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Hidetaka Akita

Section

Graduate School of Pharmaceutical Sciences

Job title

Professor

Degree

Ph.D. (Pharmaceutical Science) (The University of Tokyo)

researchmap

https://researchmap.jp/HideJ

J-GLOBAL ID

200901021400157508

Research History 8

2022/04 - Present

東北大学大学院薬学研究科教授
2021/04 - 2022/03

東北大学大学院薬学系研究科（兼任）　教授
2016/04 - 2022/03

千葉大学　大学院薬学研究院　教授
2010/12 - 2016/03

北海道大学　大学院薬学研究院　准教授
2007/04 - 2010/11

北海道大学　大学院薬学研究院　助教
2002/07 - 2007/03

Hokkaido University
2002/04 - 2002/06

日本学術振興会特別研究員(PD) 日本学術振興会特別研究員
2001/04 - 2002/03

日本学術振興会特別研究員(DC2) 日本学術振興会特別研究員

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Education 3

The University of Tokyo

1999/03 - 2002/04
The University of Tokyo

1997/03 - 1999/04
The University of Tokyo　Faculty of Pharmaceutical Sciences　Department of Pharmacy

1993/04 - 1997/03

Committee Memberships 1

日本薬剤学会　遺伝子・細胞製剤フォーカスグループメンバー

2008 -

Professional Memberships 7

日本リンパ学会
日本薬物動態学会
アメリカ遺伝子細胞治療学会
日本DDS学会
日本薬学会
日本薬剤学会
遺伝子・デリバリー研究会

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Research Interests 7

イムノエンジニアリング
DNA/RNAワクチン
リンパシステム
ナノテクノロジー
細胞内動態制御学
薬物動態学
ドラッグデリバリーシステム

Research Areas 6

Life sciences / Pharmaceuticals - analytical and physicochemistry /
Life sciences / Clinical pharmacy /
Life sciences / Biomaterials /
Life sciences / Biomedical engineering /
Nanotechnology/Materials / Nanobioscience /
Nanotechnology/Materials / Nanomaterials /

Awards 13

第11回（2023年度）パーティクルデザイン賞

2024/07　一般財団法人新製剤技術とエンジニアリング振興基金　RNA創薬を加速する環境応答性脂質様材料ssPalmの開発
令和5年度科学技術分野の文部科学大臣表彰科学技術賞（開発部門）

2023/04
第13回水島賞

2020/10　日本DDS学会
日本薬学会学術振興賞

2020/03　日本薬学会
北海道大学研究総長賞

2016/03　北海道大学
北海道大学研究総長賞

2015/03　北海道大学
第5回日本DDS学会奨励賞

2013/07
ベストプレゼンテーション賞

2010　第4回次世代を担う若手医療薬科学シンポジウム
日本薬剤学会第25年会奨励賞

2010
日本薬学会奨励賞

2010
日本薬学会北海道支部例会奨励賞

2007
コニカミノルタ画像科学奨励賞

2006
Outstanding poster賞

2006

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Papers 242

Delivery of Oligonucleotides Using a Self-Degradable Lipid-Like Material. International-journal Peer-reviewed

Hiroki Tanaka, Nae Takata, Yu Sakurai, Tokuyuki Yoshida, Takao Inoue, Shinya Tamagawa, Yuta Nakai, Kota Tange, Hiroki Yoshioka, Masatoshi Maeki, Manabu Tokeshi, Hidetaka Akita

Pharmaceutics　13　(4)　2021/04/13

DOI： 10.3390/pharmaceutics13040544 　

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The world-first success of lipid nanoparticle (LNP)-based siRNA therapeutics (ONPATTRO®) promises to accelerate developments in siRNA therapeutics/gene therapy using LNP-type drug delivery systems (DDS). In this study, we explore the optimal composition of an LNP containing a self-degradable material (ssPalmO-Phe) for the delivery of oligonucleotides. siRNA or antisense oligonucleotides (ASO) were encapsulated in LNP with different lipid compositions. The hepatic knockdown efficiency of the target genes and liver toxicity were evaluated. The optimal compositions for the siRNA were different from those for ASO, and different from those for mRNA that were reported in a previous study. Extracellular stability, endosomal escape and cellular uptake appear to be the key processes for the successful delivery of mRNA, siRNA and ASO, respectively. Moreover, the compositions of the LNPs likely contribute to their toxicity. The lipid composition of the LNP needs to be optimized depending on the type of nucleic acids under consideration if the applications of LNPs are to be further expanded.
Development of Sentinel LN Imaging with a Combination of HAase Based on a Comprehensive Analysis of the Intra-lymphatic Kinetics of LPs. International-journal Peer-reviewed

Masaki Gomi, Yu Sakurai, Takaharu Okada, Naoya Miura, Hiroki Tanaka, Hidetaka Akita

Molecular therapy : the journal of the American Society of Gene Therapy　29　(1)　225-235　2021/01/06

DOI： 10.1016/j.ymthe.2020.09.014 　

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The sentinel lymph node (LN) is the first LN to which lymph fluid flows from tumor tissue. We identified the key parameters of liposomes (LPs) that affect their accumulation in regional (primary) LNs with minimum leakage to its connecting (secondary) LNs by a comprehensive analysis of the LN-to-LN trafficking of LPs with various surface charges and various sizes. We used a lymphatic flow-modified (LFM) mouse that allows for the chronological analysis of inguinal (primary) LN-to-axillary (secondary) LN at the body surface. As a result, the anionic medium-sized LPs (130 nm on average) exhibited the highest accumulation in the primary LNs. A mechanism-based analysis revealed that CD169-positive macrophages in LNs were the dominant cell population that captures anionic LPs. Sentinel LN imaging was also performed by the intratumoral injection of fluorescent medium-sized anionic LPs using a breast cancer orthotopic model. In comparison with the typically used contrast agent indocyanine green, the anionic LPs were detected in sentinel LNs with a high sensitivity. Additionally, the co-injection of hyaluronidase significantly improved the sensitivity of detection of the fluorescent LPs in sentinel LNs. In conclusion, medium-sized anionic LPs combined with hyaluronidase represents a potent strategy for investigating sentinel LNs.
Development of lipid-like materials for RNA delivery based on intracellular environment-responsive membrane destabilization and spontaneous collapse. International-journal Peer-reviewed

Hiroki Tanaka, Yu Sakurai, Jessica Anindita, Hidetaka Akita

Advanced drug delivery reviews　154-155　210-226　2020/07

DOI： 10.1016/j.addr.2020.07.001 　

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Messenger RNA and small interfering RNA are attractive modalities for curing diseases by complementation or knock-down of proteins. For success of these RNAs, a drug delivery system (DDS) is required to control a pharmacokinetics, to enhance cellular uptake, to overcome biological membranes, and to release the cargo into the cytoplasm. Based on past research, developing nanoparticles that are neutrally charged have been the mainstream of their development. Also, the materials are further mounted with pH- and/or reducing environment-responsive units. In this review, we summarize progress made in the molecular design of these materials. We also focus on the importance of the hydrophobic scaffold for tissue/cell targeting, intracellular trafficking, and immune responses. As a practical example, the design concept of the SS-cleavable and pH-activated lipid-like material (ssPalm) and subsequent molecular modification tailored to the RNA-based medical application is discussed.
Self-Degradable Lipid-Like Materials Based on "Hydrolysis accelerated by the intra-Particle Enrichment of Reactant (HyPER)" for Messenger RNA Delivery Peer-reviewed

Hiroki Tanaka, Tatsunari Takahashi, Manami Konishi, Nae Takata, Masaki Gomi, Daiki Shirane, Ryo Miyama, Shinya Hagiwara, Yuki Yamasaki, Yu Sakurai, Keisuke Ueda, Kenjirou Higashi, Kunikazu Moribe, Eiji Shinsho, Ruka Nishida, Kaori Fukuzawa, Etsuo Yonemochi, Koji Okuwaki, Yuji Mochizuki, Yuta Nakai, Kota Tange, Hiroki Yoshioka, Shinya Tamagawa, Hidetaka Akita

ADVANCED FUNCTIONAL MATERIALS　2020/06

DOI： 10.1002/adfm.201910575 　

ISSN： 1616-301X

eISSN： 1616-3028
Vitamin E Scaffolds of pH-Responsive Lipid Nanoparticles as DNA Vaccines in Cancer and Protozoan Infection. International-journal Peer-reviewed

Mio Maeta, Naoya Miura, Hiroki Tanaka, Takashi Nakamura, Ryo Kawanishi, Yoshifumi Nishikawa, Kenichi Asano, Masato Tanaka, Shinya Tamagawa, Yuta Nakai, Kota Tange, Hiroki Yoshioka, Hideyoshi Harashima, Hidetaka Akita

Molecular pharmaceutics　17　(4)　1237-1247　2020/04/06

DOI： 10.1021/acs.molpharmaceut.9b01262 　

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DNA vaccinations are promising strategies for treating diseases that require cellular immunity (i.e., cancer and protozoan infection). Here, we report on the use of a liposomal nanocarrier (lipid nanoparticles (LNPs)) composed of an SS-cleavable and pH-activated lipidlike material (ssPalm) as an in vivo DNA vaccine. After subcutaneous administration, the LNPs containing an ssPalmE, an ssPalm with vitamin E scaffolds, elicited a higher gene expression activity in comparison with the other LNPs composed of the ssPalms with different hydrophobic scaffolds. Immunization with the ssPalmE-LNPs encapsulating plasmid DNA that encodes ovalbumin (OVA, a model tumor antigen) or profilin (TgPF, a potent antigen of Toxoplasma gondii) induced substantial antitumor or antiprotozoan effects, respectively. Flow cytometry analysis of the cells that had taken up the LNPs in draining lymph nodes (dLNs) showed that the ssPalmE-LNPs were largely taken up by macrophages and a small number of dendritic cells. We found that the transient deletion of CD169+ macrophages, a subpopulation of macrophages that play a key role in cancer immunity, unexpectedly enhanced the activity of the DNA vaccine. These data suggest that the ssPalmE-LNPs are effective DNA vaccine carriers, and a strategy for avoiding their being trapped by CD169+ macrophages will be a promising approach for developing next-generation DNA vaccines.
Development of an SS-Cleavable pH-Activated Lipid-Like Material (ssPalm) as a Nucleic Acid Delivery Device. Peer-reviewed

Hidetaka Akita

Biological & pharmaceutical bulletin　43　(11)　1617-1625　2020

DOI： 10.1248/bpb.b20-00534 　

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Gene and nucleic acid-based medication is an ultimate strategy in the field of personalized medicine. A gene or short interference RNA (siRNA) molecule needs to be delivered to the appropriate organelle (i.e., nucleus and cytoplasm, respectively). We recently focused on improving the intrinsic activity of my original material (ssPalm) in terms of endosomal/lysosomal membrane destabilization activity by chemically modifying the tertiary amine structure. In parallel, I have been expanding the range of applications of ssPalms. The first application is a DNA or RNA vaccine. My crucial finding is that the vitamin E-scaffold ssPalm (ssPalmE) is highly immune-stimulative when combined with DNA. Thereafter, I redesigned the hydrophobic scaffold structure, and found that an oleic acid-scaffold ssPalm (ssPalmO) can confer anti-inflammatory characteristics. Based on this result, I further upgraded the ssPalmO, by inserting a newly designed linker with self-degradable properties.
Development of a lipoplex-type mRNA carrier composed of an ionizable lipid with a vitamin E scaffold and the KALA peptide for use as an ex vivo dendritic cell-based cancer vaccine. International-journal Peer-reviewed

Naho Tateshita, Naoya Miura, Hiroki Tanaka, Takeshi Masuda, Sumio Ohtsuki, Kota Tange, Yuta Nakai, Hiroki Yoshioka, Hidetaka Akita

Journal of controlled release : official journal of the Controlled Release Society　310　36-46　2019/09/28

DOI： 10.1016/j.jconrel.2019.08.002 　

ISSN： 0168-3659

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A dendritic cells (DCs)-based vaccine (DC-vaccine) system is an attractive technology for eliciting antigen-specific immune responses that can protect subjects from infectious diseases and for curing various types of cancers. For the insertion of a foreign antigen to DCs, the transfection of an antigen-coding mRNA to the cells is a promising approach. In order to introduce an antigen, a carrier for mRNA transfection is required, since the mRNA molecule per se is unstable in serum-containing medium. We previously reported on an ionizable lipid-like material with vitamin E-scaffolds (ssPalmE) as a material for a lipid nanoparticle (LNP)-based carrier for nucleic acids. In the present study, we report on the development of a lipoplex-type mRNA carrier for use as a DC-vaccine by using a combination of an ssPalmE-LNP and an α-helical cationic peptide "KALA" (ssPalmE-KALA). The transfection of mRNAs complexed with the ssPalmE-KALA achieved a significantly higher protein expression and the production of proinflammatory cytokines from murine bone marrow derived DCs (BMDCs) in comparison with a lipoplex that was prepared with an ssPalm with fatty acid-scaffolds (myristic acid; ssPalmM-KALA). A cellular uptake process and a pH-responsive membrane-destabilization activity cannot explain the preferred protein expression and immune-stimulation caused by the ssPalmE-KALA. Proteomic analyses suggest that transfection with the ssPalmM-KALA stimulates a down-regulatory pathway of translation, while the transfection with the ssPalmE-KALA does not stimulate it. In the vaccination with the BMDCs that were preliminarily transfected with an ovalbumin (OVA)-encoding mRNA elicited the induction OVA specific cytotoxic T-lymphocyte activity in vivo. In parallel, the vaccination induced significant prophylactic anti-tumor effects against a model tumor that stably expressed the OVA protein. Based on the above findings, the ssPalmE-KALA appears to be a potent ex vivo DCs-based RNA vaccine platform.
Lipid nanoparticle-mediated mRNA delivery system into preimplantation embryos†. International-journal

Chihiro Emori, Hiroki Tanaka, Tatsuya Nakagawa, Hidetaka Akita, Masahito Ikawa

Biology of reproduction　114　(3)　1070-1078　2026/03/16

DOI： 10.1093/biolre/ioaf262 　

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Lipid nanoparticles (LNPs) have emerged as a nonviral mRNA delivery vehicle for both basic and clinical applications. In the present study, we showed that the LNPs passed through the zona pellucida, the extracellular matrix surrounding the egg, and efficiently transduced preimplantation embryos. We first tested different types of surface polarity in LNPs carrying Enhanced Green Fluorescent Protein (EGFP) (LNP-EGFP) by changing cholesterol and found that the LNPs composed with neutral or anionic cholesterol are less toxic and more suitable for mRNA delivery into zygotes. Next, we transferred Cre mRNA using neutral LNP (LNP-Cre) and saw the floxed allele recombination in reporter mTmG transgenic embryos. After treatment with LNP-Cre at a 10 ng/μL concentration for 20 h at the zygote stage, about 76% mTmG embryos expressed the reporter EGFP at the blastocyst stage, and about 82% of embryos expressed EGFP in both the placenta and fetus at E12.5. Finally, we treated mTmG blastocyst stage embryos with LNP-Cre at a 50 ng/μL concentration for 20 h and found that about 86% of them expressed the reporter EGFP. Intriguingly, the EGFP fluorescence was only observed in trophectoderm cells, but not in the inner cell mass. Subsequently, we observed placenta-specific reporter EGFP expression in about 65% of the later-stage embryos (E12.5). Collectively, our study shows that LNPs provide a novel mRNA delivery system into preimplantation embryos that can be used for studying gene functions in pre-, peri-, and post-implantation development.
Lipid nanoparticle-encapsulated microRNA-192: An anti-inflammatory adjuvant that enhances vaccine efficacy in aged mice. International-journal

Yuriko Takagi, Tasuku Nishimura, Suraiya Aktar, Daisuke Katayama, Ken Takashima, Tomomi Kawakita, Toshiki Sekiya, Masashi Shingai, Hiroki Tanaka, Hidetaka Akita, Yusuke Miyashita, Kimitoshi Nakamura, Takahisa Kouwaki, Hiroyuki Oshiumi

Molecular therapy. Nucleic acids　37　(1)　102784-102784　2026/03/12

DOI： 10.1016/j.omtn.2025.102784 　

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Proinflammatory cytokines are essential for initiating immune responses; however, excessive or aging-related chronic inflammation impairs immunity and reduces vaccine efficacy. In this study, we developed lipid nanoparticles (LNPs) encapsulating anti-inflammatory microRNA-192 (miR-192) to attenuate inflammation and improve vaccine performance in the elderly. Results revealed that specific proinflammatory cytokines, including interleukin (IL)-6 and tumor necrosis factor (TNF)-α at the vaccination site, diminished antigen-specific antibody production. Notably, miR-192 endowed LNPs with strong anti-inflammatory properties, markedly enhancing vaccine efficacy, especially in aged mice. Transcriptomic analyses demonstrated that miR-192 downregulated multiple pro-inflammatory cytokines, such as senescence-associated secreted phenotype factors, which hinder vaccine responses. Additionally, miR-192 inhibited key components of the JAK-STAT signaling pathway, crucial for cytokine receptor signaling in myeloid cells. Overall, these findings indicate that miR-192 effectively suppresses harmful inflammatory responses, substantially enhancing vaccine efficacy, and highlight the therapeutic potential of the anti-inflammatory microRNA-based adjuvants for improving vaccination outcomes in the elderly.
Combining Anti-Inflammatory Drugs and Cell-Specific Expression-Suppression Reduces the Adjuvant Activity of mRNA-LNPs International-journal

Hiroki Tanaka, Naomasa Nishiumi, Daiki Shirane, Yuto Kosegawa, Yves Junior Siril, Hidetaka Akita

Molecular Pharmaceutics　23　(2)　721-729　2026/02/02

DOI： 10.1021/acs.molpharmaceut.5c00789 　

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Lipid nanoparticles (LNPs)-encapsulating mRNA (mRNA-LNPs) have become an important modality for vaccine development. mRNA-LNPs also have the ability to introduce therapeutic proteins, which suggests that they should also be a promising modality for gene therapy. However, the intrinsic adjuvant activity of mRNA-LNPs is a fundamental function of the LNPs in RNA vaccines, and this has shown a potential risk of inducing anti-transgene immunity when mRNA-LNPs were used in gene therapy. Therefore, a reduction in the adjuvant activity and adaptive immunity against an expressed protein are prerequisites for therapeutic applications. In this study, two strategies were combined to modulate the adjuvant activity of subcutaneously injected mRNA-LNPs: (1) loading a lipid derivative of dexamethasone to suppress inflammation and (2) inserting a sequence complementary to microRNA-142 into the mRNA to suppress gene expression in immune cells. The combination of these strategies reduced immune reactions against the model antigen ovalbumin in mRNA-LNPs.
Combination of ionizable lipids with oleic acid and vitamin E scaffolds for RNA cancer vaccine delivery International-journal

Anindita, J., Tanaka, H., Oyama, R., Matsumaru, A., Nakai, Y., Tange, K., Nagaoka, K., Nakanishi, H., Kawamura, T., Tanaka, T., Yamashita, T., Kuroda, A., Nomura, S., Hatakeyama, H., Itaka, K., Kodama, T., Kakimi, K., Akita, H.

Journal of Controlled Release　389　114414-114414　2026

DOI： 10.1016/j.jconrel.2025.114414 　

ISSN： 0168-3659 1873-4995
Modulating Immunogenicity and Reactogenicity in mRNA-Lipid Nanoparticle Vaccines through Lipid Component Optimization International-journal

Yoshino Kawaguchi, Mari Kimura, Tatsuya Karaki, Hiroki Tanaka, Chikako Ono, Tatsuhiro Ishida, Yoshiharu Matsuura, Toshiro Hirai, Hidetaka Akita, Taro Shimizu, Yasuo Yoshioka

ACS Nano　19　(30)　27977-28001　2025/08/05

DOI： 10.1021/acsnano.5c10648 　

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Messenger RNA (mRNA) vaccines effectively induce antibody production and T cell responses. However, adverse reactions, such as fatigue and fever, following administration remain a key challenge. To modulate the immunogenicity and reactogenicity of mRNA vaccines, the optimization of lipid nanoparticle (LNP) formulations has been attempted, particularly by screening ionizable lipids. In contrast, the potential impact of modifying other LNP components─poly(ethylene glycol) (PEG)-lipids, cholesterol, and phospholipids─on overall vaccine effects and adverse reactions remains underexplored. Here, we prepared mRNA-LNP formulations with altered structures and molar ratios of these components to assess their effects on in vivo protein expression, as well as on the induction of antigen-specific immune responses and adverse reactions. Reducing the PEG chain length and molar ratio of PEG-lipids increased antigen-specific antibody and CD8+ T cell responses. LNPs with cholesterol substituted by plant sterols, or LNPs with phospholipids replaced by those with different head and tail group structures, induced antigen-specific antibody and CD8+ T cell responses comparable to the control formulation. Alternately, these LNPs significantly reduced inflammatory cytokine production and adverse reactions, including fever, compared with the control LNPs. Finally, correlation analysis revealed a positive association between protein expression in specific organs and the magnitude of immune responses and adverse reactions. These findings demonstrate that modifying PEG-lipids, cholesterol, and phospholipids is beneficial for modulating the immunogenicity and reactogenicity of the mRNA-LNP vaccine.
Neoantigen mRNA vaccines induce progenitor-exhausted T cells that support anti-PD-1 therapy in gastric cancer with peritoneal metastasis

Koji Nagaoka, Hideyuki Nakanishi, Hiroki Tanaka, Jessica Anindita, Takeshi Kawamura, Toshiya Tanaka, Takefumi Yamashita, Akihiro Kuroda, Sachiyo Nomura, Hidetaka Akita, Keiji Itaka, Tatsuhiko Kodama, Kazuhiro Kakimi

Gastric Cancer　28　(5)　825-836　2025/07/31
Publisher: Springer Science and Business Media LLC
DOI： 10.1007/s10120-025-01640-8 　

ISSN： 1436-3291

eISSN： 1436-3305

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Abstract Background Gastric cancer with peritoneal metastasis is associated with a poor prognosis. Current treatments, including the first-line therapy of combination chemotherapy with nivolumab for advanced recurrent gastric cancer, have shown limited efficacy against peritoneal dissemination. In this study, we evaluated neoantigen (neoAg)-mRNA lipid nanoparticle (LNP) as a potential agent in combination with anti-PD-1 therapy, focusing on its effects on neoAg-specific CD8⁺ T cell responses and antitumor efficacy in a murine gastric cancer model. Methods The mRNA, comprising a tandem minigene encoding three neoAgs identified from the murine gastric cancer YTN16 cell line, was synthesized by in vitro transcription and encapsulated within LNPs. NeoAg-specific CD8⁺ T cells in the spleens and tumors were assessed by flow cytometry. The antitumor efficacy of the neoAg-mRNA-LNP vaccine, alone or in combination with anti-PD-1 antibody, was evaluated in both subcutaneous and peritoneal metastasis models of YTN16. Results The neoAg-mRNA-LNP vaccine induced significantly higher frequencies of neoAg-specific CD8⁺ T cells than the neoAg-dendritic cell vaccine, confirming its enhanced immunogenicity. NeoAg-mRNA-LNP vaccination led to robust tumor regression, achieving complete eradication in all treated mice, especially when combined with anti-PD-1 therapy. This effect was associated with an increase in neoAg-specific progenitor-exhausted and intermediate-exhausted CD8⁺ T cells. In a peritoneal metastasis model, neoAg-mRNA-LNP monotherapy prevented peritoneal dissemination when administered prophylactically, and combination therapy with anti-PD-1 effectively suppressed tumor growth in a therapeutic setting. Conclusions NeoAg-mRNA-LNP vaccines elicit potent neoAg-specific CD8⁺ T cell responses and show enhanced antitumor efficacy with anti-PD-1 therapy in gastric cancer with peritoneal metastasis.
A Post-Encapsulation Method for the Preparation of mRNA-LNPs via the Nucleic Acid-Bridged Fusion of mRNA-Free LNPs. International-journal

Hiroki Tanaka, Yuka Sato, Tomoya Nakabayashi, Akari Tanaka, Kazuma Nishio, Chika Matsumoto, Atsuya Matsumaru, Takuma Yamakawa, Kota Ishizaki, Keisuke Ueda, Kenjirou Higashi, Kunikazu Moribe, Yuta Nakai, Kota Tange, Hidetaka Akita

Nano letters　25　(16)　6445-6453　2025/04/23

DOI： 10.1021/acs.nanolett.4c06643 　

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Lipid nanoparticles with encapsulated mRNA (mRNA-LNPs) have become key modalities for personalized medicines and RNA vaccines. Once the platform technology is established, the mRNA-LNPs could be applicable to a variety of protein-based therapeutic strategies. A post-encapsulation method, in which the mRNA solution is incubated with preformed mRNA-free LNPs to prepare the mRNA-LNPs, would accelerate the development of RNA-based therapeutics since even nonexperts could manufacture the mRNA-LNPs. In this study, we describe that the post-encapsulation of mRNA into mRNA-free LNPs is accompanied by "nucleic acid-bridged fusion" of them. The adsorption of mRNA onto mRNA-free LNPs via electrostatic interactions and the internalization of mRNA into the LNPs via particle-to-particle fusion are two steps that occur at different levels of pH. To complete post-encapsulation using only one-step mixing, the pH must be controlled within a limited region where both processes occur simultaneously. The size of the mRNA-free LNPs determines the effectiveness of mRNA loading.
Synthesis of new functionalized bioactive S-substituted indolyl-triazoles as cytotoxic and apoptotic agents through multi-targeted kinase inhibition

Boraei, A.T.A., Nafie, M.S., Barakat, A., Tanaka, T., Kawano, K., Tojo, T., Aoki, S., Sarhan, A.A.M.

Bioorganic Chemistry　156　2025

DOI： 10.1016/j.bioorg.2025.108154 　

ISSN： 0045-2068 1090-2120
Systemic mRNA Delivery into the Muscle of Duchenne Muscular Dystrophy Model Mice Using Alpha-Dystroglycan Binding Peptide Modified Lipid Nanoparticles.

Eri Sasaki, Yuki Itaya, Yoko Endo-Takahashi, Yusuke Yano, Nobuhito Hamano, Keisuke Hamada, Yamato Kikkawa, Kosuke Nakashima, Rui Tada, Tsuyoshi Miura, Hiroki Tanaka, Hidetaka Akita, Motoyoshi Nomizu, Yoichi Negishi

Biological & pharmaceutical bulletin　48　(5)　721-727　2025

DOI： 10.1248/bpb.b24-00898 　

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Duchenne muscular dystrophy (DMD) is a hereditary disease that requires gene or nucleic acid therapy, which involves muscle-targeted delivery of therapeutic material. We previously developed liposomes targeting muscle tissue in DMD model mice (mdx) using an A2G80 peptide, which has an affinity for α-dystroglycan abundantly expressed on the muscle cell membrane. However, these liposomes did not carry gene or nucleic acids. In this study, we aimed to develop muscle-targeting lipid nanoparticles (LNPs) encapsulating luciferase mRNA and evaluate gene expression levels after systemic administration of these LNPs. We first evaluated the efficiency of mRNA delivery based on luciferase activity using polyethylene glycol (PEG)-dimyristoyl glycerol (DMG) and PEG-distearoyl glycerol (DSG) in mdx systemic administration. PEG-DSG-LNPs showed lower luciferase expression in the liver and spleen and higher expression in mdx muscle tissue than PEG-DMG-LNPs. The addition of the A2G80 peptide to LNPs using PEG-DSG (A2G80-DSG-LNPs) significantly increased their activity in mdx but not in normal mice. These results suggest that A2G80-DSG-LNPs allow for muscle-targeted mRNA delivery and are useful tools for DMD treatment.
Antibody-Dependent Cellular Cytotoxicity of Anti-Pd-L1 Antibodies for T Cells Attenuate Their Antitumor Efficacy in a Murine Tumor Model International-journal

Yuta Tamemoto, Yoshito Nakamura, Taiki Kurino, Ruiheng Tang, Takahiro Arai, Hiroyuki Suzuki, Tomoya Uehara, Hidetaka Akita, Akihiro Hisaka, Hiroto Hatakeyama

International journal of pharmaceutics　679　125755-125755　2025
Publisher: Elsevier BV
DOI： 10.2139/ssrn.5157800 　

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We evaluated the significant determinants of the therapeutic efficacy of anti-programmed death-ligand 1 (aPD-L1) monoclonal antibodies (aPD-L1 mAbs) by comparing the differences in antibody-dependent cellular cytotoxicity (ADCC) activity of aPD-L1 mAbs of different isotypes (clones 10F.9G2 and MIH6). In MM48 tumor-bearing mice, MIH6 without ADCC activity inhibited tumor growth by more than 90 %, whereas 10F.9G2 with ADCC activity showed slight antitumor effect. The dissociation constant of PD-L1 on MM48 cell surface was approximately 100-fold lower for MIH6 than for 10F.9G2. However, antibody uptake by cells was comparable for both aPD-L1 mAbs. The half-life and amount of intact form of aPD-L1 mAbs in tumors were 2.6- and 1.6-fold higher in MIH6 cells than in 10F.9G2 cells, respectively. Therefore, differences in the pharmacokinetics of PD-L1 mAbs along with different ADCC activities are insufficient to explain their different antitumor effects. Only 10F.9G2 reduced the number of CD8+ T cells in MM48 tumor-bearing mice. These results suggest that the difference in efficacy of aPD-L1 mAbs could be because aPD-L1 mAbs recognize PD-L1 on T cells, and the number of CD8+ T cells exerting antitumor effect is depleted by 10F.9G2 through ADCC activity. In conclusion, the effect of differences in the pharmacokinetics of aPD-L1 mAbs on the antitumor effect in MM48 tumor-bearing mice was considered negligible. Additionally, the difference in ADCC activity of aPD-L1 mAbs caused a decrease in CD8+ T cells, and this off-target effect was considered a factor affecting their antitumor effects.
Low-inflammatory lipid nanoparticle-based mRNA vaccine elicits protective immunity against H5N1 high-pathogenicity avian influenza virus with reduced adverse reactions. International-journal Peer-reviewed

Atsushi Kawai, Taro Shimizu, Hiroki Tanaka, Shintaro Shichinohe, Jessica Anindita, Mika Hirose, Eigo Kawahara, Kota Senpuku, Makoto Shimooka, Le Thi Quynh Mai, Ryo Suzuki, Takuto Nogimori, Takuya Yamamoto, Toshiro Hirai, Takayuki Kato, Tokiko Watanabe, Hidetaka Akita, Yasuo Yoshioka

Molecular therapy : the journal of the American Society of Gene Therapy　33　(2)　529-547　2024/12/17

DOI： 10.1016/j.ymthe.2024.12.032 　

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Messenger RNA vaccines based on lipid nanoparticles (mRNA-LNPs) are promising vaccine modalities. However, mRNA-LNP vaccines frequently cause adverse reactions such as swelling and fever in humans, partly due to the inflammatory nature of LNP. Modification of the ionizable lipids used in LNP is one approach to avoid these adverse reactions. Herein, we report the development of mRNA-LNP vaccines with better protective immunity and reduced adverse reactions using LNP, which contains a disulfide (SS)-cleavable bond and pH-activated lipid-like materials with oleic acid (ssPalmO) as an ionizable lipid (LNPssPalmO). We used mRNA expressing H5N1 subtype high-pathogenicity avian influenza virus-derived hemagglutinin or neuraminidase to generate mRNA-LNP vaccines against H5N1 influenza. Compared with conventional LNP, mRNA-LNPssPalmO induced comparable antigen-specific antibodies and better interferon-gamma (IFN-γ)-producing T-helper type-1 (Th1) responses in mice. Both mRNA-LNPssPalmO and conventional mRNA-LNP conferred strong protection against homologous H5N1 virus challenge. In addition, mRNA-LNPssPalmO showed better cross-protection against heterologous H5N1 virus challenge compared with conventional mRNA-LNPs. Furthermore, we observed that mRNA-LNPssPalmO induced less inflammatory responses (e.g., inflammatory cytokine production and vascular hyperpermeability) and fewer adverse reactions (e.g., weight loss and fever) compared with conventional mRNA-LNP. These results suggest that mRNA-LNPssPalmO would be a safe alternative to conventional vaccines to overcome mRNA-LNP vaccine hesitancy.
Effect of Anti-PEG Antibody on Immune Response of mRNA-Loaded Lipid Nanoparticles. International-journal

Daiki Omata, Eigo Kawahara, Lisa Munakata, Hiroki Tanaka, Hidetaka Akita, Yasuo Yoshioka, Ryo Suzuki

Molecular pharmaceutics　21　(11)　5672-5680　2024/09/26

DOI： 10.1021/acs.molpharmaceut.4c00628 　

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Lipid nanoparticle-encapsulated mRNA (mRNA-LNP) vaccines have been approved for use to combat coronavirus disease 2019 (COVID-19). The mRNA-LNPs contain PEG-conjugated lipids. Clinical studies have reported that mRNA-LNPs induce the production of anti-PEG antibodies, but the anti-PEG antibodies do not affect the production of neutralizing antibodies. However, the detailed influence of anti-PEG antibodies on mRNA-LNP vaccines remains unclear. Therefore, in this study, we prepared ovalbumin (OVA) as a model antigen-encoding mRNA-loaded LNP (mRNA-OVA-LNP), and we determined whether anti-PEG antibodies could affect the antigen-specific immune response of mRNA-OVA-LNP vaccination in mice pretreated with PEG-modified liposomes to induce the production of anti-PEG antibodies. After intramuscular (i.m.) injection of the mRNA-LNP, the anti-PEG antibodies did not change the expression of protein or induction of cytokine and cellular immune response but did slightly increase the induction of antigen-specific antibodies. Furthermore, repeated mRNA-LNP i.m. injection induced the production of anti-PEG IgM and anti-PEG IgG. Our results suggest that mRNA-LNP induces the production of anti-PEG antibodies, but the priming of the antigen-specific immune response of mRNA-LNP vaccination is not notably affected by anti-PEG antibodies.
ネオアンチゲンmRNA脂質ナノ粒子ワクチンは、強力な抗腫瘍CD8+T細胞応答を誘導する(Neoantigen-mRNA-LNP Vaccine Induces Potent Anti-Tumor CD8+T cell responses in the tumor)

長岡孝治, 田中浩揮, 中西秀之, 川村猛, 田中十志也, 山下雄史, 野村幸世, 秋田英万, 位高啓史, 児玉龍彦, 垣見和宏, 垣見和宏

日本癌学会総会記事　83回　P-1165　2024/09
Publisher: (一社)日本癌学会
ISSN： 0546-0476
Author Correction: EVs-miR-17-5p attenuates the osteogenic differentiation of vascular smooth muscle cells potentially via inhibition of TGF-β signaling under high glucose conditions. International-journal

Isashi Baba, Tetsuya Matoba, Shunsuke Katsuki, Jun-Ichiro Koga, Takuro Kawahara, Mitsukuni Kimura, Hidetaka Akita, Hiroyuki Tsutsui

Scientific reports　14　(1)　19601-19601　2024/08/23

DOI： 10.1038/s41598-024-70722-x 　
NMR-based analysis of impact of siRNA mixing conditions on internal structure of siRNA-loaded LNP. International-journal

Keisuke Ueda, Yui Sakagawa, Tomoki Saito, Fumie Sakuma, Hiroki Tanaka, Hidetaka Akita, Kenjirou Higashi, Kunikazu Moribe

Journal of controlled release : official journal of the Controlled Release Society　373　738-748　2024/08/02

DOI： 10.1016/j.jconrel.2024.07.055 　

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This study aimed to assess the applicability of solution-state 1H NMR for molecular-level characterization of siRNA-loaded lipid nanoparticles (LNP). Dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA, MC3) was used as an ionizable lipid, and siRNA-loaded LNPs were prepared by pre-mixing and post-mixing methods. The pre-mixing method involved mixing an acidic solution containing siRNA with an ethanolic lipid solution using a microfluidic mixer. The pre-mixed LNP was prepared by dialyzing the mixed solution into the phosphate buffered saline (PBS, pH 7.4). The post-mixed LNP was prepared by mixing the siRNA solution with empty LNP in an acidic condition with and without ethanol, resulting in post-mixed LNP (A) and (B), respectively. Both pre-mixed and post-mixed LNPs formed LNP particles with an average diameter of approximately 50 nm. Moreover, the ratio of encapsulated siRNA to lipid content in each LNP particle remained constant regardless of the preparation method. However, small-angle X-ray scattering measurements indicated structural variations in the siRNA-MC3 stacked bilayer structure formed in the LNPs, depending on the preparation method. Solution-state 1H NMR analysis suggested that the siRNA was incorporated uniformly into the LNP core for pre-mixed LNP compared to post-mixed LNPs. In contrast, the post-mixed LNPs contained siRNA-empty regions with local enrichment of siRNA in the LNP core. This heterogeneity was more pronounced in post-mixed LNP (B) than in post-mixed LNP (A), suggesting that ethanol facilitated the homogeneous mixing of siRNA with LNP lipids. The silencing effect of each siRNA-loaded LNP was reduced in the order of pre-mixed LNP, post-mixed LNP (A), and post-mixed LNP (B). This suggested that the heterogeneity of the siRNA-loaded LNP could cause a reduction in the silencing effect of the incorporated siRNA inside LNPs. The present study highlighted that NMR-based characterization of siRNA-loaded LNP can reveal the molecular-level heterogeneity of siRNA-loaded LNP, which helps to optimize the preparation conditions of siRNA-loaded LNP formulations.
EVs-miR-17-5p attenuates the osteogenic differentiation of vascular smooth muscle cells potentially via inhibition of TGF-β signaling under high glucose conditions. International-journal

Isashi Baba, Tetsuya Matoba, Shunsuke Katsuki, Jun-Ichiro Koga, Takuro Kawahara, Mitsukuni Kimura, Hidetaka Akita, Hiroyuki Tsutsui

Scientific reports　14　(1)　16323-16323　2024/07/15

DOI： 10.1038/s41598-024-67006-9 　

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Vascular calcification, which is a major complication of diabetes mellitus, is an independent risk factor for cardiovascular disease. Osteogenic differentiation of vascular smooth muscle cells (VSMCs) is one of the key mechanisms underlying vascular calcification. Emerging evidence suggests that macrophage-derived extracellular vesicles (EVs) may be involved in calcification within atherosclerotic plaques in patients with diabetes mellitus. However, the role of macrophage-derived EVs in the progression of vascular calcification is largely unknown. In this study, we investigated whether macrophage-derived EVs contribute to the osteogenic differentiation of VSMCs under high glucose conditions. We isolated EVs that were secreted by murine peritoneal macrophages under normal glucose (EVs-NG) or high glucose (EVs-HG) conditions. miRNA array analysis in EVs from murine macrophages showed that miR-17-5p was significantly increased in EVs-HG compared with EVs-NG. Prediction analysis with miRbase identified transforming growth factor β receptor type II (TGF-β RII) as a potential target of miR-17-5p. EVs-HG as well as miR-17-5p overexpression with lipid nanoparticles inhibited the gene expression of Runx2, and TGF-β RII. Furthermore, we demonstrated that VSMCs transfected with miR-17-5p mimic inhibited calcium deposition. Our findings reveal a novel role of macrophage-derived EVs in the negative regulation of osteogenic differentiation in VSMCs under high glucose conditions.
siRNA混合条件がsiRNA封入LNPの内部構造に及ぼす影響のNMRを用いた解析

植田圭祐, 東顕二郎, 田中浩揮, 秋田英万, 森部久仁一

日本DDS学会学術集会プログラム予稿集　40回　687-687　2024/07
Publisher: 日本DDS学会
基礎と臨床の融合による新時代のがん免疫療法ネオアンチゲンmRNA脂質ナノ粒子ワクチンは、強力な抗腫瘍CD8+T細胞応答を誘導する

長岡孝治, 田中浩揮, 中西秀之, 川村猛, 田中十志也, 野村幸世, 秋田英万, 位高啓史, 児玉龍彦, 垣見和宏

日本がん免疫学会総会・日本バイオセラピィ学会学術集会総会合同大会プログラム・抄録集　28回・37回　67-67　2024/06
Publisher: 日本がん免疫学会・日本バイオセラピィ学会
The stress-responsive cytotoxic effect of diesel exhaust particles on lymphatic endothelial cells. International-journal

Yu Sakurai, Eiki Oba, Akiko Honda, Hiroki Tanaka, Hirohisa Takano, Hidetaka Akita

Scientific reports　14　(1)　10503-10503　2024/05/07

DOI： 10.1038/s41598-024-61255-4 　

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Diesel exhaust particles (DEPs) are very small (typically < 0.2 μm) fragments that have become major air pollutants. DEPs are comprised of a carbonaceous core surrounded by organic compounds such as polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs. Inhaled DEPs reach the deepest sites in the respiratory system where they could induce respiratory/cardiovascular dysfunction. Additionally, a previous study has revealed that a portion of inhaled DEPs often activate immune cells and subsequently induce somatic inflammation. Moreover, DEPs are known to localize in lymph nodes. Therefore, in this study we explored the effect of DEPs on the lymphatic endothelial cells (LECs) that are a constituent of the walls of lymph nodes. DEP exposure induced cell death in a reactive oxygen species (ROS)-dependent manner. Following exposure to DEPs, next-generation sequence (NGS) analysis identified an upregulation of the integrated stress response (ISR) pathway and cell death cascades. Both the soluble and insoluble components of DEPs generated intracellular ROS. Three-dimensional Raman imaging revealed that DEPs are taken up by LECs, which suggests internalized DEP cores produce ROS, as well as soluble DEP components. However, significant cell death pathways such as apoptosis, necroptosis, ferroptosis, pyroptosis, and parthanatos seem unlikely to be involved in DEP-induced cell death in LECs. This study clarifies how DEPs invading the body might affect the lymphatic system through the induction of cell death in LECs.
The Effect of Cholesterol Content on the Adjuvant Activity of Nucleic-Acid-Free Lipid Nanoparticles. International-journal

Jessica Anindita, Hiroki Tanaka, Takuma Yamakawa, Yuka Sato, Chika Matsumoto, Kota Ishizaki, Taiji Oyama, Satoko Suzuki, Keisuke Ueda, Kenjirou Higashi, Kunikazu Moribe, Kasumi Sasaki, Yumika Ogura, Etsuo Yonemochi, Yu Sakurai, Hiroto Hatakeyama, Hidetaka Akita

Pharmaceutics　16　(2)　2024/01/26

DOI： 10.3390/pharmaceutics16020181 　

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RNA vaccines are applicable to the treatment of various infectious diseases via the inducement of robust immune responses against target antigens by expressing antigen proteins in the human body. The delivery of messenger RNA by lipid nanoparticles (LNPs) has become a versatile drug delivery system used in the administration of RNA vaccines. LNPs are widely considered to possess adjuvant activity that induces a strong immune response. However, the properties of LNPs that contribute to their adjuvant activity continue to require clarification. To characterize the relationships between the lipid composition, particle morphology, and adjuvant activity of LNPs, the nanostructures of LNPs and their antibody production were evaluated. To simply compare the adjuvant activity of LNPs, empty LNPs were subcutaneously injected with recombinant proteins. Consistent with previous research, the presence of ionizable lipids was one of the determinant factors. Adjuvant activity was induced when a tiny cholesterol assembly (cholesterol-induced phase, ChiP) was formed according to the amount of cholesterol present. Moreover, adjuvant activity was diminished when the content of cholesterol was excessive. Thus, it is plausible that an intermediate structure of cholesterol (not in a crystalline-like state) in an intra-particle space could be closely related to the immunogenicity of LNPs.
Detection of circulating tumor cells in blood using two-step random forest

Wei, H., Natori, T., Tanaka, T., Aoki, S., Kuriyama, S., Yamada, T., Aikawa, N.

Electronics and Communications in Japan　107　(3)　2024

DOI： 10.1002/ecj.12447 　

ISSN： 1942-9533 1942-9541
Amodiaquine Analogs Are Potent Inhibitors of Interleukin-6 Production Induced by Activation of Toll-Like Receptors Recognizing Pathogen Nucleic Acids

Takenaka, Y., Tanaka, T., Otaki, S., Kanbe, A., Morita, T., Yokoi, K., Sekiguchi, S., Nakamura, K., Satoh, H., Tojo, T., Uchiumi, F., Kitabatake, K., Aoki, S., Tsukimoto, M.

Biological and Pharmaceutical Bulletin　47　(12)　2024

DOI： 10.1248/bpb.b24-00639 　

ISSN： 0918-6158 1347-5215
Detection of Circulating Tumor Cells in Blood Using Two-Step Random Forest,二段階の Random Forest を用いた血中循環がん細胞の検出

Wei, H., Natori, T., Tanaka, T., Aoki, S., Kuriyama, S., Yamada, T., Aikawa, N.

Ieej Transactions on Electronics Information and Systems　144　(3)　2024

DOI： 10.1541/ieejeiss.144.121 　

ISSN： 0385-4221 1348-8155
Lymphatic Endothelial Cells Produce Chemokines in Response to the Lipid Nanoparticles Used in RNA Vaccines.

Yi Liu, Miho Suzuoki, Hiroki Tanaka, Yu Sakurai, Hiroto Hatakeyama, Hidetaka Akita

Biological & pharmaceutical bulletin　47　(3)　698-707　2024

DOI： 10.1248/bpb.b23-00689 　

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RNA vaccines based on Lipid nanoparticles (LNP) were put into practical use within only one year after the global outbreak of the coronavirus disease 2019 (COVID-19). This success of RNA vaccine highlights the utility of an mRNA delivery system as a vaccination strategy. Potent immunostimulatory activity of LNPs (i.e., inflammation occurring at the injection site and the production of inflammatory cytokines) have recently been reported. However, we have only limited knowledge concerning which cells are responsible for responding to the LNPs. We report herein on in vitro chemokine production from non-immune cells in response to exposure to LNPs. In this study, SM-102, an ionizable lipid that is used in the approved RNA vaccine for the clinical usage of COVID-19 mRNA vaccine, was used. Immortalized mouse lymphatic endothelial cells (mLECs) or professional antigen presenting cells (APCs) such as RAW 264.7 monocyte/macrophage cells were incubated with LNPs that contained no mRNA. As a result, chemokines involved in the recruitment of monocytes/neutrophils were produced only by the mLECs following the LNP treatment. These findings indicate that LEC appear to serve as the cell that sends out initial signals to response LNPs.
Intranasal immunization with an RBD-hemagglutinin fusion protein harnesses preexisting immunity to enhance antigen-specific responses. International-journal

Atsushi Kawai, Nagisa Tokunoh, Eigo Kawahara, Shigeyuki Tamiya, Shinya Okamura, Chikako Ono, Jessica Anindita, Hiroki Tanaka, Hidetaka Akita, Sho Yamasaki, Jun Kunisawa, Toru Okamoto, Yoshiharu Matsuura, Toshiro Hirai, Yasuo Yoshioka

The Journal of clinical investigation　133　(23)　2023/12/01

DOI： 10.1172/JCI166827 　

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Intranasal vaccines are anticipated to be powerful tools for combating many infectious diseases, including SARS-CoV-2, because they induce not only systemic immunity but also mucosal immunity at the site of initial infection. However, they are generally inefficient in inducing an antigen-specific immune response without adjuvants. Here, we developed an adjuvant-free intranasal vaccine platform that utilizes the preexisting immunity induced by previous infection or vaccination to enhance vaccine effectiveness. We made RBD-HA, a fusion of the receptor-binding domain (RBD) of spike derived from SARS-CoV-2 as a vaccine target with HA derived from influenza A virus (IAV) as a carrier protein. Intranasal immunization of previously IAV-infected mice with RBD-HA without an adjuvant elicited robust production of RBD-specific systemic IgG and mucosal IgA by utilizing both HA-specific preexisting IgG and CD4+ T cells. Consequently, the mice were efficiently protected from SARS-CoV-2 infection. Additionally, we demonstrated the high versatility of this intranasal vaccine platform by assessing various vaccine antigens and preexisting immunity associated with a variety of infectious diseases. The results of this study suggest the promising potential of this intranasal vaccine platform to address problems associated with intranasal vaccines.
Development of a Ready-to-Use-Type RNA Vaccine Carrier Based on an Intracellular Environment-Responsive Lipid-like Material with Immune-Activating Vitamin E Scaffolds. International-journal

Jessica Anindita, Hiroki Tanaka, Ryotaro Oyama, Shinya Hagiwara, Daiki Shirane, Sakura Taneichi, Yuta Nakai, Kota Tange, Hiroto Hatakeyama, Yu Sakurai, Hidetaka Akita

Pharmaceutics　15　(12)　2023/11/29

DOI： 10.3390/pharmaceutics15122702 　

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Because of its efficient and robust gene transfer capability, messenger RNA (mRNA) has become a promising tool in various research fields. The lipid nanoparticle (LNP) is considered to be a fundamental technology for an mRNA delivery system and has been used extensively for the development of RNA vaccines against SARS-CoV-2. We recently developed ssPalm, an environmentally responsive lipid-like material, as a component of LNP for mRNA delivery. In this study, a self-degradable unit (phenyl ester) that confers high transfection activity and an immune stimulating unit (vitamin E scaffold) for high immune activation were combined to design a material, namely, ssPalmE-Phe-P4C2, for vaccine use. To design a simple and user-friendly form of an RNA vaccine based on this material, a freeze-drying-based preparation method for producing a ready-to-use-type LNP (LNP(RtoU)) was used to prepare the LNPssPalmE-Phe. The optimization of the preparation method and the lipid composition of the LNPssPalmE-Phe(RtoU) revealed that dioleoyl-sn-glycero phosphatidylethanolamine (DOPE) was a suitable helper lipid for achieving a high vaccination activity of the LNPssPalmE-Phe(RtoU). Other findings indicated that to maintain particle properties and vaccination activity, a 40% cholesterol content was necessary. A single administration of the LNPssPalmE-Phe(RtoU) that contained mRNA-encoding Ovalbumin (mOVA-LNPssPalmE-Phe(RtoU)) demonstrated a significant suppression of tumor progression in a tumor-bearing mouse OVA-expressing cell line (E.G7-OVA). In summary, the LNPssPalmE-Phe(RtoU) is an easy-to-handle drug delivery system (DDS) for delivering mRNA antigens in immunotherapy.
An Ionizable Lipid Material with a Vitamin E Scaffold as an mRNA Vaccine Platform for Efficient Cytotoxic T Cell Responses International-journal

Ryotaro Oyama, Harumichi Ishigame, Hiroki Tanaka, Naho Tateshita, Moeko Itazawa, Ryosuke Imai, Naomasa Nishiumi, Jun-ichi Kishikawa, Takayuki Kato, Jessica Anindita, Yoshifumi Nishikawa, Masatoshi Maeki, Manabu Tokeshi, Kota Tange, Yuta Nakai, Yu Sakurai, Takaharu Okada, Hidetaka Akita

ACS Nano　17　(19)　18758-18774　2023/09/26
Publisher: American Chemical Society (ACS)
DOI： 10.1021/acsnano.3c02251 　

ISSN： 1936-0851

eISSN： 1936-086X

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RNA vaccines based on lipid nanoparticles (LNPs) with in vitro transcribed mRNA (IVT-mRNA) encapsulated are now a currently successful but still evolving modality of vaccines. One of the advantages of RNA vaccines is their ability to induce CD8+ T-cell-mediated cellular immunity that is indispensable for excluding pathogen-infected cells or cancer cells from the body. In this study, we report on the development of LNPs with an enhanced capability for inducing cellular immunity by using an ionizable lipid with a vitamin E scaffold. An RNA vaccine that contained this ionizable lipid and an IVT-mRNA encoding a model antigen ovalbumin (OVA) induced OVA-specific cytotoxic T cell responses and showed an antitumor effect against an E.G7-OVA tumor model. Vaccination with the LNPs conferred protection against lethal infection by Toxoplasma gondii using its antigen TgPF. The vitamin E scaffold-dependent type I interferon response was important for effector CD8+ T cell differentiation induced by the mRNA-LNPs. Our findings also revealed that conventional dendritic cells (cDCs) were essential for achieving CD8+ T cell responses induced by the mRNA-LNPs, while the XCR1-positive subset of cDCs, cDC1 specialized for antigen cross-presentation, was not required. Consistently, the mRNA-LNPs were found to selectively transfect another subset of cDCs, cDC2 that had migrated from the skin to lymph nodes, where they could make vaccine-antigen-dependent contacts with CD8+ T cells. The findings indicate that the activation of innate immune signaling by the adjuvant activity of the vitamin E scaffold and the expression of antigens in cDC2 are important for subsequent antigen presentation and the establishment of antigen-specific immune responses.
A Lipid Nanoparticle-Based Method for the Generation of Liver-Specific Knockout Mice International-journal

Sumiyo Morita, Takuro Horii, Mika Kimura, Ryosuke Kobayashi, Hiroki Tanaka, Hidetaka Akita, Izuho Hatada

International Journal of Molecular Sciences　24　(18)　14299-14299　2023/09/19
Publisher: MDPI AG
DOI： 10.3390/ijms241814299 　

eISSN： 1422-0067

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Knockout mice are useful tools that can provide information about the normal function of genes, including their biochemical, developmental, and physiological roles. One problem associated with the generation of knockout mice is that the loss of some genes of interest produces a lethal phenotype. Therefore, the use of conditioned knockout mice, in which genes are disrupted in specific organs, is essential for the elucidation of disease pathogenesis and the verification of drug targets. In general, conditional knockout mice are produced using the Cre/loxP system; however, the production of the large numbers of Cre/flox knockout and control mice required for analysis requires substantial time and effort. Here, we describe the generation of liver-specific conditional knockout mice via the introduction of lipid nanoparticles encapsulating Cre mRNA into the liver of floxed mice. This technique does not require the production of offspring by mating floxed mice and is therefore more convenient than the conventional method. The results presented here demonstrate that the LNP-based method enables liver-specific gene knockout in a short period of time.
Tolerogenic Lipid Nanoparticles for Delivering Self-Antigen mRNA for the Treatment of Experimental Autoimmune Encephalomyelitis. International-journal

Masaki Gomi, Yuka Nakayama, Yu Sakurai, Ryotaro Oyama, Koki Iwasaki, Mizuki Doi, Yi Liu, Mizuho Hori, Himeka Watanabe, Kohei Hashimoto, Hiroki Tanaka, Kota Tange, Yuta Nakai, Hidetaka Akita

Pharmaceuticals (Basel, Switzerland)　16　(9)　2023/09/07

DOI： 10.3390/ph16091270 　

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Multiple sclerosis is a disease caused by autoantigen-responsive immune cells that disrupt the myelin in the central nervous system (CNS). Although immunosuppressive drugs are used to suppress symptoms, no definitive therapy exists. As in the experimental autoimmune encephalitis (EAE) model of multiple sclerosis, a partial sequence of the myelin oligodendrocyte glycoprotein (MOG35-55) was identified as a causative autoantigen. This suggests that the induction of immune tolerance that is specific to MOG35-55 would be a fundamental treatment for EAE. We previously reported that lipid nanoparticles (LNPs) containing an anionic phospholipid, phosphatidylserine (PS), in their lipid composition, can be used to deliver mRNA and that this leads to proteins of interest to be expressed in the spleen. In addition to the targeting capability of PS, PS molecules avoid activating the immune system. Physiologically, the recognition of PS on apoptotic cells suppresses immune activation against these cells by releasing cytokines, such as interleukin-10 (IL-10) and transforming growth factor (TGF)-β that negatively regulate immunity. In this study, we tested whether mRNA delivery of autoantigens to the spleen by PS-LNPs causes the expression of MOG35-55 antigens with minimal immune stimulation and whether this could be used to treat an EAE model by inducing immune tolerance.
Comprehensive Evaluation of Lipid Nanoparticles and Polyplex Nanomicelles for Muscle-Targeted mRNA Delivery. International-journal

Xuan Du, Erica Yada, Yuki Terai, Takuya Takahashi, Hideyuki Nakanishi, Hiroki Tanaka, Hidetaka Akita, Keiji Itaka

Pharmaceutics　15　(9)　2023/09/07

DOI： 10.3390/pharmaceutics15092291 　

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The growing significance of messenger RNA (mRNA) therapeutics in diverse medical applications, such as cancer, infectious diseases, and genetic disorders, highlighted the need for efficient and safe delivery systems. Lipid nanoparticles (LNPs) have shown great promise for mRNA delivery, but challenges such as toxicity and immunogenicity still remain to be addressed. In this study, we aimed to compare the performance of polyplex nanomicelles, our original cationic polymer-based carrier, and LNPs in various aspects, including delivery efficiency, organ toxicity, muscle damage, immune reaction, and pain. Our results showed that nanomicelles (PEG-PAsp(DET)) and LNPs (SM-102) exhibited distinct characteristics, with the former demonstrating relatively sustained protein production and reduced inflammation, making them suitable for therapeutic purposes. On the other hand, LNPs displayed desirable properties for vaccines, such as rapid mRNA expression and potent immune response. Taken together, these results suggest the different potentials of nanomicelles and LNPs, supporting further optimization of mRNA delivery systems tailored for specific purposes.
Logistics and distribution of small extracellular vesicles from the subcutaneous space to the lymphatic system International-journal

Yu Sakurai, Asa Ohtani, Yuka Nakayama, Masaki Gomi, Takeshi Masuda, Sumio Ohtsuki, Hiroki Tanaka, Hidetaka Akita

Journal of Controlled Release　361　77-86　2023/09
Publisher: Elsevier BV
DOI： 10.1016/j.jconrel.2023.07.043 　

ISSN： 0168-3659

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Small extracellular vesicles (sEVs) are small, cell-derived particles with sizes of approximately 100 nm. Since these particles include cargos such as host cell-derived proteins, messenger RNAs, and micro RNAs, they serve as mediators of cell-cell communication. While the analysis of the pharmacokinetic of sEVs after the intravenous injection have been reported, the lymphatic transport of sEVs remains unclear. The objective of this study was to provide insights into the intra-lymphatic trafficking and distribution of sEVs when they are injected into an interstitial space both in normal skin tissue and in cancerous tissue. When sEVs were Subcutaneously administered into the tail base and the tumor tissue, they preferably accumulated in the lymph nodes (LNs), rather than in the liver and the spleen. The findings reported herein show that the lymphatic transport of sEVs was drastically changed in model mice, in which a surgical treatment was used to modify to allow the dominant lymphatic flow from the footpad directly to the axillary LN via the inguinal LN. Based on the results, we conclude that when sEVs are injected into the subcutis space, they are preferably delivered to the LN via the lymphatic system. Further, the extent of accumulation of sEVs in the LN after subcutaneous injection was reduced when they were preliminarily incubated with Proteinase K. These results suggest that the lymphatic drainage of sEVs in normal skin tissue is regulated by membrane proteins on their surface. This reduction, however, was not observed in the case of cancer tissue. This discrepancy can be attributed to the presence of highly permeable lymphatic vessels in the tumor tissue. Further, the major cell subtypes that captured sEVs in the LN were LN-resident medullary sinus macrophages. These collective findings indicate that the lymphatic drainage of sEVs are mediated by proteins and, that they may appear to contribute to the control of the function of immune-responsive cells in the LNs.
脂質ナノ粒子の脂質組成と形態が免疫刺激特性に関与する(Lipid composition and morphology of lipid nanoparticle contributed to its immunostimulatory properties)

アニンディタ・ジェシカ, 田中浩揮, 植田圭祐, 東顕二郎, 森部久仁一, 佐々木香純, 米持悦生, 櫻井遊, 秋田英万

日本DDS学会学術集会プログラム予稿集　39回　185-185　2023/07
Publisher: 日本DDS学会
Development of an Alcohol Dilution-Lyophilization Method for the Preparation of mRNA-LNPs with Improved Storage Stability. International-journal

Daiki Shirane, Hiroki Tanaka, Yu Sakurai, Sakura Taneichi, Yuta Nakai, Kota Tange, Itsuko Ishii, Hidetaka Akita

Pharmaceutics　15　(7)　2023/06/26

DOI： 10.3390/pharmaceutics15071819 　

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The lipid nanoparticle (LNP) is one of the promising nanotechnologies for the delivery of RNA molecules, such as small interfering RNA (siRNA) and messenger RNA (mRNA). A series of LNPs that contain an mRNA encoding the antigen protein of SARS-CoV-2 were already approved as RNA vaccines against this infectious disease. Since LNP formulations are generally metastable, their physicochemical properties are expected to shift toward a more stable state during the long-time storage of suspensions. The current mRNA vaccines are supplied in the form of frozen formulations with a cryoprotectant for preventing deterioration. They must be stored in a freezer at temperatures from -80 °C to -15 °C. It is thought that therapeutic applications of this mRNA-LNP technology could be accelerated if a new formulation that permits mRNA-LNPs to be stored under milder conditions were available. We previously reported on a one-pot method for producing siRNA-encapsulated LNPs by combining freeze-drying technology with the conventional alcohol dilution method (referred to herein as the "alcohol dilution-lyophilization method"). In this study, this method was applied to the preparation of mRNA-LNPs to provide a freeze-dried formulation of mRNA LNPs. The resulting formulation can be stored at 4 °C for at least 4 months.
脂質ナノ粒子の脂質組成と形態が免疫賦活特性に及ぼす影響(Contribution of lipid composition and morphology of lipid nanoparticle to its immunostimulatory properties)

Anindita Jessica, 田中浩揮, 山川拓真, 佐藤裕果, 石崎弘汰, 松本千佳, 植田圭祐, 東顕二郎, 森部久仁一, 佐々木香純, 米持悦生, 櫻井遊, 秋田英万

日本薬剤学会年会講演要旨集　38年会　182-182　2023/05
Publisher: (公社)日本薬剤学会
脂質ナノ粒子の脂質組成と形態が免疫賦活特性に及ぼす影響(Contribution of lipid composition and morphology of lipid nanoparticle to its immunostimulatory properties)

Anindita Jessica, 田中浩揮, 山川拓真, 佐藤裕果, 石崎弘汰, 松本千佳, 植田圭祐, 東顕二郎, 森部久仁一, 佐々木香純, 米持悦生, 櫻井遊, 秋田英万

日本薬剤学会年会講演要旨集　38年会　182-182　2023/05
Publisher: (公社)日本薬剤学会
Ready-to-Use-Type Lyophilized Lipid Nanoparticle Formulation for the Postencapsulation of Messenger RNA. International-journal

Hiroki Tanaka, Shinya Hagiwara, Daiki Shirane, Takuma Yamakawa, Yuka Sato, Chika Matsumoto, Kota Ishizaki, Miho Hishinuma, Katsuyuki Chida, Kasumi Sasaki, Etsuo Yonemochi, Keisuke Ueda, Kenjirou Higashi, Kunikazu Moribe, Takashi Tadokoro, Katsumi Maenaka, Sakura Taneichi, Yuta Nakai, Kota Tange, Yu Sakurai, Hidetaka Akita

ACS nano　17　(3)　2588-2601　2023/01/31

DOI： 10.1021/acsnano.2c10501 　

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Based on the clinical success of an in vitro transcribed mRNA (IVT-mRNA) that is encapsulated in lipid nanoparticles (mRNA-LNPs), there is a growing demand by researchers to test whether their own biological findings might be applicable for use in mRNA-based therapeutics. However, the equipment and/or know-how required for manufacturing such nanoparticles is often inaccessible. To encourage more innovation in mRNA therapeutics, a simple method for preparing mRNA-LNPs is prerequisite. In this study, we report on a method for encapsulating IVT-mRNA into LNPs by rehydrating a Ready-to-Use empty freeze-dried LNP (LNPs(RtoU)) formulation with IVT-mRNA solution followed by heating. The resulting mRNA-LNPs(RtoU) had a similar intraparticle structure compared to the mRNA-LNPs prepared by conventional microfluidic mixing. In vivo genome editing, a promising application of these types of mRNA-LNPs, was accomplished using the LNPs(RtoU) containing co-encapsulated Cas9-mRNA and a small guide RNA.
Reactivation of Anticancer Immunity by Resetting Interorgan Crosstalk in Immune-Suppressive Cells with a Nanoparticulated Anti-Inflammatory Drug. International-journal

Mizuki Doi, Hiroki Tanaka, Takara Ohoto, Naoya Miura, Yu Sakurai, Hiroto Hatakeyama, Hidetaka Akita

Small (Weinheim an der Bergstrasse, Germany)　19　(16)　e2205131　2023/01/26

DOI： 10.1002/smll.202205131 　

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The reactivation of anticancer immunity is a fundamental principle in cancer immunotherapy as evidenced by the use of immune checkpoint inhibitors (ICIs). While treatment with the ICIs is shown to have remarkable and durable therapeutic effects in the responders, the low objective response rate (<40%) continues to be a major problem. Since myeloid-derived suppressor cells (MDSCs), heterogenous cells with strong immunosuppressive activity that originate in the hematopoietic system, suppress the anticancer immunity via parallel immune checkpoint-dependent and independent pathways, these cells are potential targets for improving the efficacy of cancer immunotherapy. In this study, it is demonstrated that MDSCs can be depleted by delivering synthetic glucocorticoid dexamethasone to phagocytic cells in the spleen using a lipid nanoparticle. Since the interaction of nanoparticles with T cells is intrinsically poor, this strategy also enables the "detargeting" from T cells, thus avoiding the nonspecific suppression of cytotoxic immune responses against cancer cells. In addition to the direct anticancer effect of the nanoparticulated dexamethasone, their synergistic anticancer effect with ICIs is also reported.
Electrochemical impedance simulation of porous electrodes with variously shaped pores using 3-dimensional finite element method

Loew, N., Tanaka, T., Watanabe, H., Shitanda, I., Itagaki, M.

Electrochimica Acta　440　2023

DOI： 10.1016/j.electacta.2022.141723 　

ISSN： 0013-4686
Design and Synthesis of Poly(2,2′-Bipyridyl) Ligands for Induction of Cell Death in Cancer Cells: Control of Anticancer Activity by Complexation/Decomplexation with Biorelevant Metal Cations

Balachandran, C., Hirose, M., Tanaka, T., Zhu, J.J., Yokoi, K., Hisamatsu, Y., Yamada, Y., Aoki, S.

Inorganic Chemistry　62　(36)　2023

DOI： 10.1021/acs.inorgchem.3c01738 　

ISSN： 0020-1669 1520-510X
A nasal vaccine with inactivated whole-virion elicits protective mucosal immunity against SARS-CoV-2 in mice. International-journal

Nagisa Tokunoh, Shigeyuki Tamiya, Masato Watanabe, Toru Okamoto, Jessica Anindita, Hiroki Tanaka, Chikako Ono, Toshiro Hirai, Hidetaka Akita, Yoshiharu Matsuura, Yasuo Yoshioka

Frontiers in immunology　14　1224634-1224634　2023

DOI： 10.3389/fimmu.2023.1224634 　

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INTRODUCTION: Vaccinations are ideal for reducing the severity of clinical manifestations and secondary complications of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); however, SARS-CoV-2 continues to cause morbidity and mortality worldwide. In contrast to parenteral vaccines such as messenger RNA vaccines, nasal vaccines are expected to be more effective in preventing viral infections in the upper respiratory tract, the primary locus for viral infection and transmission. In this study, we examined the prospects of an inactivated whole-virion (WV) vaccine administered intranasally against SARS-CoV-2. METHODS: Mice were immunized subcutaneously (subcutaneous vaccine) or intranasally (nasal vaccine) with the inactivated WV of SARS-CoV-2 as the antigen. RESULTS: The spike protein (S)-specific IgA level was found to be higher upon nasal vaccination than after subcutaneous vaccination. The level of S-specific IgG in the serum was also increased by the nasal vaccine, although it was lower than that induced by the subcutaneous vaccine. The nasal vaccine exhibited a stronger defense against viral invasion in the upper respiratory tract than the subcutaneous vaccine and unimmunized control; however, both subcutaneous and nasal vaccines provided protection in the lower respiratory tract. Furthermore, we found that intranasally administered inactivated WV elicited robust production of S-specific IgA in the nasal mucosa and IgG in the blood of mice previously vaccinated with messenger RNA encoding the S protein. DISCUSSION: Overall, these results suggest that a nasal vaccine containing inactivated WV can be a highly effective means of protection against SARS-CoV-2 infection.
siRNA delivery to lymphatic endothelial cells via ApoE-mediated uptake by lipid nanoparticles International-journal

Yu Sakurai, Keito Yoshikawa, Kenta Arai, Akira Kazaoka, Shigeki Aoki, Kousei Ito, Yuta Nakai, Kota Tange, Tomomi Furihata, Hiroki Tanaka, Hidetaka Akita

Journal of Controlled Release　353　125-133　2023/01
Publisher: Elsevier BV
DOI： 10.1016/j.jconrel.2022.11.036 　

ISSN： 0168-3659

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Systemically administered lipid nanoparticles (LNPs) are complexed with Apolipoprotein E (ApoE) in the bloodstream, and the complex is subsequently largely taken up by hepatocytes. Based on a previous report showing that, like blood, lymph fluid also contains ApoE, and that LECs, in turn, expresses a low density-lipoprotein receptor (LDLR), which is the receptor responsible for the ApoE-bound LNP, we hypothesized that subcutaneously administered LNPs would be taken up by LECs via an ApoE-LDLR pathway. Our in vitro studies using immortal LECs that we established in a previous study showed that LEC indeed took up LNPs in an ApoE-dependent manner. We then reported on the development of LNPs that target the lymphatic endothelium for in vivo siRNA delivery after subcutaneous administration. The key to success for in vivo LEC targeting is that the surface needs to be modified with a high density of polyethylene glycol (PEG)-conjugated lipids with short acyl chains (C14). The LNPs were drained into the lymphatic system, and then accumulated in lymphatic endothelial cells in an ApoE-dependent manner, most likely after the release of the PEG-lipid. Subcutaneous administration of optimized LNPs containing encapsulated siRNA against VEGFR3, a marker of LECs, significantly inhibited the expression of VEGFR3. These findings are the first report of a simple straightforward strategy for targeting lymphatic endothelial cells by using ionizable lipid-formulated LNPs.
Delivering mRNA to Secondary Lymphoid Tissues by Phosphatidylserine‐Loaded Lipid Nanoparticles International-journal

Masaki Gomi, Yu Sakurai, Minami Sato, Hiroki Tanaka, Yumi Miyatake, Koichi Fujiwara, Mizuki Watanabe, Satoshi Shuto, Yuta Nakai, Kota Tange, Hiroto Hatakeyama, Hidetaka Akita

Advanced Healthcare Materials　12　(9)　2202528-2202528　2022/12/25
Publisher: Wiley
DOI： 10.1002/adhm.202202528 　

ISSN： 2192-2640

eISSN： 2192-2659

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Lipid nanoparticles (LNPs) are one of the most successful technologies in messenger RNA (mRNA) delivery. While the liver is the most frequent target for LNP delivery of mRNA, technologies for delivering mRNA molecules to extrahepatic tissues are also important. Herein, it is reported on the development of an LNP that targets secondary lymphoid tissues. New types of alcohol-soluble phosphatidylserine (PS) derivatives are designed as materials that target immune cells and then incorporated into LNPs using a microfluidic technique with a high degree of scalability and reproducibility. The resulting LNP that contained the synthesized PS delivered mRNA to the spleen much more efficiently compared to a control LNP. A sub-organ analysis revealed that the PS-loaded LNP is extensively taken up by tissue-resident macrophages in the red pulp and the marginal zone of the spleen. Thus, the PS-loaded LNP reported in this study will be a promising strategy for clinical applications that involve delivering mRNA to the spleen.
Tumor-associated neutrophils and macrophages exacerbate antidrug IgG-mediated anaphylactic reaction against an immune checkpoint inhibitor International-journal

Takahiro Arai, Tomomi Kokubo, Ruiheng Tang, Hirohito Abo, Ayu Terui, Jotaro Hirakawa, Hidetaka Akita, Hiroto Kawashima, Akihiro Hisaka, Hiroto Hatakeyama

Journal for ImmunoTherapy of Cancer　10　(12)　e005657-e005657　2022/12
Publisher: BMJ
DOI： 10.1136/jitc-2022-005657 　

eISSN： 2051-1426

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Background With the increased use of immune checkpoint inhibitors (ICIs), side effects and toxicity are a great concern. Anaphylaxis has been identified as a potential adverse event induced by ICIs. Anaphylaxis is a life-threatening medical emergency. However, the mechanisms and factors that can potentially influence the incidence and severity of anaphylaxis in patients with cancer remain unclear. Methods Healthy, murine colon 26, CT26, breast 4T1, EMT6, and renal RENCA tumor-bearing mice were treated with an anti-PD-L1 antibody (clone 10F.9G2). Symptoms of anaphylaxis were evaluated along with body temperature and mortality. The amounts of antidrug antibody and platelet-activating factor (PAF) in the blood were quantified via ELISA and liquid chromatography-mass spectrometry (LC-MS/MS). Immune cells were analyzed and isolated using a flow cytometer and magnetic-activated cell sorting, respectively. Results Repeated administration of the anti-PD-L1 antibody 10F.9G2 to tumor-bearing mice caused fatal anaphylaxis, depending on the type of tumor model. After administration, antidrug immunoglobulin G (IgG), but not IgE antibodies, were produced, and PAF was released as a chemical mediator during anaphylaxis, indicating that anaphylaxis was caused by an IgG-dependent pathway. Anaphylaxis induced by 10F.9G2 was treated with a PAF receptor antagonist. We identified that neutrophils and macrophages were PAF-producing effector cells during anaphylaxis, and the tumor-bearing models with increased numbers of neutrophils and macrophages showed lethal anaphylaxis after treatment with 10F.9G2. Depletion of both neutrophils and macrophages using clodronate liposomes prevented anaphylaxis in tumor-bearing mice. Conclusions Thus, increased numbers of neutrophils and macrophages associated with cancer progression may be risk factors for anaphylaxis. These findings may provide useful insights into the mechanism of anaphylaxis following the administration of immune checkpoint inhibitors in human subjects.
Formation of reactive metabolites of benzbromarone in humanized-liver mice. International-journal

Naoki Cho, Hiroshi Suemizu, Hidetaka Kamimura, Tomoyuki Ohe, Fumi Ito, Hidetaka Akita, Kaoru Kobayashi

Drug metabolism and pharmacokinetics　47　100467-100467　2022/12

DOI： 10.1016/j.dmpk.2022.100467 　

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Benzbromarone, a uricosuric drug, has the potential to cause serious hepatotoxicity. Several studies have shown the formation of reactive metabolites of benzbromarone and their association with hepatotoxicity in mice. However, it is unknown whether those reactive metabolites are generated in humans in vivo. In the present study, we firstly investigated the pharmacokinetic profiles of benzbromarone in chimeric TK-NOG mice transplanted with human hepatocytes (humanized-liver mice) and then investigated whether reactive metabolites could be generated. The area under the plasma concentration-time curve ratio of benzbromarone and its major metabolites (benzbromarone: 1'-hydroxy benzbromarone: 6-hydroxy benzbromarone) in humanized-liver mice was 1: 1.2: 0.7, which was similar to that reported in humans. In addition, glutathione conjugates and their further metabolites derived from the epoxidation of the benzofuran ring and 1',6-dihydroxylation of benzbromarone were detected in the livers, urine and plasma. Furthermore, their peak intensities in mass spectrometry showed markedly higher levels compared with those of TK-NOG mice. These results suggested that the metabolic profiles of benzbromarone in humanized-liver mice were similar to those in humans and that the reactive metabolites detected in humanized-liver mice could be generated and are associated with the benzbromarone-induced hepatotoxicity in humans.
Delivery of aPD-L1 antibody to i.p. tumors via direct penetration by i.p. route: Beyond EPR effect International-journal Peer-reviewed

Yamamoto, M., Kurino, T., Matsuda, R., Jones, H.S., Nakamura, Y., Kanamori, T., Tsuji, A.B., Sugyo, A., Tsuda, R., Matsumoto, Y., Sakurai, Y., Suzuki, H., Sano, M., Osada, K., Uehara, T., Ishii, Y., Akita, H., Arano, Y., Hisaka, A., Hatakeyama, H.

Journal of Controlled Release　352　328-337　2022/12
Publisher: Elsevier BV
DOI： 10.1016/j.jconrel.2022.10.032 　

ISSN： 1873-4995 0168-3659
Targeted delivery of lipid nanoparticle to lymphatic endothelial cells via anti-podoplanin antibody International-journal

Yu Sakurai, Nodoka Abe, Keito Yoshikawa, Ryotaro Oyama, Satoshi Ogasawara, Takeshi Murata, Yuta Nakai, Kota Tange, Hiroki Tanaka, Hidetaka Akita

Journal of Controlled Release　349　379-387　2022/09
Publisher: Elsevier BV
DOI： 10.1016/j.jconrel.2022.06.052 　

ISSN： 0168-3659

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Lymphatic endothelial cells (LECs) that form lymphatic vessels play a pivotal role in immune regulation. It was recently reported that LECs suppress the antigen-dependent anti-tumor immunity in cancer tissues. Thus, regulating the function of LECs is a promising strategy for cancer therapy. The objective of this study was to develop a method for the selective delivery of small interfering RNA (siRNA) to LECs. For this purpose, the siRNA was formulated into nanoparticles (LNPs) to prevent them from being degraded in body fluids and to facilitate their penetration of the cell membrane. A breakthrough technology for achieving this is ONPATTRO®, a world's first siRNA drug. Since LNPs are taken up by hepatocytes relatively well via low-density lipoprotein receptors, most of the LNP systems that have been developed so far target hepatocytes. In this study, we report on the development of a new method for the rapid and convenient method for modifying LNPs with antibodies using the CLick reaction on the Interface of the nanoParticle (CLIP). The CLIP approach was faster and more versatile than the conventional method using amide coupling. As a demonstration, we report on the LEC-targeted siRNA delivery by using antibody-modified LNPs both in vitro and in vivo. The method used for the modification of LNPs is highly promising and has the potential for expanding the LNP-based delivery of nucleic acids in the future.
pH-Responsive Lipid Nanoparticles Achieve Efficient mRNA Transfection in Brain Capillary Endothelial Cells International-journal

Yu Sakurai, Himeka Watanabe, Kazuma Nishio, Kohei Hashimoto, Atsuki Harada, Masaki Gomi, Masayoshi Suzuki, Ryotaro Oyama, Takumi Handa, Risa Sato, Hina Takeuchi, Ryoga Taira, Kenta Tezuka, Kota Tange, Yuta Nakai, Hidetaka Akita, Yasuo Uchida

Pharmaceutics　14　(8)　1560-1560　2022/07/27
Publisher: MDPI AG
DOI： 10.3390/pharmaceutics14081560 　

eISSN： 1999-4923

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The blood–brain barrier (BBB), which is comprised of brain capillary endothelial cells, plays a pivotal role in the transport of drugs from the blood to the brain. Therefore, an analysis of proteins in the endothelial cells, such as transporters and tight junction proteins, which contribute to BBB function, is important for the development of therapeutics for the treatment of brain diseases. However, gene transfection into the vascular endothelial cells of the BBB is fraught with difficulties, even in vitro. We report herein on the development of lipid nanoparticles (LNPs), in which mRNA is encapsulated in a nano-sized capsule composed of a pH-activated and reductive environment-responsive lipid-like material (ssPalm). We evaluated the efficiency of mRNA delivery into non-polarized human brain capillary endothelial cells, hCMEC/D3 cells. The ssPalm LNPs permitted marker genes (GFP) to be transferred into nearly 100% of the cells, with low toxicity in higher concentration. A proteomic analysis indicated that the ssPalm-LNP had less effect on global cell signaling pathways than a Lipofectamine MessengerMAX/GFP-encoding mRNA complex (LFN), a commercially available transfection reagent, even at higher mRNA concentrations.
Human Immortalized Cell-Based Blood-Brain Barrier Spheroid Models Offer an Evaluation Tool for the Brain Penetration Properties of Macromolecules. International-journal

Keita Kitamura, Ayaka Okamoto, Hanae Morio, Ryuto Isogai, Ryo Ito, Yoshiyuki Yamaura, Saki Izumi, Takafumi Komori, Shingo Ito, Sumio Ohtsuki, Hidetaka Akita, Tomomi Furihata

Molecular pharmaceutics　19　(8)　2754-2764　2022/06/29

DOI： 10.1021/acs.molpharmaceut.2c00120 　

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Blood-brain barrier (BBB)-permeable middle- or macromolecules (middle/macromolecules) have recently attracted significant attention as new drug delivery carriers into the human brain via receptor-mediated transcytosis (RMT). During the development process of such carriers, it is necessary to thoroughly evaluate their human BBB permeability levels. In such evaluations, our recently established human immortalized cell-based multicellular spheroidal BBB models (hiMCS-BBB models) have shown high potential. However, the specifics of those capabilities have yet to be elucidated. Therefore, in this study, we characterize the ability of the hiMCS-BBB models to evaluate RMT-mediated BBB penetration properties of middle/macromolecules. More specifically, we began by validating transferrin receptor (TfR)-mediated RMT functionalities using transferrin in the hiMCS-BBB models and then examined the BBB permeability levels of MEM189 antibodies (known BBB-permeable anti-TfR antibodies). The obtained results showed that, as with the case of transferrin, temperature-dependent uptake of MEM189 antibodies was observed in the hiMCS-BBB models, and the extent of that uptake increased in a time-dependent manner until reaching a plateau after around 2 h. To further expand the evaluation applicability of the models, we also examined the BBB permeability levels of the recently developed SLS cyclic peptide and observed that peptide uptake was also temperature-dependent. To summarize, our results show that the hiMCS-BBB models possess the ability to evaluate the RMT-mediated BBB-permeable properties of antibodies and peptides and thus have the potential to provide valuable tools for use in the exploration and identification of middle/macromolecules showing excellent BBB permeability levels, thereby contributing powerfully to the development of new drug delivery carriers for transporting drugs into the human brain.
Post-complexation Functionalization of Cyclometalated Iridium(III) Complexes and Applications to Biomedical and Material Sciences

Aoki, S., Yokoi, K., Hisamatsu, Y., Balachandran, C., Tamura, Y., Tanaka, T.

Topics in Current Chemistry　380　(5)　2022

DOI： 10.1007/s41061-022-00401-w 　

ISSN： 2365-0869 2364-8961
Design, Synthesis and Biological Evaluation of Boron-Containing Macrocyclic Polyamine Dimers and Their Zinc(II) Complexes for Boron Neutron Capture Therapy

Ueda, H., Suzuki, M., Sakurai, Y., Tanaka, T., Aoki, S.

European Journal of Inorganic Chemistry　2022　(5)　2022

DOI： 10.1002/ejic.202100949 　

ISSN： 1434-1948 1099-0682
Dietary 7-ketocholesterol exacerbates myocardial ischemia–reperfusion injury in mice through monocyte/macrophage-mediated inflammation

Uchikawa, T., Matoba, T., Kawahara, T., Baba, I., Katsuki, S., Koga, J.-I., Hashimoto, Y., Yamasaki, R., Ichi, I., Akita, H., Tsutsui, H.

Scientific Reports　12　(1)　2022

DOI： 10.1038/s41598-022-19065-z 　

ISSN： 2045-2322
Lipid nanoparticles for mRNA delivery

Tanaka, H., Sakurai, Y., Akita, H.

Drug Delivery System　37　(3)　2022

DOI： 10.2745/dds.37.237 　

ISSN： 1881-2732 0913-5006
Molecular Design of In-cell Environment-responsive Lipid Like Materials for the Control of Intracellular Trafficking and Collapse,細胞内動態・崩壊性を制御するDDS 材料としての脂質様材料の分子デザイン

Tanaka, H., Akita, H.

Yuki Gosei Kagaku Kyokaishi/Journal of Synthetic Organic Chemistry　80　(1)　2022

DOI： 10.5059/yukigoseikyokaishi.80.55 　

ISSN： 0037-9980
Cellular Binding and Internalization Assay for an Anti-FcγRIIB Antibody Using Human Liver Non-parenchymal Cells.

Yuki Noguchi, Kazuhisa Ozeki, Hidetaka Akita

Biological & pharmaceutical bulletin　45　(4)　534-537　2022

DOI： 10.1248/bpb.b21-01026 　

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A cellular assay for evaluating the binding and internalization of biologics using primary human liver sinusoidal endothelial cells (LSEC) is not readily available, since human LSEC generally lose their receptor expression and internalization activity during the purifying processes and cell culturing. Here, we propose a novel cell-based assay using human liver non-parenchymal cells (NPC) as an alternative method using LSEC. To identify the LSEC population, NPC were stained with CD31 and CD45, and analyzed by flow cytometry. The expression of Fc gamma receptor IIB (FcγRIIB), one of the LSEC markers was detected in the CD31-positive and the CD45-negative fractions. The concentration-dependent binding and internalization of the anti-FcγRIIB antibody was also quantified in the LSEC fraction in human NPC. Saturated binding and internalization curves were obtained for the anti-FcγRIIB antibody. In the case of the negative control antibody, however, binding and internalization were negligible. The findings reported here indicate that cell-based assays using fresh human liver NPC will be useful for evaluating the binding and internalization of biologics as well as for determining pharmacokinetic parameters.
Toxoplasma gondii GRA15 DNA Vaccine with a Liposomal Nanocarrier Composed of an SS-Cleavable and pH-Activated Lipid-like Material Induces Protective Immunity against Toxoplasmosis in Mice. International-journal

Tanjila Hasan, Ryo Kawanishi, Hidetaka Akita, Yoshifumi Nishikawa

Vaccines　10　(1)　2021/12/24

DOI： 10.3390/vaccines10010021 　

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Toxoplasma gondii affects the health of humans and livestock and causes severe illness in the fetus and immunocompromised individuals. Because of the high incidence and severe consequences of T. gondii infection, a safe and suitable vaccine is needed. We found that lipid nanoparticles (LNPs) consisting of a series of functional materials prepared with vitamin E, such as SS-cleavable and pH-activated lipid-like materials (ssPalmE), were a safe and efficient way to develop next-generation DNA vaccines. In this study, we prepared ssPalmE-LNP to encapsulate pCpG-free-T. gondii dense granule protein 15 DNA (ssPalmE-LNPTgGRA15). Following a challenge infection with avirulent PLK strain of T. gondii, the mice immunized with ssPalmE-LNPTgGRA15 had a significantly higher survival rate and lower clinical scores compared with unimmunized and ssPalmE-LNPnon-coding-immunized mice. Immunization of mice with the ssPalmE-LNPTgGRA15 led to a significantly higher production of specific IgG1 and IG2c antibodies compared with unimmunized and ssPalmE-LNPnon-coding-immunized mice, while there was no statistically significant difference in the concentration of serum interferon-gamma at the acute stage of the infection. These findings indicate that ssPalmE-LNP is an effective cargo for the transportation of DNA vaccines for protozoan infections. To explore the mechanism of protective immunity induced by ssPalmE-LNPTgGRA15, further immunological study is needed in the future.
Improvement of mRNA Delivery Efficiency to a T Cell Line by Modulating PEG-Lipid Content and Phospholipid Components of Lipid Nanoparticles. International-journal

Hiroki Tanaka, Ryo Miyama, Yu Sakurai, Shinya Tamagawa, Yuta Nakai, Kota Tange, Hiroki Yoshioka, Hidetaka Akita

Pharmaceutics　13　(12)　2021/12/06

DOI： 10.3390/pharmaceutics13122097 　

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(1) Background: T cells are important target cells, since they exert direct cytotoxic effects on infected/malignant cells, and affect the regulatory functions of other immune cells in a target antigen-specific manner. One of the current approaches for modifying the function of T cells is gene transfection by viral vectors. However, the insertion of the exogenous DNA molecules into the genome is attended by the risk of mutagenesis, especially when a transposon-based gene cassette is used. Based on this scenario, the transient expression of proteins by an in vitro-transcribed messenger RNA (IVT-mRNA) has become a subject of interest. The use of lipid nanoparticles (LNPs) for the transfection of IVT-mRNA is one of the more promising strategies for introducing exogenous genes. In this study, we report on the development of LNPs with transfection efficiencies that are comparable to that for electroporation in a T cell line (Jurkat cells). (2) Methods: Transfection efficiency was improved by optimizing the phospholipids and polyethylene glycol (PEG)-conjugated lipid components. (3) Results: Modification of the lipid composition resulted in the 221-fold increase in luciferase activity compared to a previously optimized formulation. Such a high transfection activity was due to the efficient uptake by clathrin/dynamin-dependent endocytosis and the relatively efficient escape into the cytoplasm at an early stage of endocytosis.
Optimization of Sentinel Lymph Node Imaging Methodology Using Anionic Liposome and Hyaluronidase. International-journal

Yu Sakurai, Miho Suzuoki, Masaki Gomi, Hiroki Tanaka, Hidetaka Akita

Pharmaceutics　13　(9)　2021/09/14

DOI： 10.3390/pharmaceutics13091462 　

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The sentinel lymph node (SLN) is the first lymph node into which lymphatic fluid from tumor tissues flows. The development of a highly sensitive probe for detecting SLNs is desired for the lymph node dissection through intraoperative biopsy. We have previously shown that anionic liposomes tend to accumulate in lymph nodes and that macrophage uptake of liposomes contributes to their accumulation. In the present study, we found that among anionic lipids, phosphatidylserine (PS)-containing liposomes were substantially taken up by macrophages. We identified a new lipid composition to improve the SNL-selectivity of liposome accumulation based on Design-of-Experiment. The optimized PS-containing particles were more selectively accumulate to SLN lymph nodes than existing imaging agents indocyanine green. These results indicate the effectiveness of PS-containing anionic particles in SLN imaging.
Effects on Metabolism in Astrocytes Caused by cGAMP, Which Imitates the Initial Stage of Brain Metastasis. International-journal

Toya Okawa, Kurumi Hara, Momoko Goto, Moe Kikuchi, Masataka Kogane, Hiroto Hatakeyama, Hiroki Tanaka, Daiki Shirane, Hidetaka Akita, Akihiro Hisaka, Hiromi Sato

International journal of molecular sciences　22　(16)　2021/08/21

DOI： 10.3390/ijms22169028 　

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The second messenger 2'3'-cyclic-GMP-AMP (cGAMP) is thought to be transmitted from brain carcinomas to astrocytes via gap junctions, which functions to promote metastasis in the brain parenchyma. In the current study, we established a method to introduce cGAMP into astrocytes, which simulates the state of astrocytes that have been invaded by cGAMP around tumors. Astrocytes incorporating cGAMP were analyzed by metabolomics, which demonstrated that cGAMP increased glutamate production and astrocyte secretion. The same trend was observed for γ-aminobutyric acid (GABA). Conversely, glutamine production and secretion were decreased by cGAMP treatment. Due to the fundamental role of astrocytes in regulation of the glutamine-glutamate cycle, such metabolic changes may represent a potential mechanism and therapeutic target for alteration of the central nervous system (CNS) environment and the malignant transformation of brain carcinomas.
Development, Characterization and Potential Applications of a Multicellular Spheroidal Human Blood-Brain Barrier Model Integrating Three Conditionally Immortalized Cell Lines. Peer-reviewed

Keita Kitamura, Kenta Umehara, Ryo Ito, Yoshiyuki Yamaura, Takafumi Komori, Hanae Morio, Hidetaka Akita, Tomomi Furihata

Biological & pharmaceutical bulletin　44　(7)　984-991　2021/07/01

DOI： 10.1248/bpb.b21-00218 　

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In vitro blood-brain barrier (BBB) models are essential research tools for use in developing brain-targeted drugs and understanding the physiological and pathophysiological functions of the BBB. To develop BBB models with better functionalities, three-dimensional (3D) culture methods have gained significant attention as a promising approach. In this study, we report on the development of a human conditionally immortalized cell-based multicellular spheroidal BBB (hiMCS-BBB) model. After being seeded into non-attachment culture wells, HASTR/ci35 (astrocytes) and HBPC/ci37 cells (brain pericytes) self-assemble to form a spheroid core that is then covered with an outer monolayer of HBMEC/ci18 cells (brain microvascular endothelial cells). The results of immunocytochemistry showed the protein expression of several cellular junction and BBB-enriched transporter genes in HBMEC/ci18 cells of the spheroid model. The permeability assays showed that the hiMCS-BBB model exhibited barrier functions against the penetration of dextran (5 and 70 kDa) and rhodamine123 (a P-glycoprotein substrate) into the core. On the other hand, facilitation of 2-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)-2-deoxyglucose (2-NBDG; a fluorescent glucose analog) uptake was observed in the hiMCS-BBB model. Furthermore, tumor necrosis factor-alpha treatment elicited an inflammatory response in HBMEC/ci18 cells, thereby suggesting that BBB inflammation can be recapitulated in the hiMCS-BBB model. To summarize, we have developed an hiMCS-BBB model that possesses fundamental BBB properties, which can be expected to provide a useful and highly accessible experimental platform for accelerating various BBB studies.
In vivo evaluation of intestinal human CYP3A inhibition by macrolide antibiotics in CYP3A-humanised mice. International-journal

Genki Minegishi, Yasuhiro Kazuki, Shin-Ichiro Nitta, Atsushi Miyajima, Hidetaka Akita, Kaoru Kobayashi

Xenobiotica; the fate of foreign compounds in biological systems　51　(7)　764-770　2021/07

DOI： 10.1080/00498254.2021.1921314 　

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It is important to predict drug-drug interactions via inhibition of intestinal cytochrome P450 3A (CYP3A) which is a determinant of bioavailability of orally administered CYP3A substrates. However, inhibitory effects of macrolide antibiotics on CYP3A-mediated metabolism are not entirely identical between humans and rodents.We investigated the effects of macrolide antibiotics, clarithromycin and erythromycin, on in vitro and in vivo metabolism of triazolam, a CYP3A substrate, in CYP3A-humanised mice generated by using a mouse artificial chromosome vector carrying a human CYP3A gene.Metabolic activities of triazolam were inhibited by macrolide antibiotics in liver and intestine microsomes of CYP3A-humanised mice.The area under the plasma concentration-time curve ratios of 4-hydroxytriazolam to triazolam after oral dosing of triazolam were significantly decreased by multiple administration of macrolide antibiotics. The plasma concentrations ratios of α-hydroxytriazolam and 4-hydroxytriazolam to triazolam in portal blood were significantly decreased by multiple administration of clarithromycin in CYP3A-humanised mice.These results suggest that intestinal CYP3A activity was inhibited by macrolide antibiotics in CYP3A-humanised mice in vitro and in vivo. The plasma concentrations of triazolam and its metabolites in the portal blood of CYP3A-humanised mice would be useful for direct evaluation of intestinal CYP3A-mediated drug-drug interactions.
ビタミンE足場型pH応答性材料が誘導するワクチン効果メカニズムの解明

大山遼太朗, Anindita Jessica, 田中浩揮, 櫻井遊, 石亀晴道, 岡田隆陽, 丹下耕太, 中井裕太, 吉岡宏樹, 秋田英万

日本DDS学会学術集会プログラム予稿集　37回　97-97　2021/06
Publisher: 日本DDS学会
Broaden your horizons～がん克服に向けた薬剤学の多面的アプローチ～ビタミンE足場型新規材料を用いたmRNAがんワクチンの開発とその機能解析

大山遼太朗, 舘下菜穂, ジェシカ・アニンディタ, 田中浩揮, 三浦尚也, 櫻井遊, 石亀晴道, 岡田隆陽, 丹下耕太, 中井裕太, 吉岡宏樹, 秋田英万

日本薬剤学会年会講演要旨集　36年会　103-103　2021/05
Publisher: (公社)日本薬剤学会
A cell based assay for evaluating binding and uptake of an antibody using hepatic nonparenchymal cells. International-journal

Yuki Noguchi, Kazuhisa Ozeki, Hiroaki Takesue, Hidetaka Akita

Scientific reports　11　(1)　8383-8383　2021/04/16

DOI： 10.1038/s41598-021-87912-6 　

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Evaluation of the binding and uptake of an antibody in liver non-parenchymal cells (NPC), including liver sinusoidal endothelial cells, is important for revealing its pharmacokinetic (PK) behavior, since NPC has important roles in eliminating an antibody from the blood via the Fc fragment of IgG receptor IIB (FcγRIIB). However, there is currently no in vitro quantitative assay using NPC. This study reports on the development of a cell-based assay for evaluating the binding and uptake of such an antibody using liver NPC of mice and monkeys. In mice, the FcγRIIB-expressing cells were identified in the CD146-positive and CD45-negative fraction by flow cytometry. A titration assay was performed to determine the PK parameters, and the obtained parameter was comparable to that determined by the fitting of the in vivo PK. This approach was also extended to NPC from monkeys. The concentration-dependent binding and uptake was measured to determine the PK parameters using monkey NPC, the FcγRIIB-expressing fraction of which was identified by CD31 and CD45. The findings presented herein demonstrate that the in vitro liver NPC assay using flow cytometry is a useful tool to determine the binding and uptake of biologics and to predict the PK.
Lipid nanoparticles-encapsulated brain-derived neurotrophic factor mRNA delivered through the round window niche in the cochleae of guinea pigs. International-journal

Toru Miwa, Haruki Saito, Hidetaka Akita

Experimental brain research　239　(2)　425-433　2021/02

DOI： 10.1007/s00221-020-05970-0 　

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The treatment of sensorineural hearing loss (SNHL) may be achieved via the application of a cochlear implant (CI) that allows the electrical stimulation of spiral ganglion neurons (SGNs). Nevertheless, the efficacy of CIs is limited by the degeneration of SGNs following SNHL. Although the application of exogenous neurotrophic factors has been reported to decrease SGN degeneration, non-invasive targeted drug delivery systems are required to achieve effective results. In this study, an SS-cleavable proton-activated lipid-like material [ssPalm; a neutral lipid nanoparticle (LNP)], was loaded with mRNA, and the efficacy of this material as a delivery system was investigated. Our results showed that LNPssPalm carrying brain-derived neurotrophic factor (BDNF) mRNA was suitable for the treatment of inner ear diseases, preventing the degeneration of SGNs. In conclusion, this modern nanotechnology-based bioconjugation system, LNPssPalm, is a potential non-invasive targeted therapy allowing the delivering biomaterials to specific structures within the inner ear for the treatment of SHNL.
The structure of POMGNT2 provides new insights into the mechanism to determine the functional O-mannosylation site on α-dystroglycan

Imae, R., Kuwabara, N., Manya, H., Tanaka, T., Tsuyuguchi, M., Mizuno, M., Endo, T., Kato, R.

Genes to Cells　26　(7)　2021

DOI： 10.1111/gtc.12853 　

ISSN： 1356-9597 1365-2443
Potential anti-COVID-19 agents, cepharanthine and nelfinavir, and their usage for combination treatment

Ohashi, H., Watashi, K., Saso, W., Shionoya, K., Iwanami, S., Hirokawa, T., Shirai, T., Kanaya, S., Ito, Y., Kim, K.S., Nomura, T., Suzuki, T., Nishioka, K., Ando, S., Ejima, K., Koizumi, Y., Tanaka, T., Aoki, S., Kuramochi, K., Suzuki, T., Hashiguchi, T., Maenaka, K., Matano, T., Muramatsu, M., Saijo, M., Aihara, K., Iwami, S., Takeda, M., McKeating, J.A., Wakita, T.

Iscience　24　(4)　2021

DOI： 10.1016/j.isci.2021.102367 　

ISSN： 2589-0042
Mefloquine, a Potent Anti-severe Acute Respiratory Syndrome-Related Coronavirus 2 (SARS-CoV-2) Drug as an Entry Inhibitor in vitro

Shionoya, K., Yamasaki, M., Iwanami, S., Ito, Y., Fukushi, S., Ohashi, H., Saso, W., Tanaka, T., Aoki, S., Kuramochi, K., Iwami, S., Takahashi, Y., Suzuki, T., Muramatsu, M., Takeda, M., Wakita, T., Watashi, K.

Frontiers in Microbiology　12　2021

DOI： 10.3389/fmicb.2021.651403 　

ISSN： 1664-302X
Design, Synthesis, and Biological Evaluation of Boron-Containing Macrocyclic Polyamines and Their Zinc(II) Complexes for Boron Neutron Capture Therapy

Ueda, H., Suzuki, M., Kuroda, R., Tanaka, T., Aoki, S.

Journal of Medicinal Chemistry　64　(12)　2021

DOI： 10.1021/acs.jmedchem.1c00445 　

ISSN： 0022-2623 1520-4804
Development of metallosupramolecular phosphatases based on the combinatorial self-assembly of metal complexes and organic building blocks for the catalytic hydrolysis of phosphate monoesters

Aoki, S., Bin Rahman, A., Hisamatsu, Y., Miyazawa, Y., Zulkefeli, M., Saga, Y., Tanaka, T.

Results in Chemistry　3　2021

DOI： 10.1016/j.rechem.2021.100133 　

ISSN： 2211-7156
Foreword: Pioneer technologies of the extracelluar vesicle-based drug discovery and diagnosis

Akita, H.

Drug Delivery System　36　(2)　87-87　2021

DOI： 10.2745/dds.36.87 　

ISSN： 1881-2732 0913-5006
Proteomics Analysis of Lymphatic Metastasis-Related Proteins Using Highly Metastatic Human Melanoma Cells Originated by Sequential in Vivo Implantation.

Katsuyuki Chida, Yu Sakurai, Asa Ohtani, Takeshi Masuda, Sumio Ohtsuki, Hiroki Tanaka, Hidetaka Akita

Biological & pharmaceutical bulletin　44　(10)　1551-1556　2021

DOI： 10.1248/bpb.b21-00463 　

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Metastasis of cancer cells to lymph nodes (LN) is a common modality of metastasis in clinical settings, but the mechanisms involved in lymphatic metastasis remain unclear compared to hematogenous metastasis to bones and the brain. To elucidate the molecular mechanisms responsible for melanoma LN metastasis, we first generated LN metastasis-prone melanoma cells (C8161F2) by the sequential in vivo transplantation of parental melanoma cells (C8161F0). Although the in vitro/in vivo proliferative potential of these melanoma cells were similar, the metastatic potential of the C8161F2 for LNs was significantly enhanced. We then conducted a proteomics analysis to identify the proteins and pathways that contribute to LN metastasis. We identified six proteins (three: up-regulated and three: down-regulated) whose expressions were statistically significantly different by more than 2-fold in the two cell groups. Some of these genes are responsible for the activation of the transforming growth factor-β (TGF-β)-related pathway, a well-known inducer of epithelial-mesenchymal transition (EMT). In addition, a gene ontology analysis revealed that the enhanced cell-cell adhesion appears to be involved in lymphatic metastasis. In conclusion, we established highly lymphatic metastatic melanoma cells, which would be valuable for studies of the molecular mechanisms responsible for lymphatic metastasis.
Silencing of VEGFR2 by RGD-Modified Lipid Nanoparticles Enhanced the Efficacy of Anti-PD-1 Antibody by Accelerating Vascular Normalization and Infiltration of T Cells in Tumors. International-journal

Riki Cho, Yu Sakurai, Haleigh Sakura Jones, Hidetaka Akita, Akihiro Hisaka, Hiroto Hatakeyama

Cancers　12　(12)　2020/12/04

DOI： 10.3390/cancers12123630 　

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Despite the promising anticancer effects of immune checkpoint inhibitors, their low objective response rate remains to be resolved; thus, combination therapies have been investigated. We investigated the combination of an anti-programmed cell death 1 (aPD-1) monoclonal antibody with the knockdown of vascular endothelial factor receptor 2 (VEGFR2) on tumor endothelial cells to overcome resistance to immune checkpoint inhibitors and improve the objective response rate. The successful delivery of small interfering RNA to tumor endothelial cells was achieved by RGD peptide-modified lipid nanoparticles composed of a novel, pH-sensitive, and biodegradable ssPalmO-Phe. RGD-modified lipid nanoparticles efficiently induced the knockdown of VEGFR2 in tumor endothelial cells (TECs), which induced vascular normalization. The combination of a PD-1 monoclonal antibody with Vegfr2 knockdown enhanced CD8+ T cell infiltration into tumors and successfully suppressed tumor growth and improved response rate compared with monotherapy. Our combination approach provides a promising strategy to improve therapeutic outcomes in immune checkpoint inhibitor-resistant cancers.
Pharmacokinetic prediction of an antibody in mice based on an in vitro cell-based approach using target receptor-expressing cells. International-journal

Yuki Noguchi, Kazuhisa Ozeki, Hidetaka Akita

Scientific reports　10　(1)　16268-16268　2020/10/01

DOI： 10.1038/s41598-020-73255-1 　

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In vivo pharmacokinetics (PK) studies using mice and monkeys are the main approaches for evaluating and predicting the PK of antibodies, and there is a strong demand for methods that do not require animal experiments. In this work, we focused on quantitatively predicting the nonlinear PK of an antibody based on cell-based assays. An anti-mouse Fc gamma receptor IIB antibody was used as a model antibody. To determine the PK parameters related to nonspecific elimination in vivo, the plasma concentration profile at 100 mg/kg, at which target-specific clearance is saturated, was analyzed by a 2-compartment model. To estimate the parameters related to target-specific elimination, the Michaelis-Menten constant (Km) and the maximum elimination rate (Vmax) were determined by an uptake assay using Chinese hamster ovary (CHO) cells expressing the target receptor. Finally, the integration of all of these parameters permitted the PK to be predicted at doses ranging from 1 to 100 mg/kg regardless of whether target-specific clearance was saturated or nonsaturated. The findings presented herein show that in vitro assays using target-expressing cells are useful tools for obtaining PK parameters and predicting PK profiles and, in some cases, eliminate the need for in vivo PK studies using experimental animals.
Crystal structures of fukutin-related protein (FKRP), a ribitol-phosphate transferase related to muscular dystrophy

Kuwabara, N., Imae, R., Manya, H., Tanaka, T., Mizuno, M., Tsumoto, H., Kanagawa, M., Kobayashi, K., Toda, T., Senda, T., Endo, T., Kato, R.

Nature Communications　11　(1)　2020

DOI： 10.1038/s41467-019-14220-z 　

ISSN： 2041-1723
Development of antibody drug against mesothelioma-specific glycopeptide antigen: 13^th mizushima award, Japan society of dds

Akita, H.

Drug Delivery System　35　(5)　2020

DOI： 10.2745/dds.35.417 　

ISSN： 1881-2732 0913-5006
Targeting Tumor Endothelial Cells with Nanoparticles. International-journal Peer-reviewed

Yu Sakurai, Hidetaka Akita, Hideyoshi Harashima

International journal of molecular sciences　20　(23)　2019/11/20

DOI： 10.3390/ijms20235819 　

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Because angiogenesis is a major contributor to cancer progression and metastasis, it is an attractive target for cancer therapy. Although a diverse number of small compounds for anti-angiogenic therapy have been developed, severe adverse effects commonly occur, since small compounds can affect not only tumor endothelial cells (TECs), but also normal endothelial cells. This low selectivity for TECs has motivated researchers to develop alternate types of drug delivery systems (DDSs). In this review, we summarize the current state of knowledge concerning the delivery of nano DDSs to TECs. Their payloads range from small compounds to nucleic acids. Perspectives regarding new therapeutic targets are also mentioned.
A Human Immortalized Cell-Based Blood-Brain Barrier Triculture Model: Development and Characterization as a Promising Tool for Drug-Brain Permeability Studies. International-journal Peer-reviewed

Ryo Ito, Kenta Umehara, Shota Suzuki, Keita Kitamura, Ken-Ichi Nunoya, Yoshiyuki Yamaura, Haruo Imawaka, Saki Izumi, Naomi Wakayama, Takafumi Komori, Naohiko Anzai, Hidetaka Akita, Tomomi Furihata

Molecular pharmaceutics　16　(11)　4461-4471　2019/11/04

DOI： 10.1021/acs.molpharmaceut.9b00519 　

ISSN： 1543-8384

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Brain microvascular endothelial cells (BMEC), together with astrocytes and pericytes, form the blood-brain barrier (BBB) that strictly restricts drug penetration into the brain. Therefore, in central nervous system drug development, the establishment of an in vitro human BBB model for use in studies estimating the in vivo human BBB permeability of drug candidates has long been awaited. The current study developed and characterized a human immortalized cell-based BBB triculture model, termed the "hiBBB" model. To set up the hiBBB model, human immortalized BMEC (HBMEC/ci18) were cocultured with human immortalized astrocytes (HASTR/ci35) and brain pericytes (HBPC/ci37) in a transwell system. The trans-endothelial electrical resistance of the hiBBB model was 134.4 ± 5.5 (Ω × cm2), and the efflux ratios of rhodamine123 and dantrolene were 1.72 ± 0.11 and 1.72 ± 0.45, respectively, suggesting that the hiBBB model possesses essential cellular junction and efflux transporter functions. In BBB permeability assays, the mean value of the permeability coefficients (Pe) of BBB permeable compounds (propranolol, pyrilamine, memantine, and diphenhydramine) was 960 × 10-6 cm/s, which was clearly distinguishable from that of BBB nonpermeable compounds (sodium fluorescein and Lucifer yellow, 18 × 10-6 cm/s). Collectively, this study successfully developed the hiBBB model, which exhibits essential BBB functionality. Taking into consideration the high availability of the immortalized cells used in the hiBBB model, our results are expected to become an initial step toward the establishment of a useful human BBB model to investigate drug penetration into the human brain.
Comparison of the hepatic metabolism of triazolam in wild-type andCyp3a-knockout mice for understanding CYP3A-mediated metabolism inCYP3A-humanised mice in vivo. International-journal Peer-reviewed

Genki Minegishi, Yasuhiro Kazuki, Yuki Yamasaki, Fuka Okuya, Hidetaka Akita, Mitsuo Oshimura, Kaoru Kobayashi

Xenobiotica; the fate of foreign compounds in biological systems　49　(11)　1303-1310　2019/11

DOI： 10.1080/00498254.2018.1560516 　

ISSN： 0049-8254

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1. To investigate cytochrome P450 3A (CYP3A)-mediated metabolism in vivo, plasma concentrations of triazolam (TRZ) are often monitored as a CYP3A marker in CYP3A-humanised mice. However, it has not been determined whether plasma concentrations of TRZ after intravenous administration can reflect hepatic CYP3A activity in CYP3A-humanised mice. 2. Firstly, we investigated the pharmacokinetics of TRZ in wild-type and Cyp3a-knockout (Cyp3a-KO) mice. Plasma concentration profiles of TRZ and α-hydroxy (OH) TRZ were very similar in wild-type and Cyp3a-KO mice. On the other hand, AUC of 4-OH TRZ in Cyp3a-KO mice was significantly lower than that in wild-type mice. Pregnenolone 16α-carbonitrile (PCN) decreased the areas under the plasma concentration-time curves (AUCs) of TRZ and α-OH TRZ in both groups. There was no significant effect of PCN on AUC of 4-OH TRZ in Cyp3a-KO mice. 3. Next, we verified that AUC of 4-OH TRZ in CYP3A-humanised mice was higher than that in Cyp3a-KO mice, although the difference was not significant. 4. In conclusion, plasma concentrations of 4-OH TRZ, but not those of TRZ and α-OH TRZ, might reflect hepatic CYP3A activity in mice in vivo. These results provide important insights for in vivo studies using a CYP3A-humanised model.
Indirect activation of constitutive androstane receptor in three-dimensionally cultured HepG2 cells. International-journal Peer-reviewed

Kosuke Yokobori, Ikuko Azuma, Kan Chiba, Hidetaka Akita, Tomomi Furihata, Kaoru Kobayashi

Biochemical pharmacology　168　26-37　2019/10

DOI： 10.1016/j.bcp.2019.06.011 　

ISSN： 0006-2952

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Constitutive androstane receptor (CAR), a member of the nuclear receptor superfamily, is retained as an inactive form phosphorylated at threonine in the cytoplasm of hepatocytes. Upon activation, CAR is dephosphorylated to move into the nucleus and induces the transcription of genes. Thus, nuclear translocation is a key step for CAR activation in hepatocytes. However, this nuclear translocation has not been demonstrated in conventional two-dimensionally-cultured immortalized cell lines such as HepG2, in which CAR spontaneously accumulates in the nucleus. In this study, we showed that treatment with the indirect CAR activator phenobarbital activated transcription of the CYP3A4 gene in three-dimensionally (3D)-cultured HepG2 cells. CAR was retained as its phosphorylated form in the cytoplasm and was translocated to the nucleus in 3D-cultured HepG2 cells in response to treatment with phenobarbital. Moreover, okadaic acid and epidermal growth factor, were found to repress phenobarbital-induced CAR nuclear translocation and subsequent activation of the CYP3A4 gene promoter. These results suggested that 3D-cultured HepG2 cells properly regulated CAR activation as has been observed in hepatocytes.
A study of the endocytosis mechanism and transendothelial activity of lung-targeted GALA-modified liposomes. International-journal Peer-reviewed

Sarochin Santiwarangkool, Hidetaka Akita, Ikramy A Khalil, Mahmoud M Abd Elwakil, Yusuke Sato, Kenji Kusumoto, Hideyoshi Harashima

Journal of controlled release : official journal of the Controlled Release Society　307　55-63　2019/08/10

DOI： 10.1016/j.jconrel.2019.06.009 　

ISSN： 0168-3659

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The GALA peptide (WEAALAEALAEALAEHLAEALAEALEALAA) was originally designed to induce the destabilization of endosomal membranes based on its ability to undergo a pH-dependent conformational change from a random coil to an α-helix. We recently found that liposomes modified with GALA peptide (GALA-LPs) extensively accumulate in lung endothelial cells (ECs) after intravenous injection. However, the uptake mechanism of GALA-LPs and their ability to reach alveolar epithelium was unclear. We report herein that GALA-LPs are internalized into ECs via a clathrin-mediated pathway. Surprisingly, GALA-LPs had the ability to pass lung ECs and reach other cells through transcytosis. GALA-LPs were taken up by >70% of lung ECs, while they also accumulated in ~30% of type I alveolar epithelium (ATI). GALA-modified gold nanoparticles were detected in ECs, in the basement membrane and in other cells such as ATI, type II alveolar epithelium (ATII) and alveolar macrophages. Consistent with this result, a significant gene knockdown was achieved in lung epithelium cells using GALA-LPs encapsulating anti-podoplanin siRNA. This indicates that GALA-LPs can be used as a carrier for delivering macromolecules to parenchymal as well as to endothelial cells in the lung. Although caveolae are commonly linked to the transendothelial transport of proteins and antibodies, our data indicate that clathrin-mediated endocytosis might also participate in the transcytosis process.
ナノ粒子のリンパシステム内動態におけるサイズ・表面電荷の影響

五味昌樹, 櫻井遊, 田中浩揮, 三浦尚也, 秋田新介, 山路佳久, 三川信之, 秋田英万

日本DDS学会学術集会プログラム予稿集　35回　144-144　2019/06
Publisher: 日本DDS学会
Efficient synthesis of N- and O-linked glycopeptides using acid-labile Boc groups for the protection of carbohydrate moieties

Tanaka, T., Shiraishi, M., Matsuda, A., Mizuno, M.

Tetrahedron Letters　60　(40)　2019

DOI： 10.1016/j.tetlet.2019.151106 　

ISSN： 0040-4039 1873-3581
Design, synthesis and biological evaluation of low molecular weight CXCR4 ligands

Sakyiamah, M.M., Kobayakawa, T., Fujino, M., Konno, M., Narumi, T., Tanaka, T., Nomura, W., Yamamoto, N., Murakami, T., Tamamura, H.

Bioorganic and Medicinal Chemistry　27　(6)　2019

DOI： 10.1016/j.bmc.2019.02.013 　

ISSN： 0968-0896 1464-3391
Inhibition of the Inflammatory Pathway Enhances Both the in Vitro and in Vivo Transfection Activity of Exogenous in Vitro-Transcribed mRNAs Delivered by Lipid Nanoparticles. Peer-reviewed

Takara Ohto, Manami Konishi, Hiroki Tanaka, Koji Onomoto, Mitsutoshi Yoneyama, Yuta Nakai, Kota Tange, Hiroki Yoshioka, Hidetaka Akita

Biological & pharmaceutical bulletin　42　(2)　299-302　2019

DOI： 10.1248/bpb.b18-00783 　

ISSN： 0918-6158

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While the use of in vitro-transcribed mRNA (IVT-mRNA) in therapeutics is a rapidly expanding area, the transfection of the exogenous IVT-mRNA is accompanied by a risk of immune activation. This immunological defense mechanism suppresses cellular translation process and can reduce transfection efficiency to a considerable extent. In the present study, we investigated the in vitro effects of Integrated Stress Response Inhibitor (ISRIB), and dexamethasone, a steroidal anti-inflammatory drug, on the transfection activity of a lipid nanoparticle (LNP) that was composed of ionizable lipids and IVT-mRNA. In the case of transfection to mouse embryonic fibroblast (MEF) cells, ISRIB mainly enhanced the transfection activity at an early stage of transfection (0-6 h). In contrast, dexamethasone caused an increase in transfection activity at intermediate-late stages of transfection (4-48 h). We also investigated the in vivo effects of dexamethasone using an LNP on that the IVT-mRNA and lipid-conjugated dexamethasone (Dex-Pal) were co-loaded. The intravenous administration of the LNP successfully enhanced the protein expression in a mouse liver by up to 6.6-fold. Collectively, the co-delivery of an anti-inflammatory drug is a promising approach for enhancing transfection efficiency of IVT-mRNA.
The delivery of mRNA to colon inflammatory lesions by lipid-nano-particles containing environmentally-sensitive lipid-like materials with oleic acid scaffolds. International-journal Peer-reviewed

Hiroki Tanaka, Ayaka Watanabe, Manami Konishi, Yuta Nakai, Hiroki Yoshioka, Tatsuya Ohkawara, Hiroshi Takeda, Hideyoshi Harashima, Hidetaka Akita

Heliyon　4　(12)　e00959　2018/12

DOI： 10.1016/j.heliyon.2018.e00959 　

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An mRNA gene therapy represents a potentially promising therapeutic for curing inflammatory diseases. The transient nature of the gene expression of mRNA would be expected to be beneficial for avoiding undesired side effects. Since the mRNA is a vulnerable molecule, a development of a carrier that can deliver the mRNA to the cytoplasm has a high priority. We report herein on the development of a system for delivering mRNA to the inflammatory lesion in a dextran sulfate sodium (DSS)-induced colitis model. We modulated molecular structures of an ionizable lipid, an SS-cleavable and pH-activated lipid-like material (ssPalm). Among the fatty acids investigated, oleic acid scaffolds (ssPalmO) appeared to be more biocompatible than either myristic acid or linoleic acid scaffolds with the colitis model. The structural modification of the hydrophilic head groups from linear tertiary amines to piperazine rings (ssPalmO-Paz4-C2) resulted in a more than 10-fold higher increasing in the transgene activity in inflammatory colon. The most notable observation is that the transgene activity in the inflammatory colon is significantly higher than that in liver, the major clearance organ of lipid nanoparticles. Collectively, the ssPalmO-Paz4-C2 represents a promising material for the delivery of an mRNA to inflammatory lesions.
Characterization of P-Glycoprotein Humanized Mice Generated by Chromosome Engineering Technology: Its Utility for Prediction of Drug Distribution to the Brain in Humans. International-journal Peer-reviewed

Yuki Yamasaki, Kaoru Kobayashi, Fuka Okuya, Naoyo Kajitani, Kanako Kazuki, Satoshi Abe, Shoko Takehara, Shingo Ito, Seiryo Ogata, Tatsuki Uemura, Sumio Ohtsuki, Genki Minegishi, Hidetaka Akita, Kan Chiba, Mitsuo Oshimura, Yasuhiro Kazuki

Drug metabolism and disposition: the biological fate of chemicals　46　(11)　1756-1766　2018/11

DOI： 10.1124/dmd.118.081216 　

ISSN： 0090-9556

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P-glycoprotein (P-gp), encoded by the MDR1 gene in humans and by the Mdr1a/1b genes in rodents, is expressed in numerous tissues and performs as an efflux pump to limit the distribution and absorption of many drugs. Owing to species differences of P-gp between humans and rodents, it is difficult to predict the impact of P-gp on pharmacokinetics and the tissue distribution of P-gp substrates in humans from the results of animal experiments. Therefore, we generated a novel P-gp humanized mouse model by using a mouse artificial chromosome (MAC) vector [designated human MDR1-MAC (hMDR1-MAC) mice]. The results showed that hMDR1 mRNA was expressed in various tissues of hMDR1-MAC mice. Furthermore, the expression of human P-gp was detected in the brain capillary fraction and plasma membrane fraction of intestinal epithelial cells isolated from hMDR1-MAC mice, although the expression levels of intestinal P-gp were extremely low. Thus, we evaluated the function of human P-gp at the blood-brain barrier of hMDR1-MAC mice. The brain-to-plasma ratios of P-gp substrates in hMDR1-MAC mice were much lower than those in Mdr1a/1b-knockout mice, and the brain-to-plasma ratio of paclitaxel was significantly increased by pretreatment with a P-gp inhibitor in hMDR1-MAC mice. These results indicated that the hMDR1-MAC mice are the first P-gp humanized mice expressing functional human P-gp at the blood-brain barrier. This mouse is a promising model with which to evaluate species differences of P-gp between humans and mice in vivo and to estimate the brain distribution of drugs in humans while taking into account species differences of P-gp.
DNA-loaded nano-adjuvant formed with a vitamin E-scaffold intracellular environmentally-responsive lipid-like material for cancer immunotherapy. International-journal Peer-reviewed

Minori Kawai, Takashi Nakamura, Naoya Miura, Mio Maeta, Hiroki Tanaka, Keisuke Ueda, Kenjirou Higashi, Kunikazu Moribe, Kota Tange, Yuta Nakai, Hiroki Yoshioka, Hideyoshi Harashima, Hidetaka Akita

Nanomedicine : nanotechnology, biology, and medicine　14　(8)　2587-2597　2018/11

DOI： 10.1016/j.nano.2018.08.006 　

ISSN： 1549-9634

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Cytoplasmic DNA triggers cellular immunity via activating the stimulator of interferon genes pathway. Since DNA is degradable and membrane impermeable, delivery system would permit cytoplasmic delivery by destabilizing the endosomal membrane for the use as an adjuvant. Herein, we report on the development of a plasmid DNA (pDNA)-encapsulating lipid nanoparticle (LNP). The structural components include an SS-cleavable and pH-activated lipid-like material that mounts vitamin E as a hydrophobic scaffold, and dual sensing motifs that are responsive to the intracellular environment (ssPalmE). The pDNA-encapsulating LNP (ssPalmE-LNP) induced a high interferon-β production in Raw 264.7 cells. The subcutaneous injection of ssPalmE-LNP strongly enhanced antigen-specific cytotoxic T cell activity. The ssPalmE-LNP treatment efficiently induced antitumor effects against E.G7-OVA tumor and B16-F10 melanoma metastasis. Furthermore, when combined with an anti-programmed death 1 antibody, an extensive therapeutic antitumor effect was observed. Therefore, the ssPalmE-LNP is a promising carrier of adjuvants for cancer immunotherapy.
Cancer-type organic anion transporting polypeptide 1B3 is a target for cancer suicide gene therapy using RNA trans-splicing technology. International-journal Peer-reviewed

Yuchen Sun, Josefina Piñón Hofbauer, Manami Harada, Katharina Wöss, Ulrich Koller, Hanae Morio, Anna Stierschneider, Keita Kitamura, Mari Hashimoto, Kan Chiba, Hidetaka Akita, Naohiko Anzai, Julia Reichelt, Johann W Bauer, Christina Guttmann-Gruber, Tomomi Furihata

Cancer letters　433　107-116　2018/10/01

DOI： 10.1016/j.canlet.2018.06.032 　

ISSN： 0304-3835

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Cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3) has been identified as a cancer-specific transcript in various solid cancers, including colorectal cancer. Given its excellent cancer-specific expression profile, we hypothesized that Ct-OATP1B3 could represent a promising target for cancer-specific expression of the suicide gene, herpes simplex virus 1 thymidine kinase (HSV-tk), via a spliceosome-mediated RNA trans-splicing (SMaRT) approach. SMaRT technology is used to recombine two RNA molecules to generate a chimeric transcript. In this study, we engineered an RNA trans-splicing molecule carrying a translation-defective HSV-tk sequence (RTM44), which was capable of inducing its own trans-splicing to the desired Ct-OATP1B3 pre-mRNA target. RTM44 expression in LS180 cells resulted in generation of Ct-OATP1B3/HSV-tk fusion mRNA. A functional translation start site contributed by the target pre-mRNA restored HSV-tk protein expression, rendering LS180 cells sensitive to ganciclovir treatment in vitro and in xenografted mice. The observed effects are ascribed to accurate and efficient trans-splicing, as they were absent in cells carrying a splicing-deficient mutant of RTM44. Collectively, our data highlights Ct-OATP1B3 as an ideal target for the HSV-tk SMaRT suicide system, which opens up new translational avenues for Ct-OATP1B3-targeted cancer therapy.
Differentiated HASTR/ci35 cells: A promising in vitro human astrocyte model for facilitating CNS drug development studies. Peer-reviewed

Keita Kitamura, Ryo Ito, Kenta Umehara, Hanae Morio, Kosuke Saito, Shota Suzuki, Mari Hashimoto, Yoshiro Saito, Naohiko Anzai, Hidetaka Akita, Kan Chiba, Tomomi Furihata

Journal of pharmacological sciences　137　(4)　350-358　2018/08

DOI： 10.1016/j.jphs.2018.06.013 　

ISSN： 1347-8613

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Astrocytes have shown longstanding promise as therapeutic targets for various central nervous system diseases. To facilitate drug development targeting astrocytes, we have recently developed a new conditionally immortalized human astrocyte cell line, termed HASTR/ci35 cells. In this study, in order to further increase their chances to contribute to various astrocyte studies, we report on the development of a culture method that improves HASTR/ci35 cell differentiation status and provide several proofs related to their astrocyte characteristics. The culture method is based on the simultaneous elimination of serum effects and immortalization signals. The results of qPCR showed that the culture method significantly enhanced several astrocyte marker gene expression levels. Using the differentiated HASTR/ci35, we examined their response profiles to nucleotide treatment and inflammatory stimuli, along with their membrane fatty acid composition. Consequently, we found that they responded to ADP or UTP treatment with a transient increase of intracellular Ca2+ concentration, and that they could show reactive response to interleukin-1β treatments. Furthermore, the membrane phospholipids of the cells were enriched with polyunsaturated fatty acids. To summarize, as a unique human astrocyte model carrying the capability of a differentiation induction properties, HASTR/ci35 cells are expected to contribute substantially to astrocyte-oriented drug development studies.
A New Conditionally Immortalized Human Fetal Brain Pericyte Cell Line: Establishment and Functional Characterization as a Promising Tool for Human Brain Pericyte Studies. International-journal Peer-reviewed

Kenta Umehara, Yuchen Sun, Satoshi Hiura, Koki Hamada, Motoyuki Itoh, Keita Kitamura, Motohiko Oshima, Atsushi Iwama, Kosuke Saito, Naohiko Anzai, Kan Chiba, Hidetaka Akita, Tomomi Furihata

Molecular neurobiology　55　(7)　5993-6006　2018/07

DOI： 10.1007/s12035-017-0815-9 　

ISSN： 0893-7648

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While pericytes wrap around microvascular endothelial cells throughout the human body, their highest coverage rate is found in the brain. Brain pericytes actively contribute to various brain functions, including the development and stabilization of the blood-brain barrier (BBB), tissue regeneration, and brain inflammation. Accordingly, detailed characterization of the functional nature of brain pericytes is important for understanding the mechanistic basis of brain physiology and pathophysiology. Herein, we report on the development of a new human brain pericyte cell line, hereafter referred to as the human brain pericyte/conditionally immortalized clone 37 (HBPC/ci37). Developed via the cell conditionally immortalization method, these cells exhibited excellent proliferative ability at 33 °C. However, when cultured at 37 °C, HBPC/ci37 cells showed a differentiated phenotype that was marked by morphological alterations and increases in several pericyte-enriched marker mRNA levels, such as platelet-derived growth factor receptor β. It was also found that HBPC/ci37 cells possessed the facilitative ability of in vitro BBB formation and differentiation into a neuronal lineage. Furthermore, HBPC/ci37 cells exhibited the typical "reactive" features of brain pericytes in response to pro-inflammatory cytokines. To summarize, our results clearly demonstrate that HBPC/ci37 cells possess the ability to perform several key brain pericyte functions while also showing the capacity for extensive and continuous proliferation. Based on these findings, it can be expected that, as a unique human brain pericyte model, HBPC/ci37 cells have the potential to contribute to significant advances in the understanding of human brain pericyte physiology and pathophysiology.
A hepatic pDNA delivery system based on an intracellular environment sensitive vitamin E-scaffold lipid-like material with the aid of an anti-inflammatory drug. International-journal Peer-reviewed

Ryohei Togashi, Hiroki Tanaka, Sakiko Nakamura, Hideo Yokota, Kota Tange, Yuta Nakai, Hiroki Yoshioka, Hideyoshi Harashima, Hidetaka Akita

Journal of controlled release : official journal of the Controlled Release Society　279　262-270　2018/06/10
Publisher: Elsevier B.V.
DOI： 10.1016/j.jconrel.2018.04.022 　

ISSN： 1873-4995 0168-3659
In Vivo Introduction of mRNA Encapsulated in Lipid Nanoparticles to Brain Neuronal Cells and Astrocytes via Intracerebroventricular Administration. International-journal Peer-reviewed

Hiroki Tanaka, Taichi Nakatani, Tomomi Furihata, Kota Tange, Yuta Nakai, Hiroki Yoshioka, Hideyoshi Harashima, Hidetaka Akita

Molecular pharmaceutics　15　(5)　2060-2067　2018/05/07

DOI： 10.1021/acs.molpharmaceut.7b01084 　

ISSN： 1543-8384

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Gene therapy is a promising strategy for curing certain types of brain diseases. Supplementation of therapeutic proteins such as aromatic amino acid decarboxylase (AADC) or nerve growth factor (NGF) have been reported to be successful examples of such treatments. However, there are safety concerns because these systems are based on virus-based gene vectors. A safe and efficient artificial carrier is thus urgently needed as an alternative. In this study, an mRNA based artificial gene carrier was introduced into the mouse brain via intracerebroventricular administration. As a carrier, a lipid nanoparticle (LNP) composed of environmentally sensitive lipid-like materials called an SS-cleavable proton-activated lipid-like material is used. The apolipoprotein E mediated cellular uptake of the lipid nanoparticles is one of the key features for its superior and homogeneous transfection activity compared to commercially available transfection reagents in both in vitro and in vivo situations. Immunostaining of brain specimens suggested that exogenous proteins can be introduced into neuronal cells as well as astrocytes using the mRNA-based gene carrier. This cannot be achieved using DNA-based artificial gene carriers. The findings suggest that a combination of an mRNA and a lipid based delivery system have great promise as a platform for the treatment of brain disorders.
Development of a mouse model for the visual and quantitative assessment of lymphatic trafficking and function by in vivo imaging. International-journal Peer-reviewed

Yoshihisa Yamaji, Shinsuke Akita, Hidetaka Akita, Naoya Miura, Masaki Gomi, Ichiro Manabe, Yoshitaka Kubota, Nobuyuki Mitsukawa

Scientific reports　8　(1)　5921-5921　2018/04/12
Publisher: Nature Publishing Group
DOI： 10.1038/s41598-018-23693-9 　

ISSN： 2045-2322
Evaluation of 4β-Hydroxycholesterol and 25-Hydroxycholesterol as Endogenous Biomarkers of CYP3A4: Study with CYP3A-Humanized Mice. International-journal Peer-reviewed

Shin-Ichiro Nitta, Mari Hashimoto, Yasuhiro Kazuki, Shoko Takehara, Hiraku Suzuki, Mitsuo Oshimura, Hidetaka Akita, Kan Chiba, Kaoru Kobayashi

The AAPS journal　20　(3)　61-61　2018/04/09
Publisher: Springer New York LLC
DOI： 10.1208/s12248-018-0186-9 　

ISSN： 1550-7416
Design and synthesis of boron containing monosaccharides by the hydroboration of D-glucal for use in boron neutron capture therapy (BNCT)

Itoh, T., Tamura, K., Ueda, H., Tanaka, T., Sato, K., Kuroda, R., Aoki, S.

Bioorganic and Medicinal Chemistry　26　(22)　2018

DOI： 10.1016/j.bmc.2018.10.041 　

ISSN： 0968-0896 1464-3391
CDP-glycerol inhibits the synthesis of the functional O-mannosyl glycan of -dystroglycan

Imae, R., Manya, H., Tsumoto, H., Osumi, K., Tanaka, T., Mizuno, M., Kanagawa, M., Kobayashi, K., Toda, T., Endo, T.

Journal of Biological Chemistry　293　(31)　2018

DOI： 10.1074/jbc.RA118.003197 　

ISSN： 0021-9258 1083-351X
Development of a New Conditionally Immortalized Human Liver Sinusoidal Endothelial Cells. Peer-reviewed

Meiyan Zhu, Akira Koibuchi, Hideyuki Ide, Hanae Morio, Minaka Shibuya, Atsuko Kamiichi, Akihito Tsubota, Naohiko Anzai, Hidetaka Akita, Kan Chiba, Tomomi Furihata

Biological & pharmaceutical bulletin　41　(3)　440-444　2018

DOI： 10.1248/bpb.b17-00661 　

ISSN： 0918-6158

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Liver sinusoidal endothelial cells (LSECs), which are specialized endothelial cells that line liver sinusoids, have been reported to participate in a variety of liver functions, such as blood macromolecule clearance and factor VIII production. In addition, LSECs play crucial roles in liver regeneration following acute liver injury, as well as the development and progression of liver diseases or drug-induced hepatotoxicity. However, the molecular mechanisms underlying their roles remain mostly unknown. Therefore, in order to contribute to the clarification of those mechanisms, herein we report on the development of a new immortalized human LSEC (HLSEC) line. To produce this cell line, two immortalized genes were introduced into the primary HLSECs, which eventually resulted in the establishment of the HLSEC/conditionally immortalized, clone-J (HLSEC/ciJ). Consistent with the two-immortalized gene expression, HLSEC/ciJ showed excellent proliferation activity. Additionally, the results of gene expression analyses showed that several LSEC (as well as pan-endothelial) marker mRNAs and proteins were clearly expressed in HLSEC/ciJ. Furthermore, we found that adherence junction proteins were localized at the cell border in the HLSEC/ciJ monolayer, and that the cells exhibited a tube-like structure formation property. Taken together, the results obtained thus far indicate that we have successfully immortalized HLSECs, resulting in creation of HLSEC/ciJ, a cell line that possesses infinite proliferation ability while retaining possession of at least some HLSEC features. We believe that the HLSEC/ciJ have the potential to provide a valuable and unlimited alternative source of HLSECs for use in liver/LSEC physiology/pathophysiology, pharmacology, and toxicology studies.
Cancer-Type OATP1B3 mRNA in Extracellular Vesicles as a Promising Candidate for a Serum-Based Colorectal Cancer Biomarker. Peer-reviewed

Hanae Morio, Yuchen Sun, Manami Harada, Hideyuki Ide, Osamu Shimozato, Xujia Zhou, Kenjirou Higashi, Ryuzaburo Yuki, Naoto Yamaguchi, Josefina Piñón Hofbauer, Christina Guttmann-Gruber, Naohiko Anzai, Hidetaka Akita, Kan Chiba, Tomomi Furihata

Biological & pharmaceutical bulletin　41　(3)　445-449　2018

DOI： 10.1248/bpb.b17-00743 　

ISSN： 0918-6158

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Cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3) mRNA is a variant isoform of the liver-type OATP1B3. Because Ct-OATP1B3 mRNA shows an excellent cancer-specific expression profile in colorectal cancer (CRC), and that its expression levels are associated with CRC prognosis, it holds the potential to become a useful CRC detection and diagnosis biomarker. While the potential is currently justified only at the tissue level, if existence of Ct-OATP1B3 mRNA in CRC-derived extracellular vesicles (EVs) is validated, the findings could enhance its translational potential as a CRC detection and diagnosis biomarker. Therefore, this study aims at proving that Ct-OATP1B3 mRNA exists in CRC-derived EVs, and can be detected using serum specimens. To examine the possibility of Ct-OATP1B3 mRNA being existed in extracellular milieu, we isolated EVs from the human CRC (HCT116, HT-29, and SW480) cell lines, and prepared their cDNAs. The RT-PCR results showed that Ct-OATP1B3 mRNA was clearly present in EVs derived from the human CRC cell lines. Then, in order to further explore the possibility that Ct-OATP1B3 mRNA in CRC-derived EVs can be detected in serum, we isolated serum EVs derived from human CRC xenograft mice, and then performed RT-PCR. The results showed that Ct-OATP1B3 mRNA could be found in all serum EV and CRC tissue samples of the mice examined. Collectively, our findings, which show that Ct-OATP1B3 mRNA exists in EVs and can be detected in (at least) mouse serum, strengthen the potential use of Ct-OATP1B3 mRNA as a serum-based CRC biomarker.
Development of a Caco-2 Cell Line Carrying the Human Intestine-Type CES Expression Profile as a Promising Tool for Ester-Containing Drug Permeability Studies. Peer-reviewed

Yuma Ishizaki, Tomomi Furihata, Yusuke Oyama, Kayoko Ohura, Teruko Imai, Masakiyo Hosokawa, Hidetaka Akita, Kan Chiba

Biological & pharmaceutical bulletin　41　(5)　697-706　2018

DOI： 10.1248/bpb.b17-00880 　

ISSN： 0918-6158

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Carboxylesterase 2 (CES2), which is a member of the serine hydrolase superfamily, is primarily expressed in the human small intestine, where it plays an important role in the metabolism of ester-containing drugs. Therefore, to facilitate continued progress in ester-containing drug development, it is crucial to evaluate how CES2-mediated hydrolysis influences its intestinal permeability characteristics. Human colon carcinoma Caco-2 cells have long been widely used in drug permeability studies as an enterocyte model. However, they are not suitable for ester-containing drug permeability studies due to the fact that Caco-2 cells express CES1 (which is not expressed in human enterocytes) but do not express CES2. To resolve this problem, we created a new Caco-2 cell line carrying the human small intestine-type CES expression profile. We began by introducing short-hairpin RNA for CES1 mRNA knockdown into Caco-2 cells to generate CES1-decifient Caco-2 cells (Caco-2CES1KD cells). Then, we developed Caco-2CES1KD cells that stably express CES2 (CES2/Caco-2CES1KD cells) and their control Mock/Caco-2CES1KD cells. The results of a series of functional expression experiments confirmed that CES2-specific activity, along with CES2 mRNA and protein expression, were clearly detected in our CES2/Caco-2CES1KD cells. Furthermore, we also confirmed that CES2/Caco-2CES1KD cells retained their tight junction formation property as well as their drug efflux transporter functions. Collectively, based on our results clearly showing that CES2/Caco-2CES1KD cells carry the human intestinal-type CES expression profile, while concomitantly retaining their barrier properties, it can be expected that this cell line will provide a promising in vitro model for ester-containing drug permeability studies.
Development of an Alcohol Dilution-Lyophilization Method for Preparing Lipid Nanoparticles Containing Encapsulated siRNA. Peer-reviewed

Daiki Shirane, Hiroki Tanaka, Yuta Nakai, Hiroki Yoshioka, Hidetaka Akita

Biological & pharmaceutical bulletin　41　(8)　1291-1294　2018

DOI： 10.1248/bpb.b18-00208 　

ISSN： 0918-6158

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Systems for delivering nucleic acids are now fundamental technologies for realizing personalized medicine. Among the various nucleic acid delivery systems that are currently available, lipid-nanoparticles (LNPs) that contain short interfering RNA (siRNA) have been extensively investigated for clinical applications. LNPs are generally prepared by an alcohol dilution method. In this method, it is necessary to remove the alcohol and then concentrate the LNP sample before they can be used. In this study, we report on the development of an "alcohol dilution-lyophilization method" for preparing siRNA-encapsulating LNPs. This method involves the use of a freeze-drying (lyophilization) method to remove the residual alcohol and to simultaneously concentrate the preparation. At first, the compositions of cryoprotectants and polyethylene glycol (PEG)-lipids that were used were optimized from the point of view of particle stabilization. A combination of sucrose and 1-(monomethoxy polyethyleneglycol5000)-2,3-dimyristoylglycerol (DMG-PEG5000) was found to have the most efficient cryoprotective activity for the LNPs. The knockdown efficiency of the LNP prepared by the alcohol dilution-lyophilization method was comparable to that of an LNP prepared by the conventional ultrafiltration method.
Preparation of envelope-type lipid nanoparticles containing gold nanorods for photothermal cancer therapy. International-journal Peer-reviewed

West Kristian D Paraiso, Hiroki Tanaka, Yusuke Sato, Daiki Shirane, Noriyuki Suzuki, Yasumitsu Ogra, Kota Tange, Yuta Nakai, Hiroki Yoshioka, Hideyoshi Harashima, Hidetaka Akita

Colloids and surfaces. B, Biointerfaces　160　715-723　2017/12/01

DOI： 10.1016/j.colsurfb.2017.10.027 　

ISSN： 0927-7765

eISSN： 1873-4367
Identification and Evaluation of the Minimum Unit of a KALA Peptide Required for Gene Delivery and Immune Activation. International-journal Peer-reviewed

Naoya Miura, Kota Tange, Yuta Nakai, Hiroki Yoshioka, Hideyoshi Harashima, Hidetaka Akita

Journal of pharmaceutical sciences　106　(10)　3113-3119　2017/10

DOI： 10.1016/j.xphs.2017.05.014 　

ISSN： 0022-3549

eISSN： 1520-6017
Association of a reactive intermediate derived from 1',6-dihydroxy metabolite with benzbromarone-induced hepatotoxicity. International-journal Peer-reviewed

Mina Yoshida, Naoki Cho, Hidetaka Akita, Kaoru Kobayashi

Journal of biochemical and molecular toxicology　31　(10)　2017/10

DOI： 10.1002/jbt.21946 　

ISSN： 1095-6670

eISSN： 1099-0461
Intracellular localization of pregnane X receptor in HepG2 cells cultured by the hanging drop method. International-journal Peer-reviewed

Kosuke Yokobori, Kaoru Kobayashi, Ikuko Azuma, Hidetaka Akita, Kan Chiba

Drug metabolism and pharmacokinetics　32　(5)　265-272　2017/10
Publisher: Japanese Society for the Study of Xenobiotics
DOI： 10.1016/j.dmpk.2017.08.001 　

ISSN： 1880-0920 1347-4367
PEGylation of the GALA Peptide Enhances the Lung-Targeting Activity of Nanocarriers That Contain Encapsulated siRNA Peer-reviewed

Sarochin Santiwarangkool, Hidekata Akita, Taichi Nakatani, Kenji Kusumoto, Hiroki Kimura, Masaru Suzuki, Masaharu Nishimura, Yusuke Sato, Hideyoshi Harashima

Journal of Pharmaceutical Sciences　106　(9)　2420-2427　2017/09/01
Publisher: Elsevier B.V.
DOI： 10.1016/j.xphs.2017.04.075 　

ISSN： 1520-6017 0022-3549
The Delivery of Small Interfering RNA to Hepatic Stellate Cells Using a Lipid Nanoparticle Composed of a Vitamin A-Scaffold Lipid-Like Material. International-journal Peer-reviewed

Naoyuki Toriyabe, Yu Sakurai, Akari Kato, Shoshiro Yamamoto, Kota Tange, Yuta Nakai, Hidetaka Akita, Hideyoshi Harahsima

Journal of pharmaceutical sciences　106　(8)　2046-2052　2017/08

DOI： 10.1016/j.xphs.2017.04.042 　

ISSN： 0022-3549

eISSN： 1520-6017
Cancer-type OATP1B3 mRNA has the potential to become a detection and prognostic biomarker for human colorectal cancer. International-journal Peer-reviewed

Yuchen Sun, Manami Harada, Osamu Shimozato, Hiroaki Souda, Nobuhiro Takiguchi, Yoshihiro Nabeya, Takehiko Kamijo, Hidetaka Akita, Naohiko Anzai, Kan Chiba, Tomomi Furihata

Biomarkers in medicine　11　(8)　629-639　2017/08

DOI： 10.2217/bmm-2017-0098 　

ISSN： 1752-0363

eISSN： 1752-0371
Modifying Antigen-Encapsulating Liposomes with KALA Facilitates MHC Class I Antigen Presentation and Enhances Anti-tumor Effects. International-journal Peer-reviewed

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

Molecular therapy : the journal of the American Society of Gene Therapy　25　(4)　1003-1013　2017/04/05

DOI： 10.1016/j.ymthe.2017.01.020 　

ISSN： 1525-0016

eISSN： 1525-0024
Temperature and pH sensitivity of a stabilized self-nanoemulsion formed using an ionizable lipid-like material via an oil-to-surfactant transition. International-journal Peer-reviewed

Hiroki Tanaka, Sho Oasa, Masataka Kinjo, Kota Tange, Yuta Nakai, Hideyoshi Harashima, Hidetaka Akita

Colloids and surfaces. B, Biointerfaces　151　95-101　2017/03/01

DOI： 10.1016/j.colsurfb.2016.11.020 　

ISSN： 0927-7765

eISSN： 1873-4367
Toward Wearable Energy Storage Devices: Paper-Based Biofuel Cells based on a Screen-Printing Array Structure

Shitanda, I., Momiyama, M., Watanabe, N., Tanaka, T., Tsujimura, S., Hoshi, Y., Itagaki, M.

Chemelectrochem　4　(10)　2017

DOI： 10.1002/celc.201700561 　

ISSN： 2196-0216
Concise and versatile synthesis of sulfoquinovosyl acyl glycerol derivatives for biological applications

Tanaka, T., Sawamoto, Y., Aoki, S.

Chemical and Pharmaceutical Bulletin　65　(6)　2017

DOI： 10.1248/cpb.c17-00135 　

ISSN： 0009-2363 1347-5223
Bivalent 14-mer peptide ligands of CXCR4 with polyproline linkers with anti-chemotactic activity against Jurkat cells

Tanaka, T., Aoki, T., Nomura, W., Tamamura, H.

Journal of Peptide Science　23　(7-8)　2017

DOI： 10.1002/psc.2946 　

ISSN： 1075-2617 1099-1387
Innovative Technologies in Nanomedicines: From Passive Targeting to Active Targeting/From Controlled Pharmacokinetics to Controlled Intracellular Pharmacokinetics. International-journal Peer-reviewed

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

Macromolecular bioscience　17　(1)　2017/01

DOI： 10.1002/mabi.201600179 　

ISSN： 1616-5187

eISSN： 1616-5195
Identification of a Nucleoporin358-Specific RNA Aptamer for Use as a Nucleus-Targeting Liposomal Delivery System. International-journal Peer-reviewed

Garima Shrivastava, Mamoru Hyodo, Shige H Yoshimura, Hidetaka Akita, Hideyoshi Harashima

Nucleic acid therapeutics　26　(5)　286-298　2016/10

DOI： 10.1089/nat.2016.0604 　

ISSN： 2159-3337

eISSN： 2159-3345
Effect of particle size on their accumulation in an inflammatory lesion in a dextran sulfate sodium (DSS)-induced colitis model. International-journal Peer-reviewed

Ayaka Watanabe, Hiroki Tanaka, Yu Sakurai, Kota Tange, Yuta Nakai, Tatsuya Ohkawara, Hiroshi Takeda, Hideyoshi Harashima, Hidetaka Akita

International journal of pharmaceutics　509　(1-2)　118-122　2016/07/25

DOI： 10.1016/j.ijpharm.2016.05.043 　

ISSN： 0378-5173

eISSN： 1873-3476
Cellular environment-responsive nanomaterials for use in gene and siRNA delivery: molecular design for biomembrane destabilization and intracellular collapse. International-journal Peer-reviewed

Hiroki Tanaka, Yusuke Sato, Hideyoshi Harashima, Hidetaka Akita

Expert opinion on drug delivery　13　(7)　1015-27　2016/07

DOI： 10.1517/17425247.2016.1154531 　

ISSN： 1742-5247

eISSN： 1744-7593
Multi-nanoDDS platform based on the intracellular environment-responsive material (ssPalm) : Delivery of gene/nucleic acid delivery Peer-reviewed

Hiroki Tanaka, Hidetaka Akita

Drug Delivery System　31　(1)　24-34　2016/04/25
Publisher: Japan Society of Drug Delivery System
DOI： 10.2745/dds.31.24 　

ISSN： 1881-2732 0913-5006
Transcytosis-Targeting Peptide: A Conductor of Liposomal Nanoparticles through the Endothelial Cell Barrier. International-journal Peer-reviewed

Hidetaka Akita, Takahiro Fujiwara, Sarochin Santiwarangkool, Nazir Hossen, Kazuaki Kajimoto, Ayman El-Sayed, Yasuhiko Tabata, Hideyoshi Harashima

Small (Weinheim an der Bergstrasse, Germany)　12　(9)　1212-21　2016/03/02

DOI： 10.1002/smll.201500909 　

ISSN： 1613-6810

eISSN： 1613-6829
11B NMR/MRI Sensing of Copper(II) Ions In Vitro by the Decomposition of a Hybrid Compound of a nido-o-Carborane and a Metal Chelator

Tanaka, T., Araki, R., Saido, T., Abe, R., Aoki, S.

European Journal of Inorganic Chemistry　2016　(20)　2016

DOI： 10.1002/ejic.201600346 　

ISSN： 1434-1948 1099-0682
11B NMR Probes of Copper(II): Finding and Implications of the Cu2+-Promoted Decomposition of ortho-Carborane Derivatives

Tanaka, T., Nishiura, Y., Araki, R., Saido, T., Abe, R., Aoki, S.

European Journal of Inorganic Chemistry　2016　(12)　2016

DOI： 10.1002/ejic.201600117 　

ISSN： 1434-1948 1099-0682
Size-dependent specific targeting and efficient gene silencing in peritoneal macrophages using a pH-sensitive cationic liposomal siRNA carrier. International-journal Peer-reviewed

Hideki Matsui, Yusuke Sato, Hiroto Hatakeyama, Hidetaka Akita, Hideyoshi Harashima

International journal of pharmaceutics　495　(1)　171-178　2015/11/10

DOI： 10.1016/j.ijpharm.2015.08.044 　

ISSN： 0378-5173

eISSN： 1873-3476
Molecular Tuning of a Vitamin E-Scaffold pH-Sensitive and Reductive Cleavable Lipid-like Material for Accelerated in Vivo Hepatic siRNA Delivery. International-journal Peer-reviewed

Hidetaka Akita, Yuki Noguchi, Hiroto Hatakeyama, Yusuke Sato, Kota Tange, Yuta Nakai, Hideyoshi Harashima

ACS biomaterials science & engineering　1　(9)　834-844　2015/09/14

DOI： 10.1021/acsbiomaterials.5b00203 　

ISSN： 2373-9878
Effect of hydrophobic scaffold on the cellular uptake and gene transfection activities of DNA-encapsulating liposomal nanoparticles via intracerebroventricular administration. International-journal Peer-reviewed

Hidetaka Akita, Taichi Nakatani, Kimiko Kuroki, Katsumi Maenaka, Kota Tange, Yuta Nakai, Hideyoshi Harashima

International journal of pharmaceutics　490　(1-2)　142-5　2015/07/25

DOI： 10.1016/j.ijpharm.2015.05.043 　

ISSN： 0378-5173

eISSN： 1873-3476
Multifunctional Envelope-Type Nano Device: Evolution from Nonselective to Active Targeting System. International-journal Peer-reviewed

Yasuhiro Hayashi, Hiroto Hatakeyama, Kazuaki Kajimoto, Mamoru Hyodo, Hidetaka Akita, Hideyoshi Harashima

Bioconjugate chemistry　26　(7)　1266-76　2015/07/15

DOI： 10.1021/acs.bioconjchem.5b00184 　

ISSN： 1043-1802
Effect of the Compaction and the Size of DNA on the Nuclear Transfer Efficiency after Microinjection in Synchronized Cells. International-journal Peer-reviewed

Hidetaka Akita, Dai Kurihara, Marco Schmeer, Martin Schleef, Hideyoshi Harashima

Pharmaceutics　7　(2)　64-73　2015/06/09

DOI： 10.3390/pharmaceutics7020064 　

ISSN： 1999-4923
A neutral lipid envelope-type nanoparticle composed of a pH-activated and vitamin E-scaffold lipid-like material as a platform for a gene carrier targeting renal cell carcinoma. International-journal Peer-reviewed

Hidetaka Akita, Ryohei Ishiba, Ryohei Togashi, Kota Tange, Yuta Nakai, Hiroto Hatakeyama, Hideyoshi Harashima

Journal of controlled release : official journal of the Controlled Release Society　200　97-105　2015/02/28

DOI： 10.1016/j.jconrel.2014.12.029 　

ISSN： 0168-3659

eISSN： 1873-4995
A KALA-modified lipid nanoparticle containing CpG-free plasmid DNA as a potential DNA vaccine carrier for antigen presentation and as an immune-stimulative adjuvant. International-journal Peer-reviewed

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

Nucleic acids research　43　(3)　1317-31　2015/02/18

DOI： 10.1093/nar/gkv008 　

ISSN： 0305-1048

eISSN： 1362-4962
The RNA sensor RIG-I dually functions as an innate sensor and direct antiviral factor for hepatitis B virus. International-journal Peer-reviewed

Seiichi Sato, Kai Li, Takeshi Kameyama, Takaya Hayashi, Yuji Ishida, Shuko Murakami, Tsunamasa Watanabe, Sayuki Iijima, Yu Sakurai, Koichi Watashi, Susumu Tsutsumi, Yusuke Sato, Hidetaka Akita, Takaji Wakita, Charles M Rice, Hideyoshi Harashima, Michinori Kohara, Yasuhito Tanaka, Akinori Takaoka

Immunity　42　(1)　123-32　2015/01/20

DOI： 10.1016/j.immuni.2014.12.016 　

ISSN： 1074-7613

eISSN： 1097-4180
"Programmed packaging" for gene delivery Peer-reviewed

M. Hyodo, Y. Sakurai, H. Akita, H. Harashima

JOURNAL OF CONTROLLED RELEASE　193　316-323　2014/11

DOI： 10.1016/j.jconrel.2014.04.023 　

ISSN： 0168-3659

eISSN： 1873-4995
Advantages of ethanol dilution method for preparing GALA-modified liposomal siRNA carriers on the in vivo gene knockdown efficiency in pulmonary endothelium. International-journal Peer-reviewed

Kenji Kusumoto, Hidetaka Akita, Sarochin Santiwarangkool, Hideyoshi Harashima

International journal of pharmaceutics　473　(1-2)　144-7　2014/10/01

DOI： 10.1016/j.ijpharm.2014.07.007 　

ISSN： 0378-5173

eISSN： 1873-3476
Improvement of doxorubicin efficacy using liposomal anti-polo-like kinase 1 siRNA in human renal cell carcinomas. International-journal Peer-reviewed

Yu Sakurai, Hiroto Hatakeyama, Hidetaka Akita, Hideyoshi Harashima

Molecular pharmaceutics　11　(8)　2713-9　2014/08/04

DOI： 10.1021/mp500245z 　

ISSN： 1543-8384
Neutralized nanoparticle composed of SS-cleavable and pH-activated lipid-like material as a long-lasting and liver-specific gene delivery system. International-journal Peer-reviewed

Masami Ukawa, Hidetaka Akita, Yasuhiro Hayashi, Ryohei Ishiba, Kota Tange, Masaya Arai, Kazuhiro Kubo, Yuriko Higuchi, Kazunori Shimizu, Satoshi Konishi, Mitsuru Hashida, Hideyoshi Harashima

Advanced healthcare materials　3　(8)　1222-9　2014/08

DOI： 10.1002/adhm.201300629 　

ISSN： 2192-2640

eISSN： 2192-2659
An apolipoprotein E modified liposomal nanoparticle: ligand dependent efficiency as a siRNA delivery carrier for mouse-derived brain endothelial cells. International-journal Peer-reviewed

Mina Tamaru, Hidetaka Akita, Kazuaki Kajimoto, Yusuke Sato, Hiroto Hatakeyama, Hideyoshi Harashima

International journal of pharmaceutics　465　(1-2)　77-82　2014/04/25

DOI： 10.1016/j.ijpharm.2014.02.016 　

ISSN： 0378-5173

eISSN： 1873-3476
Neutral biodegradable lipid-envelope-type nanoparticle using vitamin A-Scaffold for nuclear targeting of plasmid DNA. International-journal Peer-reviewed

Hiroki Tanaka, Hidetaka Akita, Ryohei Ishiba, Kota Tange, Masaya Arai, Kazuhiro Kubo, Hideyoshi Harashima

Biomaterials　35　(5)　1755-61　2014/02

DOI： 10.1016/j.biomaterials.2013.11.016 　

ISSN： 0142-9612

eISSN： 1878-5905
RNAi-mediated gene knockdown and anti-angiogenic therapy of RCCs using a cyclic RGD-modified liposomal-siRNA system. International-journal Peer-reviewed

Yu Sakurai, Hiroto Hatakeyama, Yusuke Sato, Mamoru Hyodo, Hidetaka Akita, Noritaka Ohga, Kyoko Hida, Hideyoshi Harashima

Journal of controlled release : official journal of the Controlled Release Society　173　110-8　2014/01/10

DOI： 10.1016/j.jconrel.2013.10.003 　

ISSN： 0168-3659

eISSN： 1873-4995
Development of a fluoride-responsive amide bond cleavage device that is potentially applicable to a traceable linker

Yamamoto, J., Maeda, N., Komiya, C., Tanaka, T., Denda, M., Ebisuno, K., Nomura, W., Tamamura, H., Sato, Y., Yamauchi, A., Shigenaga, A., Otaka, A.

Tetrahedron　70　(34)　2014

DOI： 10.1016/j.tet.2014.05.110 　

ISSN： 0040-4020 1464-5416
Development of a traceable linker containing a thiol-responsive amino acid for the enrichment and selective labelling of target proteins

Yamamoto, J., Denda, M., Maeda, N., Kita, M., Komiya, C., Tanaka, T., Nomura, W., Tamamura, H., Sato, Y., Yamauchi, A., Shigenaga, A., Otaka, A.

Organic and Biomolecular Chemistry　12　(23)　2014

DOI： 10.1039/c4ob00622d 　

ISSN： 1477-0520
The initiatives for regulatory science researches of nanomedicines Peer-reviewed

Harashima Hideyoshi, Hidetaka Akita, Kumiko Sakai-Kato, Takehiko Ishii, Yasuhiro Matsumura, Kazunori Kataoka

Drug Delivery System　29　(3)　217-225　2014
Publisher: Japan Society of Drug Delivery System
DOI： 10.2745/dds.29.217 　

ISSN： 1881-2732 0913-5006
Application of apolipoprotein E-modified liposomal nanoparticles as a carrier for delivering DNA and nucleic acid in the brain International-journal Peer-reviewed

Mina Tamaru, Hidetaka Akita, Taichi Nakatani, Kazuaki Kajimoto, Yusuke Sato, Hiroto Hatakeyama, Hideyoshi Harashima

INTERNATIONAL JOURNAL OF NANOMEDICINE　9　4267-4276　2014

DOI： 10.2147/IJN.S65402 　

ISSN： 1178-2013
Neutralized nanoparticle composed of SS-cleavable and pH-activated lipid-like material as a long-lasting and liver-specific gene delivery system International-journal Peer-reviewed

Masami Ukawa, Hidetaka Akita, Yasuhiro Hayashi, Ryohei Ishiba, Kota Tange, Masaya Arai, Kazuhiro Kubo, Yuriko Higuchi, Kazunori Shimizu, Satoshi Konishi, Mitsuru Hashida, Hideyoshi Harashima

Advanced Healthcare Materials　3　(8)　1222-1229　2014
Publisher: Wiley-VCH Verlag
DOI： 10.1002/adhm.201300629 　

ISSN： 2192-2659 2192-2640
Application of apolipoprotein E-modified liposomal nanoparticles as a carrier for delivering DNA and nucleic acid in the brain. International-journal Peer-reviewed

Mina Tamaru, Hidetaka Akita, Taichi Nakatani, Kazuaki Kajimoto, Yusuke Sato, Hiroto Hatakeyama, Hideyoshi Harashima

International journal of nanomedicine　9　4267-76　2014

DOI： 10.2147/IJN.S65402 　

ISSN： 1176-9114

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An innovative drug delivery technology is urgently needed to satisfy unmet medical needs in treating various brain disorders. As a fundamental carrier for plasmid DNA or nucleic acids, we developed a liposomal nanoparticle (multifunctional envelope-type nano device [MEND]) containing a proton-ionizable amino lipid (YSK-MEND). Here we report on the impact of apolipoprotein E (ApoE) modification on the function of YSK-MEND in terms of targeting brain cells. The cellular uptake and function of YSK-MEND encapsulating short interference RNA or plasmid DNA were significantly improved as a result of ApoE modification in mouse neuron-derived cell lines (Neuro-2a and CAD). Intracerebroventricular administration of ApoE-modified YSK-MEND (ApoE/YSK-MEND) encapsulating plasmid DNA also resulted in higher transgene expression in comparison with YSK-MEND that was not modified with ApoE. Moreover, observation of fluorescence-labeled ApoE/YSK-MEND and expression of mCherry (fluorescence protein) derived from plasmid DNA indicated that this carrier might be useful for delivering and conferring transgene expression in neural stem cells and/or neural progenitor cells. Thus, this system may be a useful tool for the treatment of neurodegenerative disease.
Multifunctional enveloped nanodevices (MENDs). International-journal Peer-reviewed

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

Advances in genetics　88　139-204　2014

DOI： 10.1016/B978-0-12-800148-6.00006-7 　

ISSN： 0065-2660
A DNA microarray-based analysis of immune-stimulatory and transcriptional responses of dendritic cells to KALA-modified nanoparticles. International-journal Peer-reviewed

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

Biomaterials　34　(35)　8979-90　2013/11

DOI： 10.1016/j.biomaterials.2013.08.003 　

ISSN： 0142-9612
A comparative study between nanoparticle-targeted therapeutics and bioconjugates as obesity medication. International-journal Peer-reviewed

Nazir Hossen, Kazuaki Kajimoto, Hidetaka Akita, Mamoru Hyodo, Hideyoshi Harashima

Journal of controlled release : official journal of the Controlled Release Society　171　(2)　104-12　2013/10/28

DOI： 10.1016/j.jconrel.2013.07.013 　

ISSN： 0168-3659
Production of small nano-sized particles by complex formation between polycations and linearized plasmid DNA at a low pH. Peer-reviewed

Ryohei Togashi, Hidetaka Akita, Hideyoshi Harashima

Journal of bioscience and bioengineering　116　(4)　528-31　2013/10

DOI： 10.1016/j.jbiosc.2013.04.009 　

ISSN： 1389-1723

eISSN： 1347-4421
Lipid envelope-type nanoparticle incorporating a multifunctional peptide for systemic siRNA delivery to the pulmonary endothelium. International-journal Peer-reviewed

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)　7534-41　2013/09/24

DOI： 10.1021/nn401317t 　

ISSN： 1936-0851

eISSN： 1936-086X
A neutral envelope-type nanoparticle containing pH-responsive and SS-cleavable lipid-like material as a carrier for plasmid DNA. International-journal Peer-reviewed

Hidetaka Akita, Ryohei Ishiba, Hiroto Hatakeyama, Hiroki Tanaka, Yusuke Sato, Kota Tange, Masaya Arai, Kazuhiro Kubo, Hideyoshi Harashima

Advanced healthcare materials　2　(8)　1120-5　2013/08

DOI： 10.1002/adhm.201200431 　

ISSN： 2192-2640 2192-2659
Gene silencing via RNAi and siRNA quantification in tumor tissue using MEND, a liposomal siRNA delivery system. International-journal Peer-reviewed

Yu Sakurai, Hiroto Hatakeyama, Yusuke Sato, Mamoru Hyodo, Hidetaka Akita, Hideyoshi Harashima

Molecular therapy : the journal of the American Society of Gene Therapy　21　(6)　1195-203　2013/06
Publisher: Nature Publishing Group
DOI： 10.1038/mt.2013.57 　

ISSN： 1525-0024 1525-0016
Therapeutic assessment of cytochrome C for the prevention of obesity through endothelial cell-targeted nanoparticulate system. International-journal Peer-reviewed

Md Nazir Hossen, Kazuaki Kajimoto, Hidetaka Akita, Mamoru Hyodo, Taichi Ishitsuka, Hideyoshi Harashima

Molecular therapy : the journal of the American Society of Gene Therapy　21　(3)　533-41　2013/03

DOI： 10.1038/mt.2012.256 　

ISSN： 1525-0016
Particle tracking analysis for the intracellular trafficking of nanoparticles modified with African swine fever virus protein p54-derived peptide. International-journal Peer-reviewed

Hidetaka Akita, Kaoru Enoto, Hiroki Tanaka, Hideyoshi Harashima

Molecular therapy : the journal of the American Society of Gene Therapy　21　(2)　309-17　2013/02
Publisher: Nature Publishing Group
DOI： 10.1038/mt.2012.235 　

ISSN： 1525-0024 1525-0016
Cell-permeable stapled peptides based on HIV-1 integrase inhibitors derived from HIV-1 Gene products

Nomura, W., Aikawa, H., Ohashi, N., Urano, E., M{\'e}tifiot, M., Fujino, M., Maddali, K., Ozaki, T., Nozue, A., Narumi, T., Hashimoto, C., Tanaka, T., Pommier, Y., Yamamoto, N., Komano, J.A., Murakami, T., Tamamura, H.

ACS Chemical Biology　8　(10)　2013

DOI： 10.1021/cb400495h 　

ISSN： 1554-8929 1554-8937
Low-Molecular-Weight CXCR4 Ligands with Variable Spacers

Narumi, T., Aikawa, H., Tanaka, T., Hashimoto, C., Ohashi, N., Nomura, W., Kobayakawa, T., Takano, H., Hirota, Y., Murakami, T., Yamamoto, N., Tamamura, H.

Chemmedchem　8　(1)　2013

DOI： 10.1002/cmdc.201200390 　

ISSN： 1860-7179 1860-7187
Expressed protein ligation at methionine: N-terminal attachment of homocysteine, ligation, and masking

Tanaka, T., Wagner, A.M., Warner, J.B., Wang, Y.J., Petersson, E.J.

Angewandte Chemie International Edition　52　(24)　2013

DOI： 10.1002/anie.201302065 　

ISSN： 1433-7851 1521-3773
The polyethyleneglycol dilemma: advantage and disadvantage of PEGylation of liposomes for systemic genes and nucleic acids delivery to tumors. Peer-reviewed

Hiroto Hatakeyama, Hidetaka Akita, Hideyoshi Harashima

Biological & pharmaceutical bulletin　36　(6)　892-9　2013

DOI： 10.1248/bpb.b13-00059 　

ISSN： 0918-6158 1347-5215

eISSN： 1347-5215
A pH-sensitive cationic lipid facilitates the delivery of liposomal siRNA and gene silencing activity in vitro and in vivo. International-journal Peer-reviewed

Yusuke Sato, Hiroto Hatakeyama, Yu Sakurai, Mamoru Hyodo, Hidetaka Akita, Hideyoshi Harashima

Journal of controlled release : official journal of the Controlled Release Society　163　(3)　267-76　2012/11/10

DOI： 10.1016/j.jconrel.2012.09.009 　

ISSN： 0168-3659
RNA interference-based silencing reveals the regulatory role of fatty acid-binding protein 4 in the production of IL-6 and vascular endothelial growth factor in 3T3-L1 adipocytes. International-journal Peer-reviewed

Kazuaki Kajimoto, Shiharu Takayanagi, Shun Sasaki, Hidetaka Akita, Hideyoshi Harashima

Endocrinology　153　(11)　5629-36　2012/11

DOI： 10.1210/en.2012-1456 　

ISSN： 0013-7227
Vascular-targeted nanotherapy for obesity: unexpected passive targeting mechanism to obese fat for the enhancement of active drug delivery. International-journal Peer-reviewed

Md Nazir Hossen, Kazuaki Kajimoto, Hidetaka Akita, Mamoru Hyodo, Hideyoshi Harashima

Journal of controlled release : official journal of the Controlled Release Society　163　(2)　101-10　2012/10/28

DOI： 10.1016/j.jconrel.2012.09.002 　

ISSN： 0168-3659
Pretreatment of hepatocyte growth factor gene transfer mediated by octaarginine peptide-modified nanoparticles ameliorates LPS/D-galactosamine-induced hepatitis. International-journal Peer-reviewed

Yasuhiro Hayashi, Ryoichi Mizuno, Khalil A Ikramy, Hidetaka Akita, Hideyoshi Harashima

Nucleic acid therapeutics　22　(5)　360-3　2012/10

DOI： 10.1089/nat.2012.0352 　

ISSN： 2159-3337

eISSN： 2159-3345
Efficient intracellular delivery of nucleic acid pharmaceuticals using cell-penetrating peptides. International-journal Peer-reviewed

Ikuhiko Nakase, Hidetaka Akita, Kentaro Kogure, Astrid Gräslund, Ulo Langel, Hideyoshi Harashima, Shiroh Futaki

Accounts of chemical research　45　(7)　1132-9　2012/07/17

DOI： 10.1021/ar200256e 　

ISSN： 0001-4842

eISSN： 1520-4898
FRET from quantum dots to photodecompose undesired acceptors and report the condensation and decondensation of plasmid DNA. International-journal Peer-reviewed

Vasudevanpillai Biju, Abdulaziz Anas, Hidetaka Akita, Edakkattuparambil Sidharthan Shibu, Tamitake Itoh, Hideyoshi Harashima, Mitsuru Ishikawa

ACS nano　6　(5)　3776-88　2012/05/22

DOI： 10.1021/nn2048608 　

ISSN： 1936-0851

eISSN： 1936-086X
Pharmacophore-based small molecule CXCR4 ligands

Narumi, T., Tanaka, T., Hashimoto, C., Nomura, W., Aikawa, H., Sohma, A., Itotani, K., Kawamata, M., Murakami, T., Yamamoto, N., Tamamura, H.

Bioorganic and Medicinal Chemistry Letters　22　(12)　2012

DOI： 10.1016/j.bmcl.2012.04.032 　

ISSN： 0960-894X 1464-3405
A Synthetic C34 Trimer of HIV-1 gp41 Shows Significant Increase in Inhibition Potency

Nomura, W., Hashimoto, C., Ohya, A., Miyauchi, K., Urano, E., Tanaka, T., Narumi, T., Nakahara, T., Komano, J.A., Yamamoto, N., Tamamura, H.

Chemmedchem　7　(2)　2012

DOI： 10.1002/cmdc.201100542 　

ISSN： 1860-7179 1860-7187
Conjugation of cell-penetrating peptides leads to identification of anti-HIV peptides from matrix proteins

Narumi, T., Komoriya, M., Hashimoto, C., Wu, H., Nomura, W., Suzuki, S., Tanaka, T., Chiba, J., Yamamoto, N., Murakami, T., Tamamura, H.

Bioorganic and Medicinal Chemistry　20　(4)　2012

DOI： 10.1016/j.bmc.2011.12.055 　

ISSN： 0968-0896 1464-3391
[Photonics meets sensing of bio-reaction and cell dynamics]. Peer-reviewed

Yuriko Higuchi, Hidetaka Akita

Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan　132　(4)　395-395　2012

DOI： 10.1248/yakushi.132.395 　

ISSN： 0031-6903

eISSN： 1347-5231
Effect of the anchor in polyethylene glycol-lipids on the transfection activity of PEGylated cationic liposomes encapsulating DNA. Peer-reviewed

Kenji Kusumoto, Hidetaka Akita, Ayman El-Sayed, Hideyoshi Harashima

Biological & pharmaceutical bulletin　35　(4)　445-8　2012

DOI： 10.1248/bpb.35.445 　

ISSN： 0918-6158

eISSN： 1347-5215
Multifunctional envelope-type nano device (MEND) for organelle targeting via a stepwise membrane fusion process. International-journal Peer-reviewed

Yuma Yamada, Hidetaka Akita, Hideyoshi Harashima

Methods in enzymology　509　301-26　2012

DOI： 10.1016/B978-0-12-391858-1.00015-0 　

ISSN： 0076-6879
[Development of an efficient short interference RNA (siRNA) delivery system with a new pH-sensitive cationic lipid]. Peer-reviewed

Yusuke Sato, Hiroto Hatakeyama, Mamoru Hyodo, Hidetaka Akita, Hideyoshi Harashima

Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan　132　(12)　1355-63　2012

DOI： 10.1248/yakushi.12-00234-2 　

ISSN： 0031-6903

eISSN： 1347-5231
Cell penetrating peptide-mediated systemic siRNA delivery to the liver. International-journal Peer-reviewed

Yasuhiro Hayashi, Jun Yamauchi, Ikramy A Khalil, Kazuaki Kajimoto, Hidetaka Akita, Hideyoshi Harashima

International journal of pharmaceutics　419　(1-2)　308-13　2011/10/31

DOI： 10.1016/j.ijpharm.2011.07.038 　

ISSN： 0378-5173
Delivery of nucleic acids and gene delivery

H. Akita, H. Hatakeyama, I. A. Khalil, Y. Yamada, H. Harashima

Comprehensive Biomaterials　4　411-444　2011/10
Publisher: Elsevier
Autopsy Cases of Miliary Tuberculosis: Clinicopathologic Features Including Background Factors Peer-reviewed

Takuma Tajiri, Genshu Tate, Mutsuki Makino, Hidetaka Akita, Mutsuko Omatsu, Tetsuji Enosawa, Shigeharu Hamatani, Atsuko Masunaga, Toshiaki Kunimura, Toshiyuki Mitsuya, Toshio Morohoshi

JOURNAL OF NIPPON MEDICAL SCHOOL　78　(5)　305-311　2011/10

DOI： 10.1272/jnms.78.305 　

ISSN： 1345-4676
KALA-modified multi-layered nanoparticles as gene carriers for MHC class-I mediated antigen presentation for a DNA vaccine. International-journal Peer-reviewed

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

Biomaterials　32　(26)　6342-50　2011/09

DOI： 10.1016/j.biomaterials.2011.05.014 　

ISSN： 1878-5905

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DNA vaccines are a new-generation vaccines that elicit an immunological response against a wide-variety of antigens with frequent mutations. However, an effective non-viral vector for genetically engineered DNA to dendritic cells is yet to be developed. We previously reported that an octaarginine (R8)-modified tetra-lamellar multi-functional envelope-type nano device (R8-T-MEND) increases transfection efficiency in dendritic cell cultures (JAWS II). The critical structural elements of the R8-T-MEND are a DNA-polycation condensed core coated with two nuclear membrane-fusogenic inner envelopes, and two endosome-fusogenic outer envelopes. While the gene expression was drastically enhanced by R8-T-MEND, antigen presentation using an epitope-encoding plasmid DNA remains an obstacle for future non-viral vectors in DNA vaccinations. In the present study, we upgraded the function of R8-T-MEND by improving the membrane-fusion processes with endosome- and nuclear membranes by incorporating the KALA peptide, and by reducing the charge ratio (+/-), in an attempt to accelerate intra-nuclear decondensation. The resulting KALA-modified T-MEND (R8/KALA-T-MEND) showed an approximately 20-fold higher transgene expression compared with the conventional R8-T-MEND in JAWS II, and exceeded that of Lipofectamine PLUS, a commercially available transfection reagent. Furthermore, significant antigen presentation of a specific epitope (SIINFEKL) was observed for the R8/KALA-T-MEND but was not detected for the conventional T-MEND or Lipofectamine PLUS when an ovalbumin (OVA)-encoding plasmid DNA was transfected. It thus appears that the R8/KALA-T-MEND has the potential for use as a vector in DNA vaccinations.
Quantitative analysis of condensation/decondensation status of pDNA in the nuclear sub-domains by QD-FRET. International-journal Peer-reviewed

Sharif M Shaheen, Hidetaka Akita, Atsushi Yamashita, Ryo Katoono, Nobuhiko Yui, Vasudevanpillai Biju, Mitsuru Ishikawa, Hideyoshi Harashima

Nucleic acids research　39　(7)　e48-e48　2011/04

DOI： 10.1093/nar/gkq1327 　

ISSN： 1362-4962

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Recent studies indicate that controlling the nuclear decondensation and intra-nuclear localization of plasmid DNA (pDNA) would result in an increased transfection efficiency. In the present study, we established a technology for imaging the nuclear condensation/decondensation status of pDNA in nuclear subdomains using fluorescence resonance energy transfer (FRET) between quantum dot (QD)-labeled pDNA as donor, and rhodamine-labeled polycations as acceptor. The FRET-occurring pDNA/polycation particle was encapsulated in a nuclear delivery system; a tetra-lamellar multifunctional envelope-type nano device (T-MEND), designed to overcome the endosomal membrane and nuclear membrane via step-wise fusion. Nuclear subdomains (i.e. heterochromatin and euchromatin) were distinguished by Hoechst33342 staining. Thereafter, Z-series of confocal images were captured by confocal laser scanning microscopy. pDNA in condensation/decondensation status in heterochromatin or euchromatin were quantified based on the pixel area of the signals derived from the QD and rhodamine. The results obtained indicate that modulation of the supra-molecular structure of polyrotaxane (DMAE-ss-PRX), a condenser that is cleaved in a reductive environment, conferred euchromatin-preferred decondensation. This represents the first demonstration of the successful control of condensation/decondensation in specific nuclear sub-domain via the use of an artificial DNA condenser.
A multifunctional envelope type nano device (MEND) for gene delivery to tumours based on the EPR effect: a strategy for overcoming the PEG dilemma. International-journal Peer-reviewed

Hiroto Hatakeyama, Hidetaka Akita, Hideyoshi Harashima

Advanced drug delivery reviews　63　(3)　152-60　2011/03/18

DOI： 10.1016/j.addr.2010.09.001 　

ISSN： 0169-409X

eISSN： 1872-8294
Topology of octaarginines (R8) or IRQ ligand on liposomes affects the contribution of macropinocytosis- and caveolae-mediated cellular uptake Peer-reviewed

Diky Mudhakir, Hidetaka Akita, Hideyoshi Harashima

REACTIVE & FUNCTIONAL POLYMERS　71　(3)　340-343　2011/03

DOI： 10.1016/j.reactfunctpolym.2010.11.013 　

ISSN： 1381-5148

eISSN： 1873-166X
Reprint of: Nanoparticles for ex vivo siRNA delivery to dendritic cells for cancer vaccines: Programmed endosomal escape and dissociation. International-journal Peer-reviewed

Hidetaka Akita, Kentaro Kogure, Rumiko Moriguchi, Yoshio Nakamura, Tomoko Higashi, Takashi Nakamura, Satoshi Serada, Minoru Fujimoto, Tetsuji Naka, Shiroh Futaki, Hideyoshi Harashima

Journal of controlled release : official journal of the Controlled Release Society　149　(1)　58-64　2011/01/05

DOI： 10.1016/j.jconrel.2010.08.023 　

ISSN： 0168-3659
Azamacrocyclic metal complexes as CXCR4 antagonists

Tanaka, T., Narumi, T., Ozaki, T., Sohma, A., Ohashi, N., Hashimoto, C., Itotani, K., Nomura, W., Murakami, T., Yamamoto, N., Tamamura, H.

Chemmedchem　6　(5)　2011

DOI： 10.1002/cmdc.201000548 　

ISSN： 1860-7179 1860-7187
The successes and failures of HIV drug discovery

Hashimoto, C., Tanaka, T., Narumi, T., Nomura, W., Tamamura, H.

Expert Opinion on Drug Discovery　6　(10)　2011

DOI： 10.1517/17460441.2011.611129 　

ISSN： 1746-0441 1746-045X
Modulation of Endosomal Escape of IRQ-PEGylated Nano-carrier Peer-reviewed

Diky Mudhakir, Hidetaka Akita, Hideyoshi Harashima

4TH NANOSCIENCE AND NANOTECHNOLOGY SYMPOSIUM (NNS2011): AN INTERNATIONAL SYMPOSIUM　1415　2011

DOI： 10.1063/1.3667222 　

ISSN： 0094-243X
Design of Smart Nano Device for Intracellular Targeting Peer-reviewed

Diky Mudhakir, Erdal Tan, Hidetaka Akita, Hideyoshi Harashima

4TH NANOSCIENCE AND NANOTECHNOLOGY SYMPOSIUM (NNS2011): AN INTERNATIONAL SYMPOSIUM　1415　2011

DOI： 10.1063/1.3667260 　

ISSN： 0094-243X
[Regulation and analysis of intracellular trafficking of nucleic acids based on quantitative and dynamic imaging]. Peer-reviewed

Hidetaka Akita

Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan　131　(11)　1545-56　2011

DOI： 10.1248/yakushi.131.1545 　

ISSN： 0031-6903

eISSN： 1347-5231
Ligand-based targeted delivery of a peptide modified nanocarrier to endothelial cells in adipose tissue. International-journal Peer-reviewed

Md Nazir Hossen, Kazuaki Kajimoto, Hidetaka Akita, Mamoru Hyodo, Taichi Ishitsuka, Hideyoshi Harashima

Journal of controlled release : official journal of the Controlled Release Society　147　(2)　261-8　2010/10/15

DOI： 10.1016/j.jconrel.2010.07.100 　

ISSN： 0168-3659
Reporter gene expression at single-cell level characterized with real-time RT-PCR, chemiluminescence, fluorescence, and electrochemical imaging. International-journal Peer-reviewed

Hitoshi Shiku, Daisuke Okazaki, Junya Suzuki, Yasufumi Takahashi, Tatsuya Murata, Hidetaka Akita, Hideyoshi Harashima, Kosuke Ino, Tomokazu Matsue

FEBS letters　584　(18)　4000-8　2010/09/24

DOI： 10.1016/j.febslet.2010.08.008 　

ISSN： 0014-5793
2-Methacryloyloxyethyl phosphorylcholine polymer (MPC)-coating improves the transfection activity of GALA-modified lipid nanoparticles by assisting the cellular uptake and intracellular dissociation of plasmid DNA in primary hepatocytes. International-journal Peer-reviewed

Masami Ukawa, Hidetaka Akita, Tomoya Masuda, Yasuhiro Hayashi, Tomohiro Konno, Kazuhiko Ishihara, Hideyoshi Harashima

Biomaterials　31　(24)　6355-62　2010/08

DOI： 10.1016/j.biomaterials.2010.04.031 　

ISSN： 0142-9612
Isolation and culture of microvascular endothelial cells from murine inguinal and epididymal adipose tissues. International-journal Peer-reviewed

Kazuaki Kajimoto, Md Nazir Hossen, Kyoko Hida, Noritaka Ohga, Hidetaka Akita, Mamoru Hyodo, Yasuhiro Hida, Hideyoshi Harashima

Journal of immunological methods　357　(1-2)　43-50　2010/05/31

DOI： 10.1016/j.jim.2010.03.011 　

ISSN： 0022-1759
Leptin-derived peptide, a targeting ligand for mouse brain-derived endothelial cells via macropinocytosis. International-journal Peer-reviewed

Mina Tamaru, Hidetaka Akita, Takahiro Fujiwara, Kazuaki Kajimoto, Hideyoshi Harashima

Biochemical and biophysical research communications　394　(3)　587-92　2010/04/09

DOI： 10.1016/j.bbrc.2010.03.024 　

ISSN： 0006-291X
Bivalent ligands of CXCR4 with rigid linkers for elucidation of the dimerization state in cells

Tanaka, T., Nomura, W., Narumi, T., Masuda, A., Tamamura, H.

Journal of the American Chemical Society　132　(45)　2010

DOI： 10.1021/ja107447w 　

ISSN： 0002-7863 1520-5126
CD4 mimics targeting the HIV entry mechanism and their hybrid molecules with a CXCR4 antagonist

Narumi, T., Ochiai, C., Yoshimura, K., Harada, S., Tanaka, T., Nomura, W., Arai, H., Ozaki, T., Ohashi, N., Matsushita, S., Tamamura, H.

Bioorganic and Medicinal Chemistry Letters　20　(19)　2010

DOI： 10.1016/j.bmcl.2010.07.106 　

ISSN： 0960-894X
Peptidic HIV integrase inhibitors derived from HIV gene products: Structure-activity relationship studies

Suzuki, S., Maddali, K., Hashimoto, C., Urano, E., Ohashi, N., Tanaka, T., Ozaki, T., Arai, H., Tsutsumi, H., Narumi, T., Nomura, W., Yamamoto, N., Pommier, Y., Komano, J.A., Tamamura, H.

Bioorganic and Medicinal Chemistry　18　(18)　2010

DOI： 10.1016/j.bmc.2010.07.050 　

ISSN： 0968-0896
Peptide HIV-1 integrase inhibitors from HIV-1 gene products

Suzuki, S., Urano, E., Hashimoto, C., Tsutsumi, H., Nakahara, T., Tanaka, T., Nakanishi, Y., Maddali, K., Han, Y., Hamatake, M., Miyauchi, K., Pommier, Y., Beutler, J.A., Sugiura, W., Fuji, H., Hoshino, T., Itotani, K., Nomura, W., Narumi, T., Yamamoto, N., Komano, J.A., Tamamura, H.

Journal of Medicinal Chemistry　53　(14)　2010

DOI： 10.1021/jm1003528 　

ISSN： 0022-2623 1520-4804
CD4 mimics targeting the mechanism of HIV entry

Yamada, Y., Ochiai, C., Yoshimura, K., Tanaka, T., Ohashi, N., Narumi, T., Nomura, W., Harada, S., Matsushita, S., Tamamura, H.

Bioorganic and Medicinal Chemistry Letters　20　(1)　2010

DOI： 10.1016/j.bmcl.2009.10.098 　

ISSN： 0960-894X
Remodeling of dynamic structures of HIV-1 envelope proteins leads to synthetic antigen molecules inducing neutralizing antibodies

Nakahara, T., Nomura, W., Ohba, K., Ohya, A., Tanaka, T., Hashimoto, C., Narumi, T., Murakami, T., Yamamoto, N., Tamamura, H.

Bioconjugate Chemistry　21　(4)　2010

DOI： 10.1021/bc900502z 　

ISSN： 1043-1802 1520-4812
An Improvement Delivery of siRNA by IRQ Ligand-Modified Double Membranous Nano-Carrier Peer-reviewed

Diky Mudhakir, Erdal Tan, Hidetaka Akita, Hideyoshi Harashima

THIRD NANOSCIENCE AND NANOTECHNOLOGY SYMPOSIUM 2010 - NNSB2010　1284　199-+　2010

ISSN： 0094-243X
A novel nonviral gene delivery system: multifunctional envelope-type nano device. International-journal Peer-reviewed

Hiroto Hatakeyama, Hidetaka Akita, Kentaro Kogure, Hideyoshi Harashima

Advances in biochemical engineering/biotechnology　119　197-230　2010

DOI： 10.1007/10_2008_40 　

ISSN： 0724-6145
Comparison between a multifunctional envelope-type nano device and lipoplex for delivery to the liver. Peer-reviewed

Jun Yamauchi, Yasuhiro Hayashi, Kazuaki Kajimoto, Hidetaka Akita, Hideyoshi Harashima

Biological & pharmaceutical bulletin　33　(5)　926-9　2010

DOI： 10.1248/bpb.33.926 　

ISSN： 0918-6158

eISSN： 1347-5215
A Novel Technique To Investigate the Decondensation of pc-DNA3.1-luc in Nuclear Sub-Domain with QD-FRET Peer-reviewed

Shaheen Sharif M, Akita Hidetaka, Yamashita Atsushi, Katoono Ryo, Yui Nobuhiko, Biju Vasudevanpillai, Ishikawa Mitsuru, Harashima Hideyoshi

MOLECULAR THERAPY　17　S330　2009/05

ISSN： 1525-0016
Development of Efficient siRNA Delivery System to Tumor Cells by Combining Octaarginine, GALA and Enzymatically-Cleavable PEG-Lipid Peer-reviewed

Sakurai, Yu, Hatakeyama, Hiroto, Akita, Hidetaka, Oishi, Motoi, Nagasaki, Yukio, Futaki, Shiro, Harashima, Hideyoshi

MOLECULAR THERAPY　17　(Suppl. 1)　0-0　2009/05
Publisher: NATURE PUBLISHING GROUP
ISSN： 1525-0016
A pH-Sensitive Fusogenic Peptide Facilitates Endosomal Escape and Greatly Enhanced the Gene Silencing of siRNA-Containing Nanoparticles In Vitro and In Vivo Peer-reviewed

Hatakeyama Hiroto, Itho Erika, Akita Hidetaka, Oihi Motoi, Nagasaki Yukio, Futaki Shiroh, Harashima Hideyoshi

MOLECULAR THERAPY　17　S388　2009/05

ISSN： 1525-0016
Fluorogenically active leucine zipper peptides as tag-probe pairs for protein imaging in living cells

Tsutsumi, H., Nomura, W., Abe, S., Mino, T., Masuda, A., Ohashi, N., Tanaka, T., Ohba, K., Yamamoto, N., Akiyoshi, K., Tamamura, H.

Angewandte Chemie International Edition　48　(48)　2009

DOI： 10.1002/anie.200903183 　

ISSN： 1433-7851
Structure-activity relationship study on artificial CXCR4 ligands possessing the cyclic pentapeptide scaffold: The exploration of amino acid residues of pentapeptides by substitutions of several aromatic amino acids

Tanaka, T., Nomura, W., Narumi, T., Esaka, A., Oishi, S., Ohashi, N., Itotani, K., Evans, B.J., Wang, Z.-X., Peiper, S.C., Fujii, N., Tamamura, H.

Organic and Biomolecular Chemistry　7　(18)　2009

DOI： 10.1039/b908286g 　

ISSN： 1477-0520
Stereoselective synthesis of peptidomimetics based on acid-catalyzed ring-opening ofβ-Aziridinyl-α,β-enoates

Tamamura, H., Tanaka, T., Tsutsumi, H., Nemoto, K., Mizokami, S., Ohashi, N., Oishi, S., Fujii, N.

Advances in Experimental Medicine and Biology　611　2009
Publisher: Advances in Experimental Medicine and Biology
DOI： 10.1007/978-0-387-73657-0_67 　

ISSN： 0065-2598
Supramolecular control of polyplex dissociation and cell transfection: efficacy of amino groups and threading cyclodextrins in biocleavable polyrotaxanes. International-journal Peer-reviewed

Atsushi Yamashita, Daizo Kanda, Ryo Katoono, Nobuhiko Yui, Tooru Ooya, Atsushi Maruyama, Hidetaka Akita, Kentaro Kogure, Hideyoshi Harashima

Journal of controlled release : official journal of the Controlled Release Society　131　(2)　137-44　2008/10/21

DOI： 10.1016/j.jconrel.2008.07.011 　

ISSN： 1873-4995

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A novel strategy for gene delivery using biocleavable polyrotaxanes, in which dimethylaminoethyl-modified alpha-cyclodextrins (DMAE-alpha-CDs) are threaded onto a poly(ethylene glycol) (PEG) chain capped with benzyloxycarbonyl-L-tyrosine via disulfide linkages (DMAE-SS-PRX), involves the formation of a stable polyion complex (polyplex) against a counter polyanion and the intracellular plasmid DNA (pDNA) release from the polyplex accompanied by the supramolecular dissociation of DMAE-SS-PRXs. In this study, we prepared biocleavable polyrotaxanes with different numbers of threading alpha-CD and amino (DMAE) groups to enhance the transfection activity of DMAE-SS-PRXs. 29DMAE-alpha18-SS-PRX, in which the numbers of alpha-CD molecules and amino groups were 18 and 29 respectively, exhibited a high transfection activity compared with other PRXs. The transfection activity of DMAE-SS-PRXs seems to be related to the efficacy of pDNA release from those polyplexes, which was controlled by the number of alpha-CD and/or amino groups in the polyrotaxane carrier. Most of the DMAE-SS-PRX polyplexes released the pDNA only in the presence of both 10 mM DTT and of the counter-polyanion, as expected, except for 14DMAE-alpha18-SS-PRX, which released pDNA in the absence of dextran sulfate once the DTT had been added to the polyplex solution. The transfection activity of 14DMAE-alpha18-SS-PRX was significantly lower than that of 29DMAE-alpha18-SS-PRX regardless of the above features. Confocal laser scanning microscopic (CLSM) observation suggested that the specific result for 14DMAE-alpha18-SS-PRX might be due to a premature release of pDNA from the most dissociative 14DMAE-alpha18-SS-PRX polyplex in the cytosol. Therefore, transfection activity seems to be related to an appropriate timing of pDNA release.
Advances in non-viral gene delivery: using multifunctional envelope-type nano-device. International-journal Peer-reviewed

Hidetaka Akita, Hideyoshi Harashima

Expert opinion on drug delivery　5　(8)　847-59　2008/08

DOI： 10.1517/17425247.5.8.847 　

ISSN： 1742-5247

eISSN： 1744-7593
Programmed Packaging: A New Drug Delivery System and its Application to Gene Therapy

Kentaro Kogure, Hidetaka Akita, Hiroyuki Kamiya, Hideyoshi Harashima

Modern Biopharmaceuticals: Design, Development and Optimization　4　1521-1536　2008/02/05
Publisher: Wiley-VCH Verlag GmbH
DOI： 10.1002/9783527620982.ch63 　
MITO-Porter: A liposome-based carrier system for delivery of macromolecules into mitochondria via membrane fusion. International-journal

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

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

ISSN： 0006-3002

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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.
[Development of various multifunctional envelope-type nano device MEND based on novel assembly technologies]. Peer-reviewed

Kentaro Kogure, Hidetaka Akita, Hideyoshi Harashima

Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan　128　(2)　219-32　2008/02

DOI： 10.1248/yakushi.128.219 　

ISSN： 0031-6903
Fluorophore labeling enables imaging and evaluation of specific CXCR4-ligand interaction at the cell membrane for fluorescence-based screening

Nomura, W., Tanabe, Y., Tsutsumi, H., Tanaka, T., Ohba, K., Yamamoto, N., Tamamura, H.

Bioconjugate Chemistry　19　(9)　2008

DOI： 10.1021/bc800216p 　

ISSN： 1043-1802
A future perspective on the development of chemokine receptor CXCR4 antagonists

Tamamura, H., Tsutsumi, H., Nomura, W., Tanaka, T., Fujii, N.

Expert Opinion on Drug Discovery　3　(10)　2008

DOI： 10.1517/17460441.3.10.1155 　

ISSN： 1746-0441
Structure-activity relationship study of CXCR4 antagonists bearing the cyclic pentapeptide scaffold: Identification of the new pharmacophore

Tanaka, T., Tsutsumi, H., Nomura, W., Tanabe, Y., Ohashi, N., Esaka, A., Ochiai, C., Sato, J., Itotani, K., Murakami, T., Ohba, K., Yamamoto, N., Fujii, N., Tamamura, H.

Organic and Biomolecular Chemistry　6　(23)　2008

DOI： 10.1039/b812029c 　

ISSN： 1477-0520
Supramolecular control of polyplex dissociation and cell transfection: Efficacy of amine and threading cyclodextrin in biocleavable polyrotaxanes Peer-reviewed

Yamashita A, Kanda D, Katoono R, Yui N, Ooya T, Maruyama A, Akita H, Kogure K, Harashima H

8th World Biomaterials Congress 2008　4　2008
Nonviral gene delivery. International-journal Peer-reviewed

Hidetaka Akita, Hideyoshi Harashima

Contributions to nephrology　159　13-29　2008

DOI： 10.1159/000125560 　

ISSN： 0302-5144
[Development of multifunctional envelope type artificial viral-like gene delivery system]. Peer-reviewed

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

Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan　127　(10)　1655-72　2007/10

DOI： 10.1248/yakushi.127.1655 　

ISSN： 0031-6903
Therapeutic potential of the chemokine receptor CXCR4 antagonists as multifunctional agents

Tsutsumi, H., Tanaka, T., Ohashi, N., Masuno, H., Tamamura, H., Hiramatsu, K., Araki, T., Ueda, S., Oishi, S., Fujii, N.

Biopolymers Peptide Science Section　88　(2)　2007

DOI： 10.1002/bip.20653 　

ISSN： 0006-3525 1097-0282
Versatile use of acid-catalyzed ring-opening of β-aziridinyl-α,β-enoates to stereoselective synthesis of peptidomimetics

Tamamura, H., Tanaka, T., Tsutsumi, H., Nemoto, K., Mizokami, S., Ohashi, N., Oishi, S., Fujii, N.

Tetrahedron　63　(37)　2007

DOI： 10.1016/j.tet.2007.06.029 　

ISSN： 0040-4020
[Development of non-viral vector based on the quantitative comparison of intracellular trafficking with viral vector]. Peer-reviewed

Hidetaka Akita, Susumu Hama, Hiroyuki Mizuguchi, Hideyoshi Harashima

Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan　126　(11)　1047-57　2006/11

DOI： 10.1248/yakushi.126.1047 　

ISSN： 0031-6903
Cellular uptake and subsequent intracellular trafficking of R8-liposomes introduced at low temperature. International-journal

Akitada Iwasa, Hidetaka Akita, Ikramy Khalil, Kentaro Kogure, Shiroh Futaki, Hideyoshi Harashima

Biochimica et biophysica acta　1758　(6)　713-20　2006/06

ISSN： 0006-3002

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Intracellular trafficking is a determining factor in the transgene expression efficiency of gene vectors. In the present study, the mechanism of the cellular uptake of octaarginine (R8)-modified liposomes, when introduced at 37 degrees C and 4 degrees C, was investigated in living cells. Compared with 37 degrees C, the uptake of R8-liposomes was only slightly reduced at 4 degrees C. Dual imaging of liposomes and plasma membranes revealed that R8-liposomes were internalized by vesicular transport, and partially escaped to the cytosol at the perinuclear region at 37 degrees C. When introduced at 4 degrees C, intracellular liposomes were observed within a specific region close to the plasma membrane, and internalization of the plasma membrane was completely inhibited. Therefore, at 4 degrees C, R8-liposomes appear to enter cells via unique pathway, which is separate and distinct from energy-dependent vesicular transport. The subsequent nuclear delivery of encapsulated pDNA, when introduced at 4 degrees C, was less prominent compared with those introduced at 37 degrees C. Collectively, these findings demonstrate that a vesicular transport-independent pathway is responsible for the cellular uptake of liposomes. In addition, the uptake route is closely related to the subsequent nuclear delivery process; the operation of an endogenous vesicular sorting system is advantageous for the nuclear delivery of pDNA.
Cellular Uptake and Subsequent Intracellular of R8-Liposomes Introduced at Low Temperature Peer-reviewed

Iwasa Akitada, Akita Hidetaka, Khalil Ikramy, Kogure Kentaro, Futaki Shiroh, Harashima Hideyoshi

MOLECULAR THERAPY　13　S129　2006/05

DOI： 10.1016/j.ymthe.2006.08.397 　

ISSN： 1525-0016
Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery. International-journal Peer-reviewed

Ikramy A Khalil, Kentaro Kogure, Hidetaka Akita, Hideyoshi Harashima

Pharmacological reviews　58　(1)　32-45　2006/03

DOI： 10.1124/pr.58.1.8 　

ISSN： 0031-6997
[Envelope-type nano device for gene delivery]. Peer-reviewed

Kentaro Kogure, Hidetaka Akita, Hideyoshi Harashima

Nihon rinsho. Japanese journal of clinical medicine　64　(2)　258-62　2006/02

ISSN： 0047-1852

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We introduced a new concept "Programmed Packaging" for the rational development of non viral gene delivery system. There are many non-viral gene delivery system, however, their utility is limited in the in vitro experiments. The major reason is considered that it is difficult to package many functional devices into a nanoparticle. The new concept which we introduce here is named as Programmed Packaging, which is composed of the strategy to targeting, molecular design of functional devices to overcome barriers, and assembly technique to package them into a nanoparticle. In this article, we explain this concept and provide newly developed gene delivery systems based on this concept.
Sugar-displaying quantum dots for cellular stress sensing

Kenichi Niikura, Takashi Nishio, Yasutaka Matsuo, Hidetaka Akita, Kentaro Kogure, Hideyoshi Harashima, Kuniharu Ijiro

Polymer Preprints, Japan　55　(2)　5451-5452　2006
Synthesis of sugar-displaying quantum dots and their intracellular dynamics

Takashi Nishio, Kenichi Niikura, Yasutaka Matsuo, Hidetaka Akita, Kentaro Kogure, Hideyoshi Harashima, Kuniharu Ijiro

Polymer Preprints, Japan　55　(1)　2145　2006
Quantitative comparison of intracellular trafficking between artificial and viral vector: Mechanism underlying the difference in dominant rate-limiting process Peer-reviewed

Hidetaka Akita, Susumu Hama, Hiroyuki Mizuguchi, Hideyoshi Harashima

Progress on Post-Genome Technologies　521-525　2006
Effects of supramolecular structure of cationic-polyrotaxanes on pDNA compaction Peer-reviewed

Yamashita A, Ooya T, Kanda D, Choi H.S, Yui N, Maruyama A, Akita H, Kogure K, Harashima H

Polymer Preprints, Japan　55　(1)　2006
Synthesis of a biocleavable polyrotaxane-plasmid DNA (pDNA) polyplex and its use for the rapid nonviral delivery of pDNA to cell nuclei. International-journal Peer-reviewed

Atsushi Yamashita, Nobuhiko Yui, Tooru Ooya, Arihiro Kano, Atsushi Maruyama, Hidetaka Akita, Kentaro Kogure, Hideyoshi Harashima

Nature protocols　1　(6)　2861-9　2006

DOI： 10.1038/nprot.2006.438 　

ISSN： 1750-2799

eISSN： 1750-2799

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This protocol provides a method for synthesizing a biocleavable polyrotaxane/plasmid DNA (pDNA) polyplex and for using it to deliver pDNA into cell nuclei. The biocleavable polyrotaxane is synthesized in four steps: (i) introduction of disulfide linkages at both terminals of PEG, (ii) preparation of an inclusion complex between disulfide-containing PEG and alpha-cyclodextrins (alpha-CDs), (iii) synthesis of polyrotaxane and (iv) modification of alpha-CDs in the polyrotaxane with dimethylethylenediamine. A polyplex of pDNA with the polyrotaxane is formed when the two compounds are dissolved together in a phosphate buffer. Subcellular localization of rhodamine-labeled pDNA in fluorescently labeled organelles is quantified by Z-series of confocal images captured by confocal laser scanning microscopy. Significant amounts of pDNA delivered to the nucleus can be expected as well as high transfection activity of the polyplex. This protocol can be completed in 23-32 d.
Self-assembled polyglycerol dendrimer for bottom-up typed nano-fabrication Peer-reviewed

Tooru Ooya, Hidetaka Akita

Progress on Post-Genome Technologies　274-276　2006
In vitro evaluation of cationic polyrotaxanes as gene carrier Peer-reviewed

Kanda D, Yamashita A, Katoono R, Ooya T, Yui N, Maruyama A, Akita H, Kogure K, Harashima H

Polymer Preprints, Japan　55　(2)　5367-5368　2006
Pharmacokinetics of gene delivery in cells Peer-reviewed

Hidetaka Akita, Ikramy A. Khalil, Kentaro Kogure, Hideyoshi Harashima

Non-viral Gene Therapy: Gene Design and Delivery　135-154　2005
Publisher: Springer-Verlag Tokyo
DOI： 10.1007/4-431-27879-6_11 　
Factors governing the in vivo tissue uptake of transferrin-coupled polyethylene glycol liposomes in vivo. International-journal Peer-reviewed

Hiroto Hatakeyama, Hidetaka Akita, Kazuo Maruyama, Tetsuya Suhara, Hideyoshi Harashima

International journal of pharmaceutics　281　(1-2)　25-33　2004/08/20

DOI： 10.1016/j.ijpharm.2004.05.025 　

ISSN： 0378-5173
Pharmacokinetic and pharmacodynamic considerations in gene therapy. International-journal Peer-reviewed

Hiroyuki Kamiya, Hidetaka Akita, Hideyoshi Harashima

Drug discovery today　8　(21)　990-6　2003/11/01

DOI： 10.1016/s1359-6446(03)02889-7 　

ISSN： 1359-6446
Sinusoidal efflux of bile acids via Mrp3 (ABCC3) Peer-reviewed

H Akita, H Suzuki, Y Sugiyama

BILE ACIDS: FROM GENOMICS TO DISEASE AND THERAPY　129　104-110　2003
Sinusoidal efflux of taurocholate correlates with the hepatic expression level of Mrp3. International-journal Peer-reviewed

Hidetaka Akita, Hiroshi Suzuki, Yuichi Sugiyama

Biochemical and biophysical research communications　299　(5)　681-7　2002/12/20

DOI： 10.1016/s0006-291x(02)02723-7 　

ISSN： 0006-291X
Sinusoidal efflux of bile acid via multidrug resistance associated protein 3 (Mrp3)

Akita, H., Suzuki, H., Sugiyama, Y.

Japanese Pharmacology and Therapeutics　30　(SUPPL. 2)　2002

ISSN： 0386-3603
Transport activity of human MRP3 expressed in Sf9 cells: comparative studies with rat MRP3. International-journal Peer-reviewed

Hidetaka Akita, Hiroshi Suzuki, Tomoko Hirohashi, Hajime Takikawa, Yuichi Sugiyama

Pharmaceutical research　19　(1)　34-41　2002/01

DOI： 10.1023/a:1013699130991 　

ISSN： 0724-8741
Kinetic analysis of efflux of bile acids across the sinusoidal membrane: Comparison between normal and mrp2-deficient rats

H. Suzuki, H. Akita, Y. Sugiyama

Japanese Pharmacology and Therapeutics　29　(1)　S109-S111　2001

ISSN： 0386-3603
Hepatocellular extrusion of bile acids by ABC transporters Peer-reviewed

H Suzuki, H Akita, Y Sugiyama

BIOLOGY OF BILE ACIDS IN HEALTH AND DISEASE　120　105-109　2001
Characterization the mechanism for the biliary excretion of bile acids across the canalicular membrane

Hidetaka Akita, Norihito Sato, Setsuo Kinoshita, Hajime Takikawa, Junichi Goto, Takashi Iida, Hiroshi Suzuki, Yuichi Sugiyama

Japanese Pharmacology and Therapeutics　27　(7)　179-182　1999/12

ISSN： 0386-3603

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Misc. 194

Neoantigen-mRNA-LNP Vaccine Induces Potent Anti-Tumor CD8⁺ T cell responses in the tumor

Koji Nagaoka, Hiroki Tanaka, Hideyuki Nakanishi, Takeshi Kawamura, Toshiya Tanaka, Takefumi Yamashita, Sachiyo Nomura, Hidetaka Akita, Keiji Itaka, Tatsuhiko Kodama, Kazuhiro Kakimi

CANCER SCIENCE　116　502-502　2025/01

ISSN： 1347-9032

eISSN： 1349-7006
Structure and Interaction Evaluation of Nucleic Acid Encapsulated ssPalm Lipid Nanoparticles by Molecular Simulation.

小浪なおこ, 奥脇弘次, 奥脇弘次, 田中浩揮, 秋田英万, 東顕二郎, 古石誉之, 米持悦生, 福澤薫

日本薬剤学会年会講演要旨集(CD-ROM)　40th　2025
Express Hu-mAb system (1) Establishment of anti-infective human antibody discovery platform leveraging animals with humanized immune system

冨塚一磨, 香月康宏, 末次正幸, 秋田英万

日本抗体学会学術大会プログラム・抄録集(Web)　3rd　2024
mRNA-based antibody delivery into the body

原田純希, 田中浩揮, 下谷和人, 濱道修生, 金井亨輔, 景山誠二, 香月康宏, 香月康宏, 香月康宏, 冨塚一磨, 秋田英万

日本抗体学会学術大会プログラム・抄録集(Web)　3rd　2024
Express Humabシステム(3)mRNAワクチンを用いたTc-mAbマウスへの免疫

西尾一真, 田中浩揮, アニンディタジェシカ, 下谷和人, 濱道修生, 金井亨輔, 景山誠二, 香月康宏, 香月康宏, 香月康宏, 冨塚一磨, 秋田英万

日本抗体学会学術大会プログラム・抄録集(Web)　3rd　2024
Structure and interaction analysis of ssPalm lipid nanoparticle containing nucleic acids using molecular simulation

小浪なおこ, 佐々木香純, 奥脇弘次, 奥脇弘次, 奥脇弘次, 田中浩揮, 秋田英万, 古石誉之, 福澤薫, 福澤薫, 米持悦生

日本薬学会年会要旨集(Web)　144th　2024

ISSN： 0918-9823
mRNA医薬の評価の考え方 ─パネルディスカッションの論点─

吉田徳幸, 山下拓真, 山本武範, 大岡伸通, 位高啓史, 秋田英万, 武下文彦, 峰野純一, 辻畑茂朝, 山口照英, 内田恵理子, 井上貴雄

医薬品医療機器レギュラトリーサイエンス　54　(4)　322-329　2023/08

DOI： 10.51018/pmdrs.54.4_322 　
Structure and interaction evaluation of ssPalm lipid nanoparticle using computational chemistry

小浪なおこ, 氏家かれん, 佐々木香純, 奥脇弘次, 田中浩揮, 秋田英万, 古石誉之, 福澤薫, 福澤薫, 米持悦生

日本薬学会年会要旨集(Web)　143rd　2023

ISSN： 0918-9823
Structure and interaction analysis of ssPalm lipid nanoparticle containing nucleic acids using computational chemistry

小浪なおこ, 氏家かれん, 佐々木香純, 奥脇弘次, 田中浩揮, 秋田英万, 古石誉之, 福澤薫, 福澤薫, 米持悦生

日本薬剤学会年会講演要旨集(CD-ROM)　38th　2023
脳を標的とするNose-to-Brain型mRNA送達技術の開発

山本理奈, 藤田翔也, 田中浩揮, 櫻井遊, 中井悠太, 丹下耕太, 金沢貴憲, 秋田英万

遺伝子・デリバリー研究会シンポジウム要旨集　21st (CD-ROM)　2022
Ready-to-Use型mRNA内封脂質ナノ粒子製剤の物性評価

山川拓真, 田中浩揮, 萩原伸哉, 櫻井遊, 佐々木香純, 米持悦生, 福澤薫, 中井悠太, 丹下耕太, 秋田英万, 秋田英万

遺伝子・デリバリー研究会シンポジウム要旨集　21st (CD-ROM)　2022
Development of Ready-to-Use type LNPs

田中浩揮, 萩原伸哉, 山川拓真, 櫻井遊, 米持悦生, 福澤薫, 中井悠太, 丹下耕太, 秋田英万, 秋田英万

日本核酸医薬学会年会講演要旨集(CD-ROM)　7th　2022
Evaluation of Physical Properties of Ready-to-Use Formulations of Lipid Nanoparticles Enclosed in mRNA

山川拓真, 田中浩揮, 萩原伸哉, 櫻井遊, 佐々木香純, 米持悦生, 福澤薫, 中井悠太, 丹下耕太, 秋田英万, 秋田英万

日本核酸医薬学会年会講演要旨集(CD-ROM)　7th　2022
脳を標的とするNose-to-Brain型mRNA送達技術の開発

山本理奈, 藤田翔也, 田中浩揮, 櫻井遊, 中井悠太, 丹下耕太, 金沢貴憲, 秋田英万

生体膜と薬物の相互作用シンポジウム講演要旨集　43rd　2022

ISSN： 0919-2131
Regulation of CYP3A5 expression in CYP3A-humanized mice carrying CYP3A5^*3 allele

峰岸元気, 峰岸元気, 宮野綾華, 大槻純男, 宮嶋篤志, 秋田英万, 香月康宏, 香月康宏, 小林カオル

日本薬学会年会要旨集(Web)　142nd　2022

ISSN： 0918-9823
定量プロテオーム解析による新規不死化ヒトリンパ管内皮細胞の評価

古西修希, 櫻井遊, 千田克幸, 増田豪, 伊藤慎悟, 降幡知巳, 秋田英万, 秋田英万, 大槻純男

日本薬学会九州山口支部大会講演要旨集(CD-ROM)　38th　2021
Analysis of tacrolimus pharmacokinetics in CYP3A5 polymorphism model mice in vivo

峰岸元気, 峰岸元気, 香月康宏, 香月康宏, 秋田英万, 小林カオル, 小林カオル

日本薬物動態学会年会講演要旨集(Web)　35th　2021
mRNA内封脂質ナノ粒子Ready-to-Use製剤の物性評価

山川拓真, 田中浩揮, 萩原伸哉, 白根大貴, 福田翔, 福澤薫, 米持悦生, 中井悠太, 丹下耕太, 秋田英万

日本DDS学会学術集会プログラム予稿集　37th　2021
Ready-to-Use型脂質ナノ粒子製剤の物性評価

山川拓真, 田中浩揮, 萩原伸哉, 白根大貴, 高田奈依, 櫻井遊, 福田翔, 福澤薫, 米持悦生, 玉川晋也, 中井悠太, 丹下耕太, 秋田英万

日本薬学会関東支部大会講演要旨集　65th (CD-ROM)　2021
分子シミュレーションによるssPalm脂質ナノ粒子の構造と相互作用解析

小浪なおこ, 西田瑠花, 氏家かれん, 佐々木香純, 奥脇弘次, 望月祐志, 田中浩揮, 秋田英万, 古石誉之, 福澤薫, 米持悦生

日本薬学会関東支部大会講演要旨集　65th (CD-ROM)　2021
Structural analysis of ssPalm-cholesterol lipid nanoparticles using molecular simulation

氏家かれん, 西田瑠花, 奥脇弘次, 望月祐志, 田中浩揮, 秋田英万, 古石誉之, 福澤薫, 米持悦生

日本薬学会年会要旨集(Web)　141st　2021

ISSN： 0918-9823
染色体工学技術により作製した薬物代謝酵素ヒト化マウスを用いた腸管代謝評価モデル

峰岸元気, 新田真一郎, 秋田英万, 香月康宏, 香月康宏, 小林カオル

HAB研究機構学術年会プログラム・要旨集　27th　2020
Inhibition effects of macrolide antibiotics on intestinal human CYP3A in CYP3A-humanized mice.

峰岸元気, 新田真一郎, 秋田英万, 香月康宏, 香月康宏, 小林カオル

日本薬学会年会要旨集(CD-ROM)　140th　2020
Simulations for lipid bilayers and nano particles using FMO-DPD Method

奥脇弘次, 新庄永治, 西田瑠花, 氏家かれん, 望月祐志, 望月祐志, 福澤薫, 福澤薫, 福澤薫, 米持悦生, 田中浩揮, 秋田英万

応用物理学会秋季学術講演会講演予稿集(CD-ROM)　81st　2020

ISSN： 2436-7613
FMO-DPD連携による脂質ナノ粒子,ペプチドのマルチスケールシミュレーション

奥脇弘次, 西田瑠花, 氏家かれん, 土屋裕大朗, 望月祐志, 望月祐志, 福澤薫, 福澤薫, 福澤薫, 米持悦生, 田中浩揮, 秋田英万

分子シミュレーション討論会講演要旨集　34th (CD-ROM)　2020
Structural analysis of nucleic acid-encapsulated nanocarriers composed of self-degrading lipid-like substances

米持悦生, 古石誉之, 田中浩揮, 秋田英万

KEK Progress Report (Web)　(2020-6)　2020

ISSN： 1344-6320
ナノ粒子のリンパシステム内動態におけるサイズ・表面電荷の影響

五味昌樹, 櫻井遊, 田中浩揮, 三浦尚也, 秋田新介, 山路佳久, 三川信之, 秋田英万

日本DDS学会学術集会プログラム予稿集　35回　144-144　2019/06
Publisher: 日本DDS学会
ナノ粒子物性がリンパシステム内動態に及ぼす影響リンパ流改変モデルマウスを用いた検証

五味昌樹, 櫻井遊, 田中浩揮, 三浦尚也, 秋田新介, 山路佳久, 三川信之, 秋田英万

日本薬学会年会要旨集　139年会　(4)　61-61　2019/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
CYP3Aヒト化マウスを用いたヒトCYP3A特異的な薬物間相互作用の予測

峰岸元気, 山崎由貴, 奥谷風香, 秋田英万, 香月康宏, 香月康宏, 小林カオル

日本薬学会年会要旨集(CD-ROM)　139th　ROMBUNNO.22O‐pm06　2019
LC‐MS/MSを用いたマウスin vivoにおけるtriazolam代謝物の分析

峰岸元気, 香月康宏, 香月康宏, 押村光雄, 秋田英万, 小林カオル

JSBMS Letters　43　(Supplement)　109　2018/08/25

ISSN： 1881-5464
P-糖タンパク質ヒト化マウスにおける薬物の中枢移行性の解析

山崎由貴, 小林カオル, 奥谷風香, 峰岸元気, 梶谷尚世, 香月加奈子, 阿部智志, 嵩原昇子, 伊藤慎悟, 緒方星陵, 大槻純男, 秋田英万, 千葉寛, 押村光雄, 香月康宏, 香月康宏

トランスポーター研究会年会抄録集　13th　2018
新規P糖タンパク質ヒト化マウスにおける薬物の中枢移行,精巣移行および腸管吸収の解析

奥谷風香, 小林カオル, 山崎由貴, 峰岸元気, 阿部智志, 梶谷尚世, 香月加奈子, 嵩原昇子, 伊藤慎悟, 上村立記, 緒方星陵, 大槻純男, 尾崎充彦, 秋田英万, 千葉寛, 押村光雄, 香月康宏, 香月康宏

日本薬学会年会要旨集(CD-ROM)　138th　ROMBUNNO.28PA‐am076S　2018
Nanotechnology for the control of the trafficking in the whole body and intracellular space based on an intracellular environment-responsive lipid-like material

49　(12)　597-601　2017/10
Publisher: ニューサイエンス社
ISSN： 1346-7557
Nanotechnology based on an intracellular environment-responsive lipid-like material (ssPalm)

262　(2)　131-136　2017/07/08
Publisher: 医歯薬出版
ISSN： 0039-2359
三次元培養ヒト肝がん由来細胞における核内受容体CARの発現,局在および薬剤応答性

東郁子, 横堀紘祐, 千葉寛, 秋田英万, 小林カオル

HAB研究機構学術年会プログラム・要旨集　24th　63　2017/06/01
A NEW IMMORTALIZED HUMAN LIVER SINUSOIDAL ENDOTHELIAL CELL LINE: ESTABLISHMENT, CHARACTERIZATION, AND APPLICATION POTENTIAL TO DEVELOPMENT OF IN VITRO HUMAN LIVER MODELS

Meiyan Zhu, Tomomi Furihata, Akira Koibuchi, Hideyuki Ide, Hanae Morio, Minaka Shibuya, Atsuko Kamiichi, Hidetaka Akita, Kan Chiba

DRUG METABOLISM AND PHARMACOKINETICS　32　(1)　S70-S70　2017/01

ISSN： 1347-4367

eISSN： 1880-0920
エステル型プロドラッグ消化管吸収評価能の向上に向けたヒト小腸型CES発現Caco‐2細胞の樹立

石崎裕馬, 降幡知巳, 降幡知巳, 大浦華代子, 今井輝子, 細川正清, 秋田英万, 千葉寛

日本薬学会年会要旨集(CD-ROM)　137th　ROMBUNNO.25I‐pm08S　2017
薬物中枢移行評価に有用な新規P‐糖タンパク質ヒト化マウスの作製

山崎由貴, 小林カオル, 阿部智志, 梶谷尚世, 香月加奈子, 嵩原昇子, 奥谷風香, 峰岸元気, 伊藤慎悟, 緒方星陵, 大槻純男, 秋田英万, 千葉寛, 押村光雄, 香月康宏, 香月康宏

日本薬剤学会年会講演要旨集(Web)　32nd　ROMBUNNO.11‐4‐19 (WEB ONLY)　2017
Cyp3aノックアウトマウスを用いたCYP3A特異的なトリアゾラム代謝物の同定

峰岸元気, 山崎由貴, 奥谷風香, 秋田英万, 嵩原昇子, 押村光雄, 香月康宏, 香月康宏, 小林カオル

日本生化学会大会(Web)　90th　ROMBUNNO.2LBA‐097 (WEB ONLY)　2017
A Lys-Ala-Leu-Ala (KALA) repeated peptide modification in DNA nanoparticles of DOPE/CHEMS, follows GPCR mediated transgene expression in dendritic cell

Sharif Mohammad Shaheen, Hidetaka Akita, Hideyoshi Harashima

CANCER RESEARCH　75　2015/07

DOI： 10.1158/1538-7445.PANCA2014-B64 　

ISSN： 0008-5472

eISSN： 1538-7445
Nano DDS Technologies for the Gene and siRNA Delivery Based on the Intracellular Environment-responsive Lipid like Materials

15　(3)　115-122,105　2015/03
Publisher: 日本油化学会
ISSN： 1345-8949
Drug Delivery System Based on MEND

34　(10)　956-961　2015
Publisher: 学研メディカル秀潤社 ; 1982-
ISSN： 0287-3796
B型肝炎ウイルスの持続感染を再現する効率的な培養細胞評価系の開発に関する研究自然免疫認識機構の制御によるHBV複製への影響

高岡晃教, 佐藤精一, LI Kai, 亀山武志, 林隆也, 原島秀吉, 秋田英万, 櫻井遊, 小原道法, 脇田隆字, 渡士幸一

B型肝炎ウイルスの持続感染を再現する効率的な培養細胞評価系の開発に関する研究平成26年度総括・分担研究報告書　56‐61　2015
【先端的医薬品等におけるレギュラトリーサイエンス研究の現状・課題】ナノテクノロジーを基盤とした医薬品のレギュラトリーサイエンス研究への取り組み

原島秀吉, 秋田英万, 加藤くみ子, 石井武彦, 松村保広, 片岡一則

Drug Delivery System　29　(3)　217-225　2014/07
Publisher: 日本DDS学会
ISSN： 0913-5006
血管内皮バリア突破型薬物送達戦略の新展開

秋田英万

上原記念生命科学財団研究報告集　28　1-5　2014
Publisher: 上原記念生命科学財団
B型肝炎ウイルスの持続感染を再現する効率的な培養細胞評価系の開発に関する研究自然免疫認識機構の制御によるHBV複製への影響

高岡晃教, 佐藤精一, LI Kai, 亀山武志, 林隆也, 原島秀吉, 秋田英万, 櫻井遊

B型肝炎ウイルスの持続感染を再現する効率的な培養細胞評価系の開発に関する研究平成25年度総括・分担研究報告書　55‐59　2014
Development of gene delivery carrier based on the kinetic analysis of the intracellular trafficking of the nanoparticle : the 5th Young Researcher's Award in the 29th Annual Meeting of the Japan Society of Drug Delivery system

Drug delivery system : DDS : official journal of the Japan Society of Drug Delivery System　28　(4)　355-362　2013/09
Publisher: 日本DDS学会 ; 2000-
ISSN： 0913-5006
ApoEを用いたsiRNAキャリアの脳血管内皮細胞における有用性評価

田丸みな, 秋田英万, 梶本和昭, 佐藤悠介, 畠山浩人, 原島秀吉

日本DDS学会学術集会プログラム予稿集　29th　2013
腫瘍血管内皮細胞へのin vivo siRNA導入法の確立

櫻井遊, 畠山浩人, 佐藤悠介, 兵藤守, 秋田英万, 大賀則孝, 大賀則孝, 樋田京子, 原島秀吉

日本血管生物医学会学術集会プログラム・抄録集　21st　2013
樹状細胞に対するKALA修飾MENDの免疫活性化能評価

三浦尚也, 秋田英万, 石井聡一郎, シャリフシャヒーン, 原島秀吉

日本薬学会年会要旨集(CD-ROM)　133rd　2013
樹状細胞に対するKALA修飾MENDの免疫活性化能評価

三浦尚也, 秋田英万, 石井聡一郎, SHARIF Shaheen, 原島秀吉

医療薬学フォーラム講演要旨集　21st　2013

ISSN： 1348-0863
樹状細胞に対するKALA修飾MENDの免疫活性化能評価

三浦尚也, 秋田英万, 石井聡一郎, シャヒーンシャリフ, 中村孝司, 原島秀吉

日本DDS学会学術集会プログラム予稿集　29th　2013
High throughput purification devices for in vivo applications of gene-delivery multifunctional envelope-type nanodevices Peer-reviewed

Noritada Kaji, Daisuke Shigenaka, Masami Ukawa, Manabu Tokeshi, Hidetaka Akita, Hideyoshi Harashima, Yoshinobu Baba

17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013　2　1170-1172　2013
遺伝子治療用ナノ構造体精製のためのフリーフロー電気泳動マイクロデバイスの開発

重中大輔, 鵜川真美, 加地範匡, 渡慶次学, 秋田英万, 原島秀吉, 馬場嘉信

日本化学会講演予稿集　93rd　(2)　2013

ISSN： 0285-7626
A Potential non-viral vector to transfect dendritic cell and thereby MHC-Class I antigen presentation might be a potential use in carcinoma

S. Shaheen, H. Akita, I. Souichirou, N. Miura, H. Harashima

CANCER RESEARCH　72　2012/12

DOI： 10.1158/0008-5472.SABCS12-P4-04-08 　

ISSN： 0008-5472

eISSN： 1538-7445
High Throughput Purification Device for Gene Delivery Multifunctional Envelope-Type Nanodevice Peer-reviewed

Daisuke Shigenaka, Masami Ukawa, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Hidetaka Akita, Hideyoshi Harashima, Yoshinobu Baba

Micro Total Analysis Systems 2012　1546-1548-1548　2012/10
Design of Artificial Gene Carrier Based on the Quantitative Comparison of Intracellular Trafficking with Adenovirus

AKITA Hidetaka, HARASHIMA Hideyoshi

61　(8)　533-535　2012/08/01

ISSN： 0454-1138
学会報告記第28回日本DDS学会学術集会を振り返って

原島秀吉, 秋田英万

Drug delivery system : DDS : official Journal of the Japan Society of Drug Delivery System　27　(3)　218-220　2012/07
Publisher: 日本DDS学会
ISSN： 0913-5006
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

DOI： 10.1021/ar200254s 　

ISSN： 0001-4842
肺血管内皮細胞標的型siRNAキャリアの標的メカニズムとその抗腫瘍効果

楠本憲司, 秋田英万, 松本有, 野本貴大, 片岡一則, 原島秀吉

日本DDS学会学術集会プログラム予稿集　28回　119-119　2012/06
Publisher: 日本DDS学会
マイクロ流体チップによる多機能性エンベロープ型ナノ構造体の作製

加地範匡, 北添雄眞, 重中大輔, 小暮健太朗, 秋田英万, 原島秀吉, 渡慶次学, 馬場嘉信

日本DDS学会学術集会プログラム予稿集　28回　182-182　2012/06
Publisher: 日本DDS学会
siRNA delivery with multifunctional envelope-type nano device

63　(2)　113-119　2012/03
Publisher: 金原一郎記念医学医療振興財団
ISSN： 0370-9531
Stearylated INF7 peptide enhances endosomal escape and gene expression of PEGylated nanoparticles both in vitro and in vivo. International-journal

Ayman El-Sayed, Tomoya Masuda, Hidetaka Akita, Hideyoshi Harashima

Journal of pharmaceutical sciences　101　(2)　879-82　2012/02

DOI： 10.1002/jps.22807 　

ISSN： 0022-3549

eISSN： 1520-6017
siRNA搭載pH応答性MENDの構築とin vivoへの応用

佐藤悠介, 畠山浩人, 櫻井遊, 兵藤守, 秋田英万, 原島秀吉

日本薬学会年会要旨集　132nd　(1)　2012

ISSN： 0918-9823
pH応答性脂質様物質から形成される遺伝子封入中性ナノ粒子の機能および細胞内動態解析

秋田英万, 石破諒平, 畠山浩人, 佐藤悠介, 田中浩揮, 丹下耕太, 新井将也, 久保和弘, 原島秀吉

日本DDS学会学術集会プログラム予稿集　28th　2012
pH応答性カチオン性脂質を用いた腫瘍組織へのsiRNAデリバリーシステムの構築

櫻井遊, 畠山浩人, 佐藤悠介, 兵藤守, 秋田英万, 原島秀吉

日本薬剤学会年会講演要旨集(CD-ROM)　27th　2012
pH応答性脂質様物質を用いた遺伝子封入中性ナノ粒子の創製

石破諒平, 秋田英万, 畠山浩人, 佐藤悠介, 田中浩揮, 丹下耕太, 新井将也, 久保和弘, 原島秀吉

日本薬剤学会年会講演要旨集(CD-ROM)　27th　2012
siRNA搭載pH応答性MENDのin vivoにおける機能評価

佐藤悠介, 畠山浩人, 兵藤守, 秋田英万, 原島秀吉

日本薬剤学会年会講演要旨集(CD-ROM)　27th　2012
フリーフロー電気泳動法を用いた遺伝子治療用ナノデバイスの精製

重中大輔, 鵜川真美, 加地範匡, 岡本行広, 渡慶次学, 秋田英万, 原島秀吉, 馬場嘉信

日本化学会講演予稿集　92nd　(2)　2012

ISSN： 0285-7626
A DNA MICROARRAY-BASED ANALYSIS OF THE HOST RESPONSE TO A NONVIRAL GENE CARRIER: EXPLORING CPG-DEPENDENT/INDEPENDENT MECHANISMS

H. Hatakeyama, M. Yamamoto, Y. Hayashi, K. Kajimoto, H. Akita, N. Kaji, Y. Baba, H. Harashima

NUCLEIC ACID THERAPEUTICS　21　(5)　A34-A34　2011/10

ISSN： 2159-3337

eISSN： 2159-3345
IN VIVO SIRNA DELIVERY WITH PH- SENSITIVE LIPOSOME

Yusuke Sato, Hiroto Hatakeyama, Yu Sakurai, Mamoru Hyodo, Hidetaka Akita, Hideyoshi Harashima

NUCLEIC ACID THERAPEUTICS　21　(5)　A41-A42　2011/10

ISSN： 2159-3337
ENDOSOMAL ESCAPE OF LIPOSOMAL SIRNA VIA MEMBRANE FUSION AND IN VIVO DELIVERY TO TUMOR

Yu Sakurai, Hiroto Hatakeyama, Yusuke Sato, Hidetaka Akita, Hideyoshi Harashima

NUCLEIC ACID THERAPEUTICS　21　(5)　A41-A41　2011/10

ISSN： 2159-3337
KALA-modified multi-layered nanoparticles as gene carriers for MHC class-I mediated antigen presentation for a DNA vaccine International-journal

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

BIOMATERIALS　32　(26)　6342-6350　2011/09

DOI： 10.1016/j.biomaterials.2011.05.014 　

ISSN： 0142-9612
Dual imaging of mRNA and protein production: an investigation of the mechanism of heterogeneity in cationic lipid-mediated transgene expression. International-journal

Hidetaka Akita, Yuko Umetsu, Dai Kurihara, Hideyoshi Harashima

International journal of pharmaceutics　415　(1-2)　218-20　2011/08/30

DOI： 10.1016/j.ijpharm.2011.05.051 　

ISSN： 0378-5173
Functional improvement of an IRQ-PEG-MEND for delivering genes to the lung. International-journal

Taichi Ishitsuka, Hidetaka Akita, Hideyoshi Harashima

Journal of controlled release : official journal of the Controlled Release Society　154　(1)　77-83　2011/08/25

DOI： 10.1016/j.jconrel.2011.05.012 　

ISSN： 0168-3659
Endosomal escape and the knockdown efficiency of liposomal-siRNA by the fusogenic peptide shGALA. International-journal

Yu Sakurai, Hiroto Hatakeyama, Yusuke Sato, Hidetaka Akita, Kentaro Takayama, Sachiko Kobayashi, Shiroh Futaki, Hideyoshi Harashima

Biomaterials　32　(24)　5733-42　2011/08

DOI： 10.1016/j.biomaterials.2011.04.047 　

ISSN： 0142-9612
Improving in vivo hepatic transfection activity by controlling intracellular trafficking: the function of GALA and maltotriose. International-journal

Hidetaka Akita, Tomoya Masuda, Takashi Nishio, Kenichi Niikura, Kuniharu Ijiro, Hideyoshi Harashima

Molecular pharmaceutics　8　(4)　1436-42　2011/08/01

DOI： 10.1021/mp200189s 　

ISSN： 1543-8384
A DNA microarray-based analysis of the host response to a nonviral gene carrier: a strategy for improving the immune response. International-journal

Hiroto Hatakeyama, Erika Ito, Momoko Yamamoto, Hidetaka Akita, Yasuhiro Hayashi, Kazuaki Kajimoto, Noritada Kaji, Yoshinobu Baba, Hideyoshi Harashima

Molecular therapy : the journal of the American Society of Gene Therapy　19　(8)　1487-98　2011/08

DOI： 10.1038/mt.2011.24 　

ISSN： 1525-0016

eISSN： 1525-0024
Systemic delivery of siRNA to tumors using a lipid nanoparticle containing a tumor-specific cleavable PEG-lipid. International-journal

Hiroto Hatakeyama, Hidetaka Akita, Erika Ito, Yasuhiro Hayashi, Motoi Oishi, Yukio Nagasaki, Radostin Danev, Kuniaki Nagayama, Noritada Kaji, Hiroshi Kikuchi, Yoshinobu Baba, Hideyoshi Harashima

Biomaterials　32　(18)　4306-16　2011/06

DOI： 10.1016/j.biomaterials.2011.02.045 　

ISSN： 0142-9612
Quantitative analysis of condensation/decondensation status of pDNA in the nuclear sub-domains by QD-FRET International-journal

Sharif M. Shaheen, Hidetaka Akita, Atsushi Yamashita, Ryo Katoono, Nobuhiko Yui, Vasudevanpillai Biju, Mitsuru Ishikawa, Hideyoshi Harashima

NUCLEIC ACIDS RESEARCH　39　(7)　E48-U108　2011/04

DOI： 10.1093/nar/gkq1327 　

ISSN： 0305-1048
pH応答性脂質様物質を用いた遺伝子封入中性ナノ粒子の創製

石破諒平, 秋田英万, 畠山浩人, 佐藤悠介, 田中浩揮, 丹下耕太, 新井将也, 久保和弘, 原島秀吉

生体膜と薬物の相互作用シンポジウム講演要旨集　33rd　2011

ISSN： 0919-2131
pH変化に応答するsiRNA封入MENDの構築

佐藤悠介, 畠山浩人, 兵藤守, 秋田英万, 原島秀吉

日本薬剤学会年会講演要旨集　26th　2011
マウス肝プライマリー細胞における多機能性エンベロープ型ナノ構造体(MEND)のMPCポリマー修飾による遺伝子発現活性上昇メカニズムの解明

鵜川真実, 秋田英万, 増田智也, 林泰弘, 金野智浩, 石原一彦, 原島秀吉

Drug Deliv Syst　25　(3)　293-293　2010/05/28
Publisher: 日本DDS学会
ISSN： 0913-5006
Nanoparticles for ex vivo siRNA delivery to dendritic cells for cancer vaccines: programmed endosomal escape and dissociation. International-journal

Hidetaka Akita, Kentaro Kogure, Rumiko Moriguchi, Yoshio Nakamura, Tomoko Higashi, Takashi Nakamura, Satoshi Serada, Minoru Fujimoto, Tetsuji Naka, Shiroh Futaki, Hideyoshi Harashima

Journal of controlled release : official journal of the Controlled Release Society　143　(3)　311-7　2010/05/10

DOI： 10.1016/j.jconrel.2010.01.012 　

ISSN： 0168-3659
Particle tracking of intracellular trafficking of octaarginine-modified liposomes: a comparative study with adenovirus. International-journal

Hidetaka Akita, Kaoru Enoto, Tomoya Masuda, Hiroyuki Mizuguchi, Tomomi Tani, Hideyoshi Harashima

Molecular therapy : the journal of the American Society of Gene Therapy　18　(5)　955-64　2010/05

DOI： 10.1038/mt.2010.33 　

ISSN： 1525-0016
MPCポリマー修飾GALA‐MENDによる肝臓での遺伝子発現上昇と免疫応答の軽減

林泰弘, 山内順, 秋田英万, 増田智也, 鵜川真実, 金野智浩, 石原一彦, 原島秀吉

薬剤学　70　(Supplement)　184-184　2010/04/20
Publisher: (公社)日本薬剤学会
ISSN： 0372-7629
多機能性エンベロープ型ナノ構造体のMPCポリマー修飾による遺伝子発現活性上昇メカニズムの解明

鵜川真実, 秋田英万, 増田智也, 林泰弘, 金野智浩, 石原一彦, 原島秀吉

日本薬学会年会要旨集　130th　(4)　164-164　2010/03/05
Publisher: (公社)日本薬学会
ISSN： 0918-9823
遺伝子ベクターへのPEG修飾が及ぼすサイトカイン産生への影響

畠山浩人, 伊東恵里佳, 林泰弘, 秋田英万, 梶本和昭, 加地範匡, 馬場嘉信, 原島秀吉

日本薬学会年会要旨集　130年会　(4)　165-165　2010/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
Intracellular fate of octaarginine-modified liposomes in polarized MDCK cells. International-journal

Takahiro Fujiwara, Hidetaka Akita, Hideyoshi Harashima

International journal of pharmaceutics　386　(1-2)　122-30　2010/02/15

DOI： 10.1016/j.ijpharm.2009.11.005 　

ISSN： 0378-5173
多機能性エンベロープ型ナノ構造体のMPCポリマー修飾による肝実質細胞における遺伝子発現活性上昇のメカニズムの解明

鵜川真実, 秋田英万, 増田智也, 林泰弘, 金野智浩, 石原一彦, 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.13　2010
siRNA delivery by multifunctional envelope-type nano device (MEND)

Hidetaka Akita, Hiroto Hatakeyama, Hideyoshi Harashima

Drug Delivery System　25　(6)　590-597　2010
Publisher: Japan Society of Drug Delivery System
DOI： 10.2745/dds.25.590 　

ISSN： 0913-5006 1881-2732
THE FUNCTION OF A NOVEL IRQ LIGAND-MODIFIED CARRIER FOR AN EFFICIENT CYTOSOLIC DELIVERY OF SIRNA

Diky Mudhakir, Hidetaka Akita, Erdal Tan, Hideyoshi Harashima

JOURNAL OF GENE MEDICINE　11　(12)　1183-1183　2009/12

ISSN： 1099-498X
A pH-sensitive fusogenic peptide facilitates endosomal escape and greatly enhances the gene silencing of siRNA-containing nanoparticles in vitro and in vivo. International-journal

Hiroto Hatakeyama, Erika Ito, Hidetaka Akita, Motoi Oishi, Yukio Nagasaki, Shiroh Futaki, Hideyoshi Harashima

Journal of controlled release : official journal of the Controlled Release Society　139　(2)　127-32　2009/10/15

DOI： 10.1016/j.jconrel.2009.06.008 　

ISSN： 0168-3659
Envelope-type lipid nanoparticles incorporating a short PEG-lipid conjugate for improved control of intracellular trafficking and transgene transcription. International-journal

Tomoya Masuda, Hidetaka Akita, Kenichi Niikura, Takashi Nishio, Masami Ukawa, Kaoru Enoto, Radostin Danev, Kuniaki Nagayama, Kuniharu Ijiro, Hideyoshi Harashima

Biomaterials　30　(27)　4806-14　2009/09

DOI： 10.1016/j.biomaterials.2009.05.036 　

ISSN： 0142-9612
Enhanced gene expression by a novel stearylated INF7 peptide derivative through fusion independent endosomal escape. International-journal

Ayman El-Sayed, Tomoya Masuda, Ikramy Khalil, Hidetaka Akita, Hideyoshi Harashima

Journal of controlled release : official journal of the Controlled Release Society　138　(2)　160-7　2009/09/01

DOI： 10.1016/j.jconrel.2009.05.018 　

ISSN： 0168-3659

eISSN： 1873-4995
Effect of polyethyleneglycol spacer on the binding properties of nuclear localization signal-modified liposomes to isolated nucleus.

Dai Kurihara, Hidetaka Akita, Asako Kudo, Tomoya Masuda, Shiroh Futaki, Hideyoshi Harashima

Biological & pharmaceutical bulletin　32　(7)　1303-6　2009/07

DOI： 10.1248/bpb.32.1303 　

ISSN： 0918-6158
多機能性エンベロープ型ナノ構造体を用いた細胞内動態制御 : in vivo 適応型ナノキャリアの実現に向けて

秋田英万

Drug delivery system　24　(3)　238-238　2009/06/09

ISSN： 0913-5006
Novel lipidated sorbitol-based molecular transporters for non-viral gene delivery. International-journal

Tomoko Higashi, Ikramy A Khalil, Kaustabh K Maiti, Woo Sirl Lee, Hidetaka Akita, Hideyoshi Harashima, Sung-Kee Chung

Journal of controlled release : official journal of the Controlled Release Society　136　(2)　140-7　2009/06/05

DOI： 10.1016/j.jconrel.2009.01.024 　

ISSN： 0168-3659

eISSN： 1873-4995
PEG修飾による人工遺伝子ベクターの安全性向上のメカニズムの解析

畠山浩人, 伊東恵里佳, 林泰弘, 秋田英万, 梶本和昭, 加地範匡, 馬場嘉信, 原島秀吉

Drug Delivery System　24　(3)　325-325　2009/06
Publisher: 日本DDS学会
ISSN： 0913-5006

eISSN： 1881-2732
Development of Multifunctional Envelope-Type Nano-Device (MEND) for Hepatic Gene Transfer Via i.v. Administration: Impact for the Modification with Devices for Intracellular Trafficking

Hidetaka Akita, Tomoya Masuda, Kinichi Niikura, Takashi Nishio, Kuniharu Ijiro, Hideyoshi Harashima

MOLECULAR THERAPY　17　S54-S54　2009/05

ISSN： 1525-0016
Analysis of Microtubule-Dependent Transport in Lipid-Encapsulated DNA Nano-Carrier by Real-Time Imaging: Comparison with Adenovirus

Kaoru Enoto, Hidetaka Akita, Tomoya Masuda, Hiroyuki Mizuguchi, Hideyoshi Harashima

MOLECULAR THERAPY　17　S51-S51　2009/05

ISSN： 1525-0016
Efficient short interference RNA delivery to tumor cells using a combination of octaarginine, GALA and tumor-specific, cleavable polyethylene glycol system.

Yu Sakurai, Hiroto Hatakeyama, Hidetaka Akita, Motoi Oishi, Yukio Nagasaki, Shiro Futaki, Hideyoshi Harashima

Biological & pharmaceutical bulletin　32　(5)　928-32　2009/05

DOI： 10.1248/bpb.32.928 　

ISSN： 0918-6158
Multi-layered nanoparticles for penetrating the endosome and nuclear membrane via a step-wise membrane fusion process. International-journal

Hidetaka Akita, Asako Kudo, Arisa Minoura, Masaya Yamaguti, Ikramy A Khalil, Rumiko Moriguchi, Tomoya Masuda, Radostin Danev, Kuniaki Nagayama, Kentaro Kogure, Hideyoshi Harashima

Biomaterials　30　(15)　2940-9　2009/05

DOI： 10.1016/j.biomaterials.2009.02.009 　

ISSN： 0142-9612
MPCコート多機能性エンベロープ型ナノ構造体による静脈内投与型in vivo肝臓デリバリーシステムの構築

秋田英万, 増田智也, 金野智浩, 石原一彦, 原島秀吉

薬剤学　69　(Supplement)　101-101　2009/04/30
Publisher: (公社)日本薬剤学会
ISSN： 0372-7629
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
Oligosaccharide-mediated nuclear transport of nanoparticles. International-journal

Kenichi Niikura, Shota Sekiguchi, Takashi Nishio, Tomoya Masuda, Hidetaka Akita, Yasutaka Matsuo, Kentaro Kogure, Hideyoshi Harashima, Kuniharu Ijiro

Chembiochem : a European journal of chemical biology　9　(16)　2623-7　2008/11/03

DOI： 10.1002/cbic.200800464 　

ISSN： 1439-4227
Photosensitized breakage and damage of DNA by CdSe-ZnS quantum dots. International-journal

AbdulAziz Anas, Hidetaka Akita, Hideyoshi Harashima, Tamitake Itoh, Mitsuru Ishikawa, Vasudevanpillai Biju

The journal of physical chemistry. B　112　(32)　10005-11　2008/08/14

DOI： 10.1021/jp8018606 　

ISSN： 1520-6106
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

ISSN： 0913-5006
Development of multifunctional envelope-type nano device for gene delivery

59　(4)　263-268　2008/04
Publisher: 化学工業社
ISSN： 0451-2014
Multifunctional envelope-type nano device (MEND) as a non-viral gene delivery system. International-journal

Kentaro Kogure, Hidetaka Akita, Yuma Yamada, Hideyoshi Harashima

Advanced drug delivery reviews　60　(4-5)　559-71　2008/03/01

DOI： 10.1016/j.addr.2007.10.007 　

ISSN： 0169-409X
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

DOI： 10.1016/j.bbamem.2007.11.002 　

ISSN： 0005-2736
Development of lipid particles targeted via sugar-lipid conjugates as novel nuclear gene delivery system. International-journal

Tomoya Masuda, Hidetaka Akita, Takashi Nishio, Kenichi Niikura, Kentaro Kogure, Kuniharu Ijiro, Hideyoshi Harashima

Biomaterials　29　(6)　709-23　2008/02

DOI： 10.1016/j.biomaterials.2007.09.039 　

ISSN： 0142-9612
A novel IRQ ligand-modified nano-carrier targeted to a unique pathway of caveolar endocytic pathway. International-journal

Diky Mudhakir, Hidetaka Akita, Erdal Tan, Hideyoshi Harashima

Journal of controlled release : official journal of the Controlled Release Society　125　(2)　164-73　2008/01/22

DOI： 10.1016/j.jconrel.2007.10.020 　

ISSN： 0168-3659

eISSN： 1873-4995
Dynamic Cellular Stress Sensing using Sugar Displaying CdTe Quantum Dots

NISHIO Takashi, NIIKURA Kenichi, MATSUO Yasutaka, AKITA Hidetaka, KOGURE Kentaro, HARASHIMA Hideyoshi, IJIRO Kuniharu, IJIRO Kuniharu

Abstr RIES Hokudai Symp　9th　110-111　2008
Supramolecular control of polyplex dissociation and cell transfection: Efficacy of amino groups and threading cyclodextrins in biocleavable polyrotaxanes

YAMASHITA Atsushi, KANDA Daizo, KATOONO Ryo, YUI Nobuhiko, OOYA Tooru, OOYA Tooru, MARUYAMA Atsushi, AKITA Hidetaka, KOGURE Kentaro, HARASHIMA Hideyoshi

J Control Releas　131　(2)　137-144　2008

DOI： 10.1016/j.jconrel.2008.07.011 　

ISSN： 0168-3659
MITO-Porter: a liposome-based carrier system for delivery of macromolecules into mitochondria via membrane fusion.

YAMADA Yuma, AKITA Hidetaka, KAMIYA Hiroyuki, KOGURE Kentaro, YAMAMOTO Takenori, SHINOHARA Yasuo, YAMASHITA Kikuji, KOBAYASHI Hideo, KIKUCHI Hiroshi, HARASHIMA Hideyoshi

Biochimica et Biophysica Acta　1778　(2)　423-432　2008

DOI： 10.1016/j.bbamem.2007.11.002 　

ISSN： 0005-2728
Multifunctional envelope-type nano device for non-viral gene delivery: concept and application of Programmed Packaging. International-journal

Kentaro Kogure, Hidetaka Akita, Hideyoshi Harashima

Journal of controlled release : official journal of the Controlled Release Society　122　(3)　246-51　2007/10/08

DOI： 10.1016/j.jconrel.2007.06.018 　

ISSN： 0168-3659

eISSN： 1873-4995
[Development of a novel systemic gene delivery system for cancer therapy with a tumor-specific cleavable PEG-lipid].

Hiroto Hatakeyama, Hidetaka Akita, Kentaro Kogure, Hideyoshi Harashima

Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan　127　(10)　1549-56　2007/10

ISSN： 0031-6903
Tumor targeting of doxorubicin by anti-MT1-MMP antibody-modified PEG liposomes. International-journal

Hiroto Hatakeyama, Hidetaka Akita, Emi Ishida, Koichi Hashimoto, Hideo Kobayashi, Takanori Aoki, Junko Yasuda, Kenichi Obata, Hiroshi Kikuchi, Tatsuhiro Ishida, Hiroshi Kiwada, Hideyoshi Harashima

International journal of pharmaceutics　342　(1-2)　194-200　2007/09/05

DOI： 10.1016/j.ijpharm.2007.04.037 　

ISSN： 0378-5173
遺伝子の細胞内動態に化学でせまる

秋田英万, 原島秀吉

現代化学　(437)　21-26　2007/08
Publisher: 東京化学同人
ISSN： 0386-961X
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
Publisher: 日本DDS学会
ISSN： 0913-5006
MEND(multifunctional envelope-type nano device)による腫瘍への遺伝子デリバリー (特集抗がん剤のDrug Delivery System)

畠山浩人, 秋田英万, 原島秀吉

Mebio oncology　4　(2)　27-35　2007/06
Publisher: メジカルビュー社
ISSN： 1349-2179
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

ISSN： 0913-5006
血管内皮細胞標的化ペプチド搭載リポソームの細胞内取り込み機構の解析

秋田英万, ムダキュルディッキー, タンエルダル, 原島秀吉

薬剤学 = Journal of Pharmaceutical Science and Technology, Japan　67　359-359　2007/05/10

ISSN： 0372-7629
【細胞内情報デリバリーの新手法】多機能性エンベロープ型ナノ構造体による遺伝子デリバリー

秋田英万, 小暮健太朗, 原島秀吉

再生医療　6　(2)　161,122-167,122　2007/05
Publisher: (株)メディカルレビュー社
ISSN： 1347-7919
In vitro efficacy of a sterically stabilized immunoliposomes targeted to membrane type 1 matrix metalloproteinase (MT1-MMP).

Kazutaka Atobe, Tatsuhiro Ishida, Emi Ishida, Kouichi Hashimoto, Hideo Kobayashi, Jyunko Yasuda, Takanori Aoki, Ken-Ichi Obata, Hiroshi Kikuchi, Hidetaka Akita, Tomohiro Asai, Hideyoshi Harashima, Naoto Oku, Hiroshi Kiwada

Biological & pharmaceutical bulletin　30　(5)　972-8　2007/05

DOI： 10.1248/bpb.30.972 　

ISSN： 0918-6158
Octaarginine-modified multifunctional envelope-type nanoparticles for gene delivery

I. A. Khalil, K. Kogure, S. Futaki, S. Hama, H. Akita, M. Ueno, H. Kishida, M. Kudoh, Y. Mishina, K. Kataoka, M. Yamada, H. Harashima

GENE THERAPY　14　(8)　682-689　2007/04

DOI： 10.1038/sj.gt.3302910 　

ISSN： 0969-7128
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

ISSN： 0918-9823
Accumulation of O-GlcNAc-displaying CdTe quantum dots in cells in the presence of ATP. International-journal

Kenichi Niikura, Takashi Nishio, Hidetaka Akita, Yasutaka Matsuo, Ryosuke Kamitani, Kentaro Kogure, Hideyoshi Harashima, Kuniharu Ijiro

Chembiochem : a European journal of chemical biology　8　(4)　379-84　2007/03/05

DOI： 10.1002/cbic.200600496 　

ISSN： 1439-4227
Cell cycle dependent transcription, a determinant factor of heterogeneity in cationic lipid-mediated transgene expression. International-journal

Hidetaka Akita, Rie Ito, Hiroyuki Kamiya, Kentaro Kogure, Hideyoshi Harashima

The journal of gene medicine　9　(3)　197-207　2007/03

DOI： 10.1002/jgm.1010 　

ISSN： 1099-498X

eISSN： 1521-2254
Mitochondrial drug delivery and mitochondrial disease therapy - An approach to liposome-based delivery targeted to mitochondria International-journal

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

MITOCHONDRION　7　(1-2)　63-71　2007/02

DOI： 10.1016/j.mito.2006.12.003 　

ISSN： 1567-7249
Novel approach for the nuclear delivery of macromolecule via membrane fusion in non-dividing cell

Hidetaka Akita, Asako Kudo, Arisa Minoura, Kentaro Kogure, Hideyoshi Harashima

YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN　127　85-86　2007

ISSN： 0031-6903
Quantitative imaging of intracellular trafficking of siRNA vector

Tomoko Higashi, Yoshio Nakamura, Hidetaka Akita, Kentaro Kogure, Hideyoshi Harashima

YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN　127　171-172　2007

ISSN： 0031-6903
Development of multifunctional envelope-type nano-device (mend) based on the regulation of intracellular trafficking

Hidetaka Akita, Kentaro Kogure, Hideyoshi Harashima

Drug Delivery System　22　(2)　115-122　2007
Publisher: Japan Society of Drug Delivery System
DOI： 10.2745/dds.22.115 　

ISSN： 1881-2732 0913-5006
Quantitative and mechanism-based investigation of post-nuclear delivery events between adenovirus and lipoplex. International-journal

Susumu Hama, Hidetaka Akita, Shinya Iida, Hiroyuki Mizuguchi, Hideyoshi Harashima

Nucleic acids research　35　(5)　1533-43　2007

DOI： 10.1093/nar/gkl1165 　

ISSN： 0305-1048

eISSN： 1362-4962
Development of a novel systemic gene delivery system for cancer therapy with a tumor-specific cleavable PEG-lipid

H. Hatakeyama, H. Akita, K. Kogure, M. Oishi, Y. Nagasaki, Y. Kihira, M. Ueno, H. Kobayashi, H. Kikuchi, H. Harashima

GENE THERAPY　14　(1)　68-77　2007/01

DOI： 10.1038/sj.gt.3302843 　

ISSN： 0969-7128
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
Inhibition of bile acid transport across Na+/taurocholate cotransporting polypeptide (SLC10A1) and bile salt export pump (ABCB 11)-coexpressing LLC-PK1 cells by cholestasis-inducing drugs. International-journal

Sachiko Mita, Hiroshi Suzuki, Hidetaka Akita, Hisamitsu Hayashi, Reiko Onuki, Alan F Hofmann, Yuichi Sugiyama

Drug metabolism and disposition: the biological fate of chemicals　34　(9)　1575-81　2006/09

DOI： 10.1124/dmd.105.008748 　

ISSN： 0090-9556
人工ナノ遺伝子ベクターの細胞内動態制御 (特集ナノバイオロジー--ナノテクでバイオを変える)

秋田英万, 原島秀吉

細胞工学　25　(8)　899-904　2006/08
Publisher: 秀潤社
ISSN： 0287-3796
Impact of convective flow on the cellular uptake and transfection activity of lipoplex and adenovirus.

Takahiro Fujiwara, Hidetaka Akita, Katsuko Furukawa, Takashi Ushida, Hiroyuki Mizuguchi, Hideyoshi Harashima

Biological & pharmaceutical bulletin　29　(7)　1511-5　2006/07

DOI： 10.1248/bpb.29.1511 　

ISSN： 0918-6158
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

ISSN： 0913-5006
Cellular uptake and subsequent intracellular trafficking of R8-liposomes introduced at low temperature

Akitada Iwasa, Hidetaka Akita, Ikramy Khalil, Kentaro Kogure, Shiroh Futaki, Hideyoshi Harashima

BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES　1758　(6)　713-720　2006/06

DOI： 10.1016/j.bbamem.2006.04.015 　

ISSN： 0005-2736
Delivery of condensed DNA by liposomal non-viral gene delivery system into nucleus of dendritic cells.

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

Biological & pharmaceutical bulletin　29　(6)　1290-3　2006/06

DOI： 10.1248/bpb.29.1290 　

ISSN： 0918-6158

eISSN： 1347-5215
ミトコンドリアを標的としたDDSと疾患治療への応用 : ミトコンドリア融合性リポソーム、"MITO-Porter" を用いたアプローチ

山田勇麿, 秋田英万, 小暮健太朗, 紙谷浩之, 原島秀吉, 篠原康雄, 小林英夫, 菊池寛

Drug delivery system　21　(3)　275-275　2006/05/10

ISSN： 0913-5006
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開発

原島秀吉, 秋田英万, 小暮健太朗

Drug delivery system　21　(3)　254-254　2006/05/10

ISSN： 0913-5006
Mechanism of Difference in the Transcription Activity of Nucleus-Delivered Transgene Introduced by Adenovirus and Lipoplex

Hidetaka Akita, Susumu Hama, Hiroyuki Mizuguchi, Hideyoshi Harashima

MOLECULAR THERAPY　13　S199-S199　2006/05

ISSN： 1525-0016

eISSN： 1525-0024
Development of Nuclear Targeting System of Plasmid DNA; Impact of Sugar Display on the Surface of Lipid Envelope

Tomoya Masuda, Hidetaka Akita, Takashi Nishio, Kenichi Niikura, Asako Kudo, Kentaro Kogure, Akitada Iwasa, Kuniharu Ijiro, Hideyoshi Harashima

MOLECULAR THERAPY　13　S388-S388　2006/05

ISSN： 1525-0016

eISSN： 1525-0024
Development of Novel Systemic Gene Delivery System for Cancer Therapy with Tumor-Specifically Cleavable PEG-Lipid

Hiroto Hatakeyama, Hidetaka Akita, Kentaro Kogure, Motoi Oishi, Yukio Nagasaki, Hideo Kobayashi, Hiroshi Kikuchi, Hideyoshi Harashima

MOLECULAR THERAPY　13　S203-S203　2006/05

ISSN： 1525-0016

eISSN： 1525-0024
Impact of Convective Flow on the Cellular Uptake and Transfection Activity of Lipoplex and Adenovirus

Takahiro Fujiwara, Hidetaka Akita, Katsuko Furukawa, Takashi Ushida, Hideyoshi Harashima

MOLECULAR THERAPY　13　S128-S128　2006/05

ISSN： 1525-0016

eISSN： 1525-0024
Quantitative comparison of intracellular trafficking and nuclear transcription between adenoviral and lipoplex systems. International-journal

Susumu Hama, Hidetaka Akita, Rie Ito, Hiroyuki Mizuguchi, Takao Hayakawa, Hideyoshi Harashima

Molecular therapy : the journal of the American Society of Gene Therapy　13　(4)　786-94　2006/04

DOI： 10.1016/j.ymthe.2005.10.007 　

ISSN： 1525-0016
Biocleavable polyrotaxane-plasmid DNA polyplex for enhanced gene delivery. International-journal

Tooru Ooya, Hak Soo Choi, Atsushi Yamashita, Nobuhiko Yui, Yuko Sugaya, Arihiro Kano, Atsushi Maruyama, Hidetaka Akita, Rie Ito, Kentaro Kogure, Hideyoshi Harashima

Journal of the American Chemical Society　128　(12)　3852-3　2006/03/29

DOI： 10.1021/ja055868+ 　

ISSN： 0002-7863
Vectorial transport of unconjugated and conjugated bile salts by monolayers of LLC-PK1 cells doubly transfected with human NTCP and BSEP or with rat Ntcp and Bsep. International-journal

Sachiko Mita, Hiroshi Suzuki, Hidetaka Akita, Hisamitsu Hayashi, Reiko Onuki, Alan F Hofmann, Yuichi Sugiyama

American journal of physiology. Gastrointestinal and liver physiology　290　(3)　G550-6-G556　2006/03

DOI： 10.1152/ajpgi.00364.2005 　

ISSN： 0193-1857
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

ISSN： 0372-7629
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

ISSN： 0372-7629
Non-linear pharmacodynamics in a non-viral gene delivery system: positive non-linear relationship between dose and transfection efficiency. International-journal

Rumiko Moriguchi, Kentaro Kogure, Akitada Iwasa, Hidetaka Akita, Hideyoshi Harashima

Journal of controlled release : official journal of the Controlled Release Society　110　(3)　605-9　2006/02/21

DOI： 10.1016/j.jconrel.2005.10.021 　

ISSN： 0168-3659

eISSN： 1873-4995
【ナノテクノロジーと医療基礎・臨床研究と展望】ナノテクノロジーによる遺伝子治療エンベロープ型ナノ構造体による遺伝子デリバリー

小暮健太朗, 秋田英万, 原島秀吉

日本臨床　64　(2)　258-262　2006/02
Publisher: (株)日本臨床社
ISSN： 0047-1852
Improved cell viability of linear polyethylenimine through gamma-cyclodextrin inclusion for effective gene delivery. International-journal

Atsushi Yamashita, Hak Soo Choi, Tooru Ooya, Nobuhiko Yui, Hidetaka Akita, Kentaro Kogure, Hideyoshi Harashima

Chembiochem : a European journal of chemical biology　7　(2)　297-302　2006/02

DOI： 10.1002/cbic.200500348 　

ISSN： 1439-4227
Evaluation of the nuclear delivery and intra-nuclear transcription of plasmid DNA condensed with micro (mu) and NLS-micro by cytoplasmic and nuclear microinjection: a comparative study with poly-L-lysine. International-journal

Hidetaka Akita, Mitsuhide Tanimoto, Tomoya Masuda, Kentaro Kogure, Susumu Hama, Keiko Ninomiya, Shiroh Futaki, Hideyoshi Harashima

The journal of gene medicine　8　(2)　198-206　2006/02

DOI： 10.1002/jgm.839 　

ISSN： 1099-498X
Pharmacokinetics and brain uptake of lactoferrin in rats. International-journal

Bin Ji, Jun Maeda, Makoto Higuchi, Kaori Inoue, Hidetaka Akita, Hideyoshi Harashima, Tetsuya Suhara

Life sciences　78　(8)　851-5　2006/01/18

DOI： 10.1016/j.lfs.2005.05.085 　

ISSN： 0024-3205

eISSN： 1879-0631
Development of non-viral vector based on the analysis of intracellular trafficking

AKITA Hidetaka

21　(1)　76-77　2006/01/10

ISSN： 0913-5006
Challenge for the Nuclear Delivery of Plasmid DNA for the Gene Therapy

AKITA Hidetaka

66　(1)　50-55　2006/01/01

ISSN： 0372-7629
Syntheses of Sugar-displaying Quantum Dots as a Cellular Stress Sensing Probe

NISHIO Takashi, NIIKURA Kenichi, MATSUO Yasutaka, AKITA Hidetaka, KOGURE Kentaro, HARASHIMA Hideyoshi, IJIRO Kuniharu, IJIRO Kuniharu

Abstr RIES Hokudai Symp　8th　72-73　2006
Synthesis of a biocleavable polyrotaxane-plasmid DNA (pDNA) polyplex and its use for the rapid nonviral delivery of pDNA to cell nuclei International-journal

Atsushi Yamashita, Nobuhiko Yui, Tooru Ooya, Arihiro Kano, Atsushi Maruyama, Hidetaka Akita, Kentaro Kogure, Hideyoshi Harashima

NATURE PROTOCOLS　1　(6)　2861-2869　2006

DOI： 10.1038/nprot.2006.438 　

ISSN： 1754-2189

eISSN： 1750-2799
Sunflower-shaped cyclodextrin-conjugated poly(epsilon-lysine) polyplex as a controlled intracellular trafficking device. International-journal

Hak Soo Choi, Atsushi Yamashita, Tooru Ooya, Nobuhiko Yui, Hidetaka Akita, Kentaro Kogure, Rie Ito, Hideyoshi Harashima

Chembiochem : a European journal of chemical biology　6　(11)　1986-90　2005/11

DOI： 10.1002/cbic.200500242 　

ISSN： 1439-4227
【DDS研究の現状と将来展望】遺伝子治療とDDS 多機能性リポソーム

原島秀吉, 小暮健太朗, 秋田英万, 紙谷浩之

PHARM TECH JAPAN　21　(12)　2059-2063　2005/10
Publisher: (株)じほう
ISSN： 0910-4739
Development of efficient packaging method of oligodeoxynucleotides by a condensed nano particle in lipid envelope structure.

Yuma Yamada, Kentaro Kogure, Yoshio Nakamura, Kaori Inoue, Hidetaka Akita, Fumi Nagatsugi, Shigeki Sasaki, Tetsuya Suhara, Hideyoshi Harashima

Biological & pharmaceutical bulletin　28　(10)　1939-42　2005/10

DOI： 10.1248/bpb.28.1939 　

ISSN： 0918-6158
A multifunctional envelope-type nano device for novel gene delivery of siRNA plasmids. International-journal

Rumiko Moriguchi, Kentaro Kogure, Hidetaka Akita, Shiroh Futaki, Makoto Miyagishi, Kazunari Taira, Hideyoshi Harashima

International journal of pharmaceutics　301　(1-2)　277-85　2005/09/14

DOI： 10.1016/j.ijpharm.2005.05.021 　

ISSN： 0378-5173
Pharmacokinetic analysis of the tissue distribution of octaarginine modified liposomes in mice. International-journal

Diky Mudhakir, Hidetaka Akita, Ikramy A Khalil, Shiroh Futaki, Hideyoshi Harashima

Drug metabolism and pharmacokinetics　20　(4)　275-81　2005/08

DOI： 10.2133/dmpk.20.275 　

ISSN： 1347-4367

More details Close

We recently found that octaarginine modified liposomes (R8-Lip) can be efficiently internalized by cultured cells. The purpose of the present study was to quantitatively determine the effect of R8-density on the tissue distribution of R8-Lip in mice, using their clearance as an index. R8 was introduced in the form of stearylated R8 (STR-R8). The liposomes were composed of cholesterol and egg phosphatidylcholine and were labeled with [(3)H]cholesteryl hexadecyl ether. Various densities of R8 (3%, 10% and 30%) containing liposomes were prepared with a diameter of approximately 70-80 nm. The tissue distribution of R8-Lip was determined after their i.v. administration into mice and the effect of R8-density on tissue distribution was compared with uptake clearance, the calculated tissue distribution divided by the area under the blood concentration-time course. As results, R8-Lip were more rapidly eliminated from circulating blood and distributed to many tissues, especially liver depending on the R8-density. However, the tissue uptake clearance represented similar value to that of positively charge liposomes. Based on these results, we conclude that the R8-dependent increase in R8-Lip in various tissues tested indicates that positive charge, but not PTD function derived from R8 predominantly responsible for the enhancement of tissue distribution. Therefore, it is suggested that topology control of R8 is important to exhibit the PTD function.
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

ISSN： 0913-5006
遺伝子デリバリーシステムの細胞内動態の定量的解析 : ウイルスvs非ウイルス

秋田英万, 濱進, 水口裕之, 原島秀吉

Drug delivery system　20　(3)　279-279　2005/05/10

ISSN： 0913-5006
多機能性エンベロープ型ナノ構造体の開発 : Programmed Packaging の提唱

原島秀吉, 小暮健太朗, 秋田英万, 紙谷浩之

Drug delivery system　20　(3)　292-292　2005/05/10

ISSN： 0913-5006
Evaluation of nuclear transfer and transcription of plasmid DNA condensed with protamine by microinjection: the use of a nuclear transfer score. International-journal

Tomoya Masuda, Hidetaka Akita, Hideyoshi Harashima

FEBS letters　579　(10)　2143-8　2005/04/11

DOI： 10.1016/j.febslet.2005.02.071 　

ISSN： 0014-5793
Two common PFIC2 mutations are associated with the impaired membrane trafficking of BSEP/ABCB11. International-journal

Hisamitsu Hayashi, Tappei Takada, Hiroshi Suzuki, Hidetaka Akita, Yuichi Sugiyama

Hepatology (Baltimore, Md.)　41　(4)　916-24　2005/04

DOI： 10.1002/hep.20627 　

ISSN： 0270-9139
ヒトBile Salt Export Pump(BSEP)変異体を用いた進行性家族性胆汁うっ滞症2型(PFIC2)発症機構の解析

林久允, 杉山雄一, 高田龍平, 鈴木洋史, 秋田英万

薬理と治療　33　(Suppl.1)　2005

ISSN： 0386-3603
ヒトBile salt export pump(BSEP)変異体を用いた進行性家族性胆汁うっ滞症2型(PFIC2)発症機構の解析

林久允, 高田龍平, 高田龍平, 鈴木洋史, 鈴木洋史, 秋田英万, 秋田英万, 杉山雄一

日本分子生物学会年会講演要旨集　28th　2005
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
Vectorial transport of bile salts across MDCK cells expressing both rat Na+-taurocholate cotransporting polypeptide and rat bile salt export pump. International-journal

Sachiko Mita, Hiroshi Suzuki, Hidetaka Akita, Bruno Stieger, Peter J Meier, Alan F Hofmann, Yuichi Sugiyama

American journal of physiology. Gastrointestinal and liver physiology　288　(1)　G159-67-G167　2005/01

DOI： 10.1152/ajpgi.00360.2003 　

ISSN： 0193-1857
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

ISSN： 0913-5006
Quantitative three-dimensional analysis of the intracellular trafficking of plasmid DNA transfected by a nonviral gene delivery system using confocal laser scanning microscopy

H Akita, R Ito, IA Khalil, S Futaki, H Harashima

MOLECULAR THERAPY　9　(3)　443-451　2004/03

DOI： 10.1016/j.ymthe.2004.01.005 　

ISSN： 1525-0016
Factors governing tissue uptake of transferrincoupled polyethylene glycol liposomes in vivo.

Int. J. Pharm.　281　(1-2)　258-233　2004
Genipin enhances Mrp2 (Abcc2)-mediated bile formation and organic anion transport in rat liver. International-journal

Junichi Shoda, Tetsuo Miura, Hirotoshi Utsunomiya, Koji Oda, Masahiro Yamamoto, Masahito Kano, Tadashi Ikegami, Naomi Tanaka, Hidetaka Akita, Kousei Ito, Hiroshi Suzuki, Yuichi Sugiyama

Hepatology (Baltimore, Md.)　39　(1)　167-78　2004/01

DOI： 10.1002/hep.20003 　

ISSN： 0270-9139
ファルマシア９月号「生細胞におけるmRNAの動きをリアルタイムに観察する」

日本薬学会　2004
Semi-quantitative analysis of the intracellular trafficking of plasmid DNA with confocal laser scanning microscopy

H Akita, R Ito, H Kamiya, H Harashima

MOLECULAR THERAPY　7　(5)　S214-S214　2003/05

ISSN： 1525-0016
Sinusoidal efflux of taurocholate correlates with the hepatic expression level of Mrp3 International-journal

H Akita, H Suzuki, Y Sugiyama

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS　299　(5)　681-687　2002/12

DOI： 10.1016/S0006-291X(02)02723-7 　

ISSN： 0006-291X
Transport activity of human MRP3 expressed in Sf9 cells: Comparative studies with rat MRP3 International-journal

H Akita, H Suzuki, T Hirohashi, H Takikawa, Y Sugiyama

PHARMACEUTICAL RESEARCH　19　(1)　34-41　2002/01

DOI： 10.1023/A:1013699130991 　

ISSN： 0724-8741
薬物動態におけるMRP3の役割

秋田英万, 瀬田あや子, 鈴木洋史, 杉山雄一

薬物動態 = Xenobiotic metabolism and disposition　16　S216　2001/09/17

ISSN： 0916-1139
Sinusoidal efflux of taurocholate is enhanced in Mrp2-deficient rat liver

H Akita, H Suzuki, Y Sugiyama

PHARMACEUTICAL RESEARCH　18　(8)　1119-1125　2001/08

DOI： 10.1023/A:1010918825019 　

ISSN： 0724-8741
Characterization of bile acid transport mediated by multidrug resistance associated protein 2 and bile salt export pump

H Akita, H Suzuki, K Ito, S Kinoshita, N Sato, H Takikawa, Y Sugiyama

BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES　1511　(1)　7-16　2001/03

DOI： 10.1016/S0005-2736(00)00355-2 　

ISSN： 0005-2736
トランスポーターの遺伝子多型 (新世紀医療をめざして(5)Pharmacogenomics)

秋田英万, 鈴木洋史, 杉山雄一

遺伝子医学　5　(1)　32-40　2001
Publisher: メディカルドゥ
ISSN： 1343-0971
Sinusoidal efflux of bile acids is mediated by multidrug resistance associated protein 3 (MRF3) under cholestatic conditions.

H Suzuki, H Akita, Y Sugiyama

HEPATOLOGY　32　(4)　428A-428A　2000/10

ISSN： 0270-9139
Stimulatory and inhibitory effect of bile acids on the transport function of multidrug resistance associated protein 2 and bile salt export pump.

H Akita, N Sato, S Kinoshita, H Suzuki, H Takikawa, J Goto, T Iida, Y Sugiyama

HEPATOLOGY　30　(4)　530A-530A　1999/10

ISSN： 0270-9139
ABCトランスポーターによる胆汁酸輸送

鈴木洋史, 佐藤準人, 木之下節夫, 秋田英万, 伊藤晃成, 杉山雄一, 後藤順一, 飯田隆, 滝川一

BIO Clinica　14　(11)　1025-1025　1999/10
Publisher: (株)北隆館
ISSN： 0919-8237
薬物の輸送過程における薬物間相互作用薬物トランスポーターの分子特性多様性と基質特異性(2)

楠原洋之, 伊藤晃成, 秋田英万, 杉山雄一

PHARM TECH JAPAN　14　(8)　1145-1155　1998/08
Publisher: (株)じほう
ISSN： 0910-4739
哺乳類発現系を用いたラットcMOATの機能解析

木之下節夫, 伊藤晃成, 秋田英万, 鈴木洋史, 粂和彦, 清水孝雄, 杉山雄一

薬物動態 = Xenobiotic metabolism and disposition　12　221-221　1997/10/09

ISSN： 0916-1139

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Books and Other Publications 11

パートナー薬剤学

原島, 秀吉, 伊藤, 智夫, 寺田, 勝英, 伊藤, 清美

南江堂　2022/02

ISBN: 9784524403875
新薬剤学改定第3版

南江堂　2011
分子薬物動態学 17章高分子・遺伝子の体内動態の制御（細胞内動態を中心に）

南山堂　2008
ナノマシンによるナノ医療

北海道大学出版会　2007
遺伝子医学MOOK5

メディカルドゥ　2006
第２章，第２節「薬物のターゲティングとDDS技術」

技術情報協会　2005
Non-viral Therapy: Gene Design and Delivery

Springer-Verlag Tokyo, Inc.　2005
Modern Biopharmaceuticals

Wiley-VCH　2005
細胞特異的ターゲッティング

CMC出版社　2004
細胞内動態制御による新しいDDSの開発

メディカルドゥ　2003
胆汁排泄機構と胆汁酸

Bibliography in Gastroenterology　2001

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Industrial Property Rights 30

細胞内動態を改善した新規カチオン性脂質

中井悠太, 丹下耕太, 吉岡宏樹, 玉川晋也, 秋田英万, 田中浩揮, 高田奈依, 小西真奈美, 高橋達成

Property Type: Patent
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

特許第6966755号

Property Type: Patent
O/W型エマルション

丹下耕太, 中井悠太, 秋田英万, 田中浩揮, 渡邉綾香, 三浦尚也, 原島秀吉

特許第6875685号

Property Type: Patent
カチオン性脂質

中井悠太, 丹下耕太, 秋田英万, 原島秀吉, 冨樫亮平, 三浦尚也, 前田澪

特許第6640750号

Property Type: Patent
核酸送達のためのカチオン性脂質

佐々木翔太, 太田雅己, 久保和弘, 原島秀吉, 秋田英万, 畠山浩人, 野口裕生, 丹下耕太, 中井悠太, 清水那有多

特許第6605477号

Property Type: Patent
細胞内動態を改善したカチオン性脂質

丹下耕太, 新井将也, 久保和弘, 秋田英万, 原島秀吉, 畠山浩人, 石破諒平, 鵜川真実, 田中浩揮

特許第6093710号

Property Type: Patent
肺送達のためのベクター、導入剤及び使用

楠本憲司, 原島秀吉, 秋田英万, 畠山浩人, 石塚太一

特許第5850915号

Property Type: Patent
脂質膜構造体に細胞透過能を付与および/または脂質膜構造体の細胞透過能を増強するペプチド、ならびにそれらペプチドと結合した脂質を構成脂質として含む、細胞透過能を有するまたは細胞透過能が増強された脂質膜構造体

原島秀吉, 藤原孝博, 秋田英万

特許第5818319号

Property Type: Patent
核内移行性を有する脂質膜構造体

原島秀吉, 秋田英万, シャリフモハメドシャヒーン, 中村孝司, 石井聡一郎, 二木史朗

特許第5794541号

Property Type: Patent
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

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

特許第5067733号

Property Type: Patent
目的物質の脳毛細血管内皮細胞への取り込みを亢進させるための組成物

井上香織, 須原哲也, 秋田英万, 原島秀吉

特許第4904573号

Property Type: Patent
細胞に導入した核酸の標的オルガネラにおける局在量を定量する方法

原島秀吉, 秋田英万

特許第4493939号

Property Type: Patent

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特開2004-357641
核酸を細胞内に送達するための脂質膜構造体

秋田英万, 三浦尚也, 舘下菜穂, 田中浩揮

Property Type: Patent
リポソーム用生体成分抵抗性増強剤及びこれにより修飾されたリポソーム

原島秀吉, 小暮健太朗, 秋田英万, 岩朝亮忠, 中村宜央, 二木史朗

Property Type: Patent
腫瘍組織で選択的に分解性を示す血中滞留性素子

秋田英万, 畠山浩人, 長崎幸夫, 大石基, 菊池寛, 小林英夫, 原島秀吉

Property Type: Patent

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特開2007-99750
目的物質の核内送達用ベクター

増田智也, 秋田英万, 小暮健太朗, 西尾崇, 新倉謙一, 居城邦治, 原島秀吉

Property Type: Patent
目的物質を核内又は細胞内に送達するためのベクター

秋田英万, 工藤亜沙子, 原島秀吉

Property Type: Patent
目的物質を効率的に核内に送達可能なリポソーム

小暮健太朗, 中村孝司, 秋田英万, 原島秀吉, 二木史朗

Property Type: Patent
ヒト条件的不死化細胞を用いた血液脳関門モデルおよびその製造方法

降幡知巳, 梅原健太, 北村啓太, 秋田英万, 安西尚彦

Property Type: Patent
センチネルリンパ節イメージング剤

秋田英万, 櫻井遊, 田中浩揮, 五味昌樹

Property Type: Patent
結合剤及びその利用

原島秀吉, 林泰弘, 秋田英万, 鳥谷部尚之, 富樫亮平

Property Type: Patent
脂質膜構造体にpH依存性カチオン性を付与する剤、それによりpH依存性カチオン性が付与された脂質膜構造体および脂質膜構造体の製造方法

原島秀吉, 畠山浩人, 佐藤悠介, 兵藤守, 櫻井遊, 秋田英万

Property Type: Patent
機能性ポリペプチド及び当該ポリペプチドで修飾された脂質膜構造体

原島秀吉, 秋田英万, 榎戸薫, 畠山浩人, 櫻井遊

Property Type: Patent
高分子化合物またはその複合体の細胞透過性評価装置およびその細胞透過性評価方法

原島秀吉, 藤原孝博, 秋田英万, 田畑泰彦

Property Type: Patent
ゴルジ装置ターゲティング用リポソーム

原島秀吉, 秋田英万, 藤原孝博

Property Type: Patent
脂肪組織標的化ペプチド及び該ペプチドを有するリポソーム

秋田英万, ディッキームダキュル, 原島秀吉

Property Type: Patent
細胞に導入した核酸の核移行量の定量方法

秋田英万, 岩朝亮忠, 原島秀吉

Property Type: Patent

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特開2006-197824
核移行性ペプチド

秋田英万, 原島秀吉

Property Type: Patent

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特開2006-67891
目的物質の脳毛細血管内皮細胞への取り込みをこう亢進させるための組成物

Property Type: Patent

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特開2006-241107
細胞に導入した核酸の標的オルガネラにおける局在量を定量する方法

Property Type: Patent

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WO2004/108962 A1

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Research Projects 36

Development of a New Postoperative Analgesic Using Neuroregenerative Nanoparticles

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (B)

Institution: Tohoku University

2025/04 - 2030/03
Innovation of On-demand DDS platform of RNA therapeutics based on endothelial ATLAS

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (A)

Institution: Tohoku University

2023/04/01 - 2027/03/31
Ready-tp-Use RNA-drug discovery for the acceleration of personalized cancer vaccine

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Pioneering)

Category: Grant-in-Aid for Challenging Research (Pioneering)

Institution: Chiba University

2021/07/09 - 2025/03/31
自己ナノ組織化プロドラッグの創製：癌免疫サイクルの矯正と癌治療への応用展開

秋田英万

Offer Organization: 日本学術振興会

System: 科学研究費助成事業基盤研究(A)

Category: 基盤研究(A)

Institution: 千葉大学

2020/04/01 - 2023/03/31

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我々はこれまで、細胞内環境に応答して生体膜バリアを突破し、自己崩壊する脂質様材料(ssPalm)を開発し、mRNAや核酸送達システムとして利用してきた。本研究では、ssPalmから形成される脂質ナノ粒子に難水溶性薬物を統合し、①薬物同士が自発的にナノ粒子を形成し、②自らの力で体内・細胞内動態を制御し、③細胞内環境に応答して自己崩壊することで薬物を放出する『自己ナノ組織化プロドラッグ』を開発する。 2021年度においては、2020年度に開発したプロドラッグ搭載脂質ナノ粒子の核酸デリバリーシステムとしての応用可能性を検証した。核酸を細胞内に導入し、機能を発揮するためには、エンドソーム内の酸性環境に応答して正に帯電し、エンドソーム膜を不安定化することが大きな駆動力となる。様々な疎水性を有する一連のプロドラッグを搭載した脂質ナノ粒子を開発した結果、特定の薬物において、安定かつ均一性の高い脂質ナノ粒子を調製することが可能となった。プロドラッグ搭載ナノ粒子のエンドソーム膜不安定化能の指標として、pH依存的な赤血球膜の溶血活性を評価した。その結果、pHの低下に伴い、高い溶血活性が認められた。また、本技術をsiRNAの導入技術へと応用すべく、自己ナノ組織化プロドラッグにsiRNAを搭載させ、in vitroにおけるノックダウン効率を評価したところ、標的となるmRNAの発現を抑制された。このことから、本プロドラッグを搭載した脂質ナノ粒子は、核酸の送達システムとしての有用性が認められた。
リンパシステム内ナノ粒子動態・コミュニケーションの包括的制御と創薬基盤開発

秋田英万

Offer Organization: 科学技術振興機構

Category: 戦略的な研究開発の推進/戦略的創造研究推進事業/CREST

2017 - 2023

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長年にわたり、ナノ粒子を用いた血中投与型の薬物送達システムが開発されてきました。一方、体内には、もう一つの脈管系としてリンパが存在し、免疫制御の場として中心的な役割を果たすと共に、がんの転移やリンパ浮腫などの難治性疾患にも深く関与しています。本研究ではリンパにおけるナノ微粒子の動態制御技術や、リンパを構成する細胞群とのコミュニケーションを制御する技術を開発し、医療原理の開拓に貢献します。
Development of nanoDDS for cancer therapy without anti-cancer drug: Proposal of RISET therapy and its therapeutic function

Akita Hidetaka

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory)

Category: Grant-in-Aid for Challenging Research (Exploratory)

Institution: Chiba University

2019/06/28 - 2021/03/31

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In this study, we proposed "Reprogramming of Immuno-reaction in Spleen and Extra-parenchyma in Tumor (RISET) therapy" as a new cancer treatment strategy by normalizing antitumor immunity via targeting the inflammatory environment of the spleen. When nanoparticles modified with a water-soluble polymer were loaded with a lipid derivative of an anti-inflammatory drug, and administered to cancer-bearing mice, the tumor volume was significantly reduced. It was clarified that the therapeutic effect of this particle is exerted through the decrease of immunosuppressive cell group in the spleen. Furthermore, when this particle was used in combination with an immune checkpoint inhibitor, a remarkable therapeutic effect was observed in the combination group, demonstrating its usefulness in combination with existing drugs.
Percutaneous Access to the Lymphatic System Using Ultrasonography and Translymphatic DDS Using Nanoparticles

Akita Shinsuke

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

Category: Grant-in-Aid for Scientific Research (C)

Institution: Chiba University

2017/04/01 - 2020/03/31

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Changes in human lymph nodes and afferent and efferent lymphatic vessels were observed using ultrasound. There were significant differences in lymph node size and diameter of efferent lymphatic vessels according to the stage of lymphatic stasis. The skin incision was made with the aim of the lymphatic vessels searched by ultrasonography, and the lymphatic cannulation technique was successfully established under the microscope and under the small incision of the skin, and the efferent lymphatic staining method in human lymphatic vessels was established. Exported lymphatic staining was found to have different success rates depending on the stage of lymphedema. For the purpose of overcoming the technical difficulty of the lymphatic cannulation technique and stabilizing the result, the equipment for 3D printing from Computer assisted design was developed.
Development of the nanoDDS platform for the penetrarion or intracellular delivery: Develioment of the rapid ligand-identification system and in vivo control of intracellular trafficking

Akita Hidetaka

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory)

Category: Grant-in-Aid for Challenging Research (Exploratory)

Institution: Chiba University

2017/06/30 - 2019/03/31

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In this project, we have established a method for the rapid identification of targeting ligand by using a phage display library. Also, we developed a lipid-like materials that can deliver the nucleic acids via intravenous administration. As to the first project, the extracellular domain of membrane-translocating protein was fixed onto the beads, and its binding antibody was identified from the phage library that encoded a randomized domain antibody. In the second project, the lipid-like materials that mounts ternary amine and disulfide bonging as a pH-triggered protonation unit and reductive environment-triggered cleavable unit, respectively was developed as a nucleic acid delivery system. We clarified that an oleic acid is useful for the application via intravenous administration. As one example, the application to the siRNA delivery system was also demonstrated.
Development of cancer suicide gene therapy using RNA trans-splicing technology

Furihata Tomomi, Kaneda Atsushi

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory)

Category: Grant-in-Aid for Challenging Research (Exploratory)

Institution: Chiba University

2017/06/30 - 2019/03/31

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The aim of this study is to develop a cancer gene therapy using RNA trans-splicing technology. We have constructed the therapeutic gene by combining the herpes simplex virus thymidine kinase gene with an RNA trans-splicing molecule targeting cancer-type organic anion transporting polypeptide 1B3. When introduced into human colon cancer cells, the therapeutic gene shows remarkable anti-cancer effects. We have also develop ssPalm-based lipid nanoparticles as a tool for delivery of the therapeutic gene into cancer cells. Collectively, our new cancer gene therapy with RNA trans-splicing technology and ssPalm-based gene delivery system has the potential to become a promising therapeutic option to fight against various cancer types.
Innovation of multi-Drug delivery platform based on the ssPalm: Immune-engineering and cancer therapy

Hidetaka Akita

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)

Category: Grant-in-Aid for Scientific Research (A)

2015/04/01 - 2018/03/31

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For the success of the cancer immune therapy, activation of antigen-specific cytotoxic T cell (CTL) is quite important. DNA vaccine is principally useful technology to induce this CTL activity. Meanwhile, the intra-tumor region is considered to be an immune-suppressive environment. Thus, for the effective function of the cancer vaccine, the normalization/ neutralization of the immune-suppressive machinery is essential in parallel. Currently, we have developed a lipid nanoparticle composed of intracellular environment-responsive material (ss-cleavable and pH-activated lipid-like material: ssPalm) as a technology to overcome the biomembranes barrier, and spontaneous collapse. In this project, we developed a delivery system of Genes, and small compounds using ssPalm as a fundamental platform, and applied them for the immune-engineering for cancer therapy.
Debelopment of nano satelite-organized archetecture as a technology to control an intracellular trafficking

Akita Hidetaka

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research

Category: Grant-in-Aid for Challenging Exploratory Research

2015/04/01 - 2017/03/31

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For the development of efficient gene carrier, hierarchically ordered nano-architecture on which functional devices (ligand for specific tissues/cells/organelles) could be placed in desired manner was formulated. Environment sensitive lipid like material which we refer as to SS-cleavable Proton-Activated Lipid-like Material (ssPalm) was employed as a basis of nano-architecture. The hierarchical nano-architecture, Nano-size Satellite-type Organized Lipid-nanoparticle Assembly (Nano-SOLA), was fabricated via double strand formation of PEGylated complementary oligo DNA. The Nano-SOLA composed of multiple primary particles were recognized as one complex by the cells.
Challenge for treatment of refractory lung diseases using a multifunctional envelope-type nanodevice that targets the lung endothelium

Nishimura Masaharu, AKITA Hidetaka, KIMURA Hiroki, SANTIWARANGKOOL Sarochin

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research

Category: Grant-in-Aid for Challenging Exploratory Research

Institution: Hokkaido University

2015/04/01 - 2017/03/31

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We tried to knockdown lung tissue specific Keap1 mRNA using GALA-MEND containing Keap1 siRNA, but it was difficult to achieve its knockdown with good efficiency and specificity to the lung tissue. Then, we tried to improve the function of GALA-MEND by PEGylation. As a result, PEGylated GALA-MEND showed better accumulation to lung endothelial cells. This improved GALA-MEND is thought to be promising for future research.
Development of nanomachine for mitochondrial gene therapy targeted to cells derived from mitochondrial disease patients

YAMADA Yuma, AKITA Hidetaka, HYODO Mamoru

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

Category: Grant-in-Aid for Scientific Research (B)

Institution: Hokkaido University

2014/04/01 - 2017/03/31

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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

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

Offer Organization: 日本学術振興会

System: 科学研究費助成事業基盤研究(S)

Category: 基盤研究(S)

Institution: 北海道大学

2010/05/31 - 2014/03/31

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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。
Novel gene delivery system with switch function

HARASHIMA Hideyoshi, KAJIMOTO Kazuaki, HAYASHI Yasuhiro, AKITA Hidetaka

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research

Category: Grant-in-Aid for Challenging Exploratory Research

Institution: Hokkaido University

2010 - 2012

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The objective of this study is to develop a novel gene delivery system with a switch function which can interact with target cells to enhance trans geneactivities.
肺血管標的及び透過型DDSを目的としたナノ粒子のインビボ細胞内動態制御法の確立

秋田英万

Offer Organization: 日本学術振興会

System: 科学研究費助成事業若手研究(A)

Category: 若手研究(A)

Institution: 北海道大学

2010 - 2012

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多重パッケージング法の確立に関して、内膜の膜枚数制御を行うために、遺伝子とポリカチオンの凝集体コアをテトラエチレングリコール修飾コレステロール(TEG-Col)含有脂質膜でコーティングした。また、本MENDを2枚目で封入する際には、1枚膜MENDの空リポソームからの精製と長期保存を行うための凍結乾燥技術を確立することが重要である。スクロース密度勾配遠心を行うことにより、遺伝子封入ナノ粒子の精製を行い、さらに本MZNDを凍結乾燥する上で至適なバッファー条件を見出すことに成功した。また、本精製後のMENDは、1枚膜で覆われていることは、電子顕微鏡観察により明らかとした。また、2段階目の封入法の確立についても検討を行った。内膜にビオチン修飾を施し、2段階目のパッケージングに伴うアビジンの内膜への結合阻害効果を指標にパッケージングを評価する方法論を確立した。また、外膜の検討として、肺血管内皮を標的化可能な新規リガンドを探索した。本リガンド修飾リポソームに遺伝子を封入しても、初期の検討においては遺伝子発現がほとんど認められなかったが、細胞内動態(特にエンドソーム脱出)の改良により、in vivo静脈内投与によって効率的な遺伝子発現を示すナノ粒子を構築することに成功した。さらに、本粒子に対して、siRNAを封入することにより、肺血管内皮細胞における効率的な血管投与型遺伝子ノックダウンシステムを構築することに成功した。さらに、新たに脳への標的化リガンドについても探索し、レプチン由来ペプチドが脳毛細血管内皮細胞標的化に有用である事を見出した。細胞内動態の観点からは、リアルタイムイメージングシステムにより、ナノ粒子の細胞質輸送過程を定量的に評価する技術を確立した。
Control of intranuclear disposition of DNA for gene therapy

KAMIYA Hiroyuki, AKITA Hidetaka

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

Category: Grant-in-Aid for Scientific Research (B)

Institution: Ehime University

2009 - 2011

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To achieve efficient and durable transgene expression by controlled intranuclear disposition, we inserted DNA sequences that modulate binding to histones into plasmid DNAs, introduced histone-plasmid DNA complexes into cells, and developed a plasmid system that recruited transcription factors. We found that transgene expression was improved in efficiency and was maintained for a week. Moreover, we revealed that gene correction by tailed duplexes was a useful candidate in gene therapy.
Multifunctional Envelope-type Nano Device as non-viral gene delivery system for cancer therapy

HARASHIMA Hideyoshi

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Priority Areas

Category: Grant-in-Aid for Scientific Research on Priority Areas

Institution: Hokkaido University

2008 - 2009

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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 core condensed with protamine. To investigate antitumor effects, we immunized DCs which were silenced SOCS1 by GALA/DMEND to mice. As a result, SOCS1- silenced DC significantly suppressed tumor growth compared with control DC. Therefore, we succeeded in enhancing antitumor effect by silencing SOCS1 of DC with GALA/DMEND. For systemic siRNA delivery, HT1080 cells were s.c. inoculated into nude mice. After i.v. injection to tumor-bearing mice, GALA/PEG-MEND exhibited high systemic stability and accumulated in tumor tissue. Target □-actin expression in tumor tissue was knockdowned more than 60% after administration of GALA/PEG-MEND (4mg siRNA/kg), which resulted in suppression of tumor growth (特願2010-39667). Serum level of ALT remained normal value, and body weight was unchanged after i.v. administration of GALA/PEG-MEND. These results suggested that GALA/PEG-MEND should be a valuable siRNA delivery system for in vivo tumor and siRNA therapeutics.
細胞内動態の精密制御に基づいたナノキャリアのトランスサイトーシス輸送制御の新戦略

秋田英万

Offer Organization: 日本学術振興会

System: 科学研究費助成事業挑戦的萌芽研究

Category: 挑戦的萌芽研究

Institution: 北海道大学

2008 - 2009

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正常組織の血管内皮細胞は、互いに隣接する細胞間でタイトなジャンクションを形成しており、高分子ナノキャリアの血中から組織実質への輸送は著しく制限されている。従って、トランスサイトーシスによって細胞単層を透過可能なキャリアが構築できれば、血管内皮バリアの克服につながり、DDS研究の究極的な目標である血中投与を介した組織実質移行性ベクターの実現への大きなブレイクスルーとなる。本年度は、細胞内オルガネラの中でも細胞内ソーティングにおいて中心的役割を果たすゴルジ体への標的化と、本戦略とは別にトランスサイトーシス経路標的化ペプチドの探索を行った。極性細胞であるMDCK細胞などにおけるR8-MENDの細胞内動態解析を行なった結果、他の細胞における動態とは大きくことなり、エンドソーム脱出効率が悪いことが明らかとなった。また、従来のカチオン性脂質と異なり、R8-MENDの一部はゴルジ体に移行することを明らかとした。また、前年度に構築した新規トランスウェルを用い、多岐にわたるペプチドを修飾したリポソームの単層細胞培養系の透過性を解析した結果、ファージディスプレイ法によって同定されたペプチドのうちのひとつをリポソーム表面に修飾することにより、マウス脳毛細血管内皮細胞由来細胞からなる単層を透過できることを明らかとした。また、本ペプチドの長さ、配列などを種々改変し、同様にリポソームの透過実験を行った結果、ナノ粒子のトランスサイトーシスを誘起可能な新規コンセンサス配列を世界に先駆けて同定することに成功した。さらに、細胞内取り込み機構を解析した結果、本粒子はカベオラにより取り込まれ、さらに、本経路を阻害することによってトランスサイトーシスも阻害されることから、本経路がトランスサイトーシスに重要な役割を果たすことを明らかとした。また、脳毛細血管内皮細胞への標的化ペプチドとして、leptin由来ペプチドが有用であることを見出した。
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

原島秀吉, 小暮健太朗, 秋田英万, 山田勇磨

Offer Organization: 日本学術振興会

System: 科学研究費助成事業挑戦的萌芽研究

Category: 挑戦的萌芽研究

Institution: 北海道大学

2008 - 2009

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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減されるかを調べた。polyI:C搭載MEND及びpolyI:C単独をマウス尾静脈から投与し、2時間後の炎症性サイトカインIL-6の産生を調べた。その結果、polyI:C搭載MEND投与群では、polyI:C単独投与群と比較してIL-6産生の著しい抑制が認められた。以上の結果より、効率的な細胞性免疫誘導及び副作用の軽減には、アジュバントのトポロジーを考慮したワクチン設計が重要であることが明らかになった。
Development of new-generation gene vectors by a control of intracellular trafficking and dispersion based in a real-time imaging of DNA

AKITA Hidetaka

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (A)

Category: Grant-in-Aid for Young Scientists (A)

Institution: Hokkaido University

2007 - 2009

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In this project, intracellular trafficking of nano-carrier was controlled from the point of view of intracellular dynamics (i.e. vesicular transport and/or microtubule-dependent transport), dispersity and homogeneity to improve the transfection activity. These technologies will be valuable as used in DNA vaccine and liver-targeting gene carriers. Furthermore, novel peptide was discovered, which confer the activity to pass through the cell monolayer via transcytosis pathway. This device will be useful to overcome the vesicular barriers which limits the delivery to tissue parenchyma.
Development of in vivo gene delivery system for siRNA and genome wide screening of type-two diabetes related genes in mice and rats

HARASHIMA Hideyoshi, KAMIYA Hiroyuki, YAMADA Yuma, BABA Yoshinobu

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)

Category: Grant-in-Aid for Scientific Research (A)

Institution: Hokkaido University

2006 - 2009

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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.
DNA・mRNAの細胞内高感度可視化情報に基づく高性能人工遺伝子べクターの創製

秋田英万, 小暮健太朗, 谷知己, 南川典昭

Offer Organization: 日本学術振興会

System: 科学研究費助成事業特定領域研究

Category: 特定領域研究

Institution: 北海道大学

2007 - 2008

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平成20年度は、リアルタイムイメージングを駆使し、細胞内動態、特に細胞質輸送に関して人工ベクターとアデノウイルスベクター間で比較を行った。また、その輸送における微小管依存性を調べるとともに、能動的な輸送、さらには拡散速度に関してMean Square Diffusion(MSD)を算出することで定量的に解析した。さらに、小胞輸送の寄与についても解析するために人工キャリアと小胞を別々の蛍光色素によってラベルを行い、マルチカラーリアルタイムイメージングを行った。その結果、人工ベクターに関しては大部分が小胞に取り込まれた形で微小管輸送を受けていること、さらには、その速度はアデノウイルス、あるいは人工キャリアを取り込んでいない小胞と比較して優位に遅いことを明らかとした。このことは、微小管輸送は、輸送物の密度などによって速度が制限されることを示すものである。また、遺伝子発現におけるheterogeneityのメカニズムを明らかとするため、mRNAと蛋白発現に関する1細胞レベルのdual imagingを行った。蛋白発現を解析するために、ピストン蛋白とTFPとの融合蛋白発現系を用い、本mRNA発現に関しては量子ドットを用いた高感度検出の確立を行った。その結果、人工ベクターにおいては、mRNAと蛋白の発現レベルに相関が少ないことが明らかとなっている。現在、mRNAの発現レベルと蛋白の発現レベルの相関性をアデノウイルスについても解析を開始しており、本結果との比較情報をもとに論文化する予定である。さらに北陸先端大学との共同研究により、遺伝子とポロロタキサンによるナノキャリアの細胞内動態解析をおこない、律速段階の同定に成功した。
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

原島秀吉, 紙谷浩之, 秋田英万, 山田勇磨, 小暮健太朗

Offer Organization: 日本学術振興会

System: 科学研究費助成事業特定領域研究

Category: 特定領域研究

Institution: 北海道大学

2006 - 2007

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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、がん治療への道が拓かれることが期待できる。
Novel DNAs for efficient and durable transgene expression

KAMIYA Hiroyuki, AKITA Hidetaka

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

Category: Grant-in-Aid for Scientific Research (B)

Institution: Hokkaido University

2006 - 2007

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Naked luciferase-plasmid DNA was delivered into mouse liver by hydrodynamics-based injection, an modifications of the histones bound to the plasmid DNA were analyzed by a chromatin immunoprecipitation (ChIP) analysis. In addition, the effects of a second hydrodynamics-based injection on the expression from the plasmid DNA were examined The ChIP analysis revealed that the modification status of histone H3 remained constant from 4 hr t 4 weeks. Surprisingly, the injection of saline without DNA enhanced the luciferase expression from the preexisting DNA administered 4 and 14 days previously. Our results suggest that histone modification plays no role in the silencing. Instead, our data suggest that the transgene expression is activated by the hydrodynamics-based injection manipulation, and that the return from the activated status causes the silencing. The effects of a left-handedly curved sequence ([CATGTTTTT]_n, n=20-40) with high histone affinity on plasmid expression were examined in vivo. A naked luciferase-plasmid was delivered into mouse liver by a hydrodynamics-based injection, and the luciferase activities were quantitated at various time points. Plasmids containing the curved sequence markedly enhanced transgene expression and one with [CATGTTTTT]_<36> showed the highest expression. These results suggest that sequences with high histone affinity could control transgene expression from plasmids in vivo. We also examined several hundred-base single-stranded (ss) DNA fragments for gene correction. The gene correction efficiency varied (0.8-9.3%), depending on target positions and sense/antisense strands. Sense ss DNA fragments corrected the target gene with equal or higher efficiencies as compared to their antisense counterparts. The target positions corrected with high efficiency by the sense fragments also tended to be corrected efficiently by the antisense fragments. These results suggest that the sense ss DNA fragments are useful for the correction of mutated genes. The variation in the correction efficiency may depend on the sequence of the target position in double-stranded DNA.
肝臓への遺伝子導入システムの構築 Competitive

2007 -
1分子イメージングに基づく細胞内動態・解離制御型デリバリーシステムの創製

秋田英万, 小暮健太朗

Offer Organization: 日本学術振興会

System: 科学研究費助成事業特定領域研究

Category: 特定領域研究

Institution: 北海道大学

2006 - 2006

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アデノウイルスと人工ベクター間の遺伝子発現比較を行った結果、同程度の遺伝子発現を示すためには、LipofectAMINE PLUSにおいてはadenovirusと比較して、3桁以上の遺伝子コピー数を必要とすることが明らかとなっている。これまで、両ベクターの細胞内動態を解析した結果、両者の発現効率の大きな差を生み出す要因が、核に移行してから後の効率にあることを明らかとした。そこで本研急では、このような核移行後発現効率がどのようなメカニズムに基づくものであるかを明らかとした。セントラルドグマの中間産物であるmRNAをリアルタイムRT-PCRによって測定することにより、この7000-8000倍にも及ぶ核移行後発現効率差における転写、翻訳過程の寄与を解析した。この結果、転写効率、翻訳効率はそれぞれ400倍及び20倍、アデノウイルスで高い結果が明らかとなった。一方、解離型DNAのみを検出可能な高感度in situ hybridizationを用い、アデノウイルスとプラスミドDNAをTSA増感システムによって高感度に検出した結果、Adの方がLFNと比較して非常に効率的な染色が得られ、LFNの低い核内解離効率が、核移行後の発現効率の低さの原因となる事が示唆された。さらに、アデノウイルスにおいては、ユークロマチンへの特異的な局在が認められた。このような、核内の局在や解離の違いが、核内転写の違いに寄与することが示唆される。さらに、LipofectAMINE PLUSは、細胞内のmRNAと高い静電的相互作用を有し、これが、LFNにおいて低い翻訳効率をしめす一因であることが示唆された。また、リアルタイムイメージングに基づいて細胞内動態を評価した結果、遺伝子は非常にすばやく細胞内でアグリケーションを起こすことを見出し、転写移行後の発現効率の低い大きな要因であることが示唆された。
ミトコンドリアを標的とする多重型・多機能性エンベロープ型ナノ構造体の開発

原島秀吉, 紙谷浩之, 秋田英万

Offer Organization: 日本学術振興会

System: 科学研究費助成事業萌芽研究

Category: 萌芽研究

Institution: 北海道大学

2005 - 2006

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本研究は、蛋白質をミトコンドリア(MT)へ送達するための新規送達システムの開発を目的とした。研究戦略は、蛋白質をコアとするナノ粒子(直径100nm以下)を形成し、細胞膜とMT膜を突破するためにエンベロープを多重構造にする(多重型MEND)。最外層にはオクタアルギニンを、中間層にはMT targeting signal(MTS)を付加し、ナノ粒子のままMT内へ送達するシステムを開発する。 1)蛋白質を封入したMENDの構築:これまで、多機能性エンベロープ型ナノ構造体(MEND)に遺伝子を封入する方向を確立してきたが、本研究では、蛋白質を封入する方法を確立することに成功した。本MENDは、マクロピノサイトーシスで細胞内へ導入後、封入したGFPを細胞質中に放出可能であることが明らかと成った。 2)R8-MENDによるミトコンドリアへの送達システムの開発:オクタアルギニン(R8)修飾したMENDはマクロピノサイトーシスにより細胞内へ取込まれ、細胞質中へ脱出し、ミトコンドリアへGFPを送達可能であることが明らかとなった。 3)MTS結合エンベロープ体の構築:まず、脂質誘導体-MTSの合成を行った。合成は困難を極めたが、純度の高い合成品を得ることに成功した。エンベロープ化したナノ粒子に脂質誘導体一MTSを導入し、MTS結合エンベロープ体を構築した。MTS結合型多重型MENDにより、選択的なミトコンドリア送達が可能となった。
ウイルスの細胞内動態定量化情報に基づいた人工遺伝子ベクター開発の新規アプローチ

秋田英万

Offer Organization: 日本学術振興会

System: 科学研究費助成事業若手研究(B)

Category: 若手研究(B)

Institution: 北海道大学

2005 - 2006

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アデノウイルスと人工ベクター間の遺伝子発現比較を行った結果、同程度の遺伝子発現を示すためには、LipofectAMINE PLUSにおいてはadenovirusと比較して、3桁以上の遺伝子コピー数を必要とすることが明らかとなっている。H16年度おいて、両ベクターの細胞内動態を解析した結果、両者の発現効率の大きな差を生み出す要因が、核に移行してから後の効率にあることを明らかとした。H17年度は、このような核移行後発現効率がどのようなメカニズムに基づくものであるかを明らかとした。転写効率を生み出すメカニズムについて、Ad由来のコア蛋白やゲノム構造に由来する可能性や、遺伝子の核内解離の違いを解析した。Adをプレ感染してコア蛋白を核内に導入しても、プレ感染なしのものと比べて有為な上昇は認められなかつた。また、GFPをコードするAdゲノムと、プラスミドDNAを核内インジェクションした結果、GFP発現率に有為な差は認められなかった。このことから、アデノウイルス側に転写を促進する因子があるという可能性は否定された。一方、解離型DNAのみを検出可能なin situ hybridizationを用い、アデノウイルスとプラスミドDNAをTSA増感システムによって高感度に検出した結果、Adの方がLFNと比較して非常に効率的な染色が得られ、LFNの低い核内解離効率が、核移行後の発現効率の低さの原因となる事が示唆された。さらに、アデノウイルスにおいては、ユークロマチンへの特異的な局在が認められた。このような、核内の局在や解離の違いが、核内転写の違いに寄与することが示唆される。
DNAワクチンの開発 Competitive

2006 -
Development of Multifunctional Envelope type Nano Device based on Programmed Packaging

HARASHIMA Hideyoshi, KAMIYA Hiroyuki, AKITA Hidetaka

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

Category: Grant-in-Aid for Scientific Research (B)

Institution: HOKKAIDO UNIVERSITY

2003 - 2004

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A rational strategy is required to overcome cellular and intracellular barriers and to develop an innovative non-viral gene delivery system. A new concept "Programd Packaging", for the design and assembling of intelligent nano devices into an integrated, unique non-viral gene delivery system has been developed. Based on this concept, the importance of cellular entrance for a gene delivery system, using Tf as a receptor-mediated endocytic pathway and octaarginine as a non-endocytic pathway was chosen. The advantage of a non-endocytic pathway is demonstrated by an octaarginine-modified envelope type nano device, which exerts transfection activities as high as an adenovirus. Finally, a three dimensional quantitative assay for the intracellular trafficking of a gene delivery systems and its application for the pharmacokinetic optimization of a delivery system is described. These new approaches will ultimately lead to the development of a non-viral gene delivery system which is superior to viral vectors not only from the point of view of safety, but efficiency as well.
核膜透過過程の新規定量化システムの構築と核膜輸送の基質認識性、機能多様性の解析

秋田英万

Offer Organization: 日本学術振興会

System: 科学研究費助成事業若手研究(B)

Category: 若手研究(B)

Institution: 北海道大学

2003 - 2004

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遺伝子ベクターを最適化する上で、エンドソームから細胞質への脱出と、それに引き続く核膜透過過程の効率を定量化する事は重要である.本研究においては、共焦点レーザー顕微鏡により得られる画像を基に定量化を試み、細胞内動態の定量的解析法を構築した。さらに、PCRを用いて、核内への遺伝子到達量を測定する系についても確立し、これらを用いて各種遺伝子導入法の違いによる細胞内遺伝子動態の比較を行った。stearyl R8及びlipofectAMINEによる導入時においては、R8と比較してendosome/lysosomeからの脱出過程とそれに伴う核内移行過程が非常に早い事が明らかとなり、遺伝子発現活性と矛盾をしない結果となった。また、stearyl R8を用いた導入においては、エンドソームの脱出とそれに続く核膜透過が時間依存的に上昇するのに対し、lipofectAMINEを用いた際には、特に30分という非常に早い時間で細胞質や核に遺伝子が移行する事が明らかとなり、エンドサイトーシス以外の経路を介した取込み機構の存在と、効率的な核移行性が示唆された。このような細胞内遺伝子定量解析は、遺伝子ベクターの新たな細胞内挙動メカニズムを明らかにするのみでなく、核指向性新規遺伝子ベクターの開発に有用であろうと考えられる。現在、この実験系を用い、より究極的な課題として、なぜウイルスは人工ベクターと比較して遺伝子発現が悪いのかを細胞内動態の観点から明らかにする事を試みている.
腫瘍遺伝子・核酸送達システムの開発 Competitive

2004 -
肝における胆汁酸輸送担体の機能解析

秋田英万

Offer Organization: 日本学術振興会

System: 科学研究費助成事業特別研究員奨励費

Category: 特別研究員奨励費

2001 - 2002

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肝臓を介した血管側から胆汁中への胆汁酸の極性輸送には、胆汁酸の取り込み過程に関与する肝細胞血管側膜上のトランスポーター及び肝細胞内から胆汁中への排出トランスポーターの協調的な輸送が関与している。排泄に関わるトランスポーターとして硫酸抱合胆汁酸を基質とするMultidrug resistance associated protein2や一価胆汁酸を基質とするBile salt export pump(BSEP)が存在しているが、我々は、Mrp2欠損ラットであるEisai hyperbilirubinemic rat(EHBR)の肝臓よりMrp3を同定し、胆汁酸を基質とする事を明らかとした。Mrp3が胆汁うっ滞時などの病態時に肝血管側膜状に存在する事を考えると、肝細胞の胆汁酸からの保護に役立つのではないかという仮説が挙げられた。我々は、SDラットとEHBRを用いて肝潅流実験を行い、Mrp3の基質である[^3H]タウロコール酸(TC)の肝細胞から血管中側へのクリアランスがEHBRにおいて上昇している事から、Mrp3の血管側への胆汁酸排出への寄与を示唆してきた。この事をさらに実証する目的で、今回我々はMrp3の誘導が認められるフェノバルビタール処理ラットを用いてこの仮説をさらに検証した。今回我々は、Mrp3の誘導が認められるフェノバルビタール処理ラットを用いて[^3H]TCを基質とする肝潅流実験を行った結果、横軸にMrp3の肝臓における発現量を、縦軸に血管側への透過性をプロットすると良好な相関が得られた。これらの結果はMrp3が胆汁酸の血管側への排出に関与する事を強く示唆するものである。また、得られた回帰直線は原点を通らず、比較的大きなY切片を有した事から、Mrp3以外の排出メカニズムが存在すると考えられる。近年Organic anion transporting polypeptide(Oatp)ファミリートランスポーターがアニオン交換輸送担体である事を示唆する報告がなされておりOatpがMrp3以外の血管側への排出に関わる事が示唆される。
細胞内動態制御素子の開発(遺伝子の核移行性デバイスの開発）細胞内動態定量法の開発 Competitive

1999 -
Controll of itracellular trafficking of plasmid DNAEstablishment of the quantification system of intracellular destribution of pDNA Competitive

1999 -

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