducing mitochondrial oxidative stress. We also discuss other mitochondrial targeting phototherapeutic methods. In addition, we discuss anti-oxidant therapy by a mitochondrial drug delivery system (DDS) as a therapeutic strategy for suppressing oxidative stress. We also describe cell therapy for reducing oxidative stress in mitochondria. Finally, we discuss the possibilities and problems associated with clinical applications of mitochondrial DDS to regulate mitochondrial oxidative stress.
• Power of mitochondrial drug delivery systems to produce innovative nanomedicines.

  Yuma Yamada, Satrialdi, Mitsue Hibino, Daisuke Sasaki, Jiro Abe, Hideyoshi Harashima

  Advanced drug delivery reviews 154-155 187 - 209 2020 

  Mitochondria carry out various essential functions including ATP production, the regulation of apoptosis and possess their own genome (mtDNA). Delivering target molecules to this organelle, it would make it possible to control the functions of cells and living organisms and would allow us to develop a better understanding of life. Given the fact that mitochondrial dysfunction has been implicated in a variety of human disorders, delivering therapeutic molecules to mitochondria for the treatment of these diseases is an important issue. To date, several mitochondrial drug delivery system (DDS) developments have been reported, but a generalized DDS leading to therapy that exclusively targets mitochondria has not been established. This review focuses on mitochondria-targeted therapeutic strategies including antioxidant therapy, cancer therapy, mitochondrial gene therapy and cell transplantation therapy based on mitochondrial DDS. A particular focus is on nanocarriers for mitochondrial delivery with the goal of achieving mitochondria-targeting therapy. We hope that this review will stimulate the accelerated development of mitochondrial DDS.
• A mitochondrial delivery system using liposome-based nanocarriers that target myoblast cells.

  Takashi Katayama, Shintaro Kinugawa, Shingo Takada, Takaaki Furihata, Arata Fukushima, Takashi Yokota, Toshihisa Anzai, Mitsue Hibino, Hideyoshi Harashima, Yuma Yamada

  Mitochondrion 49 66 - 72 1567-7249 2019/11 [Refereed][Not invited]
   

  Mitochondrial function is reduced in skeletal muscles of many patients with systemic diseases and it is difficult to deliver medicinal substances to mitochondria in such tissue. In this study, we report on attempts to develop liposome-based carriers for mitochondrial delivery using mouse myoblasts (C2C12) by varying the lipid composition of the carriers. We found that a liposome that contains an optimal lipid modified with the KALA peptide (a cellular uptake and mitochondrial targeting device) was the most effective nanocarrier for achieving mitochondrial delivery in C2C12 cells. We also report on successful mitochondrial transgene expression using the carriers encapsulating a mitochondrial DNA vector as we previously reported.
• 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][Not 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.
• A nanocarrier for the mitochondrial delivery of nucleic acids to cardiomyocytes.

  Yamada Y, Fujishita N, Harashima H

  Nucleosides, nucleotides & nucleic acids 1 - 15 1525-7770 2019/10 [Refereed][Not invited]
  
• The Use of a Microfluidic Device to Encapsulate a Poorly Water-Soluble Drug CoQ₁₀ in Lipid Nanoparticles and an Attempt to Regulate Intracellular Trafficking to Reach Mitochondria.

  Hibino M, Yamada Y, Fujishita N, Sato Y, Maeki M, Tokeshi M, Harashima H

  Journal of pharmaceutical sciences 108 (8) 2668 - 2676 0022-3549 2019/08 [Refereed][Not invited]
   

  © 2019 A number of drugs that are currently on the market, as well as new candidates for drugs, are poorly water soluble. Because of this, a need exists to develop drug formulations that will permit the expanded use of such drugs. The use of liposomes and lipid nanoparticles for drug delivery has attracted attention as a technique for solubilizing molecules that are poorly water soluble, but this technique faces serious scale-up risks. In this study, we report on attempts to encapsulate Coenzyme Q10 (CoQ10) as a model of a poorly water-soluble drug in an MITO-Porter, a liposome for mitochondrial delivery using a microfluidic device (a CoQ10-MITO-Porter [μ]). The physical properties of the CoQ10-MITO-Porter [μ] including homogeneity, size, and preparation volume were compared with those for a CoQ10-MITO-Porter prepared by the ethanol dilution method (a CoQ10-MITO-Porter [ED]). In the case where a microfluidic device was used, a small-sized CoQ10-MITO-Porter was formed homogeneously, and it was possible to prepare it on a large scale. Intracellular observations using HeLa cells showed that the CoQ10-MITO-Porter [μ] was efficiently internalized by cells to reach mitochondria. These results indicate that the CoQ10-MITO-Porter [μ] represents a potential candidate for use in mitochondrial nanomedicine.
• Targeted mitochondrial delivery of antisense RNA-containing nanoparticles by a MITO-Porter for safe and efficient mitochondrial gene silencing.

  Kawamura E, Hibino M, Harashima H, Yamada Y

  Mitochondrion 49 178 - 188 1567-7249 2019/08 [Refereed][Not invited]
   

  Mitochondrial gene therapy will be needed to treat mitochondrial diseases. We previously demonstrated mitochondrial gene silencing by the mitochondrial delivery of antisense RNA oligonucleotide (ASO) targeting mtDNA-encoded mRNA using a MITO-Porter, a liposomal nano carrier system designed for mitochondrial delivery. Here, we report on the efficient packaging of ASO in the MITO-Porter via a nanoparticle packaging method, which showed a 10-fold higher packaging efficiency than the conventional method. The constructed carrier showed a decrease in the target mRNA levels and ATP production. These results indicate that such a MITO-Porter has potential for use in therapies designed to regulate mitochondrial function.
• Enhanced autophagy induction via the mitochondrial delivery of methylated beta-cyclodextrin-threaded polyrotaxanes using a MITO-Porter

  Yuma Yamada, Shinnosuke Daikuhara, Atsushi Tamura, Kei Nishida, Nobuhiko Yui, Hideyoshi Harashima

  CHEMICAL COMMUNICATIONS 55 (50) 7203 - 7206 1359-7345 2019/06 [Refereed][Not invited]
   

  Failure of autophagy induction results in the accumulation of abnormal mitochondria to cause neurodegenerative diseases. Artificial autophagy activation via the mitochondrial delivery of polyrotaxane with autophagy induced activity is achieved using a MITO-Porter, a nanodevice for mitochondrial delivery. This strategy can be applied to innovative research and therapy.
• オートファジー誘導能を有するミトコンドリアDDSの構築およびオートファジー誘導機構の検証

  山田 勇磨, 大工原 伸之輔, 西田 慶, 田村 篤志, 由井 伸彦, 原島 秀吉

  日本薬学会年会要旨集 (公社)日本薬学会 139年会 (4) 59 - 59 0918-9823 2019/03 [Refereed][Not invited]
  
• Future of human mitochondrial DNA editing technologies

  N. Verechshagina, N. Nikitchina, Y. Yamada, Н. Harashima, M. Tanaka, K. Orishchenko, I. Mazunin

  Mitochondrial DNA Part A 30 (2) 214 - 221 2470-1394 2019/02/17
• [A Nanocarrier System for Mitochondrial Delivery Targeted to a Pancreatic Beta Cell].

  Yamada Y

  Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan 139 (1) 41 - 45 0031-6903 2019 [Refereed][Not invited]
  
• ミトコンドリア強化幹細胞(MITO Cell)を用いた細胞移植療法の検証

  山田 勇磨, 阿部 二郎, 佐々木 大輔, 武田 充人, 原島 秀吉

  日本バイオマテリアル学会大会予稿集 日本バイオマテリアル学会 40回 180 - 180 2018/11
• Optimization of siRNA delivery to target sites: issues and future directions.

  Khalil IA, Yamada Y, Harashima H

  Expert opinion on drug delivery 1 - 13 1742-5247 2018/09 [Refereed][Not invited]
  
• The use of cardiac progenitor cells for transplantation in congenital heart disease and an innovative strategy for activating mitochondrial function in such cells.

  Abe J, Yamada Y, Harashima H

  Journal of thoracic disease 10 (Suppl 18) S2119 - S2121 2072-1439 2018/07 [Refereed][Not invited]
  
• ミトコンドリアへの分子送達技術

  山田 勇磨, 原島 秀吉

  BIO Clinica (株)北隆館 33 (7) 637 - 640 0919-8237 2018/07 

  オートファジーに関連するミトコンドリアの品質管理システム・マイトファジーが注目を集めている。人工的にマイトファジーを誘導するシステムは、進行性神経変性疾患などの疾患解明・治療に大きく貢献する事が期待される。本戦略を実現するためには、マイトファジー誘導分子をミトコンドリアに送達するDrug Delivery Systemが必要不可欠である。本稿では、ミトコンドリアを標的としたDDSを概説する。また、ミトコンドリア標的型ナノカプセル"MITO-Porter"に関する我々の最新の研究成果も紹介する。(著者抄録)
• ポリロタキサン封入MITO-Porterの調製とオートファジーの誘導評価

  田村 篤志, 西田 慶, 由井 伸彦, 山田 勇磨, 大工原 伸之輔, 原島 秀吉

  生体医歯工学共同研究拠点成果報告書 東京医科歯科大学生体材料工学研究所 平成29年度 120 - 120 2018/04 [Refereed][Not invited]
  
• Mitochondrial transgene expression via an artificial mitochondrial DNA vector in cells from a patient with a mitochondrial disease

  Takuya Ishikawa, Kana Somiya, Reina Munechika, Hideyoshi Harashima, Yuma Yamada

  Journal of Controlled Release 274 109 - 117 1873-4995 2018/03/28 [Refereed][Not invited]
   

  To achieve mitochondrial gene therapy, developing a mitochondrial transgene expression system that produces therapeutic proteins in mitochondria of disease cells is essential. We previously reported on the design of pCMV-mtLuc (CGG) containing a CMV promotor and a NanoLuc (Nluc) luciferase gene that records adjustments to the mitochondrial codon system, and showed that the mitochondrial transfection of pCMV-mtLuc (CGG) resulted in the efficient production of the Nluc luciferase protein in human HeLa cells. This mitochondrial transfection was achieved using a MITO-Porter, a liposome-based carrier for delivering a cargo to mitochondria via membrane fusion. We report herein that mitochondrial transfection using the MITO-Porter results in mitochondrial transgene expression in G625A fibroblasts obtained from a patient with a mitochondrial disease. We investigated the effect of promoters and the basic structure of pCMV-mtLuc (CGG) on gene expression efficiency, and were able to construct a high performance mitochondrial DNA vector, pCMV-mtLuc (CGG) [hND4] that contains a human mitochondrial endogenous gene. We also constructed an RP/KALA-MITO-Porter composed of the KALA peptide (cell-penetrating peptide) with a mitochondrial RNA aptamer to enhance cellular uptake and mitochondrial targeting. Finally, the mitochondrial transfection of pCMV-mtLuc (CGG) [hND4] in G625A fibroblasts using the RP/KALA-MITO-Porter resulted in strong mitochondrial transgene expression.
• Targeting the mitochondrial genome through a nanocarrier and the regulation of mitochondrial gene expression

  Yuma Yamada, Hideyoshi Harashima

  Mitochondrial Biology and Experimental Therapeutics 491 - 498 2018/03/21 [Refereed][Not invited]
   

  Mitochondria are attractive organelles that have the potential to contribute greatly to medical therapy. This organnelle is responsible for a variety of essential functions including ATP production and the regulation of apoptosis, and they have their own genome, mitochondrial DNA (mtDNA). It has recently become evident that a variety of human diseases are associated with mitochondrial dysfunctions caused by mutations and defects in mtDNA. Therefore, the ability to successfully target the mitochondrial genome and to regulate mitochondrial gene expression would contribute to mitochondrial gene therapy for various human diseases. To achieve such an innovative objective, it will be necessary to deliver various cargoes to mitochondria in living cells. However, only a limited number of approaches are available for accomplishing this. In this chapter, we discuss problems associated with mitochondrial delivery systems and mitochondrial gene expression, and propose a strategy for overcoming these problems based our current efforts. To this end, we highlight a MITO-Porter, a mitochondrial delivery system, and show some examples of the regulation of mitochondrial gene expression including mitochondrial RNA knockdown and mitochondrial transgene expression.
• In Vivo Transgene Expression in the Pancreas by the Intraductal Injection of Naked Plasmid DNA

  Yuma Yamada, Mai Tabata, Jiro Abe, Masatoshi Nomura, Hideyoshi Harashima

  Journal of Pharmaceutical Sciences 107 (2) 647 - 653 1520-6017 2018/02/01 [Refereed][Not invited]
   

  Patients with type I diabetes, which is caused by the destruction of pancreatic islets, now require regular therapeutic injections of insulin. The use of transgene therapy represents an alternate and potent strategy for the treatment of type I diabetes. However, only a limited number of studies regarding in vivo gene delivery targeting the pancreas and islets have been reported. Here, we report on the possibility of in vivo transgene expression in the pancreas by the intraductal injection of naked plasmid DNA (pDNA). Gene expression activities were detected in the pancreas of mice after the injection of naked pDNA encoding luciferase into the common bile duct. We then investigated the effects of injection dose, volume, and speed on gene delivery and determined the optimal conditions for the delivery of pDNA to the pancreas. Exogenous luciferase mRNA was detected in the pancreatic islets by reverse transcription PCR analysis. Moreover, no injury was detected in the liver, the common bile duct, or the pancreas over time after the injection. These findings indicate that the intraductal injection of naked pDNA promises to be a useful technique for in vivo gene delivery targeted to pancreatic tissue and islets.
• Cardiac progenitor cells activated by mitochondrial delivery of resveratrol enhance the survival of a doxorubicin-induced cardiomyopathy mouse model via the mitochondrial activation of a damaged myocardium

  Jiro Abe, Yuma Yamada, Atsuhito Takeda, Hideyoshi Harashima

  JOURNAL OF CONTROLLED RELEASE 269 177 - 188 0168-3659 2018/01 [Refereed][Not invited]
   

  It has been reported that transplanting native cells would lack efficiency without producing artificial cell-tissue, due to the exaggerated oxidative stress in doxorubicin-induced cardiomyopathy. We attempted to activate cardiac progenitor cells (CPCs) by delivering resveratrol to mitochondria using a mitochondrial drug delivery system (MITO-Porter system). We first evaluated the viability of H9c2 cells (a cardio myoblast cell line) after doxorubicin treatment, where H9c2 cells were co-cultured with or without the mitochondria activated CPCs (referred to herein as MITO cell). We next evaluated the survival rate of doxorubicin treated mice, with or without the injection of MITO cells into the myocardium. Finally, we examined the molecular mechanism of the cell therapy by detecting oxidative stress and the induction of apoptosis in addition to quantification of the mRNA and protein levels about oxidative phosphorylation (OXPHOS). The MITO cell transplanted mice lived significantly longer than the conventional CPC transplanted ones. Oxidative stress and massive cell death were both significantly reduced in the MITO cell transplanted hearts, in which the expression levels of OXPHOS protein and gene were also higher than the control group. In doxorubicin-induced cardiomyopathy, the transplantation of MITO cells, which possess activated mitochondria, is more efficient compared to conventional CPC transplantation.
• [Analysis by Using Roter Method of Interaction Process Analysis (RIAS) of the Ability of Pharmacy Students to Communicate after Clinical Training for Pharmacy].

  Yoh Takekuma, Ayako Mori, Masaki Kobayashi, Yuma Yamada, Yuki Sato, Katsuya Narumi, Ayako Furugen, Mitsuru Sugawara

  Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan 138 (12) 1579 - 1586 0031-6903 2018 [Refereed][Not invited]
   

  Communication education is now necessary for pharmaceutical education since the role of pharmacists has expanded from "medicine-based" to "person-based". However, a standard for assessing the effectiveness of a communication education program has not been established. Hence, the aim of this study was to determine the effectiveness of clinical training in pharmacy for enhancing the ability of pharmacy students to communicate. Role playing with simulated patients was performed by pharmacy students before and after clinical practice for pharmacy, and the effects of learning were analyzed by Roter method of interaction process analysis (RIAS). Analysis by RIAS enabled quantification and objective evaluation of communication by pharmacy students. The results showed improvement of interactive communication, decrease of "Question asking" and "Others" including "Transition words", and increase of "Partnership behaviors" and "Counsel behaviors". The pharmacy students became skillful in communication without showing hesitation. The results therefore showed that clinical training contributes to improvement in the ability of pharmacy students to communicate.
• Optical control of mitochondrial reductive reactions in living cells using an electron donor-acceptor linked molecule

  Yuta Takano, Reina Munechika, Vasudevanpillai Biju, Hideyoshi Harashima, Hiroshi Imahori, Yuma Yamada

  NANOSCALE 9 (47) 18690 - 18698 2040-3364 2017/12 [Refereed][Not invited]
   

  It has been known for decades that intracellular redox reactions control various vital functions in living systems, which include the synthesis of biomolecules, the modulation of protein functions, and cell signaling. Although there have been several reports on the control of such functions using DNA and RNA, the non-invasive optical control of biological functions is an important ongoing challenge. In this study, a hybrid of an electron donor-acceptor linked molecule based on a ferrocene(Fc)-porphyrin(ZnP)-fullerene-(C-60) analogue and an elaborately designed nano-carrier, referred to herein as a MITO-Porter, resulted in a successful photoinduced intermolecular electron transfer reaction via the long-lived intramolecular charge separation, leading to site-specific reductive reactions in the mitochondria of living HeLa cells. A Fc-ZnP-C-60 linked molecule, 1-Oct, was designed and prepared for taking advantage of the unique photophysical properties with excellent efficiency (i.e. a long lifetime and a high quantum yield) for photoinduced charge separation. The targeted delivery of 1-Oct to mitochondria was accomplished by using a combination of the Fc-ZnP-C-60 molecule and a drug delivery nano-carrier, MITO-Porter, that was recently established by our group for intracellular cargo delivery. The successful delivery of 1-Oct by the MITO-Porter permitted the optically-controlled generation of O-2(-) in the mitochondria of HeLa cells and the following induction of apoptosis as a cell signalling response was observed in confocal laser microscopy experiments. The obtained results indicate the use of an electron donor-acceptor system such as this can be a promising tool for the non-invasive triggering of redox-coupled cellular activities in living systems.
• Packaging of the Coenzyme Q(10) into a Liposome for Mitochondrial Delivery and the Intracellular Observation in Patient Derived Mitochondrial Disease Cells

  Yuma Yamada, Laila Burger, Eriko Kawamura, Hideyoshi Harashima

  BIOLOGICAL & PHARMACEUTICAL BULLETIN 40 (12) 2183 - 2190 0918-6158 2017/12 [Refereed][Not invited]
   

  While Coenzyme Q(10) (CoQ(10)) is thought to be effective for the treatment of a variety of diseases, it limits its cellular uptake. Because of the hydrophobic nature of CoQ(10), it is reasonable to assume that it could be encapsulated within a liposomal carrier. Several reports regarding the packaging of CoQ(10) in liposomes have appeared, but detailed investigations of the preparation of CoQ(10) encapsulated liposomes have not been reported. As a result, information regarding the optimal method of packaging CoQ(10) in liposomes is not available. In this study, several types of liposomes were prepared using different methods and their characteristics were compared. Since CoQ(10) is mainly located in the inner mitochondria! membrane, a liposome that targets mitochondria, a MITO-Porter, was used as a model liposome. It was possible to incorporate high levels of CoQ(10) into the carrier. Transmission electron microscopy analyses showed that an empty MITO-Porter and the CoQ(10)-MITO-Porter were structurally different from one another. Even though significant structural differences were observed, mitochondria! delivery was not affected in mitochondria! disease fibroblast cells, as evidenced by confocal laser scanning microscopy observations. The results reported herein suggest that the CoQ(10)-MITO-Porter might be a suitable candidate for the potential medical therapy of mitochondria-related diseases.
• Mitochondrial Delivery of Doxorubicin Using MITO-Porter Kills Drug-Resistant Renal Cancer Cells via Mitochondrial Toxicity

  Yuma Yamada, Reina Munechika, Eriko Kawamura, Yu Sakurai, Yusuke Sato, Hideyoshi Harashima

  JOURNAL OF PHARMACEUTICAL SCIENCES 106 (9) 2428 - 2437 0022-3549 2017/09 [Refereed][Not invited]
   

  Most anticancer drugs are intended to function in the nuclei of cancer cells. If an anticancer drug could be delivered to mitochondria, the source of cellular energy, this organelle would be destroyed, resulting in the arrest of the energy supply and the killing of the cancer cells. To achieve such an innovative strategy, a mitochondrial drug delivery system targeted to cancer cells will be required. We recently reported on the development of a MITO-Porter, a liposome for mitochondrial delivery. In this study, we validated the utility of such a cancer therapeutic strategy by delivering anticancer drugs directly to mitochondria. We succeeded in packaging doxorubicin (DOX) as a model cargo in MITO-Porter to produce a DOX-MITO-Porter. We evaluated cellular toxicity of OS-RC-2 cell, a type of DOX-resistant cancer cell, after delivering DOX to mitochondria using the MITO-Porter system. Cell viability was decreased by the DOX-MITO-Porter treatment, while cell viability was not decreased in the case of naked DOX and a conventional DOX liposomal formulation. We also found a relationship between cellular toxicity and mitochondrial toxicity. The use of a MITO-Porter system for mitochondrial delivery of a toxic agent represents a possible therapeutic strategy for treating drug-resistant cancers. (C) 2017 American Pharmacists Association (R). Published by Elsevier Inc. All rights reserved.
• Validation of the use of an artificial mitochondrial reporter DNA vector containing a Cytomegalovirus promoter for mitochondrial transgene expression

  Yuma Yamada, Takuya Ishikawa, Hideyoshi Harashima

  BIOMATERIALS 136 56 - 66 0142-9612 2017/08 [Refereed][Not invited]
   

  Mitochondria have their own gene expression system that is independent of the nuclear system, and control cellular functions in cooperation with the nucleus. While a number of useful technologies for achieving nuclear transgene expression have been reported, only a few have focused on mitochondria. In this study, we validated the utility of an artificial mitochondrial DNA vector with a virus promoter on mitochondrial transgene expression. We designed and constructed pCMV-mtLuc (CGG) that contains a CMV promotor derived from Cytomegalovirus and an artificial mitochondrial genome with a NanoLuc (Nluc) luciferase gene that records adjustments to the mitochondrial codon system. Nluc luciferase activity measurements showed that the pCMV-mtLuc (CGG) efficiently produced the Nluc luciferase protein in human HeLa cells. Moreover, we optimized the mitochondrial transfection of pCMV-mtLuc (CGG) using a MITO-Porter system, a liposome-based carrier for mitochondrial delivery via membrane fusion. As a result, we found that transfection of pCMV-mtLuc (CGG) by MITO-Porter modified with the KALA peptide (cationic amphipathic cell-penetrating peptide) showed a high mitochondrial transgene expression. The developed mitochondrial transgene expression system represents a potentially useful tool for the fields of nanoscience and nanotechnology for controlling the intracellular microenvironment via the regulation of mitochondrial function and promises to open additional innovative research fields of study. (C) 2017 Elsevier Ltd. All rights reserved.
• ドキソルビシン心筋症に対するミトコンドリアドラッグデリバリーシステムを用いた心筋前駆細胞移植の検討

  阿部 二郎, 山田 勇磨, 武田 充人, 原島 秀吉

  日本小児循環器学会雑誌 (NPO)日本小児循環器学会 33 (Suppl.1) s1 - 175 0911-1794 2017/07
• オートファジー誘導能を有する超分子構造ポリマー搭載ミトコンドリアDDSの構築

  大工原 伸之輔, 山田 勇磨, 田村 篤志, 西田 慶, 由井 伸彦, 原島 秀吉

  日本DDS学会学術集会プログラム予稿集 日本DDS学会 33回 173 - 173 2017/06 [Refereed][Not invited]
  
• MITO-Porter, multifunctional envelope-type nano device for mitochondrial delivery toward innovative nano medicine

  Yuma Yamada, Hideyoshi Harashima

  Journal of the Society of Powder Technology, Japan 54 (3) 158 - 166 1883-7239 2017 [Refereed][Not invited]
   

  A single cell contains a variety of organelles, including a nucleus, mitochondria, the golgi apparatus and others. If it were possible to prepare a nano craft that could specifically target a specific organelle, it would open a new field of research directed toward therapeutic treatments for a variety of diseases. We recently developed a new concept that we refer to as “Programmed Packaging”, by which we succeeded in creating a multifunctional envelope-type nano device (MEND) as a non-viral vector. Our attempts to target mitochondria are discussed here, mainly focusing on a MITO-Porter, a MEND for delivering cargoes to mitochondria. A variety of human diseases, including various neurodegenerative disorders, ischemic heart disease, diabetes and cancer have been reported to be associated with mitochondrial dysfunction. Because of this, mitochondrial therapy would be expected to be useful and productive in the treatment of such diseases. Our findings regarding mitochondrial drug delivery systems that are directed toward mitochondrial nano medicine development are summrized herein.
• 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][Not 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.
• MITO-porter for mitochondrial delivery and mitochondrial functional analysis

  Yuma Yamada, Hideyoshi Harashima

  Handbook of Experimental Pharmacology 240 457 - 472 1865-0325 2017 [Refereed][Not invited]
   

  Mitochondria are attractive organelles that have the potential to contribute greatly to medical therapy, the maintenance of beauty and health, and the development of the life sciences. Therefore, it would be expected that the further development of mitochondrial drug delivery systems (DDSs) would exert a significant impact on the medical and life sciences. To achieve such an innovative objective, it will be necessary to deliver various cargoes to mitochondria in living cells. However, only a limited number of approaches are available for accomplishing this. We recently proposed a new concept for mitochondrial delivery, a MITO-Porter, a liposome-based carrier that introduces macromolecular cargoes into mitochondria via membrane fusion. To date, we have demonstrated the utility of mitochondrial therapeutic strategy by MITOPorter using animal models of diseases. We also showed that the mitochondrial delivery of antisense oligo-RNA by the MITO-Porter results in mitochondrial RNA knockdown and has a functional impact on mitochondria. Here, we summarize the current state of mitochondrial DDS focusing on our research and show some examples of mitochondrial functional regulations using mitochondrial DDS.
• 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][Not 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.
• ミトコンドリア薬物送達システムを用いた心筋炎に対する新たな治療戦略

  阿部 二郎, 山田 勇磨, 武田 充人, 原島 秀吉

  日本小児循環器学会雑誌 (NPO)日本小児循環器学会 32 (Suppl.1) s1 - 208 0911-1794 2016/07 [Refereed][Not invited]
  
• A Dual-Ligand Liposomal System Composed of a Cell-Penetrating Peptide and a Mitochondrial RNA Aptamer Synergistically Facilitates Cellular Uptake and Mitochondrial Targeting

  Yuma Yamada, Ryo Furukawa, Hideyoshi Harashima

  JOURNAL OF PHARMACEUTICAL SCIENCES 105 (5) 1705 - 1713 0022-3549 2016/05 [Refereed][Not invited]
   

  It has been reported that the use of mitochondrial RNA aptamers including RNase P (RP) results in the selective mitochondrial delivery of endogenous and exogenous RNAs. The issue of whether these aptamers would be useful ligands for the mitochondrial targeting of a nanoparticle has not been demonstrated to date because nanocarriers modified with these RNA aptamers are insufficiently internalized by cells. We report here on the development of a dual-ligand liposomal system composed of octaarginine (R8), a device that enhances cellular uptake, and an RP aptamer for mitochondrial targeting to permit a nanocarrier to be efficiently delivered to mitochondria. Surprisingly, the cellular uptake of the R8-modified nanocarrier was facilitated by modification with an RP aptamer. The optimal composition of a nanocarrier needed for efficient cellular uptake and mitochondrial targeting was determined. In a confocal laser scanning microscopy analysis, the dual-ligandemodified nanocarrier was found to result in effective mitochondrial targeting through an ATP-dependent pathway and was much more effective than a single-ligand R8-modified nanocarrier. This is the first report of the regulation of intracellular trafficking by a mitochondrial RNA aptamer-modified nanocarrier system. (C) 2016 American Pharmacists Association (R). Published by Elsevier Inc. All rights reserved.
• Validation of a Strategy for Cancer Therapy: Delivering Aminoglycoside Drugs to Mitochondria in HeLa Cells

  Jiro Abe, Yuma Yamada, Hideyoshi Harashima

  JOURNAL OF PHARMACEUTICAL SCIENCES 105 (2) 734 - 740 0022-3549 2016/02 [Refereed][Not invited]
   

  Mitochondria in human cancer cells have been implicated in cancer cell proliferation, invasion, metastasis, and even drug-resistance mechanisms, making them a potential target organelle for the treatment of human malignancies. Gentamicin (GM), an aminoglycoside drug (AG), is a small molecule that functions as an antibiotic and has ototoxic and nephrotoxic characteristics. Thus, the delivery of GM to mitochondria in cancer cells would be an innovative anticancer therapeutic strategy. In this study, we attempted mitochondrial delivery of GM in HeLa cells derived from a human cervical cancer. For the mitochondrial delivery, we used MITO-Porter, a liposomal nanocarrier for mitochondrial delivery via membrane fusion. We first encapsulated GM in the aqueous phase of the carrier to construct GM-MITO-Porter. Flow cytometry analysis and fluorescent microscopy observations permitted us to confirm that the GMeMITO-Porter was efficiently taken up by HeLa cells and accumulated in mitochondria, whereas naked GM was not taken up by the cells. Moreover, cell viability assays using HeLa cells showed that the GMeMITO-Porter induced strong cytotoxic effects related to mitochondrial disorder. This finding is the first report of the mitochondrial delivery of an AG to cancer cells for cancer therapeutic strategy. (C) 2016 American Pharmacists Association (R). Published by Elsevier Inc. All rights reserved.
• Mitochondrial DDS Opens Innovative Pharmaceutics

  Yuma Yamada

  YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 136 (1) 55 - 62 0031-6903 2016/01 [Refereed][Not invited]
   

  A variety of human diseases, including neurodegenerative disorders, ischemic heart disease, diabetes, and cancer have been reported to be associated with mitochondrial dysfunction. Because of this, mitochondrial therapy is expected to be useful and productive in the treatment of such diseases. We previously reported on the development of a MITO-Porter, a liposome-based nanocarrier that permits macromolecular cargos to be delivered into mitochondria via membrane fusion. Intracellular observations using the green fluorescence protein as a model macromolecule provided confirmation that a macromolecule could be delivered to mitochondria in living cells by the MITO-Porter. Here, we present our current findings on the development of mitochondrial medicine and mitochondrial gene therapy based on our mitochondrial drug delivery system (DDS). In this review, we propose "mitochondrial DDS" as a theme for "DDS research for innovative drug development" and discuss the contribution of mitochondrial DDS to innovative drug development.
• Mitochondrial dds has a significant impact on the medical and life sciences: The 7th young researcher’s award in the 31th annual meeting of the japan society of drug delivery system

  Yuma Yamada

  Drug Delivery System 30 (4) 348 - 362 1881-2732 2015/12/25 [Refereed][Not invited]
  
• Validation of Mitochondrial Gene Delivery in Liver and Skeletal Muscle via Hydrodynamic Injection Using an Artificial Mitochondrial Reporter DNA Vector

  Yukari Yasuzaki, Yuma Yamada, Takuya Ishikawa, Hideyoshi Harashima

  MOLECULAR PHARMACEUTICS 12 (12) 4311 - 4320 1543-8384 2015/12 [Refereed][Not invited]
   

  For successful mitochondrial transgene expression, two independent processes, i.e., developing a mitochondrial gene delivery system and construction of DNA vector to achieve mitochondrial gene expression, are required. To date, very few studies dealing with mitochondrial gene delivery have been reported and, in most cases, transgene expression was not validated, because the construction of a reporter DNA vector for mitochondrial gene expression is the bottleneck. In this study, mitochondrial transgene expression by the in vivo mitochondrial gene delivery of an artificial mitochondrial reporter DNA vector via hydrodynamic injection is demonstrated. In the procedure, a large volume of naked plasmid DNA (pDNA) is rapidly injected. We designed and constructed pHSP-mtLuc (CGG) as a mitochondrial reporter DNA vector that possesses a mitochondrial heavy strand promoter (HSP) and an artificial mitochondrial genome with the reporter NanoLuc (Nluc) luciferase gene that records adjustments to the mitochondrial codon system. We delivered the pDNA into mouse liver mitochondria by hydrodynamic injection, and detected exogenous mRNA in the liver using reverse transcription PCR analysis. The hydrodynamic injection of pHSP-mtLuc (CGG) resulted in the expression of the Nluc luciferase protein in liver and skeletal muscle. Our mitochondrial transgene expression reporter system would contribute to mitochondrial gene therapy and further studies directed at mitochondrial molecular biology.
• Mitochondrial delivery of Coenzyme Q(10) via systemic administration using a MITO-Porter prevents ischemia/reperfusion injury in the mouse liver

  Yuma Yamada, Kohei Nakamura, Jiro Abe, Mamoru Hyodo, Sanae Haga, Michitaka Ozaki, Hideyoshi Harashima

  JOURNAL OF CONTROLLED RELEASE 213 86 - 95 0168-3659 2015/09 [Refereed][Not invited]
   

  We herein report on a mitochondrial therapeutic effect based on the delivery of coenzyme Q(10) (CoQ(10)), an anti-oxidant, to in vivo mitochondria using a MITO-Porter, a liposome-based mitochondrial delivery system that functions via membrane fusion. To evaluate the effects, we used a mouse liver ischemia/reperfusion injury (I/R injury) model, in which mitochondrial reactive oxygen species are overexpressed. We packaged CoQ(10) in the lipid phase of a MITO-Porter and optimized the mitochondrial fusogenic activities to produce the CoQ(10)-MITO-Porter. A histological observation of the carriers in the liver by confocal laser scanning microscopy was done and the accumulation of the carrier labeled with a radio isotope in the liver confirmed that the CoQ(10)-MITO-Porter was delivered to liver mitochondria via systemic injection. These analytical results permitted us to optimize the compositions of the CoQ(10)-MITO-Porter so as to permit it to efficiently accumulate in mouse liver mitochondria. Finally, we applied the optimized CoQ(10) -MITO-Porter to mice via tail vein injection, and hepatic I/R injury was then induced, followed by measuring serum alanine aminotransferase (ALT) levels, a marker of liver injury. We confirmed that the use of the CoQ(10)-MITO-Porter resulted in a significant decrease in serum ALT levels, indicating that in vivo mitochondrial delivery of the CoQ(10) via MITO-Porter prevents I/R injury in mice livers. This provides a demonstration of the potential use of such a delivery system in mitochondrial therapies. (C) 2015 Elsevier B.V. All rights reserved.
• An analysis of membrane fusion between mitochondrial double membranes and MITO-Porter, mitochondrial fusogenic vesicles

  Yuma Yamada, Yutaka Fukuda, Hideyoshi Harashima

  MITOCHONDRION 24 50 - 55 1567-7249 2015/09 [Refereed][Not invited]
   

  To achieve mitochondrial gene therapy, therapeutic molecules need to be transported through the outer and inner membranes of mitochondria into the innermost space (mitochondrial matrix), which contains the mtDNA pool. We previously reported on the construction of a MITO-Porter with a high fusogenic activity for the mitochondrial outer membrane for delivering molecules to the mitochondria of human cells. Here, we report on an investigation of a fusogenic lipid composition for the inner membrane, and an analysis of the fusogenic compositions for the outer and inner membranes. A significant relationship was found between fusion activity and the mitochondrial delivery of nucleic acids. (C) 2015 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
• Efficient and High-Speed Transduction of an Antibody into Living Cells Using a Multifunctional Nanocarrier System to Control Intracellular Trafficking

  Yuma Yamada, Sandra Milena Vergara Perez, Mai Tabata, Jiro Abe, Yukari Yasuzaki, Hideyoshi Harashima

  JOURNAL OF PHARMACEUTICAL SCIENCES 104 (9) 2845 - 2854 0022-3549 2015/09 [Refereed][Not invited]
   

  The transduction of antibodies into living cells would represent a major contribution to both basic and applied biomedical fields, as currently available methods suffer from limitations such as low-uptake efficiency and endosomal entrapment. In this study, a liposome-based carrier was designed to overcome these issues. Liposomes were modified with octaarginine (R8), a cell penetrating peptide and GALA, a pH-sensitive fusogenic peptide. The presence of R8 enhanced the cellular uptake of antibodies, whereas GALA reduced endosomal entrapment, resulting in antibodies being released into the cytosol within 30 min. Moreover, compared with commercially available reagents for delivering antibodies, our system was superior in both cellular uptake and endosomal escape. In addition, specific antibodies delivered by R8-GALA liposomes were found to be associated with their epitope, confirming the preservation of functionality. This system for the efficient and high-speed cytosolic delivery of an antibody provides a valuable tool that can be useful in basic and applied research for analyzing the expression and function of intracellular molecules. (c) 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:2845-2854, 2015
• Mitochondrial delivery of antisense RNA by MITO-Porter results in mitochondrial RNA knockdown, and has a functional impact on mitochondria

  Ryo Furukawa, Yuma Yamada, Eriko Kawamura, Hideyoshi Harashima

  BIOMATERIALS 57 107 - 115 0142-9612 2015/07 [Refereed][Not invited]
   

  Mitochondrial genome-targeting nucleic acids are promising therapeutic candidates for treating mitochondrial diseases. To date, a number of systems for delivering genetic information to the cytosol and the nucleus have been reported, and several successful gene therapies involving gene delivery targeted to the cytosol and the nucleus have been reported. However, much less progress has been made concerning mitochondrial gene delivery systems, and mitochondrial gene therapy has never been achieved. Here, we report on the mitochondrial delivery of an antisense RNA oligonucleotide (ASO) to perform mitochondrial RNA knockdown to regulate mitochondrial function. Mitochondrial delivery of the ASO was achieved using a combination of a MITO-Porter system, which contains mitochondrial fusogenic lipid envelopes for mitochondrial delivery via membrane fusion and D-arm, a mitochondrial import signal of tRNA to the matrix. Mitochondrial delivery of the ASO induces the knockdown of the targeted mitochondria-encoded mRNA and protein, namely cytochrome c oxidase subunit II, a component of the mitochondrial respiratory chain. Furthermore, the mitochondrial membrane potential was depolarized by the down regulation of the respiratory chain as the result of the mitochondrial delivery of ASO. This finding constitutes the first report to demonstrate that the nanocarrier-mediated mitochondrial genome targeting of antisense RNA effects mitochondrial function. (C) 2015 Elsevier Ltd. All rights reserved.
• Hyaluronic acid controls the uptake pathway and intracellular trafficking of an octaarginine-modified gene vector in CD44 positive- and CD44 negative-cells

  Yuma Yamada, Masahiro Hashida, Hideyoshi Harashima

  BIOMATERIALS 52 189 - 198 0142-9612 2015/06 [Refereed][Not invited]
   

  The cellular uptake pathway for a gene vector is an important factor in transgene expression. We previously constructed an original gene vector, multifunctional envelope-type nano device (MEND). The use of octaarginine (R8), a cell-penetrating peptide dramatically enhanced the transfection activity of the MEND since efficient cellular uptake via macropinocytosis, while the R8 should overcome its poor cell selectivity. Here we prepared an R8-MEND equipped with GALA (a peptide for endosomal escape) (R8/ GALA-MEND) coated with hyaluronic acid (HA) (HA-R8/GALA-MEND), a natural ligand for cancer cells overexpressing CD44. We investigated the cellular uptake pathway of the HA-R8/GALA-MEND and the R8/GALA-MEND using HCT116 cells overexpressing CD44. Both carriers were taken up by cells mainly via macropinocytosis, whereas only the HA-R8/GALA-MEND was partially internalized into cells via a CD44-mediated pathway. Investigation of transgene expression showed that the HA-R8/GALA-MEND had a high transfection activity in HCT116 cells via both macropinocytotic and CD44-mediated pathways. On the other hand, the value for the HA-R8/GALA-MEND was significantly decreased compared with the value for the R8/GALA-MEND in NIH3T3 cells (CD44-negative cells). These findings indicate that the HA-coating controls the intracellular pathway for R8-modified nanocarriers, and that a CD44-mediated pathway is an important route for transgene expression. (C) 2015 Elsevier Ltd. All rights reserved.
• Targeting the mitochondrial genome via a dual function MITO-porter: Evaluation of mtDNA levels and mitochondrial function

  Yuma Yamada, Hideyoshi Harashima

  Mitochondrial Medicine 2 123 - 133 1064-3745 2015/01/29 [Refereed][Not invited]
   

  Genetic mutations and defects in mitochondrial DNA (mtDNA) are associated with certain types of mitochondrial dysfunction, ultimately resulting in the occurrence of a variety of human diseases. For an effective mitochondrial gene therapy, it will be necessary to deliver therapeutic agents to the innermost mitochondrial space (the mitochondrial matrix), which contains the mtDNA pool. We recently developed a MITO-Porter, a liposome-based nano-carrier that delivers cargo to mitochondria via a membrane-fusion mechanism. Using propidium iodide, as a probe to detect mtDNA, we were able to confirm that the MITO-Porter delivered cargoes to mitochondrial matrices in living cells. More recently, we constructed a Dual Function (DF)-MITO-Porter, a liposome-based nanocarrier for mitochondrial delivery via a stepwise process. In this chapter, we describe the methodology used to deliver bioactive molecules to the mitochondrial matrix using the above DF-MITO-Porter, and the evaluation of mtDNA levels and mitochondrial activities in living cells.
• Development of a New In Vivo Optical Probe for Biological Diagnosis and Therapy

  Ozaki M, Ozawa T, Yamada Y, Nakao K, Minato N, Uemoto S

  2015 [Refereed][Not invited]
  
• A Post-Liver Transplant Girl With Recurrent Cramps in the Legs.

  Abe J, Yamada Y, Suzuki T, Shimamura T, Kobayashi I

  Global pediatric health 2 2333794X14566074  2015 [Refereed][Not invited]
  
• p62/SQSTM1 Plays a Protective Role in Oxidative Injury of Steatotic Liver in a Mouse Hepatectomy Model

  Sanae Haga, Takeaki Ozawa, Yuma Yamada, Naoki Morita, Izuru Nagashima, Hiroshi Inoue, Yuka Inaba, Natsumi Noda, Riichiro Abe, Kazuo Umezawa, Michitaka Ozaki

  ANTIOXIDANTS & REDOX SIGNALING 21 (18) 2515 - 2530 1523-0864 2014/12 [Refereed][Not invited]
   

  Aims: Liver injury and regeneration involve complicated processes and are affected by various physio-pathological factors. We investigated the mechanisms of steatosis-associated liver injury and delayed regeneration in a mouse model of partial hepatectomy. Results: Initial regeneration of the steatotic liver was significantly delayed after hepatectomy. Although hepatocyte proliferation was not significantly suppressed, severe liver injury with oxidative stress (OS) occurred immediately after hepatectomy in the steatotic liver. Fas-ligand (FasL)/Fas expression was upregulated in the steatotic liver, whereas the expression of antioxidant and anti-apoptotic molecules (catalase/MnSOD/Ref-1 and Bcl-2/Bcl-xL/FLIP, respectively) and p62/SQSTM1, a steatosis-associated protein, was downregulated. Interestingly, pro-survival Akt was not activated in response to hepatectomy, although it was sufficiently expressed even before hepatectomy. Suppression of p62/SQSTM1 increased FasL/Fas expression and reduced nuclear factor erythroid 2-related factor-2 (Nrf-2)-dependent antioxidant response elements activity and antioxidant responses in steatotic and nonsteatotic hepatocytes. Exogenously added FasL induced severe cellular OS and necrosis/apoptosis in steatotic hepatocytes, with only the necrosis being inhibited by pretreatment with antioxidants, suggesting that FasL/Fas-induced OS mainly leads to necrosis. Furthermore, p62/SQSTM1 re-expression in the steatotic liver markedly reduced liver injury and improved liver regeneration. Innovation: This study is the first which demonstrates that reduced expression of p62/SQSTM1 plays a crucial role in posthepatectomy acute injury and delayed regeneration of steatotic liver, mainly via redox-dependent mechanisms. Conclusion: In the steatotic liver, reduced expression of p62/SQSTM1 induced FasL/Fas overexpression and suppressed antioxidant genes, mainly through Nrf-2 inactivation, which, along with the hypo-responsiveness of Akt, caused posthepatectomy necrotic/apoptotic liver injury and delayed regeneration, both mainly via a redox-dependent mechanism. Antioxid. Redox Signal. 21, 2515-2530.
• Development of the MITO-Porter, a Nano Device for Mitochondrial Drug Delivery via Membrane Fusion

  Yuma Yamada

  YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 134 (11) 1143 - 1155 0031-6903 2014/11 [Refereed][Not invited]
   

  Many human diseases have been reported to be associated with mitochondrial dysfunction. Therefore, mitochondrial therapy would be expected to be useful and productive in the treatment of various diseases. To achieve such an innovative therapy, it will be necessary to deliver therapeutic agents into mitochondria. However, only a limited number of methods are available for accomplishing this. We previously developed the MITO-Porter, a liposome-based carrier that permits macromolecular cargos to be transported into mitochondria via membrane fusion. Intracellular observations using the green fluorescence protein as a model macromolecule confirmed the mitochondrial delivery of a macromolecule by the MITO-Porter. Moreover, when we attempted the mitochondrial delivery of bongkrekic acid (BKA), an antiapoptosis agent, the MITO-Porter enhanced the antiapoptosis effect compared with naked BKA. To construct a device with enhanced performance, the MITO-Porter was coated with cell membrane-fusogenic outer envelopes to produce the dual function (DF) -MITO-Porter. Intracellular observations indicated that the DF-MITO-Porter was more effective in delivering exogenous macromolecules into mitochondria than the conventional MITO-Porter. Furthermore, when biomacromolecules were delivered using the DF-MITO-Porter to estimate the mitochondrial gene targeting of the carrier, the results confirmed that the MITO-Porter system has the potential for use in therapies aimed at mitochondrial DNA. This paper sumarizes our findings on mitochondrial drug delivery systems that are directed toward mitochondrial medicine development and mitochondrial gene therapy. It is expected that the MITO-Porter system will open new research areas in mitochondrial drug delivery systems and have a significant impact on the medical and life sciences.
• 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][Not 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.
• Condensation of plasmid DNA enhances mitochondrial association in skeletal muscle following hydrodynamic limb vein injection

  Yukari Yasuzaki, Yuma Yamada, Yutaka Fukuda, Hideyoshi Harashima

  Pharmaceuticals 7 (8) 881 - 893 1424-8247 2014/08/21 [Refereed][Not invited]
   

  Mitochondrial gene therapy and diagnosis have the potential to provide substantial medical benefits. However, the utility of this approach has not yet been realized because the technology available for mitochondrial gene delivery continues to be a bottleneck. We previously reported on mitochondrial gene delivery in skeletal muscle using hydrodynamic limb vein (HLV) injection. HLV injection, a useful method for nuclear transgene expression, involves the rapid injection of a large volume of naked plasmid DNA (pDNA). Moreover, the use of a condensed form of pDNA enhances the nuclear transgene expression by the HLV injection. The purpose of this study was to compare naked pDNA and condensed pDNA for mitochondrial association in skeletal muscle, when used in conjunction with HLV injection. PCR analysis showed that the use of condensed pDNA rather than naked pDNA resulted in a more effective mitochondrial association with pDNA, suggesting that the physicochemical state of pDNA plays a key role. Moreover, no mitochondrial toxicities in skeletal muscle following the HLV injection of condensed pDNA were confirmed, as evidenced by cytochrome c oxidase activity and mitochondrial membrane potential. These findings have the potential to contribute to the development for in vivo mitochondrial gene delivery system. © 2014 by the authors licensee MDPI, Basel, Switzerland.
• A nanocarrier system for the delivery of nucleic acids targeted to a pancreatic beta cell line

  Yuma Yamada, Mai Tabata, Yukari Yasuzaki, Masatoshi Nomura, Atsushi Shibata, Yuta Ibayashi, Yosuke Taniguchi, Shigeki Sasaki, Hideyoshi Harashima

  BIOMATERIALS 35 (24) 6430 - 6438 0142-9612 2014/08 [Refereed][Not invited]
   

  Pancreatic beta cells secrete insulin in response to glucose levels and thus are involved in controlling blood glucose levels. A line of pancreatic beta cells "MIN6" has been used in studies related to the function of beta cells and diabetes therapy. Regulating gene expression in MIN6 cells could accelerate these studies, but an efficient method for the transfection of nucleic acids targeted to MINE cells is required. We report here on a liposome-based carrier targeted to pancreatic beta cells (Multifunctional envelope-type nano device for pancreatic beta cells, beta-MEND). We identified a lipid composition for use in preparing the beta-MEND, which permits the particles to be efficiently internalized into MIN6, as evidenced by flow cytometry analyses. Intracellular observation by confocal laser scanning microscopy showed that the beta-MEND efficiently delivered the oligo nucleic acids to the cytosol of MINE cells. Moreover, using a beta-MEND encapsulating a 2'-O-Methyl RNA complementary to a microRNA that suppresses insulin secretion, the knockdown of the targeted microRNA and an up-regulation of insulin secretion were observed in MINE. Thus, the beta-MEND holds promise as an efficient system for delivering nucleic acids targeted to MIN6 and can contribute to research and therapy aimed at diabetes. (C) 2014 Elsevier Ltd. All rights reserved.
• RNA Aptamers for Targeting Mitochondria Using a Mitochondria-Based SELEX Method

  Yuri Tawaraya, Mamoru Hyodo, Mst Naznin Ara, Yuma Yamada, Hideyoshi Harashima

  BIOLOGICAL & PHARMACEUTICAL BULLETIN 37 (8) 1411 - 1415 0918-6158 2014/08 [Refereed][Not invited]
   

  The use of mitochondria-based systematic evolution of ligands by exponential enrichment (SELEX) was explored. Mitochondria were isolated from rat liver and confirmed intact by respiratory control index. Isolated mitochondria and a 2'-F RNA random library were mixed and the bound RNAs collected. The counter selection was applied with nucleus and unbound RNAs were collected. After 7 rounds of selection, two sequences (Mitomer1 and Mitomer2) were verified to bind to mitochondria and the truncated Mitomer2 (short Mitomer2) showed better binding to isolated mitochondria than Mitomer1.
• Mono-cationic detergents play a critical role in the development of liposome-based gene vector via controlling its lamellarity

  Ryosuke Suzuki, Yuma Yamada, Eriko Kawamura, Hideyoshi Harashima

  JOURNAL OF NANOPARTICLE RESEARCH 16 (8) 1388-0764 2014/07 [Refereed][Not invited]
   

  Controlling the number of lipid bilayers, the lamellarity, in a liposome is a major factor in the in vivo/in vitro pharmacokinetics of drug delivery using liposome-based nanocarriers. Findings reported in a previous study indicated that a mono-cationic detergent (MCD) could be useful in controlling liposomal size via interaction with the lipid envelope. Here, we investigated controlling the lamellarity of the liposomal gene vector by MCD, using a multifunctional envelope-type nano device (MEND). The MEND consisted of a condensed plasmid DNA core and lipid envelopes. The size of the MCD-contained MEND (MCD-MEND) decreased as a function of the amount of MCD, indicating that MCD can be used to control the number of the lipid bilayers. We also developed a triple-layered MEND (TL-MEND) by packaging a di-lamellar MEND into an MCD-containing lipid bilayer. We hypothesized that the TL-MEND would efficiently deliver a gene to the nucleus, when the outer single bilayer fused with the plasma membrane and the inner double membranes then fused with the nuclear double membranes. Transfection assays showed that the TL-MEND had a high transfection activity in JAWS II cells, non-dividing cells. These results indicate that MCD has the potential for enhancing the gene delivery by controlling liposomal lamellarity.
• 細胞・臓器移植における基礎的研究の最前線 光を応用した移植細胞機能・細胞環境のモニタリングと制御の試み

  尾崎 倫孝, 芳賀 早苗, 野田 なつみ, 森田 直樹, 山田 勇磨, 小澤 岳昌

  日本外科学会雑誌 (一社)日本外科学会 115 (臨増2) 168 - 168 0301-4894 2014/03
• A method for screening mitochondrial fusogenic envelopes for use in mitochondrial drug delivery

  Yuma Yamada, Hideyoshi Harashima

  Methods in Molecular Biology 1141 57 - 66 1064-3745 2014 [Refereed][Not invited]
   

  Various types of mitochondrial dysfunctions have been implicated in a variety of human diseases. This suggests that mitochondria would be promising therapeutic drug targets and mitochondrial therapy would be expected to be useful for the treatment of various diseases. We have already reported the development of a MITO-Porter, a liposome-based nano-carrier that delivers its cargo to mitochondria via a membrane-fusion mechanism. In our strategy for delivering cargos to mitochondria using a MITO-Porter, the carriers must fuse with the organelle membrane. Here we report on methodology for screening various types of lipid envelopes that have the potential for fusing with a mitochondrial membrane. The method involves monitoring the cancellation of fluorescence resonance energy transfer (FRET) and evaluating membrane fusion between the carriers and mitochondria in living cells by FRET analysis using a spectral imaging fluorescent microscopy system. © 2014 Springer Science+Business Media, New York.
• 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.
• Intracellular observation of nanocarriers modified with a mitochondrial targeting signal peptide

  Eriko Kawamura, Yuma Yamada, Yukari Yasuzaki, Mamoru Hyodo, Hideyoshi Harashima

  JOURNAL OF BIOSCIENCE AND BIOENGINEERING 116 (5) 634 - 637 1389-1723 2013/11 [Refereed][Not invited]
   

  This study focused on the intracellular observation of nanocarriers modified with a mitochondrial targeting signal peptide (MTS). The nanocarriers showed an efficient cellular uptake, and the MTS had a positive effect on their mitochondrial targeting. This is the first report of an intracellular observation of nanocarriers modified with MTS. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.
• Mitochondrial targeting functional peptides as potential devices for the mitochondrial delivery of a DF-MITO-Porter

  Eriko Kawamura, Yuma Yamada, Hideyoshi Harashima

  MITOCHONDRION 13 (6) 610 - 614 1567-7249 2013/11 [Refereed][Not invited]
   

  To achieve mitochondrial therapy, we previously reported on the use of an octaarginine (R8) modified Dual Function (DF)-MITO-Porter for delivering molecules to mitochondria in living cells. In this study, using isolated mitochondria, homogenates and living cells, we evaluated the utility of mitochondrial targeting functional peptides as a ligand for delivering carriers. The S2 peptide modified carrier showed a high mitochondrial targeting activity in homogenates and living cells. In addition, the S2 peptide had a lower cell toxicity compared to R8 modified liposomes. The S2 peptide represents a potentially useful moiety for constructing an efficient and safe mitochondrial delivery system. (C) 2013 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
• Investigation of siRNA Nanoparticle Formation Using Mono-Cationic Detergents and Its Use in Gene Silencing in Human HeLa Cells.

  Yamada Y, Suzuki R, Harashima H

  Cancers 5 (4) 1413 - 1425 2013/11 [Refereed][Not invited]
  
• Enhancement in selective mitochondrial association by direct modification of a mitochondrial targeting signal peptide on a liposomal based nanocarrier

  Yuma Yamada, Hideyoshi Harashima

  Mitochondrion 13 (5) 526 - 532 1567-7249 2013/09 [Refereed][Not invited]
   

  The focus of this study was on the development of a nano carrier targeted to mitochondria, a promising therapeutic drug target. We synthesized a lipid derivative that is conjugated with a mitochondrial targeting signal peptide (MTS), which permits the selective delivery of certain types of proteins to mitochondria. We then explored the use of an innovative technology in which MTS and the MITO-Porter were integrated. The latter is a liposome that delivers cargos to mitochondria via membrane fusion. The results indicate that the combination of MTS and the MITO-Porter would be useful for selective mitochondrial delivery via membrane fusion. © 2012 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
• An Approach to Transgene Expression in Liver Endothelial Cells Using a Liposome-Based Gene Vector Coated with Hyaluronic Acid

  Yuma Yamada, Masahiro Hashida, Yasuhiro Hayashi, Mai Tabata, Mamoru Hyodo, Mst Naznin Ara, Noritaka Ohga, Kyoko Hida, Hideyoshi Harashima

  JOURNAL OF PHARMACEUTICAL SCIENCES 102 (9) 3119 - 3127 0022-3549 2013/09 [Refereed][Not invited]
   

  Dysfunctional sinusoidal liver endothelial cells (LECs) are associated with liver diseases, such as liver fibrosis, cirrhosis, and portal hypertension. Because of this, gene therapy targeted to LECs would be a useful and productive strategy for directly treating these diseases at the level of genes. Here, we report on the development of a transgene vector that specifically targets LECs. The vector is a liposome-based gene vector coated with hyaluronic acid (HA). HA is a natural ligand for LECs and confers desirable properties on particles, rendering them biodegradable, biocompatible, and nonimmunogenic. In this study, we constructed HA-modified carriers, and evaluated cellular uptake and transfection activity using cultured LECs from KSN nude mice (KSN-LECs). Cellular uptake analyses showed that KSN-LECs recognized the HA-modified carriers more effectively than skin endothelial cells. The transfection assay indicated that the efficient gene expression in KSN-LECs, using the HA-modified carriers, required an adequate lipid composition and a functional device to control intracellular trafficking. This finding contributes to our overall knowledge of transgene expression targeted to LECs. (C) 2013 Wiley Periodicals, Inc. and the American Pharmacists Association
• Lipid Envelope-Type Nanoparticle Incorporating a Multifunctional Peptide for Systemic siRNA Delivery to the Pulmonary Endothelium

  Kenji Kusumoto, Hidetaka Akita, Taichi Ishitsuka, Yu Matsumoto, Takahiro Nomoto, Ryo Furukawa, Ayman El-Sayed, Hiroto Hatakeyama, Kazuaki Kajimoto, Yuma Yamada, Kazunori Kataoka, Hideyoshi Harashima

  ACS NANO 7 (9) 7534 - 7541 1936-0851 2013/09 [Refereed][Not invited]
   

  A system that permits the delivery of cargoes to the lung endothelium would be extraordinarily useful in terms of curing a wide variety of lung-related diseases. This study describes the development of a multifunctional envelope-type nanodevice (MEND) that targets the lung endothelium, delivers its encapsulated siRNA to the cytoplasm, and eradicates lung metastasis. The key to the success can be attributed to the presence of a surface-modified GALA peptide that has dual functions: targeting the sialic acid-terminated sugar chains on the pulmonary endothelium and subsequently delivering the encapsulated cargoes to the cytosol via endosomal membrane fusion, analogous to the influenza virus. The active targeting of MENDs without the formation of large aggregates was verified by intravital real-time confocal laser scanning microscopy in living lung tissue. The GALA-modified MEND is a promising carrier that opens a new generation of therapeutic approaches for satisfying unmet medical needs in curing lung diseases.
• Mitochondrial delivery of bongkrekic acid using a MITO-porter prevents the induction of apoptosis in human hela cells

  Yuma Yamada, Kohei Nakamura, Ryo Furukawa, Eriko Kawamura, Takuya Moriwaki, Kenji Matsumoto, Katsuhiro Okuda, Mitsuru Shindo, Hideyoshi Harashima

  JOURNAL OF PHARMACEUTICAL SCIENCES 102 (3) 1008 - 1015 0022-3549 2013/03 [Refereed][Not invited]
   

  The fact that mitochondrial dysfunction has been implicated in a variety of human diseases suggests that they would be expected as a target organelle for these diseases. Bongkrekic acid (BKA) is a chemical that functions as a ligand of the adenine nucleotide translocator and is known to potently inhibit the mitochondrial permeability transition that is associated with apoptosis. Thus, delivering it to mitochondria would be an innovative therapy for the treatment of mitochondrial diseases that are largely associated with apoptosis. Here, we report on the use of a MITO-Porter, an innovative nanocarrier for mitochondrial delivery via mitochondrial membrane fusion, for delivering BKA to mitochondria. We first constructed a BKAMITO-Porter, in which BKA is contained in lipid envelopes of a MITO-Porter. We then confirmed that the BKAMITO-Porter was efficiently internalized into cells and is delivered to mitochondria, similar to a conventional MITO-Porter. Moreover, we evaluated the antiapoptosis effect of the BKAMITO-Porter in HeLa cells by measuring caspase 3/7 activity. The findings confirmed that the BKAMITO-Porter showed a strong antiapoptosis effect compared with naked BKA. The results reported here demonstrate its potential for the use in therapies aimed at mitochondrial diseases, as a mitochondrial medicine candidate. (c) 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:10081015, 2013
• Localization of exogenous DNA to mitochondria in skeletal muscle following hydrodynamic limb vein injection

  Yukari Yasuzaki, Yuma Yamada, Tsutomu Kanefuji, Hideyoshi Harashima

  Journal of Controlled Release 172 (3) 805 - 811 0168-3659 2013 [Refereed][Not invited]
   

  Mitochondrial genetic disorders are a major cause of mitochondrial diseases. It is therefore likely that mitochondrial gene therapy will be useful for the treatment of such diseases. Here, we report on the possibility of mitochondrial gene delivery in skeletal muscle using hydrodynamic limb vein (HLV) injection. The HLV injection procedure, a useful method for transgene expression in skeletal muscle, involves the rapid injection of a large volume of naked plasmid DNA (pDNA) into the distal vein of a limb. We hypothesized that the technique could be used to deliver pDNA not only to nuclei but also to mitochondria, since cytosolic pDNA that is internalized by the method may be able to overcome mitochondrial membrane. We determined if pDNA could be delivered to myofibrillar mitochondria by HLV injection by PCR analysis. Mitochondrial toxicity assays showed that the HLV injection had no influence on mitochondrial function. These findings indicate that HLV injection promises to be a useful technique for in vivo mitochondrial gene delivery. © 2013 Elsevier B.V.
• MITO-Porter; A Cutting-edge Technology for Mitochondrial Gene Therapy

  Ryo Furukawa, Yuma Yamada, Hideyoshi Harashima

  YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 132 (12) 1389 - 1398 0031-6903 2012/12 [Refereed][Not invited]
   

  Gene therapy is an attractive strategy, for not only targeting nuclear genome, but the mitochondrial genome as well. Human mitochondrial DNA (mtDNA) encodes 13 subunits of the electron transport chain, 22 tRNAs, and 2 rRNAs and their mutations cause a wide range of mitochondrial diseases. Each cell contains hundreds to thousands of mtDNAs, and in the case of a diseased cell, the mitochondrion possesses both mutant mtDNA and wild-type mtDNA. It is generally accepted that the disease phenotype appears when the proportion of the pathogenic mutant mtDNA exceeds a certain threshold. Therefore, the suppression of mutant mtDNA or supplementing wild-type mtDNA will control the onset of mitochondrial disease. To achieve the transfection of an exogenous therapeutic gene to the mitochondrial matrix where mtDNA is transcribed and translated, it is necessary to transfer cargos through mitochondrial outer and inner membranes. Several methods have been examined for mitochondrial transfection, but a universal, wide-ranging transfection technique has yet not been established. We recently developed a mitochondrial targeting delivery system, namely the MITO-Porter. The MITO-Porter is liposomal nanocarrier with a mitochondrial fusogenic lipid composition. We reported that the MITO-Porter could deliver chemical compounds and proteins to the mitochondrial matrix via membrane fusion. In this review, we report (1) on the pharmacological enhancement of lecithinized superoxide dismutase (PC-SOD) using MITO-Porter, (2) the transcription activation of exogenous DNA by mitochondrial transcription factor A (TFAM), and (3) perspectives on a mitochondrial targeting device.
• Neuronal injury induces microglial production of macrophage inflammatory protein-1a in rat corticostriatal slice cultures

  Toshiyuki Okamura, Takahiro Katayama, Chihiro Obinata, Yukina Iso, Yuzuho Chiba, Hayato Kobayashi, Yuma Yamada, Hideyoshi Harashima, Masabumi Minami

  JOURNAL OF NEUROSCIENCE RESEARCH 11 90 (11) 2127 - 2133 0360-4012 2012/11 [Refereed][Not invited]
   

  Chemokines are potent chemoattractants for immune and hematopoietic cells. In the central nervous system, chemokines play an important role in inflammatory responses through activation of infiltrating leukocytes and/or resident glial cells. We previously demonstrated that N-methyl-D-aspartate (NMDA)-evoked neuronal injury induced astrocytic production of monocyte chemoattractant protein-1 (MCP-1, CCL2) via sustained activation of extracellular signal-regulated kinase (ERK) in rat organotypic slice cultures. In the present study, we examined mRNA expression and protein production of macrophage inflammatory protein-1a (MIP-1a, CCL3) induced by NMDA-evoked neuronal injury in the slice cultures. MIP-1a mRNA expression was transiently increased by NMDA treatment in a concentration-dependent manner. Double-fluorescence immunohistochemistry revealed that MIP-1a was produced predominantly in microglia. Depletion of microglial cells from the slice cultures by pretreatment with liposome-encapsulated clodronate abrogated the increase in MIP-1a mRNA expression after NMDA treatment. NMDA-induced MIP-1a mRNA expression was partially but significantly inhibited by the c-Jun N-terminal kinase inhibitor SP600125; conversely, the p38 mitogen-activated protein (MAP) kinase inhibitor SB203580 enhanced it. U0126, a MAP kinase/ERK kinase inhibitor, did not affect mRNA expression. These results, combined with our previous findings, demonstrate that NMDA-evoked neuronal injury differentially induces MIP-1a and MCP-1 production in microglia and astrocytes, respectively, through activation of different intracellular signaling pathways. (c) 2012 Wiley Periodicals, Inc.
• Targeting Mitochondria: Innovation from Mitochondrial Drug Delivery System (DDS) to Mitochondrial Medicine

  Yuma Yamada, Hideyoshi Harashima

  YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 132 (10) 1111 - 1118 0031-6903 2012/10 [Refereed][Not invited]
   

  Mitochondrial dysfunction has been implicated in a variety of human diseases, including cancer and neurodegenerative disorders. Effective medical therapies for such diseases will ultimately require the targeted delivery of therapeutic agents to mitochondria. This will likely be achieved through innovations in the areas of the nanotechnology of intracellular trafficking. Mitochondrial delivery systems for a variety of cargoes have been repored to date. However, only a limited number of approaches are available for delivering macromolecules directly to mitochondria. We previously reported on the construction of a MITO-Porter, a liposome-based carrier that introduces macromolecular cargos into mitochondria via membrane fusion. Using the green fluorescence protein as a model macromolecule in conjunction with analysis by confocal laser scanning microscopy, we were able to confirm the mitochondrial delivery of a macromolecule by the MITO-Porter. Moreover, we reported that the Dual Function MITO-Porter (DF-MITO-Porter) could efficiently deliver cargo to mitochondria, through endosomal and mitochondrial membranes via step-wise membrane fusion. Here, We will present our findings on the development of our mitochondrial drug delivery system, and discuss our attempts regarding mitochondrial gene delivery and therapy. Finally, We will discuss the potential use of mitochondrial drug delivery systems in mitochondrial medicine.
• Post-nuclear gene delivery events for transgene expression by biocleavable polyrotaxanes

  Yuma Yamada, Taku Nomura, Hideyoshi Harashima, Atsushi Yamashita, Nobuhiko Yui

  Biomaterials 33 (15) 3952 - 3958 0142-9612 2012/05 [Refereed][Not invited]
   

  A quantitative comparison between nuclear DNA release from carriers and their transfection activity would be highly useful for improving the effectiveness of non-viral gene vectors. We previously reported that, for condensed DNA particles, a close relationship exists between the efficiency of DNA release and transfection activity, when biocleavable polyrotaxanes (DMAE-SS-PRX), in which the cationic density can be easily controlled. In this study, we first investigated the efficiencies of DNA release from condensed DNA particles with various types of DMAE-SS-PRX. The findings indicate that an optimal cationic density in DMAE-SS-PRX exists for DNA release. We then packaged condensed DNA particles in a multifunctional envelope-type nano device (MEND), and evaluated their transfection activities. The results showed that the transfection activity was increased and this increase was, to some extent, dependent on the efficiency of the DNA release. However, transfection activity decreased, when the value for the efficiency of DNA release was higher than a certain value. An investigation of the fate of intranuclear DNA indicated that a very high efficiency of DNA release has a positive influence on transcription, however, it would inhibit the post-transcription process nuclear mRNA export, translation and related processes. Such information provides a new viewpoint for the development of cationic polymer-based vectors. © 2012 Elsevier Ltd.
• Post-nuclear gene delivery events for transgene expression by biocleavable polyrotaxanes

  Yuma Yamada, Taku Nomura, Hideyoshi Harashima, Atsushi Yamashita, Nobuhiko Yui

  Biomaterials 33 (15) 3952 - 3958 0142-9612 2012/05 [Refereed][Not invited]
   

  A quantitative comparison between nuclear DNA release from carriers and their transfection activity would be highly useful for improving the effectiveness of non-viral gene vectors. We previously reported that, for condensed DNA particles, a close relationship exists between the efficiency of DNA release and transfection activity, when biocleavable polyrotaxanes (DMAE-SS-PRX), in which the cationic density can be easily controlled. In this study, we first investigated the efficiencies of DNA release from condensed DNA particles with various types of DMAE-SS-PRX. The findings indicate that an optimal cationic density in DMAE-SS-PRX exists for DNA release. We then packaged condensed DNA particles in a multifunctional envelope-type nano device (MEND), and evaluated their transfection activities. The results showed that the transfection activity was increased and this increase was, to some extent, dependent on the efficiency of the DNA release. However, transfection activity decreased, when the value for the efficiency of DNA release was higher than a certain value. An investigation of the fate of intranuclear DNA indicated that a very high efficiency of DNA release has a positive influence on transcription, however, it would inhibit the post-transcription process nuclear mRNA export, translation and related processes. Such information provides a new viewpoint for the development of cationic polymer-based vectors. © 2012 Elsevier Ltd.
• Different mechanisms for nanoparticle formation between pDNA and siRNA using polyrotaxane as the polycation

  Yuma Yamada, Masahiro Hashida, Taku Nomura, Hideyoshi Harashima, Yuichi Yamasaki, Kazunori Kataoka, Atsushi Yamashita, Ryo Katoono, Nobuhiko Yui

  ChemPhysChem 13 (5) 1161 - 1165 1439-4235 2012/04/10 [Refereed][Not invited]
   

  Ratios that matter: Investigations of nanoparticle formation using polyrotaxane as a polycation reveal that the formations of pDNA and siRNA nanoparticles with polycations are dependent on the nitrogen/phosphate ratio and the molar ratio, respectively. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
• Different Mechanisms for Nanoparticle Formation between pDNA and siRNA Using Polyrotaxane as the Polycation

  Yuma Yamada, Masahiro Hashida, Taku Nomura, Hideyoshi Harashima, Yuichi Yamasaki, Kazunori Kataoka, Atsushi Yamashita, Ryo Katoono, Nobuhiko Yui

  CHEMPHYSCHEM 13 (5) 1161 - 1165 1439-4235 2012/04 [Refereed][Not invited]
  
• Delivery of bioactive molecules to the mitochondrial genome using a membrane-fusing, liposome-based carrier, DF-MITO-Porter

  Yuma Yamada, Hideyoshi Harashima

  BIOMATERIALS 33 (5) 1589 - 1595 0142-9612 2012/02 [Refereed][Not invited]
   

  Mitochondrial dysfunction has been implicated in a variety of human diseases. It is now well accepted that mutations and defects in the mitochondrial genome form the basis of these diseases. Therefore, mitochondrial gene therapy and diagnosis would be expected to have great medical benefits. To achieve such a strategy, it will be necessary to deliver therapeutic agents into mitochondria in living cells. We report here on an approach to accomplish this via the use of a Dual Function (DF)-MITO-Porter, aimed at the mitochondrial genome, so-called mitochondrial DNA (mtDNA). The DF-MITO-Porter, a nano carrier for mitochondrial delivery, has the ability to penetrate the endosomal and mitochondrial membranes via step-wise membrane fusion. We first constructed a DF-MITO-Porter encapsulating DNase 1 protein as a bioactive cargo. It was expected that mtDNA would be digested, when the DNase I was delivered to the mitochondria. We observed the intracellular trafficking of the carriers, and then measured mitochondrial activity and mtDNA-levels after the delivery of DNase I by the DF-MITO-Porter. The findings confirm that the DF-MITO-Porter effectively delivered the DNase 1 into the mitochondria, and provides a demonstration of its potential use in therapies that are selective for the mitochondrial genome. (C) 2011 Elsevier Ltd. All rights reserved.
• MULTIFUNCTIONAL ENVELOPE-TYPE NANO DEVICE (MEND) FOR ORGANELLE TARGETING VIA A STEPWISE MEMBRANE FUSION PROCESS

  Yuma Yamada, Hidetaka Akita, Hideyoshi Harashima

  NANOMEDICINE: INFECTIOUS DISEASES, IMMUNOTHERAPY, DIAGNOSTICS, ANTIFIBROTICS, TOXICOLOGY AND GENE MEDICINE 509 301 - 326 0076-6879 2012 [Refereed][Not invited]
   

  A single cell contains a variety of organelles. Included among these organelles are the nucleus that regulates the central dogma, mitochondria that function as an energy plant, the Golgi apparatus that determines the destination of endogenous protein, and others. If it were possible to prepare a nano craft that could specifically target a specific organelle, this would open a new field of research directed toward therapy for various diseases. We recently developed a new concept of "Programmed Packaging," by which we succeeded in creating a multifunctional envelope-type nano device (MEND) as a nonviral gene-delivery system. Our attempts to target certain organelles (nucleus and mitochondria) are described here, mainly focusing on the construction of a tetra-lamellar MEND (T-MEND), and on methods for screening the organelle-specific fusogenic envelope. The critical structural elements of the T-MEND include an organelle-specific membrane-fusogenic inner envelope and a cellular membrane-fusogenic outer envelope. The resulting T-MEND can be utilized to overcome intracellular membrane barriers, since it involves stepwise membrane fusion. To deliver cargos into a target organelle in our strategy, the carriers must fuse with the organelle membrane. Therefore, we screened a series of lipid envelopes that have the potential for fusing with an organelle membrane by monitoring the inhibition of fluorescence resonance energy transfer and identified the optimal lipid conditions for nuclear and mitochondrial membrane fusion. Finally, we describe the delivery of a bioactive molecule targeted to the nucleus and mitochondria in living cells, demonstrating that this system can be useful for targeting various organelles.
• The manner in which DNA is packaged with TFAM has an impact on transcription activation and inhibition

  Ryo Furukawa, Yuma Yamada, Yuichi Matsushima, Yu-ichi Goto, Hideyoshi Harashima

  FEBS OPEN BIO 2 145 - 150 2211-5463 2012 [Refereed][Not invited]
   

  For successful mitochondrial transgene expression, an optimal packaging exogenous DNA is an important issue. We report herein on the effects of DNA packaged with mitochondrial transcription factor A (TFAM), which packages mitochondrial DNA (mtDNA), on the transcription process. Our initial findings indicated that the transcription of the TFAM/DNA complex was activated, when the complex was formed at an optimal ratio. We also found that TFAM has a significant advantage over protamine, a nuclear DNA packaging protein, from the viewpoint of transcription efficiency. This result indicates that TFAM can be useful packaging protein for exogenous DNA to achieve mitochondrial transgene expression. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.
• Dual Function MITO-Porter, a Nano Carrier Integrating Both Efficient Cytoplasmic Delivery and Mitochondrial Macromolecule Delivery

  Yuma Yamada, Ryo Furukawa, Yukari Yasuzaki, Hideyoshi Harashima

  MOLECULAR THERAPY 19 (8) 1449 - 1456 1525-0016 2011/08 [Refereed][Not invited]
   

  Mitochondrial dysfunction is associated with a variety of human diseases including inherited mitochondrial diseases, neurodegenerative disorders, diabetes mellitus, and cancer. Effective medical therapies for mitochondrial diseases will ultimately require an optimal drug delivery system, which will likely be achieved through innovations in the nanotechnology of intracellular trafficking. To achieve efficient mitochondrial drug delivery, two independent processes, i.e., "cytoplasmic delivery through the cell membrane" and "mitochondrial delivery through the mitochondrial membrane" are required. In previous studies, we developed an octaarginine (R8) modified nano carrier for efficient cytoplasmic delivery, showing that R8-modified liposomes were internalized into cells efficiently. On the other hand, we also constructed MITO-Porter for the mitochondrial delivery of macromolecules, a liposome-based carrier that delivers cargos to mitochondria via membrane fusion. Here, we report the development of a dual function MITO-Porter (DF-MITO-Porter), based on the concept of integrating both R8-modified liposomes and MITO-Porter. We show that the DF-MITO-Porter effectively delivers exogenous macro-biomolecules into the mitochondrial matrix, and provide a demonstration of its potential use in therapies aimed at mitochondrial DNA.
• Octaarginine-modified liposomes enhance the anti-oxidant effect of Lecithinized superoxide dismutase by increasing its cellular uptake

  Ryo Furukawa, Yuma Yamada, Mitsuko Takenaga, Rie Igarashi, Hideyoshi Harashima

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 404 (3) 796 - 801 0006-291X 2011/01 [Refereed][Not invited]
   

  The anti-oxidant enzyme superoxide dismutase (SOD) has the potential for use as a therapeutic agent in the treatment of various diseases caused by reactive oxygen species. However, achieving this would be difficult without a suitable delivery system for SOD. We previously reported that PC-SOD, in which four molecules of a phosphatidylcholine (PC) derivative were covalently bound to each dimer of recombinant human CuZnSOD, was a high affinity for the cell membrane [14]. Here, we show that an octaarginine (R8) modified liposome equipped with PC-SOD (R8-LP (PC-SOD)) enhances its anti-oxidant effect. High-density R8-modified liposomes can stimulate macropinocytosis and are taken up efficiently by cells as demonstrated in a previous study [21]. Flow cytometry analyses showed that R8-LP (PC-SOD) was taken up by cells more efficiently than PC-SOD. Moreover, R8-LP (PC-SOD) liposomes were found to scavenge superoxide anions (O(2)(-)) very efficiently. These results suggest that the efficient cytosolic delivery of PC-SOD by R8-modified liposomes would enhance the anti-oxidant effects of PC-SOD. (C) 2010 Elsevier Inc. All rights reserved.
• Intranuclear DNA Release Is a Determinant of Transfection Activity for a Non-viral Vector: Biocleavable Polyrotaxane as a Supramolecularly Dissociative Condenser for Efficient Intranuclear DNA Release

  Yuma Yamada, Taku Nomura, Hideyoshi Harashima, Atsushi Yamashita, Ryo Katoono, Nobuhiko Yui

  BIOLOGICAL & PHARMACEUTICAL BULLETIN 33 (7) 1218 - 1222 0918-6158 2010/07 [Refereed][Not invited]
   

  It has been believed that nuclear gene delivery is the most important process for gene expression, and various non-viral vectors are currently being developed with this assumption. However, some of our earlier studies revealed a surprising difference in transfection activity between viral and non-viral vectors: this difference is largely due to the result of the intranuclear disposition of DNA rather than its delivery to the nucleus (Hama S. et al. (2006), Quantitative comparison of intracellular trafficking and nuclear transcription between adenoviral and lipoplex systems. Mol. Then, 13, 786-794). Here; we report on some direct evidence that demonstrates the importance of the release of intranuclear DNA on transfection activity. The data show that transfection activity can be substantially enhanced by integrating a multifunctional envelope-type nano device (MEND) and a biocleavable polyrotaxane (DMAE-SS-PRX) as an artificial condenser. Our integration system showed significantly higher transfection activity compared to conventional gene delivery system. Moreover, this system provides a strong support for our hypothesis that intranuclear DNA disposition plays a critical role in gene expression for non-viral vectors.
• Transgene expression efficiency from plasmid DNA delivered as a complex with histone H3

  Hiroyuki Kamiya, Hitomi Goto, Genki Kanda, Yuma Yamada, Hideyoshi Harashima

  INTERNATIONAL JOURNAL OF PHARMACEUTICS 392 (1-2) 249 - 253 0378-5173 2010/06 [Refereed][Not invited]
   

  The intranuclear disposition of plasmid DNA is extremely important for transgene expression. Exogenous histones have been used as carriers of plasmid DNA in histone-mediated gene delivery. In this study, the effects of exogenous histone H3 complexed with plasmid DNA on transgene expression efficiency were examined. The plasmid-histone complexes in various ratios were transfected into HeLa cells by osmotic pressure. Histone H3 suppressed transgene expression in the nucleus in a dose-dependent manner. Our results suggest that the histone-mediated gene delivery is unlikely to be useful, from the viewpoint of the intranuclear disposition. (C) 2010 Elsevier B.V. All rights reserved.
• Mitochondrial matrix delivery using MITO-Porter, a liposome-based carrier that specifies fusion with mitochondrial membranes

  Yukari Yasuzaki, Yuma Yamada, Hideyoshi Harashima

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 397 (2) 181 - 186 0006-291X 2010/06 [Refereed][Not invited]
   

  Mitochondria are are the principal producers of energy in cells of higher organisms. It was recently reported that mutations and defects in mitochondrial DNA (mtDNA) are associated with various mitochondrial diseases including a variety of neurodegenerative and neuromuscular diseases. Therefore, an effective mitochondrial gene therapy and diagnosis would be expected to have great medical benefits. To achieve this, therapeutic agents need to be delivered into the innermost mitochondrial space (mitochondrial matrix), which contains the mtDNA pool. We previously reported on the development of MITO-Porter, a liposome-based carrier that introduces macromolecular cargos into mitochondria via membrane fusion. In this study, we provide a demonstration of mitochondrial matrix delivery and the visualization of mitochondrial genes (mtDNA) in living cells using the MITO-Porter. We first prepared MITO-Porter containing encapsulated propidium iodide (PI), a fluorescent dye used to stain nucleic acids to detect mtDNA. We then confirmed the emission of red-fluorescence from PI by conjugation with mtDNA, when the carriers were incubated in the presence of isolated rat liver mitochondria. Finally, intracellular observation by confocal laser scanning microscopy clearly verified that the MITO-Porter delivered PI to the mitochondrial matrix. (C) 2010 Elsevier Inc. All rights reserved.
• Mitochondrial drug delivery systems for macromolecule and their therapeutic application to mitochondrial diseases

  Yuma Yamada, Hideyoshi Harashima

  ADVANCED DRUG DELIVERY REVIEWS 60 (13-14) 1439 - 1462 0169-409X 2008/10 [Refereed][Not invited]
   

  Mitochondrial dysfunction has been implicated in a variety of human disorders-the so-called mitochondrial diseases. Therefore, the organelle is a promising therapeutic drug target. In this review, we describe the key role of mitochondria in living cells, a number of mitochondrial drug delivery systems and mitochondria-targeted therapeutic strategies. In particular, we discuss mitochondrial delivery of macro molecules, such as proteins and nucleic acids. The discussion of protein delivery is limited primarily to the mitochondrial import machinery. In the section on mitochondrial gene delivery and therapy, we discuss mitochondrial diseases caused by mutations in mitochondrial DNA, several gene delivery strategies and approaches to mitochondrial gene therapy. This review also summarizes our current efforts regarding liposome-based delivery system including use of a multifunctional envelope-type nano-device (MEND) and mitochondrial liposome-based delivery as anti-cancer therapies. Furthermore, we introduce the novel MITO-Porter-a liposome-based mitochondrial delivery system that functions using a membrane-fusion mechanism. (C) 2008 Elsevier B.V. All rights reserved.
• Development of small, homogeneous pDNA particles condensed with mono-cationic detergents and encapsulated in a multifunctional envelope-type nano device

  Ryosuke Suzuki, Yuma Yamada, Hideyoshi Harashima

  BIOLOGICAL & PHARMACEUTICAL BULLETIN 31 (6) 1237 - 1243 0918-6158 2008/06 [Refereed][Not invited]
   

  Non-viral DNA vectors are promising gene delivery systems and a variety of non-viral DNA vectors have been developed to date. Recently, we developed a novel non-viral gene delivery system-multifunctional envelope-type nano device (MEND). The MEND system has high transfection activity, similar to that of adenovirus vector, which is a potent viral vector. However, conventional MEND is relatively large and heterogeneous (approximately 300 nm), probably because they contain relatively large- and heterogeneous-pDNA particles condensed with polycations, such as poly-L-lysine. Small particle size is important for in vivo delivery, because large particles are rapidly eliminated from systemic circulation. Moreover, heterogeneous size of drug carriers is difficult to apply to clinical applications. Here, we describe construction of small homogeneous MEND. First, we screened mono-cationic detergents (MCD(s)) to obtain optimal pDNA condensed particles. We determined that benzyldimethylhexadecylammonium chloride (BDHAC) and thonzonium bromide (TB) were optimal pDNA condensers. Next, we packaged the condensed pDNA particles into a lipid bi-layer. The resulting lipid-encapsulated pDNA particles were then equipped with octaarginine to facilitate cell-uptake (R8-MEND (MCD)). The carrier showed high transfection activity in cultured HeLa cells. Furthermore, the R8-MEND (MCD) were small and homogeneous compared with conventional MEND. These results indicate that R8-MEND (MCD) has potential as a novel non-viral delivery system for clinical application.
• Multifunctional envelope-type nano device (MEND) as a non-viral gene delivery system

  Kentaro Kogure, Hidetaka Akita, Yuma Yamada, Hideyoshi Harashima

  ADVANCED DRUG DELIVERY REVIEWS 60 (4-5) 559 - 571 0169-409X 2008/03 [Refereed][Not invited]
   

  In this review, we describe a key role of octaarginine (R8) in developing our new concept of "Programmed Packaging", by which we succeeded in creating a multifunctional envelope-type nano device (MEND) as a non-viral gene-delivery system. This concept can be applied not only to nuclear targeting of plasmid DNA (pDNA) but also to cytosolic delivery of functional nucleic acids such as oligonucleotides or siRNA, This concept has been extended to other organelles such as mitochondria as a foundation for innovative nanomedicine. Finally, we discuss the rate-limiting step in gene delivery by comparing non-viral and viral gene delivery systems, which clearly indicates the importance of nuclear disposition of pDNA for efficient transfection. (C) 2007 Elsevier B.V. All rights reserved.
• MITO-Porter: A liposome-based carrier system for delivery of macromolecules into mitochondria via membrane fusion.

  Yamada Y, Akita H, Kamiya H, Kogure K, Yamamoto T, Shinohara Y, Yamashita K, Kobayashi H, Kikuchi H, Harashima H

  Biochimica et biophysica acta 2 1778 (2) 423 - 432 0005-2728 2008/02 [Refereed][Not invited]
  
• MITO-Porter: A liposome-based carrier system for delivery of macromolecules into mitochondria via membrane fusion.

  Yuma Yamada, Hidetaka Akita, Hiroyuki Kamiya, Kentaro Kogure, Takenori Yamamoto, Yasuo Shinohara, Kikuji Yamashita, Hideo Kobayashi, Hiroshi Kikuchi, Hideyoshi Harashima

  Biochimica et biophysica acta 1778 (2) 423 - 32 0006-3002 2008/02 

  Mitochondria are the principal producers of energy in higher cells. Mitochondrial dysfunction is implicated in a variety of human diseases, including cancer and neurodegenerative disorders. Effective medical therapies for such diseases will ultimately require targeted delivery of therapeutic proteins or nucleic acids to the mitochondria, which will be achieved through innovations in the nanotechnology of intracellular trafficking. Here we describe a liposome-based carrier that delivers its macromolecular cargo to the mitochondrial interior via membrane fusion. These liposome particles, which we call MITO-Porters, carry octaarginine surface modifications to stimulate their entry into cells as intact vesicles (via macropinocytosis). We identified lipid compositions for the MITO-Porter which promote both its fusion with the mitochondrial membrane and the release of its cargo to the intra-mitochondrial compartment in living cells. Thus, the MITO-Porter holds promise as an efficacious system for the delivery of both large and small therapeutic molecules into mitochondria.
• Efficient cytoplasmic protein delivery by means of a multifunctional envelope-type nano device

  Ryosuke Suzuki, Yuma Yamada, Hideyoshi Harashima

  Biological and Pharmaceutical Bulletin 30 (4) 758 - 762 0918-6158 2007/04 [Refereed][Not invited]
   

  The potential for protein therapy, such as the use of antibodies, and vaccines is now well accepted. However, it is difficult to enhance efficiency in protein therapy without a suitable delivery system for delivering proteins to target sites. Here we describe the development of protein delivery system, which is capable of cytoplasmic delivery as well as efficient packaging. The multifunctional envelope-type nano device (MEND), which was originally developed for the delivery of nucleic acids such as plasmid DNA and oligodeoxynucleotides, can also be applied to protein delivery. In this study, the green fluorescent protein (GFP), a model protein, was condensed with stearyl octaarginine (stearyl R8) to form a nano particle, which was then coated with a lipid membrane, thus permitting R8 to be introduced for efficient cellular uptake and controlled intracellular trafficking. The packaging efficiency of the MEND was significantly higher than that of conventional liposomes, because the GFP can be encapsulated a condensed form. According to confocal laser scanning microscopy, the MEND is internalized efficiently and escapes from the acidic compartment to efficiently release GFP into the cytosol. These results indicate that the MEND can serve as a useful cytoplasmic delivery system for protein therapy. © 2007 Pharmaceutical Society of Japan.
• Mitochondrial drug delivery and mitochondrial disease therapy - An approach to liposome-based delivery targeted to mitochondria

  Yuma Yamada, Hidetaka Akita, Kentaro Kogure, Hiroyuki Kamiya, Hideyoshi Harashima

  MITOCHONDRION 7 (1-2) 63 - 71 1567-7249 2007/02 [Refereed][Not invited]
   

  Recent progress in genetics and molecular biology has provided useful information regarding the molecular mechanisms associated with the mitochondrial diseases. Genetic approaches were initiated in the late 1980s to clarify the gene responsible for various mitochondrial diseases, and information concerning genetic mutations is currently used in the diagnosis of mitochondrial diseases. Moreover, it was also revealed that mitochondria play a central role in apoptosis, or programmed cell death, which is closely related to the loss of physiological functions of tissues. Therefore, drug therapies targeted to the mitochondria would be highly desirable. In spite of the huge amount of mechanism-based studies of mitochondrial diseases, effective therapies have not yet been established mainly because of the lack of an adequate delivery system. To date, numerous investigators have attempted to establish a mitochondrial drug delivery system. However, many problems remain to be overcome before a clinical application can be achieved. To fulfill a drug delivery targeted to mitochondria, we first need to establish a method to encapsulate various drugs, proteins, peptides, and genes into a drug carrier depending on their physical characteristics. Second, we need to target it to a specific cell. Finally, multi-processes of intracellular trafficking should be sophisticatedly regulated so as to release a drug carrier from the endosome to the cytosol, and thereafter to deliver to the mitochondria. In this review, we describe the current state of the development of mitochondrial drug delivery systems, and discuss the advantage and disadvantage of each system. Our current efforts to develop an efficient method for the packaging of macromolecules and regulating intracellular trafficking are also summarized. Furthermore, novel concept of "Regulation of intramitochondrial trafficking" is proposed herein as a future challenge to the development of a mitochondrial drug delivery system. (c) 2007 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
• Development of multifunctional envelope type artificial viral like gene delivery system

  Hideyoshi Harashima, Kentaro Kogure, Yuma Yamada, Hidetaka Akita, Hiroyuki Kamiya

  YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 127 (10) 1655 - 1672 0031-6903 2007 [Refereed][Not invited]
   

  This review introduces a new concept "Programmed Packaging" to develop a non-viral gene delivery system. Based on this concept, multifunctional envelope type nano devices (MEND) were developed for in vitro, in situ and in vivo conditions. A quantitative study to identify a rate limiting step in intracellular trafficking was also shown between viral and non-viral vectors, which indicated an important role of controlled intranuclear disposition for development a safe and efficient non-viral gene delivery system. This review will provide a future direction of non-viral gene delivery system.
• Significant and prolonged antisense effect of a multifunctional envelope-type nano device encapsulating antisense oligodeoxynucleotide

  Y Nakamura, K Kogure, Y Yamada, S Futaki, H Harashima

  JOURNAL OF PHARMACY AND PHARMACOLOGY 58 (4) 431 - 437 0022-3573 2006/04 [Refereed][Not invited]
   

  A multifunctional envelope-type nano device (MEND) was developed for use as an efficient non-viral system for the delivery of plasmid DNA (pDNA) using octaarginine (R8) as an internalizing ligand. Three types of R8-MENDs were prepared, co-encapsulating luciferase-encoding pDNA and antiluciferase oligodeoxynucleotide (ODN) condensed by three polycations, stearyl octaarginine (STR-R8), poly-L-lysine (PLL) and protamine, and the antisense effects of the ODN-encapsulated R8-MENDs (ODN-MEND) were analysed in-vitro. The ODN-MEND packaged using protamine as a condenser showed a 90% antisense effect 16 h after the transfection, and a persistent antisense effect of over 75% for up to 48 h, which was much more effective than that of LipofectAmine2000. On the other hand, the ODN-MENDs prepared using PLL and STR-R8 as condensers did not show any significant inhibition of luciferase activity. Although there was no specific relation between the physicochemical characteristics of the ODN-MENDs and their antisense effect, the pattern of the antisense effect among the ODN-MENDs was similar to that of the silencing effect of R8-MEND encapsulating plasmid DNA encoding siRNA. These results suggest that R8-MENDs are able to deliver encapsulated DNA to the cytosol as well as to the nucleus, and that protamine can also function as an efficient decondenser, not only in the nucleus but also in the cytosol. In conclusion, we successfully developed an ODN-MEND with a high antisense effect using protamine as a DNA condensing as well as a decondensing agent.
• Mitochondrial delivery of mastoparan with transferrin liposornes equipped with a pH-sensitive fusogenic peptide for selective cancer therapy

  Y Yamada, Y Shinohara, T Kakudo, S Chaki, S Futaki, H Kamiya, H Harashima

  INTERNATIONAL JOURNAL OF PHARMACEUTICS 303 (1-2) 1 - 7 0378-5173 2005/10 [Refereed][Not invited]
   

  Mastoparan (NIP), a potent facilitator of mitochondrial permeability transition (PT), could be used as an antitumor agent, if it were encapsulated in a tumor selective delivery system. We recently developed transferrin-modified liposomes (Tf-L) with a pH-sensitive fusogenic peptide (GALA), which delivers an encapsulated fluorescent marker into cytosol efficiently. In this study, we encapsulated NIP into Tf-L with GALA for the selective delivery to mitochondria of tumor cells. The NIP showed potent PT activity at concentrations above 25 mu M in a homogenate of K 562 cells as well as in isolated mitochondria in the presence of phosphate. Tf-L equipped with cholesteryl GALA can release encapsulated sulforhodamine B, while Tf-L failed, as evidenced by confocal laser scanning microscopy. The MP, which was delivered with Tf-L with GALA, released cytochrome c (cyt c) from mitochondria to the cytosol, while free NIP released cyt c not only to the cytosol but also extracellulary. These results demonstrate the utility of MP in Tf-L with GALA for cancer therapy. (c) 2005 Elsevier B.V. All rights reserved.
• Development of efficient packaging method of oligodeoxynucleotides by a condensed nano particle in lipid envelope structure

  Y Yamada, K Kogure, Y Nakamura, K Inoue, H Akita, F Nagatsugi, S Sasaki, T Suhara, H Harashima

  BIOLOGICAL & PHARMACEUTICAL BULLETIN 28 (10) 1939 - 1942 0918-6158 2005/10 [Refereed][Not invited]
   

  An efficient delivery system is required if antisense oligodeoxynucleotides (ODN) are to be utilized for gene therapy. We report herein on the development of a novel ODN delivery system, ODN-encapsulated nano particles (ODN-ENP) using an efficient and simple packaging method. The ODN-ENP consists of a condensed ODN particle and a lipid envelope, which can be equipped with various functional devices for the efficient delivery of ODN with a small diameter (150 nm). The encapsulation efficiency and ODN recovery of ODN-ENP were significantly higher than those of other packaging methods, such as a stabilized antisense-lipid particles method or a freezethaw method. Furthermore, the time required for the preparation of the ODN-ENP was shorter than the other methods. The method developed in this study is a simple and efficient packaging method for ODN with a condensed nano particle in lipid-envelope structure.
• Preparation of a novel PEG-clay hybrid as a DDS material: Dispersion stability and sustained release profiles

  T Takahashi, Y Yamada, K Kataoka, Y Nagasaki

  JOURNAL OF CONTROLLED RELEASE 107 (3) 408 - 416 0168-3659 2005/10 [Refereed][Not invited]
   

  An advanced hybrid drug carrier has been developed using porous nanocrystals of a swelling clay mineral conjugated with a block copolymer containing poly(ethylene glycol) and polyamine segments. Synthetic hectorite (Laponite) modified with ((Xacetal-poly(ethylene glycol)-block-[poly(2-(N,N-dimethylamino) ethyl methacrylate)] (Acetal-PEG-b-PAMA) produced a homogeneous dispersion of organic-inorganic hybrid in an aqueous solution, which showed flocculation-resistive properties with an elevated ionic strength. The zeta-potential measurement revealed that nonionic PEG brush layers are formed on the surface of the clay nanocrystals since negative charge of the clay surface was completely neutralized by the positive charge of the cationic PAMA segment and the entire surface charge is successfully shielded by the effect of nonionic PEG segment in the block copolymer. This charge neutralization is in good agreement with the dispersion stability in solutions of high ionic strength. The average particle size of the PEG-modified hybrid particle was estimated to be 120 nm by a dynamic light scattering (DLS) method. When pyrene was used as the model compound of hydrophobic drug, it was incorporated into the nanopore in the clay mineral without showing any remarkable expansion of the basal spacings. Fluorescence spectra and powder X-ray diffraction patterns demonstrated that pyrene molecules are captured in an amorphous state in the range of low pyrene content (< 5%), while excimer formation was seen at the higher pyrene concentration (> 5%). The PEG-clay hybrid act as a carrier for sustained release of hydrophobic substances due to the high affinity (K = 1.52 x 10(4)) between the drug and clay surface. (c) 2005 Elsevier B.V. All rights reserved.
• Kinetic analysis of protein production after DNA transfection

  Y Yamada, H Kamiya, H Harashima

  INTERNATIONAL JOURNAL OF PHARMACEUTICS 299 (1-2) 34 - 40 0378-5173 2005/08 [Refereed][Not invited]
   

  The production of an exogenous protein by the transfection of a plasmid DNA encoding the protein was kinetically analyzed, to determine the efficiency of the transfection. Cultured NIH3T3 or HeLa cells, and the luciferase protein were used as a model system in this experiment. The findings indicate that at least a 8 x 10(4)- and 4 x 10(3)-fold molar amounts of luciferase protein was produced from one copy of the plasmid DNA molecule in NIH3T3 and HeLa cells, respectively. The rate of elimination of luciferase activity upon DNA transfection was smaller than that for the luciferase protein itself (k(el) for DNA transfection < k(cl) for the luciferase protein), suggesting that a decrease in intranuclear active DNA was the main determinant of the elimination rate in this case. A preliminary pharmacokinetic model is proposed, based on the results obtained. (c) 2005 Elsevier B.V. All rights reserved.

Conference Activities & Talks

• Targeting Mitochondria Based on Mitochondrial Drug Delivery Systems (DDS)  [Invited]

  Yuma Yamada

  14th edition of the World Congress on Targeting Mitochondria 2023  2023/10
• 細胞を操るオルガネラ制御学の創出を目指して  [Invited]

  山田勇磨

  第39回日本DDS学会学術集会（若手ワークショップ）  2023/07
• ミトコンドリアを標的とした癌光治療を実践するナノカプセルの創製  [Invited]

  山田勇磨

  日本薬剤学会第38年会(旭化成創剤開発技術賞受賞講演)  2023/05
• ミトコンドリア標的型 DDS を基盤としたナノ医療の創出を目指して  [Invited]

  山田勇磨, 原島秀吉

  第43回 生体膜と薬物の相互作用シンポジウム  2022/10
• Validation of therapeutic strategies using mitochondrial drug delivery systems  [Invited]

  Yamada Y, Harashima H

  Joint Symposium of the Faculty of Pharmaceutical Sciences & WPI-ICReDD in Hokkaido University  2022/09
• 治療用rRNA送達によるミトコンドリア遺伝子治療戦略の検証  [Invited]

  山田勇磨

  日本核酸医薬学会第7回年会  2022/07
• ミトコンドリアDDSを基盤とした遺伝子細胞治療への挑戦  [Invited]

  山田勇磨

  第28回日本遺伝子細胞治療学会学術集会  2022/07
• ミトコンドリアDDSの夢： ミトコンドリア標的型ナノ医薬品の創出  [Invited]

  山田勇磨

  日本薬剤学会第37年会  2022/05
• ミトコンドリアを標的とする核酸ナノ医薬品の創製を目指して  [Invited]

  山田勇磨

  ゲノム創薬・創発フォーラム第9回シンポジウム  2022/02
• ミトコンドリアを標的とする遺伝子治療戦略の検証  [Invited]

  山田勇磨

  第20回日本ミトコンドリア学会年会  2021/12
• 未来型DDSが拓くミトコンドリアを標的とする新しいナノ医療  [Invited]

  山田勇磨

  京都大学呼吸器内科セミナー  2021/11
• Targeting mitochondria: innovation of mitochondrial drug delivery system (DDS) to mitochondrial medicine.  [Invited]

  山田勇磨

  Mitochondrial Medicine -Therapeutic Development.  2021/11
• オルガネラ標的型DDSを基盤とした核酸医薬開発研究の最前線  [Invited]

  山田勇磨

  第15回Wakoウェブ受託セミナー  2021/11
• The way to develop an innovative nanomedicine targeting mitochondria.  [Invited]

  山田勇磨

  Host Madsen Medal winner 2021 ceremony.  2021/09
• オルガネラ標的型DDSを基盤とした創剤研究の最前線  [Invited]

  山田勇磨

  第72回北日本小児科学会  2021/09
• ミトコンドリア機能を操作するDrug Delivery System  [Invited]

  山田勇磨

  第30回日本バイオイメージング学会  2021/09
• ミトコンドリア標的型 Drug Delivery System を基盤とした遺伝子治療戦略の検証  [Invited]

  山田勇磨

  日本ゲノム編集学会第6回大会  2021/06
• 希少疾患の未来について考える〜患者とともに挑むイノベーション  [Invited]

  山田勇磨

  iPark Frontier Talk  2021/02
• The challenge of developing mitochondrial nano medicine based on MITO-Porter Technology  [Invited]

  山田勇磨

  第56回日本小児循環器学会 (JSPCC-AHA 2020)  2020/11
• ミトコンドリア標的型ナノ医薬品の創製を目指して  [Invited]

  山田勇磨

  第35回日本小児神経学会北海道地方会  2020/10
• MITO-Porter, a cutting-edge mitochondrial DDS  [Invited]

  山田勇磨

  Pharmaceutical Society of Korea 2020  2020/10
• 新たな○△□治療を、ミトコンドリアに薬を運ぶ技術開発で!  [Invited]

  山田勇磨

  TAKEDA Academic WEB Salon2020  2020/08
• Mito-porter, Investigation of Drug Delivery System Targeting Mitochondria.  [Invited]

  山田勇磨

  The 84th annual scinetific meeting of the japanse circulation society (JCS2020)  2020/07
• A Mitochondrial Dds Towards An Innovative Therapy.  [Invited]

  山田勇磨

  2020 Annual Meeting & Exposition of the Controlled Release Society  2020/06
• 光よ届け！ミトコンドリアへ  [Invited]

  山田 勇磨

  第9回 生体物理化学セミナー  2020/01
• ミトコンドリアDDSが描くナノ医療の未来予想図～リポ化プロスタグランジン製剤の話題を含めて～  [Invited]

  山田勇磨

  第12回 北海道小児オータムセミナー  2019/11
• ミトコンドリア標的型ナノＤＤＳを基盤とした遺伝子・細胞治療の実装  [Invited]

  山田勇磨

  第12回 CHEMBIOハイブリッドレクチャー  2019/10
• MITO-Porter, liposomal mitochondrial delivery system: Toward Mitochondrial Nanomedicine. MITO-Porter therapy to open innovative therapies targeting mitochondria.  [Invited]

  Yuma Yamada

  16th ASMRM & 19th J-mit.  2019/10
• MITO-Porter, liposomal mitochondrial delivery system: Toward Mitochondrial Nanomedicine.  [Invited]

  Yuma Yamada

  Liposome Research Days 2019.  2019/09
• ﾐﾄｺﾝﾄﾞﾘｱDDSを用いた酸化ｽﾄﾚｽ応答制御および疾患治療への展開  [Invited]

  山田勇磨

  第72回日本酸化ストレス学会  2019/06
• ミトコンドリア標的型ナノカプセルが創る未来医療  [Invited]

  山田勇磨

  IVF JAPANセミナー  2019/02
• The MITO-Porter integrates a mitochondrial drug delivery system with a variety of other current scientific concepts with the goal of developing new innovative technologies and medicines.  [Invited]

  Yuma Yamada

  The 19th RIES-HOKUDAI International Symposium.  2018/12
• ミトコンドリア標的型DDSを基盤としたナノ医療の創出を目指して  [Invited]

  山田勇磨

  第18回 日本ミトコンドリア学会年会  2018/12
• Mitochondrial DDS for Gene & Cell Therapy.  [Invited]

  Yuma Yamada

  Cadiovascular and Metabolic Week 2018.  2018/12
• Mitochondrial DDS opens emerging therapies and novel strategy of new drug development for mitochondrial disorders.  [Invited]

  Yuma Yamada

  KSAP annual convention 2018.  2018/10
• ミトコンドリア機能を制御するナノカプセルの構築および疾患治療に向けた試み  [Invited]

  山田勇磨

  第91回 日本生化学大会  2018/09
• ミトコンドリアDDSの使い方  [Invited]

  山田勇磨

  第9回 フリーラジカルスクール  2018/08
• ミトコンドリアを標的とする遺伝子治療用ナノカプセルの創製  [Invited]

  山田勇磨

  日本核酸医薬学会第4回年会  2018/07
• 遺伝子・細胞治療に用いるミトコンドリアDDS  [Invited]

  山田勇磨

  2018/06
• ミトコンドリアDDSが拓く新しいミトコンドリア病治療戦略  [Invited]

  山田勇磨

  第60回日本小児神経学会学術集会  2018/05
• Mitochondrial Nano DDS Toward Innovative Medicine and Therapy.  [Invited]

  Yuma Yamada

  HIGO Program Cutting edge Seminar.  2018/04
• 膵島ミトコンドリアを標的とした分子送達技術の開発および糖尿病治療への展開  [Invited]

  山田勇磨

  日本薬学会 第138年会  2018/03
• ミトコンドリア病の遺伝子改変治療  [Invited]

  山田勇磨

  H29 年度 村山班ミトコンドリア病診断・診療マニュアル作成委員拡大合宿  2018/02
• ミトコンドリア機能を制御するナノカプセルの開発  [Invited]

  山田勇磨

  第6回ミトコンドリア機能研究会  2018/02
• Nano-drug delivery system for mitochondrial gene therapy.  [Invited]

  Yuma Yamada

  The 18th International Union of Materials Research Societies, International Conference in Asia (IUMRS-ICA) 2017  2017/11
• ミトコンドリアＤＤＳの開発と医療分野への展開  [Invited]

  山田勇磨

  第11回 次世代を担う若手医療薬科学シンポジウム  2017/10
• ミトコンドリア標的型ナノカプセルMITO-Porterを基盤とした遺伝子治療への挑戦  [Invited]

  山田勇磨

  第59回日本先天代謝異常学会  2017/10
• Validation of mitochondrial gene therapy using a MITO-Porter  [Invited]

  Yuma Yamada

  8th SFRR-Asia & 14th ASMRM.  2017/09
• ミトコンドリアDDSを基盤とした遺伝子・細胞治療への展開  [Invited]

  山田勇磨

  第33回日本DDS学会  2017/07
• ミトコンドリアを診る・操る・治すDrug Delivery System  [Invited]

  山田勇磨

  第2回先端ケミカルバイオロジー研究会  2017/06
• ミトコンドリア標的型ナノカプセル MITO-Porter  [Invited]

  山田勇磨

  H28 年度 村山班ミトコンドリア病診断・診療マニュアル作成委員拡大合宿  2017/02
• ミトコンドリアDDSの創製と心疾患治療への展開  [Invited]

  山田勇磨

  第3回iHFフォーラム  2016/08
• ミトコンドリア標的型ナノマシン“MITO-Porter”の開発  [Invited]

  山田勇磨

  第24回日本Cell Death学会  2015/08
• MITO-Porter, an innovative technology for mitochondrial drug delivery.  [Invited]

  Yuma Yamada

  The 1st HU-TMU-KU Joint Symposium for Pharm. Sci.  2015/08
• ミトコンドリアDDSが拓く医療・ライフサイエンス革命  [Invited]

  山田勇磨

  第31回日本DDS学会  2015/07
• ミトコンドリアDDSの創製とナノ医療への展開  [Invited]

  山田勇磨

  北海道大学病院・循環器内科主催・循環病態内科学リサーチセミナー  2015/05
• ミトコンドリアを科学するナノデバイス MITO-Porter の創製  [Invited]

  山田勇磨

  日本薬学会 第135年会  2015/03
• ミトコンドリア DDS が拓く新しい薬剤学  [Invited]

  山田勇磨

  日本薬学会 第135年会  2015/03

MISC

• Effect of Lipid Nanoparticle Properties on the Therapeutic Effects against Acetaminophen Liver Injury

  日比野光恵, 日比野光恵, 真栄城正寿, 渡慶次学, 原島秀吉, 山田勇磨  日本薬剤学会年会講演要旨集(CD-ROM)  38th-  2023
• 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
• Validation of mitochondrial-derived component supplementation strategy for the purpose of activating mitochondrial function

  伊藤百, 山田勇磨, 山田勇磨, 真栄城正寿, 渡慶次学, 原島秀吉  日本薬学会年会要旨集(Web)  143rd-  2023
• Evaluation of an innovative mitochondrial genome-editing system using mitochondria-targeted nanoparticles in vitro

  野呂田楓, 石塚宣, 廣瀬みさ, 真栄城正寿, 渡慶次学, 原島秀吉, 山田勇磨, 山田勇磨  日本薬学会年会要旨集(Web)  143rd-  2023
• 特性の異なるコエンザイムQ₁₀搭載脂質ナノ粒子を用いたアセトアミノフェン肝障害に対する治療効果の検証

  日比野光恵, 真栄城正寿, 渡慶次学, 原島秀吉, 山田勇磨  日本DDS学会学術集会プログラム予稿集  39th-  2023
• CRISPR/Cas9システムを用いたmtDNAゲノム編集の検証

  野呂田楓, 野呂田楓, 野呂田楓, 石塚宣, 廣瀬みさ, 真栄城正寿, 渡慶次学, 原島秀吉, 山田勇磨  日本DDS学会学術集会プログラム予稿集  39th-  2023
• 癌光治療用ミトコンドリア標的型ナノカプセルの殺細胞効果向上を目指した試み

  長縄莉奈, 高野勇太, 高野勇太, 趙韓俊, 真栄城正寿, 渡慶次学, 原島秀吉, 山田勇磨  日本DDS学会学術集会プログラム予稿集  39th-  2023
• Construction of preparation method using microfluidic device and validation of therapeutic effect for mitochondrial targeted coenzyme Q₁₀ encapsulated nanocapsule injectable drug

  日比野光恵, 山田勇磨, 真栄城正寿, 渡慶次学, 原島秀吉  日本薬学会年会要旨集(Web)  142nd-  2022
• Validation of mitochondrial component supplementation strategy to activate mitochondrial function

  伊藤百, 山田勇磨, 山田勇磨, 日比野光恵, 佐々木大輔, 真栄城正寿, 渡慶次学, 渡慶次学, 太田善浩, 原島秀吉, 原島秀吉  日本薬学会年会要旨集(Web)  142nd-  2022
• ミトコンドリア活性化ヒト由来心筋幹細胞を用いた細胞移植療法の検証

  白石真大, 白石真大, 山田勇磨, 山田勇磨, 佐々木大輔, 日比野光恵, 真栄城正寿, 渡慶次学, 武田充人, 原島秀吉  日本DDS学会学術集会プログラム予稿集  38th-  2022
• Establishment of Preparation Method for Lipid Nanoparticles Encapsulating Poorly Water-Soluble Drug by Using Microfluidic Device

  日比野光恵, 日比野光恵, 山田勇磨, 山田勇磨, 原島秀吉  製剤機械技術学会誌  31-  (4)  2022
• イメージングと操作で見るミトコンドリア機能 ミトコンドリア機能を操作するDrug Delivery System

  山田 勇磨, 原島 秀吉  バイオイメージング  30-  (2)  38  -38  2021/08
• 心筋虚血再灌流モデルラットに対するヒトミトコンドリア強化心筋幹細胞移植療法の治療効果の検討

  佐々木 大輔, 山田 勇磨, 後藤 悠太, 白石 真大, 武田 充人, 原島 秀吉  日本DDS学会学術集会プログラム予稿集  37回-  99  -99  2021/06
• ミトコンドリア人工共生による細胞機能制御の試み

  山田 勇磨, 日比野 光恵, 伊藤 百, 荒井 愛永, 佐々木 大輔, 真栄城 正寿, 渡慶次 学, 太田 善浩, 原島 秀吉  日本DDS学会学術集会プログラム予稿集  37回-  111  -111  2021/06
• 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  95-  (2)  S123  -S123  2021/03
• ミトコンドリア強化ヒト心筋幹細胞(hMITO Cell)の製造および心虚血再灌流モデルを用いた細胞移植療法の検証

  後藤 悠太, 山田 勇磨, 日比野 光恵, 佐々木 大輔, 武田 充人, 真栄城 正寿, 渡慶次 学, 原島 秀吉  日本薬学会年会要旨集  141年会-  29V09  -pm09S  2021/03
• オンライン会議システムを活用した医療面接実習の実践

  森 綾子, 加藤 いづみ, 柏木 仁, 今井 俊吾, 鳴海 克哉, 佐藤 夕紀, 古堅 彩子, 山田 勇磨, 小林 正紀  日本薬学会年会要旨集  141年会-  29V11  -pm04  2021/03
• Trans MIT system

  山田勇磨, 山田勇磨, 日比野光恵, 伊藤百, 荒井愛永, 佐々木大輔, 真栄城正寿, 渡慶次学, 渡慶次学, 太田善浩, 原島秀吉, 原島秀吉  日本薬剤学会年会講演要旨集(CD-ROM)  36th-  2021
• Fundamental study on preparation of polymer nanoparticles by microfluidic device

  飯岡真吾, 日比野光恵, 山田勇磨, 真栄城正寿, 渡慶次学, 照喜名孝之, 金沢貴憲, 近藤啓  日本薬学会年会要旨集(Web)  141st-  2021
• ミトコンドリア標的型ナノ医薬品の研究開発

  山田 勇磨  薬剤学: 生命とくすり  80-  (6)  280  -285  2020/11
• MITO-Porter Technologyに基づいたmitochondrial nano medicine開発への挑戦(The challenge of developing mitochondrial nano medicine based on MITO-Porter Technology)

  山田 勇磨  日本小児循環器学会雑誌  36-  (Suppl.2)  s2  -103  2020/11
• ミトコンドリア病(Leigh脳症)モデルマウスの心機能の評価とモデルマウス由来心筋前駆細胞のミトコンドリア機能の検討

  佐々木 大輔, 武田 充人, 山田 勇磨, 原島 秀吉  日本小児循環器学会雑誌  36-  (Suppl.2)  s2  -358  2020/11
• ミトコンドリアを標的とする遺伝子治療用RNAナノカプセルの創製

  山田 勇磨, 原島 秀吉  遺伝子医学  10-  (4)  74  -79  2020/10  

  多彩な機能を有するミトコンドリアの機能破綻は様々な疾患を誘発する。これらの疾患の一部は,ミトコンドリアDNA(mtDNA)の変異・欠失が原因であることが報告されている。そのため,ミトコンドリアを標的とした遺伝子治療研究は,革新的医薬品の創製につながると期待されている。本稿では,われわれが創製したミトコンドリア標的型drug delivery system(DDS)"MITO-Porter"を基盤とした「ミトコンドリアを標的とする遺伝子治療用RNAナノカプセル」に関する研究を中心に紹介する。(著者抄録)
• 核酸医薬とナノメディシン 多機能性エンベロープ型ナノ構造体の開発とナノ医療への展開

  原島 秀吉, 佐藤 悠介, 中村 孝司, 山田 勇磨  日本癌学会総会記事  79回-  ML13  -ML13  2020/10
• コエンザイムQ10封入ミトコンドリア標的型DDSの開発およびアセトアミノフェン肝障害モデルへの治療効果の検証

  日比野 光恵, 山田 勇磨, 真栄城 正寿, 渡慶次 学, 石塚 洋一, 原島 秀吉  日本DDS学会学術集会プログラム予稿集  36回-  163  -163  2020/08
• ミトコンドリアDDSを用いたビタミンB1の脳内送達・治療効果の検証

  山田 勇磨, 石丸 拓也, 原島 秀吉  日本DDS学会学術集会プログラム予稿集  36回-  169  -169  2020/08
• ミトコンドリア強化幹細胞(MITO Cell)の製造および細胞移植療法への展開

  山田 勇磨, 阿部 二郎, 佐々木 大輔, 原島 秀吉  BIO Clinica  35-  (5)  448  -450  2020/05  

  心筋幹細胞(Cardiac Progenitor Cell[CP Cell])移植は、心筋症に対する治療法として大規模臨床試験において、その有用性が認められている。一方で、持続的な移植効果維持が困難とされており、移植細胞のミトコンドリア機能不全などが原因として報告されている。我々はこれまでに、酸化ストレス発生源であり細胞内ATP産生を担うミトコンドリアを強化した心筋幹細胞(MITO Cell)の製造を行い、細胞移植療法の治療有効性を報告している。MITO Cellの構築は、MITO-Porter(ミトコンドリア標的型ナノカプセル)を用いてCP Cellのミトコンドリアに機能性分子を送達し構築した。本稿では、ミトコンドリア毒性を有するドキソルビシン心筋症モデルマウスを用いた。MITO Cellの細胞移植療法の心筋症治療の成績向上に関する我々の研究成果を紹介する。(著者抄録)
• MELAS A3243G変異型ミトコンドリアDNAを標的とした遺伝子治療戦略の検証

  山田 勇磨, 宗宮 加奈, 佐々木 大輔, 武田 充人, 原島 秀吉  日本薬剤学会年会講演要旨集  35年会-  169  -169  2020/05
• 【核酸創薬に貢献するバイオマテリアル】バイオマテリアルに基づいた核酸ナノ医療の創製

  山田 勇磨, 中村 孝司, 佐藤 悠介, 原島 秀吉  バイオマテリアル-生体材料-  38-  (2)  92  -99  2020/04  

  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)
• 光増感分子rTPAを搭載したミトコンドリア標的型ナノカプセルの構築および担癌モデルマウスを用いた癌光治療戦略の検証

  山田 勇磨, Satrialdi, 高野 勇太, Biju Vasudevanpillai, 原島 秀吉  日本薬学会年会要旨集  140年会-  26X  -am11  2020/03
• 【ミトコンドリアと疾患・老化 細胞内代謝プラントとしての役割を知り、ミトコンドリアを標的とした創薬に挑む】(第3章)ミトコンドリア疾患の診断技術と治療戦略 治療技術・治療薬開発 ミトコンドリア標的型ナノDDSが創る未来医療

  山田 勇磨, 原島 秀吉  実験医学  37-  (12)  2067  -2073  2019/08  [Not refereed][Not invited]
   

  多彩な機能を有するミトコンドリアへ目的分子を送達するdrug delivery system(DDS)は、医療・ライフサイエンス分野の発展に大きく貢献すると期待されている。本稿では、DDS研究者の立場から、「ミトコンドリアDDS研究の魅力を伝えること」を目標とし、さらに、「ミトコンドリアを標的とする未来医療の可能性」も提示したい。ここでは、ミトコンドリアを標的とするナノDDSについて概説するとともに、われわれが創製したミトコンドリア標的型ナノDDS"MITO-Porter"の開発研究に関して紹介する。(著者抄録)
• 心不全病態に対する多面的アプローチ 基礎から治療へ 虚血再灌流モデルマウスを用いた心不全に対するミトコンドリア活性化心筋幹細胞を用いた細胞移植療法の治療効果の検討

  佐々木 大輔, 阿部 二郎, 武田 充人, 原島 秀吉, 山田 勇磨  日本小児循環器学会雑誌  35-  (Suppl.1)  s1  -125  2019/06
• ミトコンドリアを標的とする遺伝子治療用ナノカプセルの創製

  山田 勇磨  日本核酸医薬学会誌  23-  (1)  12  -21  2019/06  [Not refereed][Not invited]
  
• MITO-Porterを用いた治療用mRNA送達によるミトコンドリア遺伝子治療戦略の検証

  山田 勇磨, 宗宮 加奈, 原島 秀吉  日本薬剤学会年会講演要旨集  34年会-  179  -179  2019/05
• 【小児科医に知ってほしいミトコンドリア病UPDATE】治療の展望 ミトコンドリアDDSが拓く新しいミトコンドリア病治療戦略

  山田 勇磨, 原島 秀吉  小児科診療  82-  (4)  523  -528  2019/04  [Not refereed][Not invited]
   

  ●ミトコンドリアの機能異常は種々の疾患(進行性変性疾患、虚血性疾患、糖尿病、ミトコンドリア遺伝病、など)を誘発することが報告されている。●そのため、ミトコンドリアを標的とした医薬品の開発研究は、新たな疾患治療法を提供する次世代医薬品の創製につながると期待される。●本稿では、筆者らが創製したミトコンドリア標的型drug delivery system(DDS)"MITO-Porter"の開発研究に関して、特に「ミトコンドリアを標的とする遺伝子治療戦略」および「ミトコンドリア強化幹細胞を用いた移植療法」に焦点をあて紹介する。(著者抄録)
• ミトコンドリア標的型DDSを基盤とした革新的ナノ医薬品の創製

  山田 勇磨  ファルマシア  55-  (4)  330  -334  2019/04  [Not refereed][Not invited]
  
• 薬学実務実習前後における薬学生のコミュニケーション分析 RIAS(Roter method of interaction process analysis)を用いて

  武隈 洋, 森 綾子, 小林 正紀, 山田 勇磨, 佐藤 夕紀, 鳴海 克哉, 古堅 彩子, 菅原 満  日本薬学会年会要旨集  139年会-  (4)  212  -212  2019/03  [Not refereed][Not invited]
  
• 実務実習前後における薬学生のRIASによるコミュニケーション分析

  森 綾子, 武隈 洋, 小林正紀, 山田勇磨, 佐藤夕紀, 鳴海克哉, 古堅彩子, 菅原 満  総合技術研究会2017東京大学  2017/03/08  [Not refereed][Not invited]
  
• Research history of mitochondrial drug delivery system

  山田 勇磨  医学のあゆみ  260-  (1)  42  -48  2017/01/07
• 多機能性エンベロープ型ナノ構造体の創世とナノ医療への展開

  佐藤悠介, 山田勇磨, 梶本和昭, 原島秀吉  化学とマイクロ・ナノシステム  14-  (1)  15  -23  2015  [Not refereed][Invited]
  
• 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
• INNOVATIVE TECHNOLOGY FOR IN VIVO MITOCHONDRIAL GENE DELIVERY USING HYDRODYNAMIC LIMB VEIN INJECTION

  Yukari Yasuzaki, Yuma Yamada, Tsutomu Kanefuji, Dexi Liu, Hideyoshi Harashima  JOURNAL OF GENE MEDICINE  16-  (7-8)  275  -275  2014/07  [Not refereed][Not invited]
  
• Development of Nanotechnology for Mitochondrial Drug Delivery

  山田 勇磨, 原島 秀吉  化學工業  65-  (6)  418  -423  2014/06
• PD-1-1 光を応用した移植細胞機能・細胞環境のモニタリングと制御の試み(PD-1 パネルディスカッション(1)細胞・臓器移植における基礎的研究の最前線,第114回日本外科学会定期学術集会)

  尾崎 倫孝, 芳賀 早苗, 野田 なつみ, 森田 直樹, 山田 勇磨, 小澤 岳昌  日本外科学会雑誌  115-  (2)  168  -168  2014/03/05  [Not refereed][Not invited]
  
• 膵島移植とミトコンドリア標的型drug delivery system(DDS)

  山田勇磨, 野村政壽, 原島秀吉  人工臓器(日本人工臓器学会)  41-  (3)  212  -214  2012/12/15  [Not refereed][Not invited]
  
• MITO-Porter; A Cutting-edge Technology for Mitochondrial Gene Therapy

  Ryo Furukawa, Yuma Yamada, Hideyoshi Harashima  YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN  132-  (12)  1389  -1398  2012/12  [Not refereed][Not invited]
   

  Gene therapy is an attractive strategy, for not only targeting nuclear genome, but the mitochondrial genome as well. Human mitochondrial DNA (mtDNA) encodes 13 subunits of the electron transport chain, 22 tRNAs, and 2 rRNAs and their mutations cause a wide range of mitochondrial diseases. Each cell contains hundreds to thousands of mtDNAs, and in the case of a diseased cell, the mitochondrion possesses both mutant mtDNA and wild-type mtDNA. It is generally accepted that the disease phenotype appears when the proportion of the pathogenic mutant mtDNA exceeds a certain threshold. Therefore, the suppression of mutant mtDNA or supplementing wild-type mtDNA will control the onset of mitochondrial disease. To achieve the transfection of an exogenous therapeutic gene to the mitochondrial matrix where mtDNA is transcribed and translated, it is necessary to transfer cargos through mitochondrial outer and inner membranes. Several methods have been examined for mitochondrial transfection, but a universal, wide-ranging transfection technique has yet not been established. We recently developed a mitochondrial targeting delivery system, namely the MITO-Porter. The MITO-Porter is liposomal nanocarrier with a mitochondrial fusogenic lipid composition. We reported that the MITO-Porter could deliver chemical compounds and proteins to the mitochondrial matrix via membrane fusion. In this review, we report (1) on the pharmacological enhancement of lecithinized superoxide dismutase (PC-SOD) using MITO-Porter, (2) the transcription activation of exogenous DNA by mitochondrial transcription factor A (TFAM), and (3) perspectives on a mitochondrial targeting device.
• 医療現場で新しい薬剤師職能を開拓する活動(病院オペ室での活動)と教員としての研究教育活動

  小林正紀, 山田勇磨, 佐藤夕紀, 井関健  日本医療薬学会年会講演要旨集  22nd-  221  -221  2012/10/10  [Not refereed][Not invited]
  
• Mitochondrial-targeted DNA delivery using a DF-MITO-Porter, an innovative nano carrier with cytoplasmic and mitochondrial fusogenic envelopes

  Yuma Yamada, Eriko Kawamura, Hideyoshi Harashima  JOURNAL OF NANOPARTICLE RESEARCH  14-  (8)  2012/08  [Not refereed][Not invited]
   

  Mitochondrial gene therapy has the potential for curing a variety of diseases that are associated with mitochondrial DNA mutations and/or defects. To achieve this, it will be necessary to deliver therapeutic agents into the mitochondria in diseased cells. A number of mitochondrial drug delivery systems have been reported to date. However, reports of mitochondrial-targeted DNA delivery are limited. To achieve this, the therapeutic agent must be taken up by the cell (1), after which, the multi-processes associated with intracellular trafficking must be sophisticatedly regulated so as to release the agent from the endosome and deliver it to the cytosol (2) and to pass through the mitochondrial membrane (3). We report herein on the mitochondrial delivery of oligo DNA as a model therapeutic using a Dual Function (DF)-MITO-Porter, an innovative nano carrier designed for mitochondrial delivery. The critical structural elements of the DF-MITO-Porter include mitochondria-fusogenic inner envelopes and endosome-fusogenic outer envelopes, modified with octaarginine which greatly assists in cellular uptake. Inside the cell, the carrier passes through the endosomal and mitochondrial membranes via step-wise membrane fusion. When the oligo DNA was packaged in the DF-MITO-Porter, cellular uptake efficiency was strongly enhanced. Intracellular observation using confocal laser scanning microscopy showed that the DF-MITO-Porter was effectively released from endosomes. Moreover, the findings confirmed that the mitochondrial targeting activity of the DF-MITO-Porter was significantly higher than that of a carrier without outer endosome-fusogenic envelopes. These results support the conclusion that mitochondrial-targeted DNA delivery using a DF-MITO-Porter can be achieved when intracellular trafficking is optimally regulated.
• Mitochondrial-targeted DNA delivery using a DF-MITO-Porter, an innovative nano carrier with cytoplasmic and mitochondrial fusogenic envelopes

  Yuma Yamada, Eriko Kawamura, Hideyoshi Harashima  JOURNAL OF NANOPARTICLE RESEARCH  14-  (8)  1  -15  2012/08  [Not refereed][Not invited]
   

  Mitochondrial gene therapy has the potential for curing a variety of diseases that are associated with mitochondrial DNA mutations and/or defects. To achieve this, it will be necessary to deliver therapeutic agents into the mitochondria in diseased cells. A number of mitochondrial drug delivery systems have been reported to date. However, reports of mitochondrial-targeted DNA delivery are limited. To achieve this, the therapeutic agent must be taken up by the cell (1), after which, the multi-processes associated with intracellular trafficking must be sophisticatedly regulated so as to release the agent from the endosome and deliver it to the cytosol (2) and to pass through the mitochondrial membrane (3). We report herein on the mitochondrial delivery of oligo DNA as a model therapeutic using a Dual Function (DF)-MITO-Porter, an innovative nano carrier designed for mitochondrial delivery. The critical structural elements of the DF-MITO-Porter include mitochondria-fusogenic inner envelopes and endosome-fusogenic outer envelopes, modified with octaarginine which greatly assists in cellular uptake. Inside the cell, the carrier passes through the endosomal and mitochondrial membranes via step-wise membrane fusion. When the oligo DNA was packaged in the DF-MITO-Porter, cellular uptake efficiency was strongly enhanced. Intracellular observation using confocal laser scanning microscopy showed that the DF-MITO-Porter was effectively released from endosomes. Moreover, the findings confirmed that the mitochondrial targeting activity of the DF-MITO-Porter was significantly higher than that of a carrier without outer endosome-fusogenic envelopes. These results support the conclusion that mitochondrial-targeted DNA delivery using a DF-MITO-Porter can be achieved when intracellular trafficking is optimally regulated.
• 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
• ミトコンドリア標的型核酸デリバリーシステムの開発

  古川亮, 山田勇磨, 原島秀吉  日本DDS学会学術集会プログラム予稿集  28th-  134  2012/06/05  [Not refereed][Not invited]
  
• 骨格筋ミトコンドリアを標的とした外来遺伝子送達技術の開発

  安崎友香理, 山田勇磨, 兼藤努, LIU Dexi, 原島秀吉  日本DDS学会学術集会プログラム予稿集  28th-  119  2012/06/05  [Not refereed][Not invited]
  
• Post-nuclear gene delivery events for transgene expression by biocleavable polyrotaxanes

  Yuma Yamada, Taku Nomura, Hideyoshi Harashima, Atsushi Yamashita, Nobuhiko Yui  BIOMATERIALS  33-  (15)  3952  -3958  2012/05  [Not refereed][Not invited]
   

  A quantitative comparison between nuclear DNA release from carriers and their transfection activity would be highly useful for improving the effectiveness of non-viral gene vectors. We previously reported that, for condensed DNA particles, a close relationship exists between the efficiency of DNA release and transfection activity, when biocleavable polyrotaxanes (DMAE-SS-PRX), in which the cationic density can be easily controlled. In this study, we first investigated the efficiencies of DNA release from condensed DNA particles with various types of DMAE-SS-PRX. The findings indicate that an optimal cationic density in DMAE-SS-PRX exists for DNA release. We then packaged condensed DNA particles in a multifunctional envelope-type nano device (MEND), and evaluated their transfection activities. The results showed that the transfection activity was increased and this increase was, to some extent, dependent on the efficiency of the DNA release. However, transfection activity decreased, when the value for the efficiency of DNA release was higher than a certain value. An investigation of the fate of intranuclear DNA indicated that a very high efficiency of DNA release has a positive influence on transcription, however, it would inhibit the post-transcription process; nuclear mRNA export, translation and related processes. Such information provides a new viewpoint for the development of cationic polymer-based vectors. (C) 2012 Elsevier Ltd. All rights reserved.
• Different Mechanisms for Nanoparticle Formation between pDNA and siRNA Using Polyrotaxane as the Polycation

  Yuma Yamada, Masahiro Hashida, Taku Nomura, Hideyoshi Harashima, Yuichi Yamasaki, Kazunori Kataoka, Atsushi Yamashita, Ryo Katoono, Nobuhiko Yui  CHEMPHYSCHEM  13-  (5)  1161  -1165  2012/04  [Not refereed][Not invited]
  
• 手術部サテライトファーマシーにおける麻酔薬の無菌混合調製体制の確立

  山崎浩二郎, 須田範行, 新沼朋美, 水口貴史, 小林正紀, 山田勇磨, 山下美妃, 柴山良彦, 山田武宏, 井関健, 井関健  日本薬学会年会要旨集  132nd-  (4)  218  -218  2012/03/05  [Not refereed][Not invited]
  
• ミトコンドリアを標的とした遺伝子治療に向けて

  古川亮, 山田勇磨, 原島秀吉  日本薬学会年会要旨集  132nd-  (1)  246  2012/03/05  [Not refereed][Not invited]
  
• ミトコンドリア標的型DDSが拓くミトコンドリア創薬への道

  山田勇磨  日本薬学会年会要旨集  132nd-  (1)  158  2012/03/05  [Not refereed][Not invited]
  
• ボンクレキン酸搭載MITO‐Porterの構築およびミトコンドリア送達・抗アポトーシス効果の検証

  中村宏平, 山田勇磨, 古川亮, 河村恵理子, 奥田勝博, 新藤充, 原島秀吉  日本薬学会年会要旨集  132nd-  (4)  209  2012/03/05  [Not refereed][Not invited]
  
• Targeting Mitochondria: Innovation from Mitochondrial Drug Delivery System (DDS) to Mitochondrial Medicine

  Yamada Yuma, Harashima Hideyoshi  YAKUGAKU ZASSHI  132-  (10)  1111  -1118  2012  [Not refereed][Not invited]
   

  Mitochondrial dysfunction has been implicated in a variety of human diseases, including cancer and neurodegenerative disorders. Effective medical therapies for such diseases will ultimately require the targeted delivery of therapeutic agents to mitochondria. This will likely be achieved through innovations in the areas of the nanotechnology of intracellular trafficking. Mitochondrial delivery systems for a variety of cargoes have been repored to date. However, only a limited number of approaches are available for delivering macromolecules directly to mitochondria. We previously reported o...
• Drug Delivery System and Mitochondria

  YAMADA Yuma  薬剤学 = The archives of practical pharmacy  72-  (1)  53  -57  2012/01/01  [Not refereed][Not invited]
  
• Dual Function MITO-Porter, a Nano Carrier Integrating Both Efficient Cytoplasmic Delivery and Mitochondrial Macromolecule Delivery

  Yuma Yamada, Ryo Furukawa, Yukari Yasuzaki, Hideyoshi Harashima  MOLECULAR THERAPY  19-  (8)  1449  -1456  2011/08  [Not refereed][Not invited]
   

  Mitochondrial dysfunction is associated with a variety of human diseases including inherited mitochondrial diseases, neurodegenerative disorders, diabetes mellitus, and cancer. Effective medical therapies for mitochondrial diseases will ultimately require an optimal drug delivery system, which will likely be achieved through innovations in the nanotechnology of intracellular trafficking. To achieve efficient mitochondrial drug delivery, two independent processes, i.e., "cytoplasmic delivery through the cell membrane" and "mitochondrial delivery through the mitochondrial membrane" are required. In previous studies, we developed an octaarginine (R8) modified nano carrier for efficient cytoplasmic delivery, showing that R8-modified liposomes were internalized into cells efficiently. On the other hand, we also constructed MITO-Porter for the mitochondrial delivery of macromolecules, a liposome-based carrier that delivers cargos to mitochondria via membrane fusion. Here, we report the development of a dual function MITO-Porter (DF-MITO-Porter), based on the concept of integrating both R8-modified liposomes and MITO-Porter. We show that the DF-MITO-Porter effectively delivers exogenous macro-biomolecules into the mitochondrial matrix, and provide a demonstration of its potential use in therapies aimed at mitochondrial DNA.
• ミトコンドリア転写因子TFAM‐プラスミドDNA複合体のin vitro転写評価

  古川亮, 山田勇磨, 松島雄一, 後藤雄一, 原島秀吉  Drug Deliv Syst  26-  (3)  317  2011/05/28  [Not refereed][Not invited]
  
• ミトコンドリア遺伝子を標的とした機能分子送達の試み

  山田勇磨, 原島秀吉  Drug Deliv Syst  26-  (3)  285  2011/05/28  [Not refereed][Not invited]
  
• ミトコンドリア(Mt)を標的とした遺伝子治療への挑戦―Dual Function MITO‐Porterを用いたMtマトリクスへの核酸送達―

  山田勇磨, 河村恵理子, 安崎友香理, 古川亮, 原島秀吉  日本薬剤学会年会講演要旨集  26th-  102  2011/05/01  [Not refereed][Not invited]
  
• 新たなパッケージングコンセプトに基づく多機能性エンベロープ型ナノ構造体の創製

  山田勇磨, 鈴木亮佑, 原島秀吉  日本薬学会年会要旨集  131st-  (4)  159  2011/03/05  [Not refereed][Not invited]
  
• ミトコンドリア標的型DDS,MITO‐Porterを用いたオリゴ核酸送達の試み

  河村恵理子, 山田勇磨, 原島秀吉  日本薬学会年会要旨集  131st-  (4)  159  2011/03/05  [Not refereed][Not invited]
  
• AN INNOVATIVE APPROACH FOR MITOCHONDRIAL GENE DELIVERY: MITOCHONDRIAL DELIVERY OF OLIGO DNA USING MITO-PORTER, A LIPOSOME-BASED CARRIER FOR MITOCHONDRIAL DELIVERY VIA MEMBRANE FUSION

  KAWAMURA Eriko, YAMADA Yuma, HARASHIMA Hideyoshi  日本ミトコンドリア学会年会要旨集  11th-  178  2011  [Not refereed][Not invited]
  
• TECHNICAL APPLICATION OF HYDRODYNAMIC LIMB VEIN INJECTION FOR IN VIVO MITOCHONDRIAL GENE DELIVERY

  YASUZAKI Yukari, YAMADA Yuma, KANEFUJI Tsutomu, LIU Dexi, HARASHIMA Hideyoshi  日本ミトコンドリア学会年会要旨集  11th-  170  2011  [Not refereed][Not invited]
  
• EFFECT OF DNA PACKAGED WITH TFAM ON TRANSCRIPTION ACTIVATION

  FURUKAWA Ryo, YAMADA Yuma, MATSUSHIMA Yuichi, GOTO Yu‐ichi, HARASHIMA Hideyoshi  日本ミトコンドリア学会年会要旨集  11th-  179  2011  [Not refereed][Not invited]
  
• MITOCHONDRIAL GENOME TARGETING STRATEGY: MITOCHONDRIAL MATRIX DELIVERY OF BIO-MACROMOLECULE USING DF-MITO-PORTER, AN INNOVATIVE NANO CARRIER WITH CYTOPLASMIC AND MITOCHONDRIAL FUSOGENIC ENVELOPES.

  YAMADA Yuma, HARASHIMA Hideyoshi  日本ミトコンドリア学会年会要旨集  11th-  149  2011  [Not refereed][Not invited]
  
• Intranuclear DNA Release Is a Determinant of Transfection Activity for a Non-viral Vector: Biocleavable Polyrotaxane as a Supramolecularly Dissociative Condenser for Efficient Intranuclear DNA Release

  Yuma Yamada, Taku Nomura, Hideyoshi Harashima, Atsushi Yamashita, Ryo Katoono, Nobuhiko Yui  BIOLOGICAL & PHARMACEUTICAL BULLETIN  33-  (7)  1218  -1222  2010/07  [Not refereed][Not invited]
   

  It has been believed that nuclear gene delivery is the most important process for gene expression, and various non-viral vectors are currently being developed with this assumption. However, some of our earlier studies revealed a surprising difference in transfection activity between viral and non-viral vectors: this difference is largely due to the result of the intranuclear disposition of DNA rather than its delivery to the nucleus (Hama S. et al. (2006), Quantitative comparison of intracellular trafficking and nuclear transcription between adenoviral and lipoplex systems. Mol. Then, 13, 786-794). Here; we report on some direct evidence that demonstrates the importance of the release of intranuclear DNA on transfection activity. The data show that transfection activity can be substantially enhanced by integrating a multifunctional envelope-type nano device (MEND) and a biocleavable polyrotaxane (DMAE-SS-PRX) as an artificial condenser. Our integration system showed significantly higher transfection activity compared to conventional gene delivery system. Moreover, this system provides a strong support for our hypothesis that intranuclear DNA disposition plays a critical role in gene expression for non-viral vectors.
• ボンクレキン酸(抗アポトーシス作動薬)搭載ミトコンドリア標的型DDS(MITO‐Porter)の開発

  山田勇磨, 河村恵理子, 安崎友香理, 新藤充, 原島秀吉  Drug Deliv Syst  25-  (3)  307  2010/05/28  [Not refereed][Not invited]
  
• DDS技術を基盤とした生細胞ミトコンドリアDNAの可視化

  安崎友香理, 山田勇磨, 原島秀吉  薬剤学  70-  (Supplement)  104  2010/04/20  [Not refereed][Not invited]
  
• 効率的な細胞内導入を目的としたレシチン化SOD(PC‐SOD)搭載R8リポソームの構築およびその機能評価

  古川亮, 山田勇磨, 武永美津子, 五十嵐理慧, 原島秀吉  薬剤学  70-  (Supplement)  103  2010/04/20  [Not refereed][Not invited]
  
• プラスミド‐ヒストン複合体からの外来遺伝子発現効率

  紙谷浩之, 紙谷浩之, 後藤仁美, 後藤仁美, 神田元紀, 神田元紀, 山田勇磨, 山田勇磨, 原島秀吉, 原島秀吉  薬剤学  70-  (Supplement)  148  -148  2010/04/20  [Not refereed][Not invited]
  
• 革新的遺伝子発現ベクター『ポリロタキサン/pDNAナノ粒子搭載型MEND』の構築および核内動態・遺伝子発現活性評価

  山田勇磨, 野村卓, 原島秀吉, 山下敦, 上遠野亮, 由井信彦  日本薬学会年会要旨集  130th-  (4)  198  2010/03/05  [Not refereed][Not invited]
  
• PC‐SOD搭載型リポソームの構築および細胞内動態・抗酸化効果の評価

  古川亮, 山田勇磨, 武永美津子, 五十嵐理慧, 原島秀吉  日本薬学会年会要旨集  130th-  (4)  198  2010/03/05  [Not refereed][Not invited]
  
• 生分解性ポリロタキサンを用いたpDNA,siRNAのナノ粒子の調製および核酸ナノ粒子形成機構に関する検討

  橋田真裕, 山田勇磨, 原島秀吉, 山下敦, 上遠野亮, 由井信彦  日本薬学会年会要旨集  130th-  (4)  198  2010/03/05  [Not refereed][Not invited]
  
• 北海道大学薬学部における実務実習事前実習の取り組みとその評価

  武隈洋, 小林正紀, 山田勇磨, 板垣史郎, 吉田和幸, 井関健, 菅原満  日本薬学会年会要旨集  130年会-  (4)  346  -346  2010/03  [Not refereed][Not invited]
  
• 生細胞ミトコンドリアDNAセンシング技術の開発

  安崎友香理, 山田勇磨, 原島秀吉  遺伝子・デリバリー研究会シンポジウム要旨集  10th-  P.38  2010  [Not refereed][Not invited]
  
• 効率的な遺伝子送達を実現する非ウィルス性ベクターの創製

  鈴木亮佑, 山田勇磨, 原島秀吉  遺伝子・デリバリー研究会シンポジウム要旨集  10th-  P.33  2010  [Not refereed][Not invited]
  
• MgO,Al₂O₃/Mg₂Si複合材料の組織におけるSiO₂,Al₂O₃粉末粒径の影響

  山田勇磨, 小橋眞, 金武直幸  日本金属学会講演概要  145th-  296  2009/09/15  [Not refereed][Not invited]
  
• Mono‐cationic detergentによるsiRNAナノ粒子パッケージング法の構築

  鈴木亮佑, 山田勇磨, 原島秀吉  Drug Deliv Syst  24-  (3)  326  2009/06/09  [Not refereed][Not invited]
  
• 細胞内分解性ポリロタキサン搭載多機能性エンベロープ型ナノ構造体の構築

  野村卓, 山田勇磨, 原島秀吉, 山下敦, 上遠野亮, 由井伸彦  Drug Deliv Syst  24-  (3)  326  2009/06/09  [Not refereed][Not invited]
  
• ミトコンドリア標的型ナノデバイス“多重型MITO‐Porter”の製剤化に関する検討

  山田勇磨, 古川亮, 安崎友香理, 原島秀吉  Drug Deliv Syst  24-  (3)  302  2009/06/09  [Not refereed][Not invited]
  
• 核内pDNA放出制御によるトランスフェクション活性の向上

  山田勇磨, 野村卓, 原島秀吉, 山下敦, 上遠野亮, 由井伸彦  高分子学会予稿集(CD-ROM)  58-  (1 Disk1)  1PG147  2009/05/12  [Not refereed][Not invited]
  
• Development of Small, Homogenous Non-Viral Gene Vector Using Mono-Cationic Detergents

  Ryosuke Suzuki, Yuma Yamada, Hideyoshi Harashima  MOLECULAR THERAPY  17-  S52  -S52  2009/05  [Not refereed][Not invited]
  
• ミトコンドリア標的型ナノデバイス“多重型MITO‐Porter”の有用性の検証

  山田勇磨, 古川亮, 安崎友香理, 原島秀吉  薬剤学  69-  (Supplement)  150  2009/04/30  [Not refereed][Not invited]
  
• 効率的な核内遺伝子放出を可能とする多機能性エンベロープ型ナノ構造体の創製

  山田勇磨, 野村卓, 原島秀吉, 山下敦, 上遠野亮, 由井伸彦  日本薬学会年会要旨集  129th-  (4)  177  2009/03/05  [Not refereed][Not invited]
  
• 細胞内分解性ポリロタキサンの構造因子が与える遺伝子送達過程への影響

  山下敦, 上遠野亮, 由井伸彦, 山田勇磨, 秋田英万, 原島秀吉  遺伝子・デリバリー研究会シンポジウム要旨集  9th-  P.24  2009  [Not refereed][Not invited]
  
• ミトコンドリア標的型ナノデバイス“多重型MITO‐Porter”の創製

  山田勇磨, 古川亮, 安崎友香理, 原島秀吉  日本ミトコンドリア学会年会要旨集  9th-  86  2009  [Not refereed][Not invited]
  
• 多重型MITO‐Porterによるミトコンドリアを標的とした薬物送達戦略

  山田勇磨, 秋田英万, 紙谷浩之, 原島秀吉  Drug Deliv Syst  23-  (3)  374  2008/06/06  [Not refereed][Not invited]
  
• Development of small, homogeneous pDNA particles condensed with mono-cationic detergents and encapsulated in a multifunctional envelope-type nano device

  Ryosuke Suzuki, Yuma Yamada, Hideyoshi Harashima  BIOLOGICAL & PHARMACEUTICAL BULLETIN  31-  (6)  1237  -1243  2008/06  [Not refereed][Not invited]
   

  Non-viral DNA vectors are promising gene delivery systems and a variety of non-viral DNA vectors have been developed to date. Recently, we developed a novel non-viral gene delivery system-multifunctional envelope-type nano device (MEND). The MEND system has high transfection activity, similar to that of adenovirus vector, which is a potent viral vector. However, conventional MEND is relatively large and heterogeneous (approximately 300 nm), probably because they contain relatively large- and heterogeneous-pDNA particles condensed with polycations, such as poly-L-lysine. Small particle size is important for in vivo delivery, because large particles are rapidly eliminated from systemic circulation. Moreover, heterogeneous size of drug carriers is difficult to apply to clinical applications. Here, we describe construction of small homogeneous MEND. First, we screened mono-cationic detergents (MCD(s)) to obtain optimal pDNA condensed particles. We determined that benzyldimethylhexadecylammonium chloride (BDHAC) and thonzonium bromide (TB) were optimal pDNA condensers. Next, we packaged the condensed pDNA particles into a lipid bi-layer. The resulting lipid-encapsulated pDNA particles were then equipped with octaarginine to facilitate cell-uptake (R8-MEND (MCD)). The carrier showed high transfection activity in cultured HeLa cells. Furthermore, the R8-MEND (MCD) were small and homogeneous compared with conventional MEND. These results indicate that R8-MEND (MCD) has potential as a novel non-viral delivery system for clinical application.
• 効率的な遺伝子送達を目指した1遺伝子パッケージング法の構築

  鈴木亮佑, 山田勇磨, 原島秀吉  薬剤学  68-  (Supplement)  194  2008/04/30  [Not refereed][Not invited]
  
• MITO-Porter: A liposome-based carrier system for delivery of macromolecules into mitochondria via membrane fusion

  Yuma Yamada, Hidetaka Akita, Hiroyuki Kamiya, Kentaro Kogure, Takenori Yamamoto, Yasuo Shinohara, Kikuji Yamashita, Hideo Kobayashi, Hiroshi Kikuchi, Hideyoshi Harashima  BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES  1778-  (2)  423  -432  2008/02  [Not refereed][Not invited]
   

  Mitochondria are the principal producers of energy in higher cells. Mitochondrial dysfunction is implicated in a variety of human diseases, including cancer and neurodegenerative disorders. Effective medical therapies for such diseases will ultimately require targeted delivery of therapeutic proteins or nucleic acids to the mitochondria, which will be achieved through innovations in the nanotechnology of intracellular trafficking. Here we describe a liposome-based carrier that delivers its macromolecular cargo to the mitochondrial interior via membrane fusion. These liposome particles, which we call MITO-Porters, carry octaarginine surface modifications to stimulate their entry into cells as intact vesicles (via macropinocytosis). We identified lipid compositions for the MITO-Porter which promote both its fusion with the mitochondrial membrane and the release of its cargo to the intra-mitochondrial compartment in living cells. Thus, the MITO-Porter holds promise as an efficacious system for the delivery of both large and small therapeutic molecules into mitochondria. (c) 2007 Elsevier B.V All rights reserved.
• Development of Multifunctional Envelope Type Artificial Viral like Gene Delivery System

  HARASHIMA Hideyoshi, KOGURE Kentaro, YAMADA Yuma, AKITA Hidetaka, KAMIYA Hiroyuki  Journal of the Pharmaceutical Society of Japan  127-  (10)  1655  -1672  2007/10/01  [Not refereed][Not invited]
   

  This review introduces a new concept "Programmed Packaging" to develop a non-viral gene delivery system. Based on this concept, multifunctional envelope type nano devices (MEND) were developed for in vitro, in situ and in vivo conditions. A quantitative study to identify a rate limiting step in intracellular trafficking was also shown between viral and non-viral vectors, which indicated an important role of controlled intranuclear disposition for development a safe and efficient non-viral gene delivery system. This review will provide a future direction of non-viral gene delivery system.
• 多機能性エンベロープ型ナノ構造体による人工遺伝子デリバリーシステムの創製 第7回 日本DDS学会永井賞受賞によせて

  原島秀吉, 小暮健太朗, 秋田英万, 山田勇磨, 紙谷浩之  Drug Deliv Syst  22-  (5)  569  -577  2007/09/26  [Not refereed][Not invited]
  
• Development of in vivo gene delivery system by multifunctional envelope-type nano device : Awarded with Nagai Prize in 7th Annual Meeting of the Japan Society of Drug Delivery System

  HARASHIMA Hideyoshi, KOGURE Kentaro, AKITA Hidetaka, YAMADA Yuma, KAMIYA Hiroyuki  Drug delivery system  22-  (5)  569  -577  2007/06/10  [Not refereed][Not invited]
  
• 細胞内FRET法を用いたMITO‐Porter(ミトコンドリア膜融性リポソーム)の膜融合能評価

  山田勇磨, 秋田英万, 紙谷浩之, 小暮健太朗, 篠原康雄, 原島秀吉  Drug Deliv Syst  22-  (3)  315  2007/05/30  [Not refereed][Not invited]
  
• 多機能性エンベロープ型ナノ構造体(MEND)を用いたタンパク質の効率的な細胞質送達

  鈴木亮佑, 山田勇磨, 原島秀吉  薬剤学  67-  (Supplement)  280  2007/05/10  [Not refereed][Not invited]
  
• 多機能性エンベロープ型ナノ構造体(MEND)を用いたタンパク質の効率的な細胞質送達

  鈴木 亮佑, 山田 勇磨, 原島 秀吉  薬剤学 = Journal of Pharmaceutical Science and Technology, Japan  67-  (0)  2007/05/10  [Not refereed][Not invited]
  
• Efficient cytoplasmic protein delivery by means of a multifunctional envelope-type nano device

  Ryosuke Suzuki, Yuma Yamada, Hdeyoshi Harashima  BIOLOGICAL & PHARMACEUTICAL BULLETIN  30-  (4)  758  -762  2007/04  [Not refereed][Not invited]
   

  The potential for protein therapy, such as the use of antibodies, and vaccines is now well accepted. However, it is difficult to enhance efficiency in protein therapy without a suitable delivery system for delivering proteins to target sites. Here we describe the development of protein delivery system, which is capable of cytoplasmic delivery as well as efficient packaging. The multifunctional envelope-type nano device (MEND), which was originally developed for the delivery of nucleic acids such as plasmid DNA and oligodeoxynucleotides, can also be applied to protein delivery. In this study, the green fluorescent protein (GFP), a model protein, was condensed with stearyl octaarginine (stearyl R8) to form a nano particle, which was then coated with a lipid membrane, thus permitting R8 to be introduced for efficient cellular uptake and controlled intracellular trafficking. The packaging efficiency of the MEND was significantly higher than that of conventional liposomes, because the GFP can be encapsulated a condensed form. According to confocal laser scanning microscopy, the MEND is internalized efficiently and escapes from the acidic compartment to efficiently release GFP into the cytosol. These results indicate that the MEND can serve as a useful cytoplasmic delivery system for protein therapy.
• Efficient cytoplasmic protein delivery by means of a multifunctional envelope-type nano device

  Ryosuke Suzuki, Yuma Yamada, Hdeyoshi Harashima  BIOLOGICAL & PHARMACEUTICAL BULLETIN  30-  (4)  758  -762  2007/04  [Not refereed][Not invited]
   

  The potential for protein therapy, such as the use of antibodies, and vaccines is now well accepted. However, it is difficult to enhance efficiency in protein therapy without a suitable delivery system for delivering proteins to target sites. Here we describe the development of protein delivery system, which is capable of cytoplasmic delivery as well as efficient packaging. The multifunctional envelope-type nano device (MEND), which was originally developed for the delivery of nucleic acids such as plasmid DNA and oligodeoxynucleotides, can also be applied to protein delivery. In this study, the green fluorescent protein (GFP), a model protein, was condensed with stearyl octaarginine (stearyl R8) to form a nano particle, which was then coated with a lipid membrane, thus permitting R8 to be introduced for efficient cellular uptake and controlled intracellular trafficking. The packaging efficiency of the MEND was significantly higher than that of conventional liposomes, because the GFP can be encapsulated a condensed form. According to confocal laser scanning microscopy, the MEND is internalized efficiently and escapes from the acidic compartment to efficiently release GFP into the cytosol. These results indicate that the MEND can serve as a useful cytoplasmic delivery system for protein therapy.
• ミトコンドリア選択的な高分子薬物送達を可能とする薬物送達システムの開発

  山田勇磨, 秋田英万, 紙谷浩之, 小暮健太朗, 篠原康雄, 小林英夫, 菊池寛, 原島秀吉  日本薬学会年会要旨集  127th-  (1)  284  2007/03/05  [Not refereed][Not invited]
  
• タンパク質を効率的に細胞質へ送達するナノ構造体の開発

  鈴木亮佑, 山田勇磨, 原島秀吉  日本薬学会年会要旨集  127th-  (3)  87  2007/03/05  [Not refereed][Not invited]
  
• Mitochondrial drug delivery and mitochondrial disease therapy - An approach to liposome-based delivery targeted to mitochondria

  Yuma Yamada, Hidetaka Akita, Kentaro Kogure, Hiroyuki Kamiya, Hideyoshi Harashima  Mitochondrion  7-  (1-2)  63  -71  2007/02  [Not refereed][Not invited]
   

  Recent progress in genetics and molecular biology has provided useful information regarding the molecular mechanisms associated with the mitochondrial diseases. Genetic approaches were initiated in the late 1980s to clarify the gene responsible for various mitochondrial diseases, and information concerning genetic mutations is currently used in the diagnosis of mitochondrial diseases. Moreover, it was also revealed that mitochondria play a central role in apoptosis, or programmed cell death, which is closely related to the loss of physiological functions of tissues. Therefore, drug therapies targeted to the mitochondria would be highly desirable. In spite of the huge amount of mechanism-based studies of mitochondrial diseases, effective therapies have not yet been established mainly because of the lack of an adequate delivery system. To date, numerous investigators have attempted to establish a mitochondrial drug delivery system. However, many problems remain to be overcome before a clinical application can be achieved. To fulfill a drug delivery targeted to mitochondria, we first need to establish a method to encapsulate various drugs, proteins, peptides, and genes into a drug carrier depending on their physical characteristics. Second, we need to target it to a specific cell. Finally, multi-processes of intracellular trafficking should be sophisticatedly regulated so as to release a drug carrier from the endosome to the cytosol, and thereafter to deliver to the mitochondria. In this review, we describe the current state of the development of mitochondrial drug delivery systems, and discuss the advantage and disadvantage of each system. Our current efforts to develop an efficient method for the packaging of macromolecules and regulating intracellular trafficking are also summarized. Furthermore, novel concept of "Regulation of intramitochondrial trafficking" is proposed herein as a future challenge to the development of a mitochondrial drug delivery system. © 2007 Mitochondria Research Society.
• Effective drug delivery toward mitochondria by combination of mitochondrial membrane fusion system and mitochondrial targeting signal peptide

  YAMADA Yuma, AKITA Hidetaka, KAMIYA Hiroyuki, KOGURE Kentaro, SHINOHARA Yasuo, KOBAYASHI Hideo, KIKUCHI Hiroshi, HARASHIMA Hideyoshi  日本ミトコンドリア学会年会要旨集  6th-  31  2006/12/13  [Not refereed][Not invited]
  
• Report on The First FIP-APSTJ Joint Workshop on Gene Delivery

  YAMADA Yuma, HATAKEYAMA Hiroto  薬剤学 = Journal of Pharmaceutical Science and Technology, Japan  66-  (6)  451  -453  2006/11/01  [Not refereed][Not invited]
  
• 膜融合を介してミトコンドリアへの高分子薬物送達を可能とする,“MITO‐Porter”の細胞内での膜融合能評価

  山田勇磨, 秋田英万, 紙谷浩之, 小暮健太朗, 山本武範, 篠原康雄, 山下菊治, 小林英夫, 菊池寛, 原島秀吉  Drug Deliv Syst  21-  (3)  298  2006/06/20  [Not refereed][Not invited]
  
• オリゴデオキシヌクレオチドの機能向上を目指した多機能性エンベロープ型ナノ構造体への封入

  小暮健太朗, 小暮健太朗, 中村宣央, 中村宣央, 山田勇磨, 山田勇磨, 原島秀吉, 原島秀吉  日本薬学会年会要旨集  126th-  (2)  138  2006/03/06  [Not refereed][Not invited]
  
• 核膜融合に基づく新規核内送達デバイスの構築

  工藤亜沙子, 秋田英万, 増田智也, 山田勇磨, 小暮健太朗, 箕浦ありさ, 原島秀吉  薬剤学  66-  (Supplement)  113  2006/02/24  [Not refereed][Not invited]
  
• オリゴDNAの多機能性エンベロープ型ナノ構造体へのパッケージングの確立とその機能評価

  中村宜央, 小暮健太朗, 山田勇磨, 原島秀吉  薬剤学  66-  (Supplement)  114  2006/02/24  [Not refereed][Not invited]
  
• ミトコンドリアを標的とする膜融合性リポソーム,MITO‐Porterを用いた生細胞ミトコンドリアへの高分子薬物送達―共焦点レーザースキャン顕微鏡を用いた細胞内動態観察―

  山田勇磨, 秋田英万, 紙谷浩之, 小暮健太朗, 篠原康雄, 小林英夫, 菊池寛, 原島秀吉  薬剤学  66-  (Supplement)  109  2006/02/24  [Not refereed][Not invited]
  
• Development of efficient packaging method of oligodeoxynucleotides by a condensed nano particle in lipid envelope structure

  Y Yamada, K Kogure, Y Nakamura, K Inoue, H Akita, F Nagatsugi, S Sasaki, T Suhara, H Harashima  BIOLOGICAL & PHARMACEUTICAL BULLETIN  28-  (10)  1939  -1942  2005/10  [Not refereed][Not invited]
   

  An efficient delivery system is required if antisense oligodeoxynucleotides (ODN) are to be utilized for gene therapy. We report herein on the development of a novel ODN delivery system, ODN-encapsulated nano particles (ODN-ENP) using an efficient and simple packaging method. The ODN-ENP consists of a condensed ODN particle and a lipid envelope, which can be equipped with various functional devices for the efficient delivery of ODN with a small diameter (150 nm). The encapsulation efficiency and ODN recovery of ODN-ENP were significantly higher than those of other packaging methods, such as a stabilized antisense-lipid particles method or a freezethaw method. Furthermore, the time required for the preparation of the ODN-ENP was shorter than the other methods. The method developed in this study is a simple and efficient packaging method for ODN with a condensed nano particle in lipid-envelope structure.
• 膜融合を介してミトコンドリアへの送達を可能とするリポソーム,“Mitosome”の開発

  山田勇磨, 秋田英万, 小暮健太朗, 紙谷浩之, 篠原康雄, 小林英夫, 菊池寛, 原島秀吉  Drug Deliv Syst  20-  (3)  312  2005/07/06  [Not refereed][Not invited]
  
• 外来DNAによる転写・翻訳効率の新規定量的評価法

  山田勇磨, 紙谷浩之, 原島秀吉  日本薬学会年会要旨集  125th-  (2)  147  2005/03/05  [Not refereed][Not invited]
  
• MITO-Porter, a new-type carrier for mitochondrial macromolecules delivery system via a membrane fusion mechanism

  YAMADA Yuma, AKITA Hidetaka, KAMIYA Hiroyuki, KOGURE Kentaro, SHINOHARA Yasuo, KOBAYASHI Hideo, KIKUCHI Hiroshi, HARASHIMA Hideyoshi  日本ミトコンドリア学会年会要旨集  5th-  69  2005  [Not refereed][Not invited]
  
• 直接導入法とDNAトランスフェクション法による蛋白質送達の定量的評価

  山田勇磨, 紙谷浩之, 秋田英万, 原島秀吉  Drug Deliv Syst  19-  (3)  290  2004/05/10  [Not refereed][Not invited]
  

Industrial Property Rights

• 特開2024-041813:ミトコンドリアのリンパ器官への移植およびそのための組成物  2024/03/27

  菅沼 正司, 原島 秀吉, 山田 勇磨, 佐々木 大輔, 横田 貴史  ルカ・サイエンス株式会社  202403012899987552
• 特表2023-508779:サイズがより小さい単離されたミトコンドリアおよび単離されたミトコンドリアを内包している脂質膜ベースの小胞  2023/03/03

  菅沼 正司, 山田 勇磨, 原島 秀吉, 佐々木 大輔, 日比野 光恵, 太田 善浩  ルカ・サイエンス株式会社  202303002180247864
• 特許第7218897号:心不全の治療及び/又は予防に用いるための心筋幹細胞の製造方法  

  原島 秀吉, 山田 勇磨, 阿部 二郎, 武田 充人  ルカ・サイエンス株式会社  202303007576008740
• 特開2022-105226:ミトコンドリアリボソームRNA変異検出用プライマーDNAセットを含むキット、ミトコンドリアリボソームRNA発現用核酸、前記核酸を封入してなる脂質膜構造体及びそれらの利用  2022/07/13

  山田 勇磨, 原島 秀吉, 丸山 美菜子  ルカ・サイエンス株式会社  202203010989256665
• 特開2022-084962:リポソーム様ナノカプセル及びその製造方法  2022/06/08

  山田 勇磨, 原島 秀吉, 日比野 光恵, 佐藤 悠介, 渡慶次 学, 真栄城 正寿  ルカ・サイエンス株式会社  202203010499832250
• 特開2021-168625:ミトコンドリアDNAの3243位のアデニンからグアニンへの変異による変異tRNA Leuを検出する方法  2021/10/28

  山田 勇磨, 原島 秀吉  国立大学法人北海道大学  202103000075425377
• 特許第6966755号:組換え発現ベクター及び当該ベクターを封入した脂質膜構造体  

  原島 秀吉, 山田 勇磨, 石川 卓哉, 秋田 英万  ルカ・サイエンス株式会社  202103004447787780
• 特開2021-167289:水溶性化合物を内包した脂質ナノ粒子  2021/10/21

  山田 勇磨, 原島 秀吉  国立大学法人北海道大学  202103014885420380
• WO2020-054829:ミトコンドリアのリンパ器官への移植およびそのための組成物  2020/03/19

  菅沼 正司, 原島 秀吉, 山田 勇磨, 佐々木 大輔, 横田 貴史  ルカ・サイエンス株式会社  202103020804951832
• WO2017-090763:組換え発現ベクター及び当該ベクターを封入した脂質膜構造体  2017/06/01

  原島 秀吉, 山田 勇磨, 石川 卓哉, 秋田 英万  国立大学法人北海道大学  201803005641031779
• 特開2015-110522:機能性タンパク質を細胞内に送達するためのキャリア  2015/06/18

  原島 秀吉, 山田 勇磨, サンドラ, ミレーナ, ベルガラ ペレッツ  国立大学法人北海道大学  201503019802774309
• WO2012-117971:脂質膜構造体、脂質膜構造体の製造方法および1の目的物質を1枚の脂質膜で封入する方法  2012/09/07

  原島 秀吉, 山田 勇磨, 鈴木 亮佑  国立大学法人北海道大学  201403021176716730
• 特許第5067733号:目的物質をミトコンドリア内に送達可能な脂質膜構造体  2012/08/24

  山田 勇磨, 秋田 英万, 小暮 健太朗, 紙谷 浩之, 原島 秀吉, 菊池 寛, 小林 英夫  国立大学法人北海道大学  201303043484260566
• 特開2010-126505:核酸の放出性に優れたリポソームベクター  2010/06/10

  原島 秀吉, 山田 勇磨, 野村 卓, 由井 伸彦, 上遠野 亮, 山下 敦  国立大学法人北海道大学, 国立大学法人北陸先端科学技術大学院大学  201003008997969186
• 特開2009-221165:一遺伝子ナノ粒子のパッケージング法  2009/10/01

  山田 勇磨, 鈴木 亮佑, 原島 秀吉  国立大学法人 北海道大学  200903046276417326
• WO2006-095837:目的物質をミトコンドリア内に送達可能な脂質膜構造体  2006/09/14

  山田 勇磨, 秋田 英万, 小暮 健太朗, 紙谷 浩之, 原島 秀吉, 菊池 寛, 小林 英夫  国立大学法人 北海道大学, 第一三共株式会社  200903005554646163

Awards & Honors

• 2023/11 株式会社コーセー コーセーコスメトロジー奨励賞
   

  受賞者: 山田勇磨
• 2023/05 日本薬剤学会 日本薬剤学会旭化成創剤開発技術賞
   

  受賞者: 山田勇磨
• 2022/03 The American Pharmacists Association Ebert Prize Award
   Mitochondrial Delivery of an Anticancer Drug Via Systemic Administration Using a Mitochondrial Delivery System That Inhibits the Growth of Drug-Resistant Cancer Engrafted on Mice 

  受賞者: Yuma Yamada
• 2018/07 日本核酸医薬学会奨励賞
   

  受賞者: 山田勇磨
• 2018/05 日本酸化ストレス学会学術奨励賞
   

  受賞者: 山田勇磨
• 2017/02 北海道科学技術奨励賞（知事表彰）
   

  受賞者: 山田勇磨
• 2016/01 インテリジェント材料・システムシンポジウム 高木賞
   

  受賞者: 山田勇磨
• 2015/07 日本DDS学会奨励賞
   

  受賞者: 山田勇磨
• 2014/05 日本薬剤学会奨励賞
   

  受賞者: 山田勇磨
• 2014/03 日本薬学会奨励賞
   

  受賞者: 山田勇磨
• 2014 J. Control. Release: Elsevier 2014 Outstanding Reviewer
   

  受賞者: 山田勇磨
• 2011/10 NINA PHARM ISANH Award
   

  受賞者: Yuma Yamada
• 2011/06 第27回日本ＤＤＳ学会 優秀発表者賞
   

  受賞者: 山田勇磨
• 2006/03 日本薬剤学会永井財団大学院学生スカラシップ賞
   

  受賞者: 山田勇磨
• 2005/12 Asian Mitochondrial Young Investigator Award
   

  受賞者: 山田勇磨
• 2004/05 PSWC 2004 Student Travel grant
   

  受賞者: 山田勇磨

Research Grants & Projects

• ミトコンドリア遺伝子治療を実現するゲノム編集技術の創出

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2023/04 -2026/03 

  Author : 山田 勇磨, 真栄城 正寿, 武田 充人, 佐々木 大輔, 日比野 光恵
• 脳疾患治療を実現するミトコンドリア標的型ナノカプセルの開発

  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 : 山田 勇磨
• Functional background of mitochondrial genome evolution unraveled from the atp8 gene of Polypedates leucomystax

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

  Date (from‐to) : 2022/04 -2025/03 

  Author : 安岡 有理, 山田 勇磨
• Development of Phototherapeutic Drugs and Elucidation of Single Molecule Dynamics Using Highly Bright Stable Quantum Dots and Innovative 3D cell system

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

  Date (from‐to) : 2021/04 -2025/03 

  Author : 高野 勇太, 宮武 由甲子, 平田 恵理, 山田 勇磨, 繁富 香織

   

  本年度は、当初計画通り「(I):3D培養系で高い浸透性・発光性・安定性をもつ機能化量子ドットの合成と培養組織内動態の解明（研究１～2年目計画）」のために細胞標的指向性分子-量子ドット複合体の開発に着手した。量子ドットに対してポリエチレングリコール鎖を介して水酸基（中性）およびカルボン酸（アニオン性）を接合した量子ドットを合成した。また、量子ドットに対して癌細胞マーカーとして良く知られているCD44抗体およびEpCAM抗体を接合した複合体も作成し、血液モデルのPBMC中から癌細胞を選択的に癌細胞認識できることを確認した。当該量子ドット-抗体複合体は現在、膵がん細胞における取り込み挙動を、2次元培養および3次元培養にて検討中である。また、量子ドット自体の光安定性についても検討を行い、光照射強度に応じて表面エッチングが起きる一方、光強度の制御や量子ドット表面組成によって細胞毒性を抑え得ることを見出した。 また「(II):マイクロ組織内の分子動態解明を利用した高効果光がん治療薬の開発（研究２～３年目計画）」に関して、その前段階として光増感剤の開発検討を行った。我々がこれまでも研究に用いているπ拡張型ポルフィリン分子(rTPA)について、量子ドット表面への担持やナノカーボン表面への担持を行い、その光増感性能（＝一重項酸素発生能）の検証を行った。その結果、量子ドット表面への直接不可は、分子凝集などが原因となり十分な一重項酸素発生能を発揮しない一方、ナノカーボンの1種であるカーボンナノホーンに担持したrTPAは十分な一重項酸素発生能と殺がん効果を発揮し得ることを見出した。
• 造血幹細胞移植後の免疫寛容と組織寛容の機序の解明とその制御法の開発研究

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  Date (from‐to) : 2021/04 -2025/03 

  Author : 豊嶋 崇徳, 冨塚 一磨, 山田 勇磨

   

  マウス同種造血幹細胞移植後にcyclosporin A (CSP)を投与することによって, ドナーT細胞疲弊の進行が停止し, 疲弊T細胞 (Tex)の前駆細胞 (precursor Tex: pTex)が増加することを示した。pTexが保たれることによって, 移植後の免疫チェックポイント阻害剤の抗腫瘍効果が増強された。一方, 移植後のT細胞を回収して, 別のレシピエントに輸注したところ, 1回目の移植後にCSPを利用していると, ２回目の移植後に慢性移植片対宿主病(慢性GVHD)が発症することが示された。一方, 移植後大量シクロフォスファミド（Posttransplant cyclophosphamide: PTCY）法を行ってからCSPを移植後day5から投与しても, pTexは誘導されなかった。CSPによるGVHD予防は長期的には慢性GVHDの発症に繋がる可能性が示された。 移植後にFLT3阻害剤 (gilteritinib)投与によって, 移植後にFLT3変異陽性急性白血病細胞からIL-15の産生が促進され, ドナーT細胞のTexの分化が抑制され, 白血病に対する細胞傷害活性が増強されることを示した。移植後day5からday14まで短期間のgilteritinib投与で, GVHDの増悪無しでGVL効果が増強されて, マウスの生存が延長されることを示し, Bone marrow transplantation誌に発表した。 ミトコンドリアナノメディシンを利用した悪性腫瘍治療法の開発のため, ミトコンドリア外膜の活性を保った単離ミトコンドリア (Q)を利用した。新規キメラ抗原受容体T細胞（CAR-T細胞）作成時にQを加えることによって, CAR-T細胞内にQを取り込ませ, CAR-T細胞の作成効率を改善することが可能となった。Qを利用することによって臨床的に問題となる, CAR-T細胞の作成失敗を防ぐことが期待される。
• Development of Trojan horse-type cancer therapeutics by elucidating and utilizing the predation mechanism of pancreatic cancer cells

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

  Date (from‐to) : 2021/07 -2023/03 

  Author : 高野 勇太, 宮武 由甲子, 山田 勇磨, 繁富 香織

   

  本年度は当初計画通り、「[段階-I] 捕食誘引物質の粗分離、観察、構造決定による同定」にとりかかり、膵がん細胞の死細胞を調製した上で捕食誘引物質の分離を開始した。まず、死細胞について大きく「アポトーシス様死細胞」と「ネクローシス様死細胞」に分けて調製し、それぞれ膵がん細胞より再構築したミクロ組織3次元と共培養しながらリアルタイム顕微鏡観察を行い、取り込まれる挙動の観察を行った。この際、コントロール条件として1μm（大体アポトーシス死細胞と同サイズ）のCOOH修飾蛍光ビーズを用いた。結果、COOH修飾蛍光ビーズでは細胞への取り込み、特に細胞集団が触手を伸ばすなどの能動的な取り込みは起きないことを確認した。そして「アポトーシス様死細胞」と「ネクローシス様死細胞」は両者とも取り込まれることを確認したが、ネクローシス様死細胞では取り込みがやや少ない傾向にあった。これはネクローシスでは死細胞構造がランダムであるとともに、細胞内容物が維持できないことが一因であると考えられた。よって、今後の検討は主にアポトーシス様死細胞で行うこととした。現在は、アポトーシス様死細胞の大量調製と、粗分離を開始したところである。 また、「[段階-II]捕食誘引物質を複合化した高薬効性の光がん治療薬の合成開発」の準備段階として、量子ドットに近赤外光殺がん分子(rTPA)を接合した複合体や、ドラッグデリバリキャリアとrTPAの複合化も行い、その一重項酸素発生能および光殺がん効果を確認した。本研究の遂行によってドラッグデリバリキャリアとrTPAの相性により、内容分子の機能性（本研究の場合は光殺がん性能）が変化することが分かった。今後、最適化を進め最大効率で殺がん効果を示す複合体プロトタイプを作成し、本研究の目的とするトロイの木馬型殺がん化合物を完成させる。
• Production of novel drug derivery system with light-induced disruption of liposomes (LiDL)

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

  Date (from‐to) : 2020/07 -2023/03 

  Author : 須藤 雄気, 山田 勇磨

   

  【目的】物理化学（代表者）と薬剤学（分担者・協力者）の融合による光誘起崩壊リポソーム：Light-induced Disruption of Liposomes（LiDL）の開発と、それに基づく新奇薬物送達手法の確立。【背景】狙った時間と場所に薬物を届け・働かせることは、薬学における大きな『夢』である。【計画】光受容タンパク質・ロドプシンとpH感受性ポリマーおよび任意の化合物（薬物）を内封させたリポソームを開発することで、時空間制御性に優れた『光』により、狙った時間・場所で薬物を放出させる新奇手法を確立する。【意義】薬学における『夢』の一つを叶える手法となり、大きな波及効果をもたらす。
  具体的には、光受容タンパク質「(1) ロドプシン（H+ポンプ・チャネル）」と「(2) pH 感受性ポリマー」を含む「(3) リポソーム」を作成する。その際、「(4) 化合物 (薬物)」を内封させる。このリポソームに「(5) 光」を照射すると、ロドプシンが活性化され、リポソーム内外のpH が大きく（> 5 ユニット）変化する。これにより、pH 感受性ポリマーの物理的形状が変化し、リポソームが崩壊し、化合物が「(6) 放出」される。LiDL と命名するこの手法は、時空間分解能に優れた「光」により薬物を放出させるという、新奇かつ独創性・汎用性の高い薬物送達（DDS）手法になる。本年度は、ロドプシンを組み込んだリポソームの作成と、光によるpH変化を定量的に測定し、ロドプシン組み込みリポソームが狙い通りに機能することを明らかにした。今後は、このリポソームにpH感受性分子を組み込むとともに、光により崩壊するかを内部に導入する蛍光分子の蛍光変化により確認する。
• Development of a nano device for mitochondrial gene editing

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

  Date (from‐to) : 2020/04 -2023/03 

  Author : 山田 勇磨

   

  ミトコンドリアのゲノム変異と種々の疾患との関連が報告されており、本オルガネラを標的とした遺伝子治療が期待されている。本申請研究では、ミトコンドリア標的型ナノカプセル (MITO-Porter)を用いて標的ミトコンドリアへゲノム編集装置を運搬し、ミトコンドリア機能を遺伝子レベルで治療する事を目的とする。2021年度は、1. ゲノム編集の検証および2. ミトコンドリア機能の評価について、下記に記載した計画で研究を進めた。 1. ゲノム編集の検証： 疾患細胞が保有するtRNA領域のmtDNA変異をゲノム編集可能な装置をパッケージングしたMITO-Porterを調製し、疾患細胞ミトコンドリア内部にゲノム編集装置を導入した。単離したミトコンドリアを用いて、定量的PCR法を利用してミトコンドリア内部のmtDNA変異の切断効率を測定し、ゲノム編集装置搭載MITO-Porterが変異mtDNAを効率的に切断することを確認した。さらに、疾患細胞での評価を行い、キャリアの最適化を図った。 2. ミトコンドリア機能の評価： 実験に用いる疾患細胞は、ミトコンドリア呼吸活性複合体の活性低下が観察されている。そのため、治療効果の指標としてミトコンドリア呼吸活性 （細胞外フラックスアナライザー）を評価する。2022年は、疾患細胞のミトコンドリア呼吸活性を測定する手法を確立した。現在、ゲノム編集装置搭載MITO-Porter投与時の疾患細胞ミトコンドリア呼吸活性を測定し、投与プロトコルの最適化を図っている。
• 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 Grant-in-Aid for Scientific Research (A)

  Date (from‐to) : 2019/04 -2023/03 

  Author : 原島 秀吉, 佐藤 悠介, 中村 孝司, 山田 勇磨

   

  １ siRNA送達（佐藤）：siRNAは細胞質中でリン酸化酵素Clp1により5’末端のリン酸化修飾を受け、RISCを形成し、標的mRNAのサイレンシングを引き起こす。我々の予備検討結果から、siRNAの多くが上述の一般的な経路をたどっていない可能性が強く示唆された。そこでブラックボックスとなっているsiRNAの細胞内運命を①5’リン酸化・RISC形成速度、②細胞質内・核内局在、の観点から解明する。 ２ pDNA送達（原島）：転写(TC)/翻訳(TL)過程に大きな差が認められたナノDDSにおいて、TCとTLのどちらの寄与が大きいかを、mRNAを定量することにより識別する。 ①TLの寄与が大きい場合、ナノDDSのエンドソーム脱出時にエンドソーム内物質が漏出したり、あるいはpDNA導入に伴う各種細胞内センサーの刺激によって、導入細胞におけるTL活性が低下する、という仮説を検証する。 ３ がん免疫ナノ療法（中村）：患者個人の腫瘍関連微小環境の免疫状態を把握することは治療戦略の決定に不可欠である。そこで、免疫状態が異なる担がんマウスモデルから腫瘍組織、所属リンパ節、脾臓を採取し、遺伝子発現解析を行うことで腫瘍関連微小環境の免疫状態情報を得る。文献情報からヒトで確認されている腫瘍関連微小環境の免疫状態をカテゴリー化し、それらを反映する担がんマウスモデルを5種類以上選定する。 ４ Mt送達（山田）：核酸・遺伝子をコアとするナノ粒子を形成し、細胞膜とMt膜 を突破するために、脂質膜コーティングを施す。脂質膜表面には細胞導入素子およびミトコンドリア移行性アプタマー(負電荷)を修飾したDual ligand MITO-Porterを基盤骨格とする。また、細胞内での流動性の向上を目指し、ポリエチレングリコール（PEG）などの親水性ポリマーの修飾や、微小な粒子径も検討する。
• Development of artificial mitochondria with cell-symbiosis ability

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

  Date (from‐to) : 2020/07 -2022/03 

  Author : Yamada Yuma

   

  Mitochondria transplantation therapy, in which mitochondria are transplanted into diseased cells, could be a very useful, but there is a problem in that optimal symbiosis (cellular internalization and mitochondrial communication) for therapeutic efficacy has not been achieved. The aim of this research was to develop artificial mitochondria with enhanced symbiotic ability for use in therapeutic applications. This research proposal includes (i) the formulation of artificial mitochondria and the optimization of their intracellular dynamics, (ii) validation of their therapeutic efficiency using a model of mitochondrial disease cells. A number of results regarding the development of artificial mitochondria have been achieved and evaluated in preliminary experiments.
• Establishment of the new diagnostic method in mitochondrial cardiomyopathy

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

  Date (from‐to) : 2019/04 -2022/03 

  Author : Takeda Atsuhito

   

  Mitochondrial cardiomyopathy (MCM), one of the secondary cardiomyopathies, is diagnosed definitively by pathology, tissue biochemistry, and genetic testing, but there are no clear criteria for myocardial pathology. Therefore, we measured mitochondrial volume density in electron microscopy images and performed immunohistochemistry using respiratory chain enzyme antibodies in MCMs diagnosed definitively by clinical symptoms, genetic testing, and tissue biochemistry, and compared them with other cardiomyopathies other than MCM. Mitochondrial volume density was significantly higher in the MCM group, and immunohistochemistry showed decreased staining for Complex I or IV in the MCM group. This study allowed the pathological diagnosis of MCM using electron microscopy and immunohistochemistry, which is very useful for the pathological criteria of MCM.
• Drug discovery of nano-molechule mitochondria targeted agent and impact on post cardiac arrest brain injury

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

  Date (from‐to) : 2017/04 -2021/03 

  Author : Kohei Ikeda

   

  Mitochondrial dysfunction following ischemia/reperfusion injury including cardiac arrest and CPR is characterized by an impairment of mitochondrial function which leads to pro-apoptotic signaling and cell death. No pharmacological agent has yet been found to improve clinical outcomes after cardiac arest. We developed a new mitochondria targeted nano-particle involving active form vitamin B1, VB1-MITO-Porter. This VB1-MITO-Porter statistically increased ATP turn over of SHSY5Y cell's mitochondria in-vitro. However, VB1-MITO-Porter did not improve 10-day survival and histological brain injury on murine cardiac arrest model.
• 異常ミトコンドリアを認識する遺伝子治療用ナノカプセルの構築

  文部科学省：科学研究費助成事業（基盤研究(B)）

  Date (from‐to) : 2017/04 -2020/03 

  Author : 山田勇磨
• Investigation into unraveling the mechanism of drug delivery system to the inner ear

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

  Date (from‐to) : 2016/04 -2019/03 

  Author : Kita Tomoko

   

  The final aim of this study was to achieve the effective drug delivery systems to the inner ear. First, we tried to establish the culture system of blood-labyrinth barrier but unfortunately failed. And second, to clarify the contribution of each drug transporter expressed in this inner ear barrier, we have established the preparation protocols of cDNA library from samples obtained by laser capture micro dissection. Third, using several model drugs for each drug transporter and their inhibitors, it was shown that several tissue type specific transporters are localized in the inner ear barrier, but there was no significant change in the distribution of each model drug.
• ミトコンドリアの品質管理を科学するナノデバイスの開発

  文部科学省：科学研究費補助金(挑戦的萌芽研究)

  Date (from‐to) : 2017/04 -2019/03 

  Author : 山田勇磨
• Establishment of diagnostic procedure and the new diagnostic criteria of mitochondrial cardiomyopathy based on an Imaging technology

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

  Date (from‐to) : 2015/04 -2018/03 

  Author : Takeda Atsuhito, ABE JIRO

   

  Mitochondrial disease, which is a designated intractable disease, has not yet been established as a treatment method, and it is urgent to establish a diagnostic method leading to early detection. We have aimed to establish mitochondrial cardiomyopathy diagnostic criteria including mitochondrial cardiomyopathy model and its evaluation system. In the basic experiments, the results were confirmed in the preparation and evaluation system of doxorubicin mitochondrial cardiomyopathy-induced model animals and the rescue experiment using mitochondrial DDS. Clinically, as diagnostic criteria for mitochondrial cardiomyopathy, we identified three points of (1) myocardial pathology tissue: mitochondrial abnormality in electron microscopic image, (2) tissue biochemistry: myocardial tissue respiratory chain enzyme activity reduction, and (3) gene mutation as necessary elements.
• Development of optical control method of cell organelle function by photoindeuced charge separation molecule

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

  Date (from‐to) : 2015/04 -2018/03 

  Author : Takano Yuta, IMAHORI Hiroshi, MURAKAMI Tatsuya, YAMADA Yuma

   

  In this study, novel functional molecules utilizing the photoinduced charge separation were developed for a fundamental technology to develope photocontrol methods of cellular activities. We investigated intracellular optical functionality mainly for amphipathic ferrocene-porphyrin-fullerene linked molecules and aryl-acridinium molecules. As research outcomes, we succeeded in photoinduced depolarization of the cell membrane by high charge separation efficiency due to development of molecules with enhanced cell membrane fluidity. In addition, we succeeded in mitochondria selective molecular transport in living cells by utilization of drug transport carriers or optimization of molecular structure. The molecules demonstrated successful method for triggering photooxidation/reduction reactions in cells by irradiation with light.
• Elucidation of organelle network mechanism controlled by mitochondrial dynamics and its application.

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

  Date (from‐to) : 2014/04 -2017/03 

  Author : Nomura Masatoshi, ISHIHARA Naotada, YAMADA Yuma

   

  Mitochondria are highly dynamic organelles that frequently fuse and divide in response to cellular energy demands. In an excessive energy state such as a high fat diet, the organelle network between Mt and ER is impaired, followed by the induction of ER stress. As a result, FGF21 is secreted from the liver by activation of PERK-eIF2a-ATF4 pathway, and energy expenditure is accelerated in the peripheral organs such as skeletal muscle and adipose tissue. In other words, mitochondrial dynamics-ER stress-FGF21 axis function as a biological defense system for energy excess. Therefore, mitochondrial dynamics is a therapeutic target for the obese diabetes.
• New approach for the mechanism of ototoxic drugs against the mtDNA1555AG mutation - human iPSC and nanocarieer-

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

  Date (from‐to) : 2014/04 -2017/03 

  Author : Kitajiri Shinichiro, KITA Tomoko, IKI Takehiro, MIZUKOSHI Akifumi

   

  To clarify the mechanism of ototoxicity with aminoglycoside antibiotice (AG), we have established the experiment systems of ototoxicity for MtDNA1555AG iPSC. In the recent technology, it is impossible to make transgenic mouse for this mitochondrial SNP mutation. Compared with hair cells, most cells don't have any transport systems for taking AG into the cells. We therefore use nanocarrier encapsulated AG. When we use this nanocarrier for Hela cell line, only mitochondria specific carrier showed cell death dose-dependently. We established patient drived iPSC cell lines and characterized them. We further optimized the composition of nanocarrier for iPSC, and Ag encapsulated nanocarrier are now preparing.
• Development of a nanoparticle to release nucleic acids in a mitochondrial environment

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

  Date (from‐to) : 2015 -2016 

  Author : YAMADA Yuma, HYODO Mamoru

   

  It was recently reported that mutations in mitochondrial DNA (mtDNA) form the basis of mitochondria (Mt) related diseases. Therefore, the delivery of nucleic acids targeting mutant mtDNA would be expected to be an innovative therapeutic strategy for such diseases. To date, we developed a MITO-Porter, a nano carrier for mitochondrial delivery. The aim of this study is to develop a nanoparticle that can release nucleic acids in response to the Mt environment following its delivery to the Mt. In this study, we evaluated nanoparticles with various Mt environment responsive peptides, and we succeeded in constructing a nanoparticle that was capable of releasing nucleic acids in response to conditions that mimic the Mt environment. In addition, we confirmed successful regulation of the mitochondrial gene expression by transfection of nanoparticles using the MITO-Porter. Our findings can contribute to development nanomedicine for mitochondrial gene therapy.
• Development of nanomachine for mitochondrial gene therapy targeted to cells derived from mitochondrial disease patients

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

  Date (from‐to) : 2014 -2016 

  Author : YAMADA Yuma, AKITA Hidetaka, HYODO Mamoru

   

  In order to establish a therapeutic strategy targeting genes in mitochondria (Mt), we examined Mt gene delivery and mitochondrial exogenous gene expression in cells derived from Mt disease patients. To date, we have been able to develop a MITO-Porter, a nano carrier for Mt delivery. In this study, we report on the successful Mt gene delivery in disease cells that have a heteroplasmic mutation in mtDNA, using MITO-Porter system. In addition, we designed a pHSP-mtLuc (CGG) analog, an artificial Mt gene expression plasmid DNA vector that contains a promotor for Mt transcription and an artificial Mt genome with a reporter gene that records adjustments to the Mt codon system. Collectively, we succeeded in achieving Mt exogenous gene expression by the Mt delivery of a pHSP-mtLuc (CGG) analog using the developed MITO-Porter system. Our Mt exogenous gene expression carriers promises to be a useful approach to Mt gene therapy and research regarding Mt molecular biology.
• 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。
• Development of nano device for regulating mitochondrial gene expression

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

  Date (from‐to) : 2013 -2014 

  Author : YAMADA Yuma

   

  Mitochondria have attracted interest as an organelle that can be used as a target in medical therapy, in the maintenance of beauty & health and the development of the life sciences. The objectives of the research supported by this grant is to develop a nano device that can be used for regulating mitochondrial gene expression including mitochondrial gene silencing and mitochondrial transgene expression. In this study, we report on the successful mitochondrial delivery of nucleic acids in human cells using a MITO-Porter, which is a liposome-based carrier that is capable of introducing macromolecular cargos into mitochondria via membrane fusion. Moreover, we achieved mitochondrial RNA knockdown and mitochondrial transgene expression using such a MITO-Porter system. Our system for regulating mitochondrial gene expression system promises to have a significant impact on the medical and life sciences.
• Development of in vivo mitochondrial gene delivery system and validation of mitochondrial exogenous gene expression in skeletal muscle

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

  Date (from‐to) : 2011 -2012 

  Author : YAMADA Yuma

   

  Mitochondrial genetic disorders are a major cause of mitochondrial diseases including mitochondrial inherited diseases, neurodegenerative diseases and diabetes. It is therefore likely that mitochondrial gene therapy will be useful for the treatment of such diseases. To achieve such an innovative therapy, successful mitochondrial exogenous gene expression, two independent processes, i.e., "development of in vivo mitochondrial gene delivery system" and "construction of DNA vector to achieve mitochondrial gene expression" are required. In this study, we report on successful mitochondrial gene delivery in skeletal muscle using hydrodynamic limb vein (HLV) injection. Moreover, we could construct the DNA vector which can express on mitochondria through hydrodynamic gene delivery. Our mitochondrial exogenous gene expression system promises to be a useful technique for mitochondrial gene therapy and research regarding mitochondrial molecular biology.
• Development of Multi-layered nano-device for mitochondrial drug delivery and an innovative approach for mitochondrial disease' therapy

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(若手研究(B))

  Date (from‐to) : 2009 -2010 

  Author : Yuma YAMADA

   

  Mitochondrial dysfunction is associated with a variety of human diseases. Effective medical therapies for mitochondrial diseases will ultimately require an optimal drug delivery system, which will likely be achieved through innovations in the nanotechnology of intracellular trafficking. To achieve efficient mitochondrial drug delivery, two independent processes, i.e., "cytoplasmic delivery through the cell membrane" and "mitochondrial delivery through the mitochondrial membrane" are required. Here, we report the development of a Dual Function MITO-Porter (DF-MITO-Porter), a nano carrier int...
• Multifunctional Envelope-type Nano Device as non-viral gene delivery system for cancer therapy

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(特定領域研究)

  Date (from‐to) : 2008 -2009 

  Author : Hideyoshi HARASHIMA, 秋田 英万, 紙谷 浩之, 山田 勇磨, 畠山 浩人, 馬場 嘉信

   

  In this study, we developed a multifunctional envelope-type nano device (MEND) as a non-viral siRNA delivery system for cancer, which can induce the knockdown of tumor specific genes and anti-tumor effect with no toxicity. First, we measured antigen presentation of OVA encapsulated R8-MEND in dendritic cell (DC). As a result, R8-MEND showed specific MHC class I presentation, although showed low MHC class II presentation. Moreover, mice subcutaneously immunized by R8-MEND were showed significant antitumor effect compared with control mice. Next, we prepared GALA/DMEND encapsulating siRNA cor...
• 抗原提示バランスとアジュバントの制御に基づいたオーダーメイド癌ワクチン

  文部科学省：科学研究費補助金(萌芽研究, 挑戦的萌芽研究)

  Date (from‐to) : 2008 -2009 

  Author : 原島 秀吉, 小暮 健太朗, 秋田 英万, 山田 勇磨

   

  本年度は、アジュバントのトポロジー制御の観点からアジュバント搭載型ナノ構造体の構築とその機能評価を行った。まず認識受容体の局在が異なる2種類のアジュバントを搭載したMENDの構築を行い、細胞性免疫誘導効率への影響を調べた。polyI:Cはエンドソーム内のTLR3及び細胞質中のMda5により認識され、CpG-ODNはエンドソーム内のTLR9によって認識される。それ故、細胞への取り込み後に認識されるように、MENDの内部に抗原と共に内封した。polyI:CもしくはCpG-ODNを内封したMENDをマウスに皮下免疫し、CTL活性を測定した結果、polyI:C内封MENDを免疫したマウス群では、CpG-ODN搭載MENDを免疫した群と比較して著しく高いCTL活性の誘導が認められた。次にpolyI:C搭載MENDとpolyI:C/R8複合体のCTL活性誘導能を比較した。その結果、polyI:C搭載MENDを免疫したマウス群の方が高いCTL活性及び抗腫瘍活性を示した。MEND内にpolyI:Cを内封することで、効率良くTLR3及びMda5に認識され、強力な細胞性免疫が誘導されたと考えられる。このことからアジュバントの機能を十分に発揮させるためには、そのトポロジーを考慮することが重要であることが示唆された。またpolyI:CをMEND内に内封することでアジュバント投与時の炎症等の副作用が軽...
• Development of in vivo gene delivery system for siRNA and genome wide screening of type-two diabetes related genes in mice and rats

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(A))

  Date (from‐to) : 2006 -2009 

  Author : Hideyoshi HARASHIMA, 中谷 明弘, Hiroyuki KAMIYA, 小暮 健太朗, 秋田 英万, Yoshinobu BABA, Yuma YAMADA

   

  We performed genome-wide screening of type-2 diabetes related genes which can be detectable in blood cells before disease state or after disease occurred. In parallel, we succeeded in developing in vivo gene delivery system for liver..In addition, we constructed an original assay system for nano carriers to evaluate transcytosis of endothelial cells and finally succeeded in finding a selective ligand for transcytosis of nano carriers.
• Development of Multi-layered nano-device for mitochondrial drugdelivery and mitochondrial protein delivery for cancer therapy

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(若手研究(スタートアップ))

  Date (from‐to) : 2007 -2008 

  Author : Yuma YAMADA

   

  ミトコンドリア(Mt)は様々な疾患と密接に関わっており、Mtを標的とした薬物治療が注目を集めている。これらの治療を実現するためには、Mtへ治療薬物を送達する必要があるが、有用なシステムは報告されていない。本研究では、Mtを標的とした新規送達システムを開発する事に成功し、癌細胞Mtへの薬物送達および薬理効果を確認した。本システムを用いる事で、今までは不可能であった難治性疾患の治療が期待できる。
• がん選択的多機能性エンベロープ型ナノ構造体の開発とがん遺伝子治療への応用

  文部科学省：科学研究費補助金(特定領域研究)

  Date (from‐to) : 2006 -2007 

  Author : 原島 秀吉, 紙谷 浩之, 小暮 健太朗, 秋田 英万, 山田 勇磨

   

  我々は、多機能性エンベロープ型ナノ構造体(MEND)の開発に成功し、細胞膜透過機能を有するオクタアル,ギニン(R8)修飾したR8-MENDは、培養細胞系においてアデノウイルスと同等の遺伝子発現を示すことを明らかにした(Khalil,et al.Gene Ther.2007)。さらに、in vivoでがん組織選択的な遺伝子デリバリーシステムを開発するために、がん組織内で特異的に発現するペプチダーゼ(MMP)により切断されてPEGを脱離し、がん細胞内へ侵入するという新しい戦略(PEG-peptide-DOPE(PPD)-MEND)を設計し,本コンセプトが機能するかをin vitro系で検証した。その結果、PPD-MENDはMMP依存的にPEGが解離し、高い遺伝子導入を誘起できることが明らかとなった。さらに、本PPD-MENDをマウスに静脈内投与すると、血中滞留性を示すと同時に、腫瘍組織へ有為な送達が可能であり、in vivoにおける遺伝子発現にも成功した(Hatakeyama,et al.Gene Ther.2007)。さらに、PPD-MENDにsiRNAを搭載し、in vivoで腫瘍組織へ標的化可能、かつ、がん細胞で発現している遺伝子(ルシフェラーゼ遺伝子)を選択的にノックダウンすることにも成功した。本、PPD-MENDに抗腫瘍効果を誘起するsiRNAを搭載することにより、がん...

Educational Activities

Teaching Experience

Social Contribution

Social Contribution

Media Coverage

• 北大、ミトコンドリアを活性化させ心機能改善効果を強化したヒト心筋前駆細胞を開発

  Date : 2024/02

  Program, newspaper magazine: 日経バイオテク

  Internet
• 北海道大の山田氏、LNPに封入した補酵素をミトコンドリアに送達

  Date : 2023/11

  Program, newspaper magazine: 日経バイオテク

  Internet
• ミトコンドリアに直接コエンザイムを届けて肝障害克服 北大がカプセル開発

  Date : 2023/07

  Program, newspaper magazine: YAHOO! JAPAN: ニュース

  Internet