2021年03月, 公益財団法人 日本薬学会, 2021年度日本薬学会奨励賞　　　　　　　　　　　　　　　
脂質分子デザインに基づく核酸搭載脂質ナノ粒子製剤の開発
国内学会・会議・シンポジウム等の賞, 日本国

2016年02月, 北海道大学, 平成27年度北海道大学研究総長賞奨励賞　　　　　　　　　　　　　　　
佐藤悠介, その他の賞, 日本国

2013年07月, 日本DDS学会, 第29回日本DDS学会学術集会 優秀発表賞　　　　　　　　　　　　　　　
佐藤悠介, 国内学会・会議・シンポジウム等の賞, 日本国

Selective vascular disrupting therapy by lipid nanoparticle-mediated Fas ligand silencing and stimulation of STING.
Rikito Endo, Tomoki Ueda, Takumi Nagaoki, Yusuke Sato, Nako Maishi, Kyoko Hida, Hideyoshi Harashima, Takashi Nakamura
Biomaterials, 321, 123297, 123297, 2025年10月, ［国際誌］
英語, 研究論文（学術雑誌）, Although recent therapeutic developments have greatly improved the outcomes of patients with cancer, it remains on ongoing problem, particularly in relation to acquired drug resistance. Vascular disrupting agents (VDAs) directly damage tumor blood vessels, thus promoting drug efficacy and reducing the development of drug resistance; however, their low molecular weight and resulting lack of selectivity for tumor endothelial cells (TECs) lead to side effects that can hinder their practical use. Here, we report a novel tumor vascular disrupting therapy using nucleic acid-loaded lipid nanoparticles (LNPs). We prepared two LNPs: a small interfering RNA (siRNA) against Fas ligand (FasL)-loaded cyclic RGD modified LNP (cRGD-LNP) to knock down FasL in TECs and a stimulator of interferon genes (STING) agonist-loaded LNP to induce systemic type I interferon (IFN) production. The combination therapy disrupted the tumor vasculature and induced broad tumor cell apoptosis within 48 h, leading to rapid and strong therapeutic effects in various tumor models. T cells were not involved in these antitumor effects. Furthermore, the combination therapy demonstrated a significantly superior therapeutic efficacy compared with conventional anti-angiogenic agents and VDAs. RNA sequencing analysis suggested that reduced collagen levels may have been responsible for TEC apoptosis. These findings demonstrated a potential therapeutic method for targeting the tumor vasculature, which may contribute to the development of a new class of anti-cancer drugs.

Lipid nanoparticle delivery of the CRISPR/Cas9 system directly into the mitochondria of cells carrying m.7778G>T mutation in MtDNA (mt-Atp8).
Kaede Norota, Sen Ishizuka, Misa Hirose, Yusuke Sato, Masatoshi Maeki, Manabu Tokeshi, Saleh M Ibrahim, Hideyoshi Harashima, Yuma Yamada
Scientific reports, 15, 1, 18717, 18717, 2025年06月19日, ［国際誌］
英語, 研究論文（学術雑誌）, Mitochondrial genome mutations are associated with various diseases and gene therapy targeted to mitochondria has the potential to effectively treat such diseases. Here, we targeted a point mutation in mitochondrial DNA (mtDNA) that can cause mitochondrial diseases via delivery of the clustered, regularly interspaced, short palindromic repeats/Cas9 (CRISPR/Cas9) system to mitochondria using an innovative lipid nanoparticle (LNP) delivery system. To overcome the major barrier of the mitochondrial membrane structure, we investigated a strategy to deliver ribonucleoprotein (RNP) directly to mitochondria via membrane fusion using MITO-Porter, a mitochondria-targeting lipid nanoparticle. First, we constructed RNP-MITO-Porter, in which an RNP was loaded into MITO-Porter using a microfluidic device. Sequence-specific double-strand breaks were confirmed when the constructed RNP-MITO-Porter was applied to isolated mitochondria. Next, the RNP-MITO-Porter was applied to HeLa cells, and a portion of the RNP-MITO-Porter was colocalized with mitochondria and caused sequence-specific double-strand breaks in mtDNA. Finally, RNP-MITO-Porter was successfully delivered to mitochondria of cells derived from a mouse carrying a point mutation (m.7778G > T) in mtDNA (mt-Atp8) (LMSF-N-MTFVB cells), and created double-strand breaks at the target sequence. RNP-MITO-Porter is expected to contribute significantly to the clinical application of mitochondrion-targeted gene therapy.

Microfluidic Production of Exosome-Mimicking Lipid Nanoparticles for Enhanced RNA Delivery: Role of Exosomal Proteins.
Masatoshi Maeki, Ayuka Niwa, Shota Oyama, Kyoko Aratani, Rina Ito, Yuichi Suzuki, Yusuke Sato, Akihiko Ishida, Hideyoshi Harashima, Manabu Tokeshi
ACS applied materials & interfaces, 17, 29, 41666, 41679, 2025年06月16日, ［国際誌］
英語, 研究論文（学術雑誌）, Exosomes, which are cell-secreted lipid-based nanoparticles, play a crucial role in intercellular communication by encapsulating and delivering various biomolecules such as DNA, mRNA, miRNA, and proteins. They offer potential as drug delivery systems (DDSs) based on their ability to cross biological barriers, use natural communication mechanisms, and minimize immunogenicity. However, the heterogeneity of exosomes presents a bottleneck for functional analysis and the development of exosome-based DDSs. Therefore, engineering techniques are needed to produce exosomes or exosome-mimicking nanoparticles with controlled characteristics, including the presentation of specific exosomal proteins on their surface. Here, a one-step microfluidic method for producing exosome-mimicking lipid-based nanoparticles decorated with specific exosomal proteins was developed, enabling control over the composition and characteristics of the resulting exosomes. Exosome-mimicking nanoparticles decorated with tetraspanin proteins (CD9, CD63, CD81) and integrins (ITG αVβ5, ITG α6β4), which are involved in cell signaling and organ targeting, were thereby generated. Investigating the impact of these exosomal proteins on RNA delivery efficiency revealed that ITG αVβ5-decorated exosome-mimicking nanoparticles significantly enhance RNA delivery both in vitro and in vivo. This study provides an approach for producing precisely decorated exosome-mimicking nanoparticles, which may be applied to elucidate the functions of exosomal proteins and develop targeted DDSs.

Cubic Phase-Inducible Zwitterionic Phospholipids Improve the Functional Delivery of mRNA.
Kazuki Iwakawa, Rikako Sato, Mariko Konaka, Yuma Yamada, Hideyoshi Harashima, Yusuke Sato
Advanced science (Weinheim, Baden-Wurttemberg, Germany), 12, 17, e2413016, 2025年05月, ［国際誌］
英語, 研究論文（学術雑誌）, Lipid nanoparticles (LNPs) are clinically advanced delivery systems for RNA. The extensively developed structure of ionizable lipids greatly contributes to the functional delivery of mRNA. However, endosomal escape is one of the severe biological barriers that continue to render this process inefficient (e.g., less than 10%). Although LNPs contain phospholipids, their role is poorly understood, and there have been few attempts to perform the chemical engineering required to improve their functionality. Herein, a cubic phase-inducible fusogenic zwitterionic phospholipid derived from 1,2-dioleoyl-3-sn-glycero-phosphoethanolamine (DOPE), DOPE-Cx is described, that is designed to correct this problem. The orientation of a zwitterionic head group of DOPE is engineered by attaching a series of hydrophobic moieties for zwitterionic intermolecular interaction with the head structure of phosphatidylcholine (PC), and this is followed by a lipid-phase transition into non-lamellar phases to facilitate membrane fusion-mediated endosomal escape. A structure-activity relationship study reveals that DOPE-Cx lipids with small hydrophobic chains induce cubic phases instead of a hexagonal phase when mixed with PC, which enhances the functional delivery of mRNA in the liver as opposed to the action of the typically utilized and naturally occurring phospholipids. Engineered functionalized phospholipids will be of great value for the therapeutic applications of mRNAs.

Splenic B cell-targeting lipid nanoparticles for safe and effective mRNA vaccine delivery.
Yuichi Suzuki, Mai Yakuwa, Mina Sato, Eleni Samaridou, Moritz Beck-Broichsitter, Masatoshi Maeki, Manabu Tokeshi, Yuma Yamada, Hideyoshi Harashima, Yusuke Sato
Journal of controlled release : official journal of the Controlled Release Society, 382, 113687, 113687, 2025年04月03日, ［国際誌］
英語, 研究論文（学術雑誌）, mRNA-loaded lipid nanoparticles (LNPs) have emerged as a potent and versatile platform that underpins the success of mRNA vaccines, but guidelines for designing safe and effective formulations with minimal off-target effects remain unclear. In this study, we focused on a rational design for a novel ionizable lipid library that is based on ionizable tri-oleoyl-tris (iTOT) compounds with a high yield via a simple 2-step synthesis. To further enhance the efficacy and safety of this potent library for vaccine applications, we identified the optimal composition for a vaccine by focusing on the molar ratio of specific lipid excipients in the formulation. This composition brought about a shift in delivery to the spleen, and the LNP formulation, which contained 15 mol% DSPC (15%DSPC-LNPs), was thoroughly taken up by both B cells and other splenic immune cells. This formulation requires neither additional lipid components nor targeting ligand modifications, and it is accompanied by antigen-specific cytotoxic T lymphocyte responses. The rigid, hydrophobic, and charge-neutral surface of 15%DSPC-LNPs minimizes apolipoprotein E-dependent hepatic uptake and maximizes complement receptor-mediated B-cell targeting. Furthermore, as an intramuscularly administered vaccine, 15%DSPC-LNPs induce antigen-specific immune responses and, importantly, results in significantly lower levels of hepatotoxicity compared with that of the mRNA vaccine formulations currently being marketed. In summary, this study demonstrated how the passive targeting of mRNA-LNPs to organs and cells could be regulated by designing novel ionizable lipids combined with adjusting the relative proportions of lipid components. The results of this study also emphasize how selective mRNA delivery to the spleen could avoid the liver, which highlights a promising strategy for the development of safe and effective vaccines.

Harnessing the composition of lipid nanoparticles to selectively deliver mRNA to splenic immune cells for anticancer vaccination
Mahmoud A. Younis, Yusuke Sato, Yaser H. A. Elewa, Hideyoshi Harashima
Drug Delivery and Translational Research, Springer Science and Business Media LLC, 2025年03月07日, ［国際誌］
英語, 研究論文（学術雑誌）, Herein, we report a design for lipid nanoparticles (LNPs) that specifically delivers mRNA to splenic immune cells post intravenous administration for potential anticancer vaccination applications. A diverse library of ionizable lipids was screened in vivo, in combination with various helper lipids, where the composition of LNPs was tweaked to control their in vivo performance. The biodistribution of the LNPs was then investigated at both organ and sub-organ levels. Subsequently, the LNPs were recruited to deliver an anticancer mRNA-based vaccine to mice. The in vivo tropism of the LNPs was dramatically affected by the chemical structure of the ionizable lipids in question, where a model lipid, CL15H6, was recognized as displaying high affinity for the spleen. Further optimization of the composition of the LNPs enabled highly efficient and spleen-selective mRNA delivery, where the optimized CL15H6 LNPs demonstrated a high capacity for homing to splenic antigen-presenting cells (APCs). Furthermore, loading the LNPs with a low dose of ovalbumin-encoding mRNA (mOVA), as a model antigen, protected the mice against OVA-expressing tumor challenges and suppressed the tumor growth in tumor-bearing mice by ~ 75%, which was superior to the results of a clinically-relevant formulation. The CL15H6 LNPs proved to be biosafe upon either acute dose escalation or repeated administrations. The novel and scalable platform reported herein is promising for clinical translation as a neoantigen vaccine.

Impact of Lipid Tail Length on the Organ Selectivity of mRNA-Lipid Nanoparticles.
Kazuki Hashiba, Masamitsu Taguchi, Sachiko Sakamoto, Ayaka Otsu, Yoshiki Maeda, Yuichi Suzuki, Hirofumi Ebe, Arimichi Okazaki, Hideyoshi Harashima, Yusuke Sato
Nano letters, 24, 41, 12758, 67, 2024年10月07日, ［国際誌］
英語, 研究論文（学術雑誌）, The delivery of mRNA molecules to organs beyond the liver is valuable for therapeutic applications. Functionalized lipid nanoparticles (LNPs) using exogenous mechanisms can regulate in vivo mRNA expression profiles from hepatocytes to extrahepatic tissues but lead to process complexity and cost escalation. Here, we report that mRNA expression gradually shifts from the liver to the spleen in an ionizable lipid tail length-dependent manner. Remarkably, this simple chemical strategy held true even when different ionizable lipid head structures were employed. As a potential mechanism underlying this discovery, our data suggest that 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) is enriched on the surface of mRNA/LNPs with short-tail lipids. This feature limits their interaction with biological components, avoiding their rapid hepatic clearance. We also show that spleen-targeting LNPs loaded with SARS-CoV-2 receptor-binding domain (RBD) mRNA can efficiently induce immune responses and neutralize activity following intramuscular vaccination priming and boosting.

Impact of in vivo fate of STING agonist-loaded lipid nanoparticles on antitumor immunity.
Rikito Endo, Tomoki Ueda, Takumi Nagaoki, Natsumi Shima, Yusuke Sato, Hideyoshi Harashima, Takashi Nakamura
Journal of controlled release : official journal of the Controlled Release Society, 372, 609, 618, 2024年08月, ［国際誌］
英語, 研究論文（学術雑誌）, Therapeutically manipulating the stimulator of interferon genes (STING) pathway has promising potential for enhancing antitumor immunity. Agonists of this pathway (STING agonists) are being evaluated in clinical trials. Loading the STING agonists into lipid nanoparticles (LNPs) increases their safety and efficacy. We previously developed STING agonists loaded LNPs consisting of the ionizable lipid YSK12-C4 (YSK12-LNPs), which showed significant antitumor effects. However, it is largely unclear how the in vivo fate of STING agonists loaded LNPs affects the antitumor immune responses. In this study, we compared the YSK12-LNPs with LNPs composed of DLin-MC3-DMA (MC3-LNPs) showing different in vivo fates. Biodistribution and flow cytometry analyses of mouse tissues revealed that the MC3-LNPs delivered higher amounts of STING agonists to the liver than the YSK12-LNPs. Additionally, significantly more liver leukocytes internalized the MC3-LNPs than the YSK12-LNPs. In contrast, the YSK12-LNPs delivered higher amounts of STING agonists to the liver leukocytes than the MC3-LNPs, leading to the effective induction of innate immunity and inflammation in the tumors. However, the antitumor effects in the B16-F10 lung metastasis and CT26 tumor models were comparable. Interestingly, flow cytometry analyses suggested that the YSK12-LNPs were more likely to activate natural killer cells and M1 macrophages, while the MC3-LNPs were more likely to activate CD8+ T cells. Our data suggest that different antitumor immune response mechanisms may operate depending on the characteristics and distribution of the LNPs.

The impact of, and expectations for, lipid nanoparticle technology: From cellular targeting to organelle targeting.
Yusuke Sato, Takashi Nakamura, Yuma Yamada, Hideyoshi Harashima
Journal of controlled release : official journal of the Controlled Release Society, 370, 516, 527, 2024年06月, ［国際誌］
英語, 研究論文（学術雑誌）, The success of mRNA vaccines against COVID-19 has enhanced the potential of lipid nanoparticles (LNPs) as a system for the delivery of mRNA. In this review, we describe our progress using a lipid library to engineer ionizable lipids and promote LNP technology from the viewpoints of safety, controlled biodistribution, and mRNA vaccines. These advancements in LNP technology are applied to cancer immunology, and a potential nano-DDS is constructed to evaluate immune status that is associated with a cancer-immunity cycle that includes the sub-cycles in tumor microenvironments. We also discuss the importance of the delivery of antigens and adjuvants in enhancing the cancer-immunity cycle. Recent progress in NK cell targeting in cancer immunotherapy is also introduced. Finally, the impact of next-generation DDS technology is explained using the MITO-Porter membrane fusion-based delivery system for the organelle targeting of the mitochondria. We introduce a successful example of the MITO-Porter used in a cell therapeutic strategy to treat cardiomyopathy.

Overcoming thermostability challenges in mRNA-lipid nanoparticle systems with piperidine-based ionizable lipids.
Kazuki Hashiba, Masamitsu Taguchi, Sachiko Sakamoto, Ayaka Otsu, Yoshiki Maeda, Hirofumi Ebe, Arimichi Okazaki, Hideyoshi Harashima, Yusuke Sato
Communications biology, 7, 1, 556, 556, 2024年05月10日, ［国際誌］
英語, 研究論文（学術雑誌）, Lipid nanoparticles (LNPs) have emerged as promising platforms for efficient in vivo mRNA delivery owing to advancements in ionizable lipids. However, maintaining the thermostability of mRNA/LNP systems remains challenging. While the importance of only a small amount of lipid impurities on mRNA inactivation is clear, a fundamental solution has not yet been proposed. In this study, we investigate an approach to limit the generation of aldehyde impurities that react with mRNA nucleosides through the chemical engineering of lipids. We demonstrated that piperidine-based lipids improve the long-term storage stability of mRNA/LNPs at refrigeration temperature as a liquid formulation. High-performance liquid chromatography analysis and additional lipid synthesis revealed that amine moieties of ionizable lipids play a vital role in limiting reactive aldehyde generation, mRNA-lipid adduct formation, and loss of mRNA function during mRNA/LNP storage. These findings highlight the importance of lipid design and help enhance the shelf-life of mRNA/LNP systems.

Vaccination with a combination of STING agonist-loaded lipid nanoparticles and CpG-ODNs protects against lung metastasis via the induction of CD11bhighCD27low memory-like NK cells.
Alaa M Khalifa, Takashi Nakamura, Yusuke Sato, Hideyoshi Harashima
Experimental hematology & oncology, 13, 1, 36, 36, 2024年03月29日, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Natural killer (NK) cells are effective in attacking tumor cells that escape T cell attack. Memory NK cells are believed to function as potent effector cells in cancer immunotherapy. However, knowledge of their induction, identification, and potential in vivo is limited. Herein, we report on the induction and identification of memory-like NK cells via the action of a combination of a stimulator of interferon genes (STING) agonist loaded into lipid nanoparticles (STING-LNPs) and cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG-ODNs), and the potential of the inducted memory-like NK cells to prevent melanoma lung metastasis. METHODS: The antitumor effects of either the STING-LNPs, CpG-ODNs, or the combination therapy were evaluated using a B16-F10 lung metastasis model. The effect of the combined treatment was evaluated by measuring cytokine production. The induction of memory-like NK cells was demonstrated via flow cytometry and confirmed through their preventative effect. RESULTS: The combination of STING-LNPs and CpG-ODNs tended to enhance the production of interleukin 12 (IL-12) and IL-18, and exerted a therapeutic effect against B16-F10 lung metastasis. The combination therapy increased the population of CD11bhighCD27low NK cells. Although monotherapies failed to show preventative effects, the combination therapy induced a surprisingly strong preventative effect, which indicates that CD11bhighCD27low cells could be a phenotype of memory-like NK cells. CONCLUSION: As far as could be ascertained, this is the first report of the in vivo induction, identification, and confirmation of a phenotype of the memory-like NK cells through a prophylactic effect via the use of an immunotherapeutic drug. Our findings provide novel insights into the in vivo induction of CD11bhighCD27low memory-like NK cells thus paving the way for the development of efficient immunotherapies.

Development of Polymer-Lipid Hybrid Nanoparticles for Large-Sized Plasmid DNA Transfection.
Masatoshi Maeki, Shuya Uno, Kaisei Sugiura, Yusuke Sato, Yoichiro Fujioka, Akihiko Ishida, Yusuke Ohba, Hideyoshi Harashima, Manabu Tokeshi
ACS applied materials & interfaces, 16, 2, 2110, 2119, 2024年01月17日, ［国際誌］
英語, 研究論文（学術雑誌）, RNA and DNA delivery technologies using lipid nanoparticles (LNPs) have advanced significantly, as demonstrated by their successful application in mRNA vaccines. To date, commercially available RNA therapeutics include Onpattro, a 21 bp siRNA, and mRNA vaccines comprising 4300 nucleotides for COVID-19. However, a significant challenge remains in achieving efficient transfection, as the size of the delivered RNA and DNA increases. In contrast to RNA transfection, plasmid DNA (pDNA) transfection requires multiple steps, including cellular uptake, endosomal escape, nuclear translocation, transcription, and translation. The low transfection efficiency of large pDNA is a critical limitation in the development of artificial cells and their cellular functionalization. Here, we introduce polymer-lipid hybrid nanoparticles designed for efficient, large-sized pDNA transfection. We demonstrated that LNPs loaded with positively charged pDNA-polycation core nanoparticles exhibited a 4-fold increase in transfection efficiency for 15 kbp pDNA compared with conventional LNPs, which encapsulate a negatively charged pDNA-polycation core. Based on assessments of the size and internal structure of the polymer-lipid nanoparticles as well as hemolysis and cellular uptake analysis, we propose a strategy to enhance large-sized pDNA transfection using LNPs. This approach holds promise for accelerating the in vivo delivery of large-sized pDNA and advancing the development of artificial cells.

Cancer Immunotherapy with Lipid Nanoparticles Loaded with a Stimulator of Interferon Genes Agonist against Renal Tumor Lung Metastasis.
Takashi Nakamura, Shun Sasaki, Yusuke Sato, Hideyoshi Harashima
Pharmaceutics, 16, 1, 2023年12月26日, ［国際誌］
英語, 研究論文（学術雑誌）, Metastatic renal cell carcinoma (RCC) has a poor prognosis, and the major organ of metastasis is the lung. Immunotherapy with immune checkpoint inhibitors (ICIs) is the first-line therapy, but the response rates are low. Thus, the development of a more effective immunotherapy against metastatic RCC would be highly desirable. We previously demonstrated how a stimulator of an interferon gene (STING) agonist-loaded lipid nanoparticles (STING-LNPs) significantly activates natural killer (NK) cells and induces an antitumor effect against cases of melanoma lung metastasis that have shown ICI resistance. In this study, we evaluated the potential of using STING-LNPs in the treatment of lung metastatic RCC (Renca). An intravenous injection of STING-LNPs drastically decreased the amount of Renca tumor colonies. In contrast, monotherapies using ICIs showed no antitumor effect, and even a combination of ICI and STING-LNP therapies failed to enhance the antitumor effects. The main effector cells would be NK cells, and the activation of NK cells by the STING-LNPs may avoid the increased expression of immune checkpoint molecules. These findings provide useful insights into the development of an effective immunotherapy against metastatic RCC.

Genetically engineered transfusable platelets using mRNA lipid nanoparticles.
Jerry Leung, Colton Strong, Katherine E Badior, Madelaine Robertson, Xiaowu Wu, Michael A Meledeo, Emma Kang, Manoj Paul, Yusuke Sato, Hideyoshi Harashima, Andrew P Cap, Dana V Devine, Eric Jan, Pieter R Cullis, Christian J Kastrup
Science advances, 9, 48, eadi0508, 2023年12月, ［国際誌］
英語, 研究論文（学術雑誌）, Platelet transfusions are essential for managing bleeding and hemostatic dysfunction and could be expanded as a cell therapy due to the multifunctional role of platelets in various diseases. Creating these cell therapies will require modifying transfusable donor platelets to express therapeutic proteins. However, there are currently no appropriate methods for genetically modifying platelets collected from blood donors. Here, we describe an approach using platelet-optimized lipid nanoparticles containing mRNA (mRNA-LNP) to enable exogenous protein expression in human and rat platelets. Within the library of mRNA-LNP tested, exogenous protein expression did not require nor correlate with platelet activation. Transfected platelets retained hemostatic function and accumulated in regions of vascular damage after transfusion into rats with hemorrhagic shock. We expect this technology will expand the therapeutic potential of platelets.

Erratum: Branching Ionizable Lipids Can Enhance the Stability, Fusogenicity, and Functional Delivery of mRNA.
Kazuki Hashiba, Yusuke Sato, Masamitsu Taguchi, Sachiko Sakamoto, Ayaka Otsu, Yoshiki Maeda, Takuya Shishido, Masao Murakawa, Arimichi Okazaki, Hideyoshi Harashima
Small science, 3, 12, 2300285, 2300285, 2023年12月, ［国際誌］
英語, [This corrects the article DOI: 10.1002/smsc.202200071.].

Reprogramming activated hepatic stellate cells by siRNA-loaded nanocarriers reverses liver fibrosis in mice
Mahmoud A. Younis, Yusuke Sato, Yaser H.A. Elewa, Hideyoshi Harashima
Journal of Controlled Release, 361, 592, 603, 2023年09月, ［国際誌］
英語, 研究論文（学術雑誌）

Harnessing Topology and Stereochemistry of Glycidylamine‐Derived Lipid Nanoparticles for in Vivo mRNA Delivery to Immune Cells in Spleen and Their Application for Cancer Vaccination
Mahmoud M. Abd Elwakil, Ryota Suzuki, Alaa M. Khalifa, Rania M. Elshami, Takuya Isono, Yaser H.A. Elewa, Yusuke Sato, Takashi Nakamura, Toshifumi Satoh, Hideyoshi Harashima
Advanced Functional Materials, Wiley, 2023年07月26日
研究論文（学術雑誌）, Abstract

mRNA lipid nanoparticles (LNPs) have reached an inflection point and are now paving the way for a new wave of precision therapies. The design of nonhepatocyte RNA delivery systems without targeting ligands, however, remains a challenge. It is reported that the development of ligand‐free glycidylamine (GA) derivatives containing LNPs (GA‐LNPs) that preferentially deliver mRNA to immune cells in the spleen. Notably, it is demonstrated that the stereochemistry of GA‐lipids has a significant impact on their self‐assembly and in vitro and in vivo RNA delivery efficiency and tropism. This impact is dependent on the monomeric structure of GA and number of stereogenic centers. Furthermore, the nonlinear topology of GA lipid derivatives induced a sevenfold improvement in mRNA delivery efficiency. The top‐performing estriol‐GA05‐30 LNPs elicited strong antitumor activity in a therapeutic and prophylactic cancer model and are well tolerated in mice. These results highlight the significance of the chemistry of ionizable lipids for extrahepatic RNA delivery and indicated a promising direction for the development of next‐generation mRNA immunotherapies.

Delivering mRNA to a human NK cell line, NK-92 cells, by lipid nanoparticles.
Takashi Nakamura, Taisei Nakade, Yusuke Sato, Hideyoshi Harashima
International journal of pharmaceutics, 636, 122810, 122810, 2023年04月05日, ［国際誌］
英語, 研究論文（学術雑誌）, In cancer immunotherapy, therapeutic methods targeting NK are highly expected. NK cell-based therapy using NK-92, a human NK cell line, has been clinically evaluated. Delivering mRNA into NK-92 cells is a potent strategy for enhancing its functions. However, the use of lipid nanoparticles (LNP) for this purpose has not yet been evaluated. We previously developed a LNP that was composed of CL1H6 (CL1H6-LNP) for the efficient delivery of siRNA to NK-92 cells, and the use of this material for delivering mRNA to NK-92 cells is reported in this study. Compared with a DLin-MC3-DMA based LNP, used as a benchmark, the CL1H6-LNP caused a high mRNA expression intensity and a cell transfection efficiency of 100%. The efficient mRNA delivery by this CL1H6-LNP is attributed to the high affinity for NK-92 cells and the intense, rapid fusion with the endosomal membrane. It therefore appears that the CL1H6-LNP could be a useful non-viral vector for modifying the NK-92 functions by mRNA. Our findings also provide some insights into the design and development of LNPs for delivering mRNA to NK-92 and NK cells.

Mass production system for RNA-loaded lipid nanoparticles using piling up microfluidic devices
Masatoshi Maeki, Yuto Okada, Shuya Uno, Kaisei Sugiura, Yuichi Suzuki, Kento Okuda, Yusuke Sato, Masao Ando, Hiroyuki Yamazaki, Masaki Takeuchi, Akihiko Ishida, Hirofumi Tani, Hideyoshi Harashima, Manabu Tokeshi
Applied Materials Today, 2023年04月, ［査読有り］
研究論文（学術雑誌）

Self-homing nanocarriers for mRNA delivery to the activated hepatic stellate cells in liver fibrosis
Mahmoud A. Younis, Yusuke Sato, Yaser H.A. Elewa, Yasuhiro Kon, Hideyoshi Harashima
Journal of Controlled Release, 353, 685, 698, Elsevier BV, 2023年01月
研究論文（学術雑誌）

Non-Viral Gene Therapy in Trabecular Meshwork Cells to Prevent Fibrosis in Minimally Invasive Glaucoma Surgery
Jinyuan Luo, Greymi Tan, Kai Xin Thong, Konstantinos N. Kafetzis, Neeru Vallabh, Carl M. Sheridan, Yusuke Sato, Hideyoshi Harashima, Aristides D. Tagalakis, Cynthia Yu-Wai-Man
Pharmaceutics, 2022年11月
研究論文（学術雑誌）

Interval- and cycle-dependent combined effect of STING agonist loaded lipid nanoparticles and a PD-1 antibody.
Alaa M Khalifa, Takashi Nakamura, Yusuke Sato, Takanori Sato, Mamoru Hyodo, Yoshihiro Hayakawa, Hideyoshi Harashima
International journal of pharmaceutics, 624, 122034, 122034, 2022年08月25日, ［国際誌］
英語, 研究論文（学術雑誌）, Programmed cell death 1 (PD-1) blockade combination to other drugs have attracted the interest of scientists for treating tumors resistant to PD-1 blockade. In this study, the impact of the interval, order of administration, and number of cycles of immunotherapeutic combination of stimulator of interferon genes (STING) pathway agonist loaded lipid nanoparticle (STING-LNP) and PD-1 antibody for inducing the optimal combined antitumor activity against a melanoma lung metastasis is reported. One cycle had no effect, but two and three cycles resulted in a combinedantitumor effect. The interval between the administration was found to influence the induction of the combined effect. The second and third doses increased the gene expression of the NK cell activation marker, interferon γ (IFN-γ), PD-1 and a ligand of PD-1 (PD-L1), whereas the first dose failed. NK cells in the lung showed an increase in the expression of the activation markers and PD-1 after the second dose. The combined antitumor effect of this combination therapy against melanoma lung metastasis model could be dependent on the interval as well as the number of doses of STING-LNP.These findings suggest the importance of the protocol setting when combining a nano system loaded with an immune adjuvant and PD-1 antibody.

Extrahepatic targeting of lipid nanoparticles in vivo with intracellular targeting for future nanomedicines.
Takashi Nakamura, Yusuke Sato, Yuma Yamada, Mahmoud M Abd Elwakil, Seigo Kimura, Mahmoud A Younis, Hideyoshi Harashima
Advanced drug delivery reviews, 188, 114417, 114417, 2022年07月03日, ［国際誌］
英語, 研究論文（学術雑誌）, A new era of nanomedicines that involve nucleic acids/gene therapy has been opened after two decades in 21st century and new types of more efficient drug delivery systems (DDS) are highly expected and will include extrahepatic delivery. In this review, we summarize the possibility and expectations for the extrahepatic delivery of small interfering RNA/messenger RNA/plasmid DNA/genome editing to the spleen, lung, tumor, lymph nodes as well as the liver based on our studies as well as reported information. Passive targeting and active targeting are discussed in in vivo delivery and the importance of controlled intracellular trafficking for successful therapeutic results are also discussed. In addition, mitochondrial delivery as a novel strategy for nucleic acids/gene therapy is introduced to expand the therapeutic dimension of nucleic acids/gene therapy in the liver as well as the heart, kidney and brain.

mRNA-Loaded Lipid Nanoparticles Targeting Dendritic Cells for Cancer Immunotherapy
Kosuke Sasaki, Yusuke Sato, Kento Okuda, Kazuki Iwakawa, Hideyoshi Harashima
Pharmaceutics, 2022年07月
研究論文（学術雑誌）

On the size-regulation of RNA-loaded lipid nanoparticles synthesized by microfluidic device.
Kento Okuda, Yusuke Sato, Kazuki Iwakawa, Kosuke Sasaki, Nana Okabe, Masatoshi Maeki, Manabu Tokeshi, Hideyoshi Harashima
Journal of controlled release : official journal of the Controlled Release Society, 348, 648, 659, 2022年06月18日, ［国際誌］
英語, 研究論文（学術雑誌）, The use of lipid nanoparticles (LNPs) for nucleic acid delivery is now becoming a promising strategy with a number of clinical trials as vaccines or as novel therapies against a variety of genetic and infectious diseases. The use of microfluidics for the synthesis of the LNPs has attracted interest because of its considerable advantages over other conventional synthetic methods including scalability, reproducibility, and speed. However, despite the potential usefulness of large particles for nucleic acid delivery to dendritic cells (DCs) as a vaccine, the particle size of the LNPs prepared using microfluidics is typically limited to approximately from 30 to 100 nm. In this study, focusing on Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, the effect of some synthetic parameters, including total flow rate, flow rate ratio, buffer pH, lipid concentration, molar ratio of PEG-lipid as well as salt concentration, on particle size was systematically examined by means of the design of experiment approaches. The findings indicated that the simple addition of salt (e.g. NaCl) to a buffer containing nucleic acids contributed greatly to the synthesis of large LNPs over 200 nm and this effect was concentration-dependent with respect to the salt. The effect of salt on particle size was consistent with a Hofmeister series. The systemic injection of larger mRNA-loaded LNPs resulted in a higher transgene expression in mouse splenic DCs, a higher activation of various splenic immune cells, and had a superior effect as a therapeutic cancer vaccine in a syngeneic mouse model compared to the smaller-sized counterpart with constant lipid composition prepared with lower NaCl concentration. Collectively, size-regulation by the simple addition of salt is a promising strategy for developing potent LNPs.

Retrograde Axonal Transport of Liposomes from Peripheral Tissue to Spinal Cord and DRGs by Optimized Phospholipid and CTB Modification.
Takafumi Fukui, Hironao Tateno, Takashi Nakamura, Yuma Yamada, Yusuke Sato, Norimasa Iwasaki, Hideyoshi Harashima, Ken Kadoya
International journal of molecular sciences, 23, 12, 2022年06月15日, ［国際誌］
英語, 研究論文（学術雑誌）, Despite recent advancements in therapeutic options for disorders of the central nervous system (CNS), the lack of an efficient drug-delivery system (DDS) hampers their clinical application. We hypothesized that liposomes could be optimized for retrograde transport in axons as a DDS from peripheral tissues to the spinal cord and dorsal root ganglia (DRGs). Three types of liposomes consisting of DSPC, DSPC/POPC, or POPC in combination with cholesterol (Chol) and polyethylene glycol (PEG) lipid were administered to sciatic nerves or the tibialis anterior muscle of mature rats. Liposomes in cell bodies were detected with infrared fluorescence of DiD conjugated to liposomes. Three days later, all nerve-administered liposomes were retrogradely transported to the spinal cord and DRGs, whereas only muscle-administered liposomes consisting of DSPC reached the spinal cord and DRGs. Modification with Cholera toxin B subunit improved the transport efficiency of liposomes to the spinal cord and DRGs from 4.5% to 17.3% and from 3.9% to 14.3% via nerve administration, and from 2.6% to 4.8% and from 2.3% to 4.1% via muscle administration, respectively. Modification with octa-arginine (R8) improved the transport efficiency via nerve administration but abolished the transport capability via muscle administration. These findings provide the initial data for the development of a novel DDS targeting the spinal cord and DRGs via peripheral administration.

Innovative cancer nanomedicine based on immunology, gene editing, intracellular trafficking control.
Yuma Yamada, Yusuke Sato, Takashi Nakamura, Hideyoshi Harashima
Journal of controlled release : official journal of the Controlled Release Society, 348, 357, 369, 2022年06月09日, ［国際誌］
英語, 研究論文（学術雑誌）, The recent rapid progress in the area of drug delivery systems (DDS) has opened a new era in medicine with a strong linkage to understanding the molecular mechanisms associated with cancer survival. In this review, we summarize new cancer strategies that have recently been developed based on our DDS technology. Cancer immunotherapy will be improved based on the concept of the cancer immunity cycle, which focuses on dynamic interactions between various types of cancer and immune cells in our body. The new technology of genome editing will also be discussed with reference to how these new DDS technologies can be used to introduce therapeutic cargoes into our body. Lastly, a new organelle, mitochondria will be the focus of creating a new cancer treatment strategy by a MITO-Porter which can deliver macromolecules directly to mitochondria of cancer cells via a membrane fusion approach and the impact of controlled intracellular trafficking will be discussed.

Combined nano cancer immunotherapy based on immune status in a tumor microenvironment.
Takashi Nakamura, Kyoko Kawakami, Momoka Nomura, Yusuke Sato, Mamoru Hyodo, Hiroto Hatakeyama, Yoshihiro Hayakawa, Hideyoshi Harashima
Journal of controlled release : official journal of the Controlled Release Society, 345, 200, 213, 2022年05月, ［国際誌］
英語, 研究論文（学術雑誌）, Since the effect of cancer immunotherapy is largely dependent on the status of the immune system in the tumor microenvironment (TME), choice of therapy and the development of new therapies based on the immune status in the TME would be predicted to be effective. Unfortunately, the development of delivery systems for such therapy has been slow. Here, we defined a parameter of immune status in TME showing antitumor effects and demonstrated the cancer immunotherapy with an adjuvant loaded lipid nanoparticle (LNP), which was taken advantage the parameter. An analysis was carried out to determine the relationship between antitumor effects and gene expression (22 target genes) in tumors (MC38 and E.G7-OVA) that respond to the programmed cell death 1 (PD-1) antibody and non-responding tumors (B16-F10 and 4T1). The immune status showing an effective antitumor effect, which consisted of 10 genes, was then extracted. Treatment with the adjuvant loaded LNP caused a significant antitumor effect against an E.G7-OVA tumor, and the gene expression in the E.G7-OVA tumor was completely within the range of gene expression for showing an effective antitumor effect, as defined by the identified immune status panel (IS-panel-10). Although the treatment with the adjuvant loaded LNP failed to induce a sufficient antitumor effect against the 4T1 tumor, we succeeded in enhancing the antitumor effect by using a combination therapy that was adopted based on the analysis by the IS-panel-10 in the TME. The 10 genes were found to affect the prognosis in a variety of human cancers. Collectively, the findings reported herein demonstrate the potential of immune status analysis in the TME for developing cancer immunotherapies using a delivery system.

The hydrophobic tail of a pH-sensitive cationic lipid influences siRNA transfection activity and toxicity in human NK cell lines
Takashi Nakamura, Taisei Nakade, Koharu Yamada, Yusuke Sato, Hideyoshi Harashima
International Journal of Pharmaceutics, 609, 121140, 121140, Elsevier BV, 2021年11月
研究論文（学術雑誌）

Maximizing the Oral Bioavailability of Poorly Water-Soluble Drugs Using Novel Oil-Like Materials in Lipid-Based Formulations.
Saed Abbasi, Haruki Higashino, Yusuke Sato, Keiko Minami, Makoto Kataoka, Shinji Yamashita, Hideyoshi Harashima
Molecular pharmaceutics, 18, 9, 3181, 3189, 2021年08月05日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Lipid-based formulations, such as self-microemulsifying drug-delivery systems (SMEDDSs), are promising tools for the oral delivery of poorly water-soluble drugs. However, failure to maintain adequate aqueous solubility after coming into contact with gastrointestinal fluids is a major drawback. In this study, we examined the use of a novel cinnamic acid-derived oil-like material (CAOM) that binds drugs with a high affinity through π-π stacking and hydrophobic interactions, as an oil core in a SMEDDS for the oral delivery of fenofibrate in rats. The use of the CAOM in the SMEDDS resulted in an unprecedented enhancement in fenofibrate bioavailability, which exceeded the bioavailability values obtained using SMEDDSs based on corn oil, a conventional triglyceride oil, or Labrasol, an enhancer of intestinal permeation. Further characterization revealed that the CAOM SMEDDS does not alter the intestinal permeability and has no inhibitory activity on P-glycoprotein-mediated drug efflux. The results reported herein demonstrate the strong potential of CAOM formulations as new solubilizers for the efficient and safe oral delivery of drugs that have limited water solubility.

STING agonist loaded lipid nanoparticles overcome anti-PD-1 resistance in melanoma lung metastasis via NK cell activation.
Takashi Nakamura, Takanori Sato, Rikito Endo, Shun Sasaki, Naomichi Takahashi, Yusuke Sato, Mamoru Hyodo, Yoshihiro Hayakawa, Hideyoshi Harashima
Journal for immunotherapy of cancer, 9, 7, e002852, 2021年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Resistance to an immune checkpoint inhibitor (ICI) is a major obstacle in cancer immunotherapy. The causes of ICI resistance include major histocompatibility complex (MHC)/histocompatibility locus antigen (HLA) class I loss, neoantigen loss, and incomplete antigen presentation. Elimination by natural killer (NK) cells would be expected to be an effective strategy for the treatment of these ICI-resistant tumors. We previously demonstrated that a lipid nanoparticle containing a stimulator of an interferon gene (STING) agonist (STING-LNP) efficiently induced antitumor activity via the activation of NK cells. Thus, we evaluated the potential of reducing ICI resistance by STING-LNPs. METHODS: Lung metastasis of a B16-F10 mouse melanoma was used as an anti-programmed cell death 1 (anti-PD-1)-resistant mouse model. The mice were intravenously injected with the STING-LNP and the mechanism responsible for the improvement of anti-PD-1 resistance by the STING-LNPs was analyzed by RT-qPCR and flow cytometry. The dynamics of STING-LNP were also investigated. RESULTS: Although anti-PD-1 monotherapy failed to induce an antitumor effect, the combination of the STING-LNP and anti-PD-1 exerted a synergistic antitumor effect. Our results indicate that the STING-LNP treatment significantly increased the expression of CD3, CD4, NK1.1, PD-1 and interferon (IFN)-γ in lung metastases. This change appears to be initiated by the type I IFN produced by liver macrophages that contain the internalized STING-LNPs, leading to the systemic activation of NK cells that express PD-1. The activated NK cells appeared to produce IFN-γ, resulting in an increase in the expression of the PD ligand 1 (PD-L1) in cancer cells, thus leading to a synergistic antitumor effect when anti-PD-1 is administered. CONCLUSIONS: We provide a demonstration to show that a STING-LNP treatment can overcome PD-1 resistance in a B16-F10 lung metastasis model. The mechanism responsible for this indicates that NK cells are activated by stimulating the STING pathway which, in turn, induced the expression of PD-L1 on cancer cells. Based on the findings reported herein, the STING-LNP represents a promising candidate for use in combination therapy with anti-PD-1-resistant tumors.

Interferon signaling suppresses the unfolded protein response and induces cell death in hepatocytes accumulating hepatitis B surface antigen.
Ian Baudi, Masanori Isogawa, Federica Moalli, Masaya Onishi, Keigo Kawashima, Yuji Ishida, Chise Tateno, Yusuke Sato, Hideyoshi Harashima, Hiroyasu Ito, Tetsuya Ishikawa, Takaji Wakita, Matteo Iannacone, Yasuhito Tanaka
PLoS pathogens, 17, 5, e1009228, 2021年05月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Virus infection, such as hepatitis B virus (HBV), occasionally causes endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) is counteractive machinery to ER stress, and the failure of UPR to cope with ER stress results in cell death. Mechanisms that regulate the balance between ER stress and UPR are poorly understood. Type 1 and type 2 interferons have been implicated in hepatic flares during chronic HBV infection. Here, we examined the interplay between ER stress, UPR, and IFNs using transgenic mice that express hepatitis B surface antigen (HBsAg) (HBs-Tg mice) and humanized-liver chimeric mice infected with HBV. IFNα causes severe and moderate liver injury in HBs-Tg mice and HBV infected chimeric mice, respectively. The degree of liver injury is directly correlated with HBsAg levels in the liver, and reduction of HBsAg in the transgenic mice alleviates IFNα mediated liver injury. Analyses of total gene expression and UPR biomarkers' protein expression in the liver revealed that UPR is induced in HBs-Tg mice and HBV infected chimeric mice, indicating that HBsAg accumulation causes ER stress. Notably, IFNα administration transiently suppressed UPR biomarkers before liver injury without affecting intrahepatic HBsAg levels. Furthermore, UPR upregulation by glucose-regulated protein 78 (GRP78) suppression or low dose tunicamycin alleviated IFNα mediated liver injury. These results suggest that IFNα induces ER stress-associated cell death by reducing UPR. IFNγ uses the same mechanism to exert cytotoxicity to HBsAg accumulating hepatocytes. Collectively, our data reveal a previously unknown mechanism of IFN-mediated cell death. This study also identifies UPR as a potential target for regulating ER stress-associated cell death.

Novel PEGylated Lipid Nanoparticles Have a High Encapsulation Efficiency and Effectively Deliver MRTF-B siRNA in Conjunctival Fibroblasts.
Amisha Sanghani, Konstantinos N Kafetzis, Yusuke Sato, Salsabil Elboraie, Julia Fajardo-Sanchez, Hideyoshi Harashima, Aristides D Tagalakis, Cynthia Yu-Wai-Man
Pharmaceutics, 13, 3, 2021年03月13日, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, The master regulator of the fibrosis cascade is the myocardin-related transcription factor/serum response factor (MRTF/SRF) pathway, making it a key target for anti-fibrotic therapeutics. In the past, inhibitors and small interfering RNAs (siRNAs) targeting the MRTF-B gene have been deployed to counter fibrosis in the eye, with the latter showing promising results. However, the biggest challenge in implementing siRNA therapeutics is the method of delivery. In this study, we utilised the novel, pH-sensitive, cationic lipid CL4H6, which has previously demonstrated potent targeting of hepatocytes and endosomal escape, to safely and efficiently deliver an MRTF-B siRNA into human conjunctival fibroblasts. We prepared two lipid nanoparticle (LNP) formulations, incorporating targeting cleavable peptide cY in one of them, and measured their physicochemical properties and silencing effect in human conjunctival fibroblasts. Both proved to be non-cytotoxic at a concentration of 50 nM and effectively silenced the MRTF-B gene in vitro, with the targeting cleavable peptide not affecting the silencing efficiency [LNP with cY: 62.1% and 81.5% versus LNP without cY: 77.7% and 80.2%, at siRNA concentrations of 50 nM (p = 0.06) and 100 nM (p = 0.09), respectively]. On the other hand, the addition of the targeting cleavable peptide significantly increased the encapsulation efficiency of the LNPs from 92.5% to 99.3% (p = 0.0005). In a 3D fibroblast-populated collagen matrix model, both LNP formulations significantly decreased fibroblast contraction after a single transfection. We conclude that the novel PEGylated CL4H6-MRTF-B siRNA-loaded LNPs represent a promising therapeutic approach to prevent conjunctival fibrosis after glaucoma filtration surgery.

リポソームを応用した末梢組織から脊髄への低侵襲薬物輸送システムの開発
福井 隆史, 角家 健, 舘野 寛直, 中村 孝司, 山田 勇磨, 佐藤 悠介, 岩崎 倫政, 原島 秀吉
日本整形外科学会雑誌, 95, 2, S123, S123, (公社)日本整形外科学会, 2021年03月
日本語

Engineered ε-decalactone lipomers bypass the liver to selectively in vivo deliver mRNA to the lungs without targeting ligands
Mahmoud M. Abd Elwakil, Tianle Gao, Takuya Isono, Yusuke Sato, Yaser H. A. Elewa, Toshifumi Satoh, Hideyoshi Harashima
Materials Horizons, Royal Society of Chemistry (RSC), 2021年
研究論文（学術雑誌）,

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

Three-dimensional, symmetrically assembled microfluidic device for lipid nanoparticle production
Niko Kimura, Masatoshi Maeki, Kosuke Sasaki, Yusuke Sato, Akihiko Ishida, Hirofumi Tani, Hideyoshi Harashima, Manabu Tokeshi
RSC ADVANCES, 11, 3, 1430, 1439, 2021年01月
英語, 研究論文（学術雑誌）

The nanomedicine rush: New strategies for unmet medical needs based on innovative nano DDS.
Yusuke Sato, Takashi Nakamura, Yuma Yamada, Hideyoshi Harashima
Journal of controlled release : official journal of the Controlled Release Society, 330, 305, 316, 2020年12月20日, ［査読有り］, ［招待有り］, ［筆頭著者］, ［国際誌］
英語, 研究論文（学術雑誌）, The era of Nanomedicine has arrived with the approval of ONPATTRO™ by the FDA in 2018. Lipid nanoparticle (LNP) technology has succeeded in delivering siRNA to the human liver in genetic diseases and has also been applied to mRNA vaccinations for COVID-19 using a similar LNP technology. In this review, we focus on the current status of new lipids for use in LNP formulations including our original lipids (CL4H6/CL4C6/CL4D6) as well as mechanisms of targeting without a ligand. Clinical applications of nano DDS are moving forward rapidly in the field of cancer immunology since the successful introduction of OPDIVO™ in 2014. Antigen presentation and the maturation of immune cells can be controlled by nano DDS for cancer immunotherapy. YSK12-C4, a newly designed ionizable amino lipid can induce successful immune activation by silencing mRNA in DC and NK cells, which are expected to be evaluated for clinical use. Finally, new cancer therapy by targeting mitochondria involving the use of a MITO-Porter, a membrane fusion-type mitochondrial delivery system, has been introduced. The importance of delivering a photo sensitizer to mitochondria was clearly demonstrated in photodynamic cancer therapy. Clinical applications of MITO-Porters started in collaborative efforts with LUCA Science Co., Ltd. And was established in 2018. The future direction of Nanomedicine is discussed.

Lipid nanoparticles loaded with ribonucleoprotein-oligonucleotide complexes synthesized using a microfluidic device exhibit robust genome editing and hepatitis B virus inhibition.
Yuichi Suzuki, Haruno Onuma, Risa Sato, Yusuke Sato, Akari Hashiba, Masatoshi Maeki, Manabu Tokeshi, Mohammad Enamul Hoque Kayesh, Michinori Kohara, Kyoko Tsukiyama-Kohara, Hideyoshi Harashima
Journal of controlled release : official journal of the Controlled Release Society, 330, 61, 71, 2020年12月14日, ［査読有り］, ［筆頭著者, 責任著者］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system has considerable therapeutic potential for use in treating a wide range of intractable genetic and infectious diseases including hepatitis B virus (HBV) infections. While non-viral delivery technologies for the CRISPR/Cas system are expected to have clinical applications, difficulties associated with the clinically relevant synthesis of formulations and the poor efficiency of delivery severely hinder therapeutic genome editing. We report herein on the production of a lipid nanoparticle (LNP)-based CRISPR/Cas ribonucleoprotein (RNP) delivery nanoplatform synthesized using a clinically relevant mixer-equipped microfluidic device. DNA cleavage activity and the aggregation of Cas enzymes was completely avoided under the optimized synthetic conditions. The optimized formulation, which was identified through 2 steps of design of experiments, exhibited excellent gene disruption (up to 97%) and base substitution (up to 23%) without any apparent cytotoxicity. The addition of negative charges to the RNPs by complexing single-stranded oligonucleotide (ssON) significantly enhanced the delivery of both Cas9 and Cpf1 RNPs. The optimized formulation significantly suppressed both HBV DNA and covalently closed circular DNA (cccDNA) in HBV-infected human liver cells compared to adeno-associated virus type 2 (AAV2). These findings represent a significant contribution to the development of CRISPR/Cas RNP delivery technology and its practical applications in genome editing therapy.

The use of design of experiments with multiple responses to determine optimal formulations for in vivo hepatic mRNA delivery.
Akari Hashiba, Manaya Toyooka, Yusuke Sato, Masatoshi Maeki, Manabu Tokeshi, Hideyoshi Harashima
Journal of controlled release : official journal of the Controlled Release Society, 327, 467, 476, 2020年11月10日, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Although great advances have been made in the delivery of short RNAs by lipid nanoparticles (LNPs), the optimal formulation composition and physicochemical properties of LNPs for long RNA (including mRNA) remain unclear. In the present study, we optimized the lipid composition of liver-targeted mRNA-loaded LNPs that were prepared with pH-sensitive cationic lipids that had been previously designed for siRNA delivery through a two stepped design of experiment (DoE). Multiple responses including physicochemical properties, gene expression, and liver-specificity were analyzed in order, not only to understand the role of each formulation parameter, but also to examine parameters that would be difficult to predict. We found that particle size and the PEG-to-phospholipid (PEG/PL) ratio were additional key factors for liver-specific gene expression in addition to the other formulation factors. The optimized formulation showed a better gene expression compared to other lipid formulations from industry leaders. These findings suggest that a "DoE with multiple responses" approach can be used to predict significant parameters and permit optimized formulations to be prepared more efficiently.

Evolution of drug delivery system from viewpoint of controlled intracellular trafficking and selective tissue targeting toward future nanomedicine.
Yuma Yamada, Yusuke Sato, Takashi Nakamura, Hideyoshi Harashima
Journal of controlled release : official journal of the Controlled Release Society, 327, 533, 545, 2020年11月10日, ［査読有り］, ［招待有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Due to the rapid changes that have occurred in the field of drug discovery and the recent developments in the early 21st century, the role of drug delivery systems (DDS) has become increasingly more important. For the past 20 years, our laboratory has been developing gene delivery systems based on lipid-based delivery systems. One of our efforts has been directed toward developing a multifunctional envelope-type nano device (MEND) by modifying the particle surface with octaarginine, which resulted in a remarkably enhanced cellular uptake and improved intracellular trafficking of plasmid DNA (pDNA). When we moved to in vivo applications, however, we were faced with the PEG-dilemma and we shifted our strategy to the incorporation of ionizable cationic lipids into our system. This resulted in some dramatic improvements over our original design and this can be attributed to the development of a new lipid library. We have also developed a mitochondrial targeting system based on a membrane fusion mechanism using a MITO-Porter, which can deliver nucleic acids/pDNA into the matrix of mitochondria. After the appearance of antibody medicines, Opdivo, an immune checkpoint inhibitor, has established cancer immunology as the 4th strategy in cancer therapy. Our DDS technologies can also be applied to this new field of cancer therapy to cure cancer by controlling our immune mechanisms. The latest studies are summarized in this review article.

New Design Strategies for Controlling the Rate of Hydrophobic Drug Release from Nanoemulsions in Blood Circulation.
Saed Abbasi, Yusuke Sato, Kazuaki Kajimoto, Hideyoshi Harashima
Molecular pharmaceutics, 17, 10, 3773, 3782, 2020年10月05日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The intravenous administration of drug-loaded nanoparticles (NPs) is needed to achieve passive or active targeting in disease tissues. However, when the loaded drug is a hydrophobic small molecule, the NPs fail to reach adequate plasma drug concentrations mainly because of premature drug release. The pharmacokinetics of such drugs can be controlled by covalent modification, but this approach could compromise the safety or potency of the drug. In this study, we investigated two formulation parameters that could be used to improve the plasma concentrations of unmodified drugs that are loaded in a nanoemulsion (NE), a core-shell type NP. The first parameter is the loading ratio, and the second is the affinity of the drug to the core. Optimized NEs with reduced drug loading and with a high drug-core affinity resulted in a 12.4- and 11.2-fold increase in the plasma retention of curcumin and paclitaxel, respectively. Our strategy for enhancing the drug-core interaction affinity relied on mixing oils and surfactants to achieve cooperativity in noncovalent interactions, such as hydrophobic interactions, hydrogen bonding, and π-π stacking, which was further confirmed by theoretical calculations of interaction affinities. Finally, we report on the development of a cinnamic acid-derived oil-like material as a novel drug vehicle with exceptional solubilizing ability that could be used in intravenous formulations of NEs.

核酸医薬とナノメディシン 多機能性エンベロープ型ナノ構造体の開発とナノ医療への展開　　　　　　　　　　　　　　　
原島 秀吉, 佐藤 悠介, 中村 孝司, 山田 勇磨
日本癌学会総会記事, 79回, ML13, ML13, (一社)日本癌学会, 2020年10月
英語

Inhibitory Effect of a Human MicroRNA, miR-6133-5p, on the Fibrotic Activity of Hepatic Stellate Cells in Culture.
Susumu Hamada-Tsutsumi, Masaya Onishi, Kentaro Matsuura, Masanori Isogawa, Keigo Kawashima, Yusuke Sato, Yasuhito Tanaka
International journal of molecular sciences, 21, 19, 7251, 2020年10月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: We recently identified 39 human microRNAs, which effectively suppress hepatitis B virus (HBV) replication in hepatocytes. Chronic HBV infection often results in active, hepatitis-related liver fibrosis; hence, we assessed whether any of these microRNAs have anti-fibrotic potential and predicted that miR-6133-5p may target several fibrosis-related genes. METHODS: The hepatic stellate cell line LX-2 was transfected with an miR-6133-5p mimic and subsequently treated with Transforming growth factor (TGF)-β. The mRNA and protein products of the COL1A1 gene, encoding collagen, and the ACTA2 gene, an activation marker of hepatic stellate cells, were quantified. RESULTS: The expression of COL1A1 and ACTA2 was markedly reduced in LX-2 cells treated with miR-6133-5p. Interestingly, phosphorylation of c-Jun N-terminal kinase (JNK) was also significantly decreased by miR-6133-5p treatment. The expression of several predicted target genes of miR-6133-5p, including TGFBR2 (which encodes Transforming Growth Factor Beta Receptor 2) and FGFR1 (which encodes Fibroblast Growth Factor Receptor 1), was also reduced in miR-6133-5p-treated cells. The knockdown of TGFBR2 by the corresponding small interfering RNA greatly suppressed the expression of COL1A1 and ACTA2. Treatment with the JNK inhibitor, SP600125, also suppressed COL1A1 and ACTA2 expression, indicating that TGFBR2 and JNK mediate the anti-fibrotic effect of miR-6133-5p. The downregulation of FGFR1 may result in a decrease of phosphorylated Akt, ERK (extracellular signal-regulated kinase), and JNK. CONCLUSION: miR-6133-5p has a strong anti-fibrotic effect, mediated by inactivation of TGFBR2, Akt, and JNK.

Lipid nanoparticles fuse with cell membranes of immune cells at low temperatures leading to the loss of transfection activity.
Takashi Nakamura, Koharu Yamada, Yusuke Sato, Hideyoshi Harashima
International journal of pharmaceutics, 587, 119652, 119652, 2020年09月25日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Delivering nucleic acid using a non-viral vector is a potent strategy for gene modification and controlling gene expression in immune cell therapy. Since the low-temperature storage (0-4 °C) or cryopreservation of cells are indispensable for performing immune cell therapy, we investigated the interactions between an siRNA-loaded lipid nanoparticle (LNP), a multifunctional envelope-type nanodevice (MEND) containing YSK12-C4 (YSK12-MEND), and human immune cell lines (NK-92 and Jurkat) at low-temperature and its effect on transfection activity. The YSK12-MEND readily bound to the cell membrane of NK-92 cells at low-temperature, but no internalization of the YSK12-MEND by cells was observed, even after returning the temperature to 37 °C. Gene silencing activity was completely impaired. The cause of this inhibition appears to be membrane fusion between the YSK12-MEND and cell membrane at the low-temperature. Collectively, our results suggest that the exposure of siRNA-loaded LNPs to cells at low-temperature should be avoided in defining transfection protocols in immune cell therapy.

Manipulating the function of tumor-associated macrophages by siRNA-loaded lipid nanoparticles for cancer immunotherapy.
Nour Shobaki, Yusuke Sato, Yuichi Suzuki, Nana Okabe, Hideyoshi Harashima
Journal of controlled release : official journal of the Controlled Release Society, 325, 235, 248, 2020年09月10日, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, The tumor-microenvironment contains large numbers of tumor-associated macrophages (TAMs) which are largely M2 phenotypes and are involved in pro-tumorous functions. Targeting TAMs so as to manipulate them and to modify their functions could be a novel immunotherapy for the treatment of cancer. Such a strategy would involve targeting TAMs with short interfering RNA (siRNA) to modify their functions by silencing certain genes that are responsible for their M2 polarization. In this study, a lipid nanoparticle (LNP) formulation was used to target and deliver siRNA to TAMs. The LNP was mainly composed of a novel, pH-sensitive cationic lipid, referred to as the CL4H6 lipid, which had previously been optimized to target hepatocytes. The optimized siRNA-loaded CL4H6-LNPs were selectively and efficiently taken up and showed strong gene silencing activity in TAMs in a human tumor xenograft model in nude mice. Furthermore, an anti-tumor therapeutic response in the same tumor model was obtained by targeting TAMs using the optimized siRNA-loaded CL4H6-LNPs. The anti-tumor therapeutic response was obtained through the silencing of the signal transducer and activator of transcription 3 (STAT3) and hypoxia inducible factor 1 α (HIF-1α), which resulted in an increase in the level of infiltrated macrophage (CD11b+ cells) into the tumor-microenvironment (TME) as well as a tendency to increase the concentration of M1 macrophages (CD169+ cells). The treatment also resulted in reversing the pro-tumorous functions of TAMs -mainly angiogenesis and tumor cell activation-, as evidenced by a decrease in the related gene expression at the mRNA level. This research has promising clinical and pharmaceutical applications for achieving novel macrophage-based cancer immunotherapy.

Mitochondrial Delivery of an Anticancer Drug Via Systemic Administration Using a Mitochondrial Delivery System That Inhibits the Growth of Drug-Resistant Cancer Engrafted on Mice.
Yuma Yamada, Reina Munechika, Satrialdi, Fumika Kubota, Yusuke Sato, Yu Sakurai, Hideyoshi Harashima
Journal of pharmaceutical sciences, 109, 8, 2493, 2500, 2020年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Mitochondrial delivery of an anticancer drug targeting cancer cells would eventually result in cell death. To achieve this, a drug delivery system targeting mitochondria is needed. We recently developed a MITO-Porter, a liposome that delivers its cargo to mitochondria. We reported that such a MITO-Porter could deliver doxorubicin (DOX), an anticancer drug, to mitochondria in OS-RC-2 cells, a drug resistant cancer cell, resulting in inhibiting the cell growth, based in in vitro experiments. Herein, we report on validating the benefit of such a therapeutic strategy for treating drug resistant cancers by the in vivo targeting of mitochondria. We prepared a DOX-MITO-Porter, in which DOX was encapsulated in the MITO-Porter and optimized its retention in blood circulation. When the DOX-MITO-Porter was administered to mice bearing OS-RC-2 cells via tail vein injection, tumor size was significantly decreased, compared to DOX itself and to the DOX-encapsulated polyethylene glycol-modified liposome (DOX-PEG-LP). Intracellular observation confirmed that the DOX-MITO-Porter had accumulated in tumor mitochondria. It was also found a relationship between anti-tumor effect and the mitochondrial function, as indicated by the depolarization of mitochondrial membrane potential. This study provides support for the utility of an in vivo mitochondrial delivery system in drug resistant cancer therapies.

Development of a Microfluidic-Based Post-Treatment Process for Size-Controlled Lipid Nanoparticles and Application to siRNA Delivery.
Niko Kimura, Masatoshi Maeki, Yusuke Sato, Akihiko Ishida, Hirofumi Tani, Hideyoshi Harashima, Manabu Tokeshi
ACS applied materials & interfaces, 12, 30, 34011, 34020, 2020年07月29日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Microfluidic methodologies for preparation of lipid nanoparticles (LNPs) based on an organic solvent injection method enable precise size control of the LNPs. After preparation of LNPs, the organic solvent injection method needs some post-treatments, such as overnight dialysis or direct dilution with a buffer solution. LNP production using the microfluidic-based organic solvent injection method is dominated by kinetics rather than thermodynamics. Kinetics of ethanol removal from the inner and outer membranes of LNPs could induce a structural change in the membrane that could lead to fusion of LNPs. However, the effects of microfluidic post-treatment on the final size of LNPs have not been sufficiently understood. Herein, we investigated the effect of the post-treatment processes on the final product size of LNPs in detail. A simple baffle device and a model lipid system composed of a neutral phospholipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC) and cholesterol were used to produce the LNPs. We demonstrated that flow conditions of the post-treatment diluting the remaining ethanol in the LNP suspension affected the final product size of LNPs. Based on the findings, we developed an integrated baffle device composed of an LNP production region and a post-treatment region for a microfluidic-based LNP production system; this integrated baffle device prevented the undesirable aggregation or fusion of POPC LNPs even for the high-lipid-concentration condition. Finally, we applied our concept to small interfering RNA (siRNA) delivery and confirmed that no significant effects due to the continuous process occurred on the siRNA encapsulation efficiency, biological distribution, and knockdown activity. The microfluidic post-treatment method is expected to contribute to the production of LNPs for practical applications and the development of novel LNP-based nanomedicines.

Different kinetics for the hepatic uptake of lipid nanoparticles between the apolipoprotein E/low density lipoprotein receptor and the N-acetyl-d-galactosamine/asialoglycoprotein receptor pathway.
Yusuke Sato, Yoshiyuki Kinami, Kazuki Hashiba, Hideyoshi Harashima
Journal of controlled release : official journal of the Controlled Release Society, 322, 217, 226, 2020年06月10日, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Lipid nanoparticles (LNPs) are one of the more promising technologies for efficiently delivering nucleic acids in vivo. Hepatocytes are the primary target cells of LNPs that are delivered via the apolipoprotein E (ApoE)-low density lipoprotein receptor (LDLR) pathway, an endogenous targeting pathway. This robust targeting mechanism results in the specific and efficient delivery of nucleic acids to hepatocytes. Trivalent N-acetyl-D-galactosamine (GalNAc) is known to be a high-affinity exogenous ligand against the asialoglycoprotein receptor (ASGPR), which is highly expressed on hepatocytes. In this study, we report that the kinetics of the hepatic uptake process between the two types of targeting pathways are different. Rapid blood clearance, accumulation to the space of Disse and a subsequent slow cellular uptake was observed in the case of the endogenous ApoE-LDLR pathway. On the other hand, both blood clearance and cellular uptake were more gradual in the case of the exogenous GalNAc-ASGPR pathway. Interactions between ApoE-bound LNPs and hepatic heparan sulfate proteoglycans (HSPGs) were involved in the rapid blood clearance and accumulation to the space of Disse in the case of the endogenous pathway. The findings presented here contribute to a more precise understanding of the mechanism of hepatic uptake and to the rational design of hepatocyte-targeting nanoparticles.

脂質ナノ粒子の特性がリンパ節送達とリンパ節内分布へ与える影響　　　　　　　　　　　　　　　
中村 孝司, 河合 美典, 佐藤 悠介, 真栄城 正寿, 渡慶次 学, 原島 秀吉
日本薬剤学会年会講演要旨集, 35年会, 171, 171, (公社)日本薬剤学会, 2020年05月
日本語

【核酸創薬に貢献するバイオマテリアル】バイオマテリアルに基づいた核酸ナノ医療の創製　　　　　　　　　　　　　　　
山田 勇磨, 中村 孝司, 佐藤 悠介, 原島 秀吉
バイオマテリアル-生体材料-, 38, 2, 92, 99, 日本バイオマテリアル学会, 2020年04月
日本語

The Effect of Size and Charge of Lipid Nanoparticles Prepared by Microfluidic Mixing on Their Lymph Node Transitivity and Distribution.
Takashi Nakamura, Minori Kawai, Yusuke Sato, Masatoshi Maeki, Manabu Tokeshi, Hideyoshi Harashima
Molecular pharmaceutics, 17, 3, 944, 953, 2020年03月02日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Because the lymph node (LN) is a critical organ for inducing immune responses against pathogens and cancers, the transport of immune functional molecules such as antigens and adjuvants to LNs by delivery systems is a useful strategy for the effective outcome of an immune response. The size and charge of a delivery system largely affect the transitivity to and distribution within LN. Although pH-sensitive lipid nanoparticles (LNPs) prepared by microfluidic mixing are the latest delivery system to be applied clinically, the effects of their size and charge on the transitivity to and distribution within LN are currently unknown. We investigated the size and charge effect of LNPs prepared by microfluidic mixing on transitivity to and distribution within LNs. A 30 nm-sized LNP (30-LNP) was efficiently translocated to LNs and was taken up by CD8+ dendritic cells, while the efficiency was drastically decreased in the cases of 100 and 200 nm-sized LNPs. Furthermore, a comparative study between neutral, positively, and negatively charged 30-LNP revealed that the negative 30-LNP moved to the LN more efficiently than the other LNPs. Interestingly, the negative 30-LNP reached the deep cortex, namely, the T cell zone. Our findings provide informative insights for designing LN-targeting LNPs prepared by microfluidic mixing and for the translocation of nanoparticles in LNs.

Hydrophobic scaffolds of pH-sensitive cationic lipids contribute to miscibility with phospholipids and improve the efficiency of delivering short interfering RNA by small-sized lipid nanoparticles.
Yusuke Sato, Nana Okabe, Yusuke Note, Kazuki Hashiba, Masatoshi Maeki, Manabu Tokeshi, Hideyoshi Harashima
Acta biomaterialia, 102, 341, 350, Elsevier {BV}, 2020年01月15日, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Despite the fact that small-sized lipid nanoparticles (LNPs) are important for improved tissue penetration and efficient drug delivery, their poor stability and intracellular trafficking significantly hinders their use as potent small-sized LNPs. It has been reported that both the diffusion of lipid components from LNPs and the adsorption of proteins on the surface of LNPs are responsible for their decreased potency. To overcome this issue, we focused on the chemical structure of hydrophobic scaffolds of pH-sensitive cationic lipids with various lengths and shapes. LNPs composed of a pH-sensitive cationic lipid with long, linear scaffolds induced gene silencing in a dose-dependent manner, while LNPs with a classical scaffold length (C18) failed. Replacing the helper lipid from cholesterol to egg sphingomyelin (ESM) resulted in the formation of smaller LNPs with a diameter of ~22 nm and enhanced gene silencing activity. Most of the ESMs were located in the outer layer and functioned to stabilize the LNPs. Long, linear scaffolds contributed to immiscibility with phosphocholine-containing lipids including ESM. This contribution was dependent on the scaffold length of pH-sensitive cationic lipids. Although phosphocholine-containing lipids usually inhibit membrane fusion-mediated endosomal escape, long, linear scaffolds contributed to avoiding the inhibitory effect and to enhance the potency of the LNPs. These findings provide useful information needed for the rational design of pH-sensitive cationic lipid structures and the selection of appropriate helper lipids and will facilitate the development of highly potent small-sized LNPs. STATEMENT OF SIGNIFICANCE: Despite the fact that small-sized lipid nanoparticles (LNPs) are important for improved tissue penetration and efficient drug delivery, the size reduction-associated decrease in the stability and intracellular trafficking significantly hinders the development of potent small-sized LNPs. Our limited understanding of the mechanism underlying the reduced potency has also hindered the development of more potent small-sized LNPs. The findings of the present study indicate that long and linear hydrophobic scaffolds of pH-sensitive cationic lipids could overcome the loss of efficiency for nucleic acid delivery. In addition, the long hydrophobic scaffolds led to immiscibility with neutral phospholipids, resulting in efficient endosomal escape. These findings provide useful information needed for the rational design of pH-sensitive cationic lipid structures and will facilitate the development of highly potent small-sized LNPs.

Recent advances in the targeting of systemically administered non-viral gene delivery systems.
Ikramy A Khalil, Yusuke Sato, Hideyoshi Harashima
Expert opinion on drug delivery, 16, 10, 1037, 1050, 2019年10月, ［査読有り］, ［招待有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Introduction: Systemically administered non-viral gene delivery systems face multiple biological barriers that decrease their efficiency. These systems are rapidly cleared from the circulation and sufficient concentrations do not accumulate in diseased tissues. A number of targeting strategies can be used to provide for sufficient accumulation in the desired tissues to achieve a therapeutic effect. Areas covered: We discuss recent advances in the targeting of non-viral gene delivery systems to different tissues after systemic administration. We compare passive and active targeting applied for tumor delivery and propose some strategies that can be used to overcome the drawbacks of each case. We also discuss targeting the liver and lungs as two particularly important organs in gene therapy. Expert opinion: There is currently no optimum non-viral gene delivery system for targeting genes to specific tissues. The dose delivered to tumor tissues using passive targeting is low and shows a high patient variation. Although active targeting can enhance binding to specific cells, only a few reports are available to support its value in vivo. The design of smart nanocarriers for promoting active targeting is urgently needed and targeting the endothelium is a promising strategy for gene delivery to tumors as well as other organs.

Innovative nanotechnologies for enhancing nucleic acids/gene therapy: Controlling intracellular trafficking to targeted biodistribution.
Takashi Nakamura, Yuma Yamada, Yusuke Sato, Ikramy A Khalil, Hideyoshi Harashima
Biomaterials, 218, 119329, 119329, 2019年10月, ［査読有り］, ［招待有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Nanomedicine promises to play an important role in next generation therapy, including Nucleic acid/Gene therapy. To accomplish this, innovative nanotechnologies will be needed to support nanomedicine by controlling not only the biodistribution but also the intracellular trafficking of macromolecules such as RNA/DNA. A multifunctional envelope-type nano device (MEND) was developed to meet this requirement. We herein provide an update regarding the functions of the MEND system focusing on the introduction of different functional biomaterials that enhance efficiency. The octaarginine (R8) peptide enhances cellular uptake and controls intracellular trafficking to induce synergism in transgene expression. The R8 was also used for developing a MITO-Porter system for mitochondrial targeting. The function of the MITO-Porter system was extended by developing a mitochondrial reporter gene for mitochondrial gene therapy. For efficient in vivo gene delivery, new pH-sensitive lipids have been introduced to achieve controlled biodistribution and to enhance endosomal escape. For example, the CL4H6 lipid exerts a more efficient in vivo gene silencing than that of ONPATTROTM, a preparation that has been approved by the US Food and Drug Administration. We further summarize new technologies that have been successfully applied to cancer immunotherapy leading to the introduction of a new strategy based on the concept of the Cancer-Immunity Cycle.

A study of the endocytosis mechanism and transendothelial activity of lung-targeted GALA-modified liposomes.
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年08月10日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

The silencing of indoleamine 2,3-dioxygenase 1 (IDO1) in dendritic cells by siRNA-loaded lipid nanoparticles enhances cell-based cancer immunotherapy.
Rikito Endo, Takashi Nakamura, Kyoko Kawakami, Yusuke Sato, Hideyoshi Harashima
Scientific reports, 9, 1, 11335, 11335, 2019年08月05日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Cell-based therapy using dendritic cells (DC) represents a potent cancer immunotherapy. However, activated DC express indoleamine 2,3-dioxygenase 1 (IDO1), a counter-regulatory and tolerogenic molecule, leading to the inhibition of T cell activation and the promotion of T cell differentiation into regulatory T cells. Silencing the IDO1 gene in DC by small interfering RNA (siRNA) represents a potent therapeutic strategy. We report on the successful and efficient introduction of a siRNA targeting IDO1 into mouse DCs by a means of a multifunctional envelope-type nanodevice (MEND) containing a YSK12-C4 (YSK12-MEND). The YSK12-C4 has both fusogenic and cationic properties. The YSK12-MEND induced an effective level of gene silencing of IDO1 at siRNA doses in the range of 1-20 nM, a concentration that commercially available transfection reagents are not able to silence. The YSK12-MEND mediated IDO1 silencing had no effect on the characteristic determinants of DC phenotype such as CD11c, CD80 and MHC class II. The silencing of IDO1 in DC by the YSK12-MEND significantly enhanced the antitumor effect against E.G7-OVA tumor. Moreover, a decrease in the numbers of regulatory T cells in the tumor was observed in mice that were treated with the IDO1-silenced DC. The YSK12-MEND appears to be a potent delivery system for IDO1-silenced DC based cancer immunotherapy.

The Use of a Microfluidic Device to Encapsulate a Poorly Water-Soluble Drug CoQ10 in Lipid Nanoparticles and an Attempt to Regulate Intracellular Trafficking to Reach Mitochondria.
Mitsue Hibino, Yuma Yamada, Naoki Fujishita, Yusuke Sato, Masatoshi Maeki, Manabu Tokeshi, Hideyoshi Harashima
Journal of pharmaceutical sciences, 108, 8, 2668, 2676, 2019年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）

Lung-Endothelium-Targeted Nanoparticles Based on a pH-Sensitive Lipid and the GALA Peptide Enable Robust Gene Silencing and the Regression of Metastatic Lung Cancer
Mahmoud M. Abd Elwakil, Ikramy A. Khalil, Yaser H. A. Elewa, Kenji Kusumoto, Yusuke Sato, Nour Shobaki, Yasuhiro Kon, Hideyoshi Harashima
Advanced Functional Materials, 29, 18, Wiley-VCH Verlag, 2019年05月02日
英語, 研究論文（学術雑誌）

Understanding structure-activity relationships of pH-sensitive cationic lipids facilitates the rational identification of promising lipid nanoparticles for delivering siRNAs in vivo.
Yusuke Sato, Kazuki Hashiba, Kosuke Sasaki, Masatoshi Maeki, Manabu Tokeshi, Hideyoshi Harashima
Journal of controlled release : official journal of the Controlled Release Society, 295, 140, 152, 2019年02月10日, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）

【加速する核酸医薬開発】siRNAのDDS開発の現状と展望
佐藤 悠介, 原島 秀吉
医薬ジャーナル, 55, 2, 615, 619, (株)医薬ジャーナル社, 2019年02月
日本語

Two modes of toxicity of lipid nanoparticles containing a pH-sensitive cationic lipid on human A375 and A375-SM melanoma cell lines,　　　　　　　　　　　　　　　
アロバロウル アーメッド, 佐藤 悠介, 間石 奈湖, 樋田 京子
BPB Reports, 2, 48, 55, 2019年, ［査読有り］, ［責任著者］
英語, 研究論文（学術雑誌）

Neutralization of negative charges of siRNA results in improved safety and efficient gene silencing activity of lipid nanoparticles loaded with high levels of siRNA.
Sato Y, Matsui H, Sato R, Harashima H
Journal of controlled release : official journal of the Controlled Release Society, 284, 179, 187, 2018年08月, ［査読有り］, ［筆頭著者］

Reducing the Cytotoxicity of Lipid Nanoparticles Associated with a Fusogenic Cationic Lipid in a Natural Killer Cell Line by Introducing a Polycation-Based siRNA Core.
Nakamura T, Yamada K, Fujiwara Y, Sato Y, Harashima H
Molecular pharmaceutics, 15, 6, 2142, 2150, 2018年06月, ［査読有り］

微小化脂質ナノ粒子によるアジュバントのリンパ節送達　　　　　　　　　　　　　　　
中村 孝司, 河合 美典, 佐藤 悠介, 真栄城 正寿, 渡慶次 学, 原島 秀吉
日本薬剤学会年会講演要旨集, 33年会, 193, 193, (公社)日本薬剤学会, 2018年05月
日本語

Development of the iLiNP Device: Fine Tuning the Lipid Nanoparticle Size within 10 nm for Drug Delivery
N. Kimura, M. Maeki, Y. Sato, Y. Note, A. Ishida, H. Tani, H. Harashima, M. Tokeshi
ACS Omega, 3, 5, 5044, 5051, 2018年05月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The precise size control of the lipid nanoparticle (LNP)-based nanodrug delivery system (DDS) carriers, such as 10 nm size tuning of LNPs, is one major challenge for the development of next-generation nanomedicines. Size-controlled LNPs would realize size-selective tumor targeting and deliver DNA and RNA to target tumor tissues effectively by passing through the stromal cells. Herein, we developed a baffle mixer device named the invasive lipid nanoparticle production device, or iLiNP device for short, which has a simple two-dimensional microchannel and mixer structure, and we achieved the first reported LNP size tuning at 10 nm intervals in the size range from 20 to 100 nm. In comparison with the conventional LNP preparation methods and reported micromixer devices, our iLiNP device showed better LNP size controllability, robustness of device design, and LNP productivity. Furthermore, we prepared 80 nm sized LNPs with encapsulated small interfering RNA (siRNA) using the iLiNP device; these LNPs effectively performed as nano-DDS carriers in an in vivo experiment. We expect iLiNP devices will become novel apparatuses for LNP production in nano-DDS applications.

Synergism between a cell penetrating peptide and a pH-sensitive cationic lipid in efficient gene delivery based on double-coated nanoparticles.
Khalil IA, Kimura S, Sato Y, Harashima H
Journal of controlled release : official journal of the Controlled Release Society, 275, 107, 116, Elsevier BV, 2018年04月, ［査読有り］
研究論文（学術雑誌）

がん組織における浸潤性向上を目指した極小脂質ナノ粒子の開発　　　　　　　　　　　　　　　
岡部 奈々, 佐藤 悠介, 真栄城 正寿, 渡慶次 学, 原島 秀吉
日本薬学会年会要旨集, 138年会, 4, 78, 78, (公社)日本薬学会, 2018年03月
日本語

Advances in microfluidics for lipid nanoparticles and extracellular vesicles and applications in drug delivery systems.
Maeki M, Kimura N, Sato Y, Harashima H, Tokeshi M
Advanced drug delivery reviews, 128, 84, 100, 2018年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Lipid-based nanobiomaterials as liposomes and lipid nanoparticles (LNPs) are the most widely used nanocarriers for drug delivery systems (DDSs). Extracellular vesicles (EVs) and exosomes are also expected to be applied as DDS nanocarriers. The performance of nanomedicines relies on their components such as lipids, targeting ligands, encapsulated DNA, encapsulated RNA, and drugs. Recently, the importance of the nanocarrier sizes smaller than 100nm is attracting attention as a means to improve nanomedicine performance. Microfluidics and lab-on-a chip technologies make it possible to produce size-controlled LNPs by a simple continuous flow process and to separate EVs from blood samples by using a surface marker, ligand, or electric charge or by making a mass or particle size discrimination. Here, we overview recent advances in microfluidic devices and techniques for liposomes, LNPs, and EVs and their applications for DDSs.

A post-treatment methodology for precise size control of lipid nanoparticles by stepwise and rapid ethanol dilution
Niko Kimura, Masatoshi Maeki, Nana Okabe, Yusuke Sato, Akihiko Ishida, Hirofumi Tani, Hideyoshi Harashima, Manabu Tokeshi
22nd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2018, 3, 1404, 1405, 2018年
研究論文（国際会議プロシーディングス）

Mixing lipids to manipulate the ionization status of lipid nanoparticles for specific tissue targeting.
Nour Shobaki, Yusuke Sato, Hideyoshi Harashima
International journal of nanomedicine, 13, 8395, 8410, 2018年, ［査読有り］, ［筆頭著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Introduction: The development of targeted drug delivery systems is a rapidly growing area in the field of nanomedicine. Methods: We report herein on optimizing the targeting efficiency of a lipid nanoparticle (LNP) by manipulating the acid dissociation constant (pKa) value of its membrane, which reflects its ionization status. Instead of changing the chemical structure of the lipids to achieve this, we used a mixture of two types of pH-sensitive cationic lipids that show different pKa values in a single LNP. We mixed various ratios of YSK05 and YSK12-C4 lipids, which have pKa values of 6.50 and 8.00, respectively, in one formulation (referred to as YSK05/12-LNP). Results: The pKa of the YSK05/12-LNP was dependent not only on the molar ratio of each lipid but also on the individual contribution of each lipid to the final pKa (the YSK12-C4 lipid showed a higher contribution). Furthermore, we succeeded in targeting and delivering short interfering RNA to liver sinusoidal endothelial cells using one of the YSK05/12-LNPs which showed an optimum pKa value of 7.15 and an appropriate ionization status (~36% cationic charge) to permit the particles to be taken up by liver sinusoidal endothelial cells. Conclusion: This strategy has the potential for preparing custom LNPs with endless varieties of structures and final pKa values, and would have poten tial applications in drug delivery and ionic-based tissue targeting.

Preparation of envelope-type lipid nanoparticles containing gold nanorods for photothermal cancer therapy.
Paraiso WKD, Tanaka H, Sato Y, Shirane D, Suzuki N, Ogra Y, Tange K, Nakai Y, Yoshioka H, Harashima H, Akita H
Colloids and surfaces. B, Biointerfaces, 160, 715, 723, 2017年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The use of gold nanorods (AuNRs) that produce heat in response to near infrared (NIR) irradiation is an attractive approach to cancer photothermal therapy. AuNRs are usually prepared by using a highly toxic detergent: cetyltrimethylammonium bromide (CTAB). Thus, the removal of CTAB from the reaction mixture, and further stabilization of the surface of the AuNRs is required. In the present study, AuNRs were encapsulated in a multifunctional envelope-type nano device (AuNR-MEND) formed with an SS-cleavable and pH-activated lipid-like material. In the process of encapsulation, AuNRs were first stabilized with bovine serum albumin (AuNR-BSA), and then further encapsulated in the lipid envelope by the ethanol dilution method. The in vitro photothermal cytotoxicity of AuNR-MEND was further demonstrated on 4T1 breast cancer cells. After NIR radiation, the temperature of the medium was increased to approximately 60°C, and cell viability was drastically decreased to approximately 11%. However, this cytotoxic effect cannot simply be explained by medium heating. It therefore appears that intracellular delivery of the AuNRs is a key factor for achieving a high degree of cytotoxicity. Dose dependent cytotoxicity data revealed that a higher dose of AuNR-MEND resulted in the complete destruction of the cells when they were subjected to NIR irradiation, while the cell survival rate reached a plateau at 30% in the case of AuNR-BSA. Apoptosis was induced after treatment with the nanoparticles. AuNR-MEND showed superior cellular uptake activity over AuNR-BSA. Thus, delivering AuNR by means of functionalized lipid nanoparticles represents a promising approach to induce NIR-triggered apoptosis.

Highly specific delivery of siRNA to hepatocytes circumvents endothelial cell-mediated lipid nanoparticle-associated toxicity leading to the safe and efficacious decrease in the hepatitis B virus.
Sato Y, Matsui H, Yamamoto N, Sato R, Munakata T, Kohara M, Harashima H
Journal of controlled release : official journal of the Controlled Release Society, 266, 216, 225, Elsevier BV, 2017年11月, ［査読有り］, ［筆頭著者］
研究論文（学術雑誌）

pH-labile PEGylation of siRNA-loaded lipid nanoparticle improves active targeting and gene silencing activity in hepatocytes.
Hashiba K, Sato Y, Harashima H
Journal of controlled release : official journal of the Controlled Release Society, 262, 239, 246, 2017年09月, ［査読有り］, ［筆頭著者］

Mitochondrial Delivery of Doxorubicin Using MITO-Porter Kills Drug-Resistant Renal Cancer Cells via Mitochondrial Toxicity
Yuma Yamada, Reina Munechika, Eriko Kawamura, Yu Sakurai, Yusuke Sato, Hideyoshi Harashima
JOURNAL OF PHARMACEUTICAL SCIENCES, 106, 9, 2428, 2437, 2017年09月, ［査読有り］
英語, 研究論文（学術雑誌）

PEGylation of the GALA Peptide Enhances the Lung-Targeting Activity of Nanocarriers That Contain Encapsulated siRNA
Sarochin Santiwarangkool, Hidekata Akita, Taichi Nakatani, Kenji Kusumoto, Hiroki Kimura, Masaru Suzuki, Masaharu Nishimura, Yusuke Sato, Hideyoshi Harashima
JOURNAL OF PHARMACEUTICAL SCIENCES, 106, 9, 2420, 2427, 2017年09月, ［査読有り］
英語, 研究論文（学術雑誌）

Size-Dependency of the Surface Ligand Density of Liposomes Prepared by Post-insertion
Shang-Hsuan Lee, Yusuke Sato, Mamoru Hyodo, Hideyoshi Harashima
BIOLOGICAL & PHARMACEUTICAL BULLETIN, 40, 7, 1002, 1009, 2017年07月, ［査読有り］
英語, 研究論文（学術雑誌）

リンパ節内樹状細胞を標的とした極小ナノキャリアシステムの開発　　　　　　　　　　　　　　　
河合 美典, 中村 孝司, 佐藤 悠介, 真栄城 正寿, 原島 秀吉
日本DDS学会学術集会プログラム予稿集, 33回, 175, 175, 日本DDS学会, 2017年06月
日本語

Understanding the formation mechanism of lipid nanoparticles in microfluidic devices with chaotic micromixers.
Maeki M, Fujishima Y, Sato Y, Yasui T, Kaji N, Ishida A, Tani H, Baba Y, Harashima H, Tokeshi M
PloS one, 12, 11, e0187962, 2017年, ［査読有り］
研究論文（学術雑誌）

Innovative Technologies in Nanomedicines: From Passive Targeting to Active Targeting/From Controlled Pharmacokinetics to Controlled Intracellular Pharmacokinetics
Yusuke Sato, Yu Sakurai, Kazuaki Kajimoto, Takashi Nakamura, Yuma Yamada, Hidetaka Akita, Hideyoshi Harashima
MACROMOLECULAR BIOSCIENCE, 17, 1, 1600179, 2017年01月, ［査読有り］, ［招待有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）

Development of a multifunctional envelope-type nano device and its application to nanomedicine
Yusuke Sato, Takashi Nakamura, Yuma Yamada, Hideyoshi Harashima
JOURNAL OF CONTROLLED RELEASE, 244, Pt B, 194, 204, 2016年12月, ［査読有り］, ［招待有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）

Topology of Surface Ligands on Liposomes: Characterization Based on the Terms, Incorporation Ratio, Surface Anchor Density, and Reaction Yield
Shang-Hsuan Lee, Yusuke Sato, Mamoru Hyodo, Hideyoshi Harashima
BIOLOGICAL & PHARMACEUTICAL BULLETIN, 39, 12, 1983, 1994, 2016年12月, ［査読有り］
英語, 研究論文（学術雑誌）

Small-sized, stable lipid nanoparticle for the efficient delivery of siRNA to human immune cell lines
Takashi Nakamura, Moeka Kuroi, Yuki Fujiwara, Shota Warashina, Yusuke Sato, Hideyoshi Harashima
SCIENTIFIC REPORTS, 6, 37849, 2016年11月, ［査読有り］
英語, 研究論文（学術雑誌）

Liver-Specific Silencing of Lipin1 Reduces Fat Mass as Well as Hepatic Triglyceride Biosynthesis in Mice
Kazuaki Kajimoto, Erina Suemitsu, Yusuke Sato, Yu Sakurai, Hideyoshi Harashima
BIOLOGICAL & PHARMACEUTICAL BULLETIN, 39, 10, 1653, 1661, 2016年10月, ［査読有り］
英語, 研究論文（学術雑誌）

Cellular environment-responsive nanomaterials for use in gene and siRNA delivery: molecular design for biomembrane destabilization and intracellular collapse.
Tanaka H, Sato Y, Harashima H, Akita H
Expert opinion on drug delivery, 13, 7, 1015, 1027, 2016年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, INTRODUCTION: The development of gene and nucleic acid-based medication is one of the ultimate strategies in the research field of personalized medicine. For the desired function of a gene or siRNA, these molecules need to be delivered to the appropriate organelle (i.e. nucleus and cytoplasm, respectively). AREAS COVERED: The topics covered herein are rational design in order to control the pharmacokinetics, intracellular trafficking and release (decondensation or decapsulation) of the intended material. Since the endosome and cytoplasm are acidic (endosome) and reducing (cytoplasm) environments, respectively, a large variety of the materials have been developed that induce destabilization of endosome via its protonation, or are spontaneously collapsed in the cytoplasm. Finally, we propose materials (SS-cleavable and pH-activated lipid-like materials: ssPalm) that mount these sensing motifs, i.e., a positive charging unit in response to the acid environment (tertiary amines) and a cleavage unit (disulfide bonding) that is responsive to an reducing environment, respectively. EXPERT OPINION: Currently, the main target of the nanocarrier-mediated siRNA delivery systems is liver. The targeting of non-hepatic tissue is the next challenge. In this case, the design of neutral particle with well-organized intracellular trafficking, as well as an identification of the promising ligand is needed.

Anti-tumor effect via passive anti-angiogenesis of PEGylated liposomes encapsulating doxorubicin in drug resistant tumors
Golam Kibria, Hiroto Hatakeyama, Yusuke Sato, Hideyoshi Harashima
INTERNATIONAL JOURNAL OF PHARMACEUTICS, 509, 1-2, 178, 187, 2016年07月, ［査読有り］
英語, 研究論文（学術雑誌）

Elucidation of the physicochemical properties and potency of siRNA-loaded small-sized lipid nanoparticles for siRNA delivery
Yusuke Sato, Yusuke Note, Masatoshi Maeki, Noritada Kaji, Yoshinobu Baba, Manabu Tokeshi, Hideyoshi Harashima
JOURNAL OF CONTROLLED RELEASE, 229, 48, 57, 2016年05月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）

Relationship Between the Physicochemical Properties of Lipid Nanoparticles and the Quality of siRNA Delivery to Liver Cells
Yusuke Sato, Hiroto Hatakeyama, Mamoru Hyodo, Hideyoshi Harashima
MOLECULAR THERAPY, 24, 4, 788, 795, 2016年04月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）

Novel pH-sensitive multifunctional envelope-type nanodevice for siRNA-based treatments for chronic HBV infection
Naoki Yamamoto, Yusuke Sato, Tsubasa Munakata, Masakazu Kakuni, Chise Tateno, Takahiro Sanada, Yuichi Hirata, Shuko Murakami, Yasuhito Tanaka, Kazuaki Chayama, Hiroto Hatakeyama, Mamoru Hyodo, Hideyoshi Harashima, Michinori Kohara
JOURNAL OF HEPATOLOGY, 64, 3, 547, 555, 2016年03月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）

A lipid nanoparticle for the efficient delivery of siRNA to dendritic cells
Shota Warashina, Takashi Nakamura, Yusuke Sato, Yuki Fujiwara, Mamoru Hyodo, Hiroto Hatakeyama, Hideyoshi Harashima
JOURNAL OF CONTROLLED RELEASE, 225, 183, 191, 2016年03月, ［査読有り］
英語, 研究論文（学術雑誌）

A Multifunctional Envelope-Type Nano Device Containing a pH-Sensitive Cationic Lipid for Efficient Delivery of Short Interfering RNA to Hepatocytes In Vivo.
Sato Y, Harashima H, Kohara M
Methods in molecular biology (Clifton, N.J.), 1364, 71, 78, 2016年, ［査読有り］, ［筆頭著者］

Size-dependent specific targeting and efficient gene silencing in peritoneal macrophages using a pH-sensitive cationic liposomal siRNA carrier
Hideki Matsui, Yusuke Sato, Hiroto Hatakeyama, Hidetaka Akita, Hideyoshi Harashima
INTERNATIONAL JOURNAL OF PHARMACEUTICS, 495, 1, 171, 178, 2015年11月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）

The intracellular pharmacodynamics of siRNA is responsible for the low gene silencing activity of siRNA-loaded nanoparticles in dendritic cells
Takashi Nakamura, Yuki Fujiwara, Shota Warashina, Hideyoshi Harashima
INTERNATIONAL JOURNAL OF PHARMACEUTICS, 494, 1, 271, 277, 2015年10月, ［査読有り］
英語, 研究論文（学術雑誌）

Liposomes loaded with a STING pathway ligand, cyclic di-GMP, enhance cancer immunotherapy against metastatic melanoma
Takashi Nakamura, Hiroko Miyabe, Mamoru Hyodo, Yusuke Sato, Yoshihiro Hayakawa, Hideyoshi Harashima
JOURNAL OF CONTROLLED RELEASE, 216, 149, 157, 2015年10月, ［査読有り］
英語, 研究論文（学術雑誌）

Molecular Tuning of a Vitamin E-Scaffold pH-Sensitive and Reductive Cleavable Lipid-like Material for Accelerated in Vivo Hepatic siRNA Delivery
Hidetaka Akita, Yuki Noguchi, Hiroto Hatakeyama, Yusuke Sato, Kota Tange, Yuta Nakai, Hideyoshi Harashima
ACS BIOMATERIALS SCIENCE & ENGINEERING, 1, 9, 834, 844, 2015年09月, ［査読有り］
英語, 研究論文（学術雑誌）

フォスファゼン塩基を触媒に用いた置換エポキシドのリビング開環重合系の開発
磯野拓也, 佐藤悠介, 覚知豊次, 佐藤敏文
高分子論文集, 72, 5, 295, 305, 2015年
日本語, 研究論文（学術雑誌）

Microfluidic approach for production of lipid nanoparticles-based nano medicine
M. Maeki, T. Saito, Y. Node, Y. Sato, T. Yasui, N. Kaji, A. Ishida, H. Tani, Y. Baba, H. Harashima, M. Tokeshi
MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 838, 840, 2015年
研究論文（国際会議プロシーディングス）

A Strategy for Synthesis of Lipid Nanoparticles Using a Microfluidic Device with a Mixer Structure
Masatoshi Maeki, Tatsuyoshi Saito, Yusuke Sato, Takao Yasui, Noritada Kaji, Akihiko Ishida, Hirofumi Tani, Yoshinobu Baba, Hideyoshi Harashima, Manabu Tokeshi
RSC Advances, 5, 57, 46181, 46185, The Royal Society of Chemistry, 2015年, ［査読有り］
英語, 研究論文（学術雑誌）, Formation behavior of lipid nanoparticles (LNPs) in microfluidic devices with a staggered herringbone micromixer (SHM) structure was investigated. The fundamental role for SHMs in LNP formation was demonstrated by determining such factors as the limiting SHM cycle numbers and the effect of flow rate. The SHM cycle numbers and the position of the first SHM were as significant as factors as the flow rate condition for producing the small-size LNPs.

The RNA Sensor RIG-I Dually Functions as an Innate Sensor and Direct Antiviral Factor for Hepatitis B Virus
Seiichi Sato, Kai Li, Takeshi Kameyama, Takaya Hayashi, Yuji Ishida, Shuko Murakami, Tsunamasa Watanabe, Sayuki Iijima, Yu Sakurai, Koichi Watashi, Susumu Tsutsumi, Yusuke Sato, Hidetaka Akita, Takaji Wakita, Charles M. Rice, Hideyoshi Harashima, Michinori Kohara, Yasuhito Tanaka, Akinori Takaoka
IMMUNITY, 42, 1, 123, 132, 2015年01月, ［査読有り］, ［国際共著］
英語, 研究論文（学術雑誌）

Multifunctional envelope-type nano device for controlled intracellular trafficking and selective targeting in vivo
Kazuaki Kajimoto, Yusuke Sato, Takashi Nakamura, Yuma Yamada, Hideyoshi Harashima
JOURNAL OF CONTROLLED RELEASE, 190, 593, 606, 2014年09月, ［査読有り］
英語

A new adjuvant delivery system 'cyclic di-GMP/YSK05 liposome' for cancer immunotherapy
Hiroko Miyabe, Mamoru Hyodo, Takashi Nakamura, Yusuke Sato, Yoshihiro Hayakawa, Hideyoshi Harashima
JOURNAL OF CONTROLLED RELEASE, 184, 20, 27, 2014年06月, ［査読有り］
英語, 研究論文（学術雑誌）

An apolipoprotein E modified liposomal nanoparticle: Ligand dependent efficiency as a siRNA delivery carrier for mouse-derived brain endothelial cells
Mina Tamaru, Hidetaka Akita, Kazuaki Kajimoto, Yusuke Sato, Hiroto Hatakeyama, Hideyoshi Harashima
INTERNATIONAL JOURNAL OF PHARMACEUTICS, 465, 1-2, 77, 82, 2014年04月, ［査読有り］
英語, 研究論文（学術雑誌）

In vivo therapeutic potential of Dicer-hunting siRNAs targeting infectious hepatitis C virus.
Tsunamasa Watanabe, Hiroto Hatakeyama, Chiho Matsuda-Yasui, Yusuke Sato, Masayuki Sudoh, Asako Takagi, Yuichi Hirata, Takahiro Ohtsuki, Masaaki Arai, Kazuaki Inoue, Hideyoshi Harashima, Michinori Kohara
SCIENTIFIC REPORTS, 4, 4750, 2014年04月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）

Hepatic Monoacylglycerol O-acyltransferase 1 as a Promising Therapeutic Target for Steatosis, Obesity, and Type 2 Diabetes
Yasuhiro Hayashi, Erina Suemitsu, Kazuaki Kajimoto, Yusuke Sato, Afsana Akhter, Yu Sakurai, Hiroto Hatakeyama, Mamoru Hyodo, Noritada Kaji, Yoshinobu Baba, Hideyoshi Harashima
MOLECULAR THERAPY-NUCLEIC ACIDS, 3, e154, 2014年03月, ［査読有り］
英語, 研究論文（学術雑誌）

The systemic administration of an anti-miRNA oligonucleotide encapsulated pH-sensitive liposome results in reduced level of hepatic microRNA-122 in mice.
Hiroto Hatakeyama, Manami Murata, Yusuke Sato, Mayumi Takahashi, Noriaki Minakawa, Akira Matsuda, Hideyoshi Harashima
Journal of controlled release, Vol.173, 1, 43, 50, 2014年01月10日, ［査読有り］
英語, 研究論文（学術雑誌）

Application of apolipoprotein E-modified liposomal nanoparticles as a carrier for delivering DNA and nucleic acid in the brain.
Tamaru M, Akita H, Nakatani T, Kajimoto K, Sato Y, Hatakeyama H, Harashima H
International journal of nanomedicine, 9, 4267, 4276, 2014年, ［査読有り］
英語, 研究論文（学術雑誌）

RNAi-mediated gene knockdown and anti-angiogenic therapy of RCCs using a cyclic RGD-modified liposomal-siRNA system.
Sakurai Y, Hatakeyama H, Sato Y, Hyodo M, Akita H, Ohga N, Hida K, Harashima H
Journal of controlled release : official journal of the Controlled Release Society, 173, 110, 118, 2014年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Angiogenesis is one of crucial processes associated with tumor growth and development, and consequently a prime target for cancer therapy. Although tumor endothelial cells (TECs) play a key role in pathological angiogenesis, investigating phenotypical changes in neovessels when a gene expression in TEC is suppressed is a difficult task. Small interfering RNA (siRNA) represents a potential agent due to its ability to silence a gene of interest. We previously developed a system for in vivo siRNA delivery to cancer cells that involves a liposomal-delivery system, a MEND that contains a unique pH-sensitive cationic lipid, YSK05 (YSK-MEND). In the present study, we report on the development of a system that permits the delivery of siRNA to TECs by combining the YSK-MEND and a ligand that is specific to TECs. Cyclo(Arg-Gly-Asp-D-Phe-Lys) (cRGD) is a well-known ligand to αVβ3 integrin, which is selectively expressed at high levels in TECs. We incorporated cRGD into the YSK-MEND (RGD-MEND) to achieve an efficient gene silencing in TECs. Quantitative RT-PCR and the 5' rapid amplification of cDNA ends PCR indicated that the intravenous injection of RGD-MEND at a dose of 4.0mg/kg induced a significant RNAi-mediated gene reduction in TEC but not in endothelial cells of other organs. Finally, we evaluated the therapeutic potency of the RGD-MEND encapsulating siRNA against vascular endothelial growth factor receptor 2. A substantial delay in tumor growth was observed after three sequential RGD-MEND injections on alternate days. In conclusion, the RGD-MEND represents a new approach for the characterization of TECs and for us in anti-angiogenic therapy.

Multifunctional Enveloped Nanodevices (MENDs)
Yusuke Sato, Takashi Nakamura, Yuma Yamada, Hidetaka Akita, Hideyoshi Harashima
NONVIRAL VECTORS FOR GENE THERAPY LIPID- AND POLYMER-BASED GENE TRANSFER, 88, 139, 204, 2014年, ［査読有り］
英語, 論文集(書籍)内論文

In vitro optimization of 2 '-OMe-4 '-thioribonucleoside-modified anti-microRNA oligonucleotides and its targeting delivery to mouse liver using a liposomal nanoparticle
Mayumi Takahashi, Naoki Yamada, Hiroto Hatakeyama, Manami Murata, Yusuke Sato, Noriaki Minakawa, Hideyoshi Harashima, Akira Matsuda
NUCLEIC ACIDS RESEARCH, 41, 22, 10659, 10667, 2013年12月, ［査読有り］
英語, 研究論文（学術雑誌）

A Neutral envelope-type nanoparticle containing pH-responsive and ss-cleavable lipid-like material as a carrier for plasmid DNA
Hidetaka Akita, Ryohei Ishiba, Hiroto Hatakeyama, Hiroki Tanaka, Yusuke Sato, Kota Tange, Masaya Arai, Kazuhiro Kubo, Hideyoshi Harashima
Advanced Healthcare Materials, 2, 8, 1120, 1125, 2013年08月, ［査読有り］
英語, 研究論文（学術雑誌）

Gene silencing via RNAi and siRNA quantification in tumor tissue using MEND, a liposomal siRNA delivery system.
Sakurai Y, Hatakeyama H, Sato Y, Hyodo M, Akita H, Harashima H
Molecular therapy : the journal of the American Society of Gene Therapy, 21, 6, 1195, 1203, 2013年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Small interfering RNA (siRNA) would be predicted to function as a cancer drug, but an efficient siRNA delivery system is required for clinical development. To address this issue, we developed a liposomal siRNA carrier, a multifunctional envelope-type nanodevice (MEND). We previously reported that a MEND composed of a pH-sensitive cationic lipid, YSK05, showed significant knockdown in both in vitro and in tumor tissue by intratumoral injection. Here, we report on the development of an in vivo siRNA delivery system that is delivered by systemic injection and an analysis of the pharmacokinetics of an intravenously administered siRNA molecule in tumor tissue. Tumor delivery of siRNA was quantified by means of stem-loop primer quantitative reverse transcriptase PCR (qRT-PCR) method. PEGylation of the YSK-MEND results in the increase in the accumulation of siRNA in tumor tissue from 0.0079% ID/g tumor to 1.9% ID/g tumor. The Administration of the MEND (3 mg siRNA/kg body weight) showed about a 50% reduction in the target gene mRNA and protein. Moreover, we verified the induction of RNA interference by 5' RACE-PCR method. The collective results reported here indicate that an siRNA carrier was developed that can deliver siRNA to a target cell in tumor tissue through an improved siRNA bioavailability.

A pH-sensitive cationic lipid facilitates the delivery of liposomal siRNA and gene silencing activity in vitro and in vivo
Yusuke Sato, Hiroto Hatakeyama, Yu Sakurai, Mamoru Hyodo, Hidetaka Akita, Hideyoshi Harashima
JOURNAL OF CONTROLLED RELEASE, 163, 3, 267, 276, 2012年11月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）

Endosomal escape and the knockdown efficiency of liposomal-siRNA by the fusogenic peptide shGALA
Yu Sakurai, Hiroto Hatakeyama, Yusuke Sato, Hidetaka Akita, Kentaro Takayama, Sachiko Kobayashi, Shiroh Futaki, Hideyoshi Harashima
BIOMATERIALS, 32, 24, 5733, 5742, 2011年08月, ［査読有り］
英語, 研究論文（学術雑誌）

Ornithine and Tryptophan Analogs as Efficient Polycations for Short Interference RNA Delivery to Tumor Cells
Yusuke Sato, Hiroto Hatakeyama, Hideyoshi Harashima
BIOLOGICAL & PHARMACEUTICAL BULLETIN, 33, 7, 1246, 1249, 2010年07月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）

ポリカチオン/DNA複合体搭載脂質ナノ粒子による効率的な長鎖DNAトランスフェクション
宇野秀哉, 真栄城正寿, 真栄城正寿, 真栄城正寿, 佐藤悠介, 石田晃彦, 日比野光恵, 谷博文, 原島秀吉, 渡慶次学, 化学系学協会北海道支部冬季研究発表会(Web), 2023, 2023年

薬剤送達ナノ粒子作製用ガラス製マイクロ流体システムの開発
岡田悠斗, 真栄城正寿, 真栄城正寿, 佐藤悠介, 石田晃彦, 谷博文, 原島秀吉, 渡慶次学, 化学系学協会北海道支部冬季研究発表会(Web), 2022, 2022年

蛍光ナノダイヤモンド搭載セラノスティクス用脂質ナノ粒子の作製
杉浦魁星, 真栄城正寿, 真栄城正寿, 佐藤悠介, 石田晃彦, 谷博文, 原島秀吉, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集(CD-ROM), 45th, 2022年

マイクロ流体デバイスを用いた長鎖プラスミドDNA搭載ナノ粒子の作製
宇野秀哉, 真栄城正寿, 真栄城正寿, 佐藤悠介, 石田晃彦, 原島秀吉, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集(CD-ROM), 46th, 2022年

マイクロ流体デバイスを用いたプラスミドDNA搭載脂質ナノ粒子の作製
宇野秀哉, 真栄城正寿, 真栄城正寿, 佐藤悠介, 石田晃彦, 谷博文, 原島秀吉, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 43rd, 2021年

リンパ節送達とリンパ節内分布に影響を与える脂質ナノ粒子特性
中村孝司, 河合美典, 佐藤悠介, 真栄城正寿, 渡慶次学, 原島秀吉, 日本薬剤学会年会講演要旨集(CD-ROM), 36th, 2021年

Lipid nanoparticles for cell-specific in vivo targeted delivery of nucleic acids
Ikramy A. Khalil, Mahmoud A. Younis, Seigo Kimura, Hideyoshi Harashima, Biological and Pharmaceutical Bulletin, 43, 4, 584, 595, 2020年04月01日
書評論文,書評,文献紹介等

脂質ナノ粒子のサイズと電荷がリンパ節移行とリンパ節内分布に与える影響
中村孝司, 河合美典, 佐藤悠介, 真栄城正寿, 渡慶次学, 原島秀吉, 日本薬学会年会要旨集(CD-ROM), 140th (Web), 2020年

CRISPR/Casタンパク質搭載脂質ナノ粒子の開発
佐藤悠介, 鈴木裕一, 小沼はるの, 佐藤里咲, 真栄城正寿, 渡慶次学, 原島秀吉, 日本DDS学会学術集会プログラム予稿集, 36th, 2020年

ガラス製マイクロ流体デバイスを用いたsiRNA搭載脂質ナノ粒子の作製と大量生産用集積化デバイスの開発
真栄城正寿, 真栄城正寿, 岡田悠斗, 佐藤悠介, 石田晃彦, 谷博文, 原島秀吉, 渡慶次学, 日本DDS学会学術集会プログラム予稿集, 36th, 2020年

DDSナノ粒子作製用ガラス製マイクロ流体デバイスの開発
岡田悠斗, 真栄城正寿, 真栄城正寿, 佐藤悠介, 石田晃彦, 谷博文, 原島秀吉, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 42nd (Web), 2020年

DEVELOPMENT OF A THREE-DIMENSIONAL MICROMIXER DEVICE FOR PRODUCTION OF VARIOUS LIPID-BASED NUCLEIC ACID NANOCARRIERS
Niko Kimura, Masatoshi Maeki, Yusuke Sato, Kosuke Sasaki, Akihiko Ishida, Hirofumi Tani, Hideyoshi Harashima, Manabu Tokeshi, Proc. Micro TAS 2019, 368, 369, 2019年10月, ［査読有り］
英語

実験計画法によるmRNA送達キャリアの効率的な最適化
橋場月, 佐藤悠介, 真栄城正寿, 渡慶次学, 原島秀吉, 日本核酸医薬学会年会講演要旨集, 5th, 2019年

ゲノム編集タンパク質搭載脂質ナノ粒子の開発
佐藤悠介, 鈴木裕一, 佐藤里咲, 真栄城正寿, 渡慶次学, 原島秀吉, 日本核酸医薬学会年会講演要旨集, 5th, 2019年

脂質ナノ粒子形成後の二次希釈操作が粒子サイズへ与える影響の解明
木村笑, 真栄城正寿, 岡部奈々, 佐藤悠介, 石田晃彦, 谷博文, 原島秀吉, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 39th, 2019年

Optimization of siRNA delivery to target sites: issues and future directions
Ikramy A. Khalil, Yuma Yamada, Hideyoshi Harashima, Expert Opinion on Drug Delivery, 15, 11, 1053, 1065, 2018年11月02日
Taylor and Francis Ltd, 英語, 書評論文,書評,文献紹介等

Microfluidic stepwise and rapid ethanol dilution for precise size control of lipid nanoparticles　　　　　　　　　　　　　　　
Niko Kimura, Masatoshi Maeki, Nana Okabe, Yusuke Sato, Akihiko Ishida, Hirofumi Tani, Hideyoshi Harashima, Proc. Micro TAS 2018, 1404, 1405, 2018年11月, ［査読有り］
英語

iLiNPデバイスによるpH応答性カチオン性脂質ナノ粒子の粒径制御
木村笑, 真栄城正寿, 岡部奈々, 佐藤悠介, 石田晃彦, 谷博文, 原島秀吉, 渡慶次学, 渡慶次学, 渡慶次学, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 37th, 2018年

A Small-Sized Lipid Nanoparticles Production Method Using Microfluidic Devices with Baffle Structures
N. Kimura, M. Maeki, Y. Sato, A. Ishida, H. Tani, H. Harashima, M. Tokeshi, Proc. Micro TAS 2017, 965, 966, 2017年10月
英語

pH応答性カチオン性脂質－効率的な核酸送達と肝疾患治療への応用　　　　　　　　　　　　　　　
佐藤悠介, 医学のあゆみ, 262, 2, 127, 130, 2017年06月

マイクロデバイスを用いた脂質ナノ粒子作製と生体内動態挙動
真栄城正寿, 木村笑, 佐藤悠介, 石田晃彦, 谷博文, 原島秀吉, 渡慶次学, 化学工学会秋季大会研究発表講演要旨集(CD-ROM), 49th, 2017年

pH感受性脂質を基盤とした脂質ナノ粒子の開発と核酸ナノメディシンへの応用 (特集 ナノメディシンの研究開発と応用)
佐藤 悠介, 原島 秀吉, 化學工業, 67, 11, 821, 827, 2016年11月
小峰工業出版, 日本語

pH感受性脂質を基盤とした脂質ナノ粒子の開発と核酸ナノメディシンへの応用　　　　　　　　　　　　　　　
佐藤悠介, 原島秀吉, 化学工業, 67, 11, 21, 27, 2016年11月

Effect of the Grooved Structures and the Ethanol Concentration on the Small-sized Lipid Nanoparticles Formation
Yuka Fujishima, Masatoshi Maeki, Yusuke Sato, Takao Yasui, Akihiko Ishida, Hirofumi Tani, Yoshinobu Baba, Hideyoshi Harashima, Manabu Tokeshi, Proceedings of MicroTAS 2016, 1412, 1413, 2016年10月, ［査読有り］
英語, 記事・総説・解説・論説等（国際会議プロシーディングズ）

マイクロ流体デバイスによる脂質ナノ粒子作製とDDSへの応用
真栄城正寿, 佐藤悠介, 原島秀吉, 渡慶次学, 機能材料, 36, 7, 15, 21, 2016年07月
シーエムシー出版, 日本語

脂質ナノ粒子の粒径制御のためのマイクロ流体デバイス設計
真栄城正寿, 藤島由佳, 佐藤悠介, 石田晃彦, 谷博文, 原島秀吉, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 32nd, 2015年

多機能性エンベロープ型ナノ構造体(MEND)を基盤としたDDS (特集 ナノDDS革命 : 革新的ドラッグデリバリーシステムが難治疾患治療に光をもたらす)
秋田 英万, 佐藤 悠介, 中村 孝司, 細胞工学, 34, 10, 956, 961, 2015年
学研メディカル秀潤社 ; 1982-, 日本語

多機能性エンベロープ型ナノ構造体の創世とナノ医療への展開
佐藤悠介, 山田勇磨, 梶本和昭, 原島秀吉, 化学とマイクロ・ナノシステム, 14, 1, 15, 23, 2015年, ［招待有り］
化学とマイクロ・ナノシステム研究会, 日本語, 記事・総説・解説・論説等（学術雑誌）

MENDで拓く遺伝子治療への道 (特集 Drug delivery system(DDS)の最新展望)
佐藤 悠介, 中村 孝司, 山田 勇磨, 医薬ジャーナル, 50, 7, 83, 87, 2014年07月
医薬ジャーナル社, 日本語

肝臓におけるDrp1の発現抑制は糖代謝を改善する　　　　　　　　　　　　　　　
井林 雄太, 畠山 浩人, 佐藤 悠介, 王 麗華, 原島 秀吉, 高柳 涼一, 野村 政壽, 日本内分泌学会雑誌, 90, 1, 348, 348, 2014年04月
(一社)日本内分泌学会, 日本語

DNAマイクロアレイとin vivo siRNA送達システムの融合による新規2型糖尿病治療薬創出　　　　　　　　　　　　　　　
末光 永理奈, 林 泰弘, 梶本 和昭, 佐藤 悠介, Akhter Afsana, 櫻井 遊, 畠山 浩人, 兵藤 守, 加地 範匡, 馬場 嘉信, 原島 秀吉, 日本薬剤学会年会講演要旨集, 28年会, 237, 237, 2013年04月
(公社)日本薬剤学会, 日本語

新規pH応答性カチオン性脂質を用いた効率的なshort interference RNA (siRNA) デリバリーシステムの開発
佐藤 悠介, 畠山 浩人, 兵藤 守, 秋田 英万, 原島 秀吉, YAKUGAKU ZASSHI, 132, 12, 1355, 1363, 2012年12月
日本語, 書評論文,書評,文献紹介等

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年10月
英語, 研究発表ペーパー・要旨（国際会議）

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年10月
英語, 研究発表ペーパー・要旨（国際会議）

薬剤学IV　　　　　　　　　　　　　　　
北海道大学
2024年10月 - 現在

薬剤学II　　　　　　　　　　　　　　　
北海度大学
2024年10月 - 現在

フレッシュマンセミナー　　　　　　　　　　　　　　　
北海道大学
2024年10月 - 2025年03月

分析化学実習　　　　　　　　　　　　　　　
北海道大学

薬剤学実習　　　　　　　　　　　　　　　
北海道大学

先端生物科学実験法II　　　　　　　　　　　　　　　
北海道大学

医療薬学特論　　　　　　　　　　　　　　　
北海道大学

2020年04月 - 現在
日本ゲノム編集学会　　　　　　　　　　　　　　　

LNPとスマートポリマーが創るコアシェル構造を有する活性型mRNA送達システム
科学研究費助成事業
2023年04月12日 - 2028年03月31日
原島 秀吉, 佐藤 悠介, 中村 孝司, 山田 勇磨, 西山 伸宏, 内田 智士
日本学術振興会, 基盤研究(S), 北海道大学, 23H05451

全身投与型Cas9 RNP搭載脂質ナノ粒子製剤による脳でのゲノム編集
科学研究費助成事業
2023年04月01日 - 2026年03月31日
佐藤 悠介
日本学術振興会, 基盤研究(B), 北海道大学, 23K28421

ライソゾーム栄養感知機構を標的としたNASH肝がん抑止を目指した創薬研究
科学研究費助成事業
2023年04月01日 - 2026年03月31日
田中 靖人, 馬場 理也, 佐藤 悠介, 田中 義正
コリン欠乏高脂肪食によるNASH肝癌モデルにおいて、FLCN-KO （マウス）により、NASHや肝発癌が抑制されることが証明された。NASH肝癌はmTOR pathwayと深い関わりがあることが示唆されており、FLCN抑制によるmTOR経路への影響を明らかにする。さらに、FLCN-FNIP1及びFLCN-FNIP2の蛋白質間相互作用機構を明らかにするため、分子動力学シミュレーション(MDシミュレーション)を行った結果、ヘリックス2とBの間にFLCN-FNIPの結合を阻害する分子が入り込む余地があると考えられた。MDシミュレーションで明らかにしたFLCN-FNIP1/2相互作用に重要な部位を標的としてインシリコスクリーニングを開始した。
本研究では、新たにFLCNを標的としたin vivoゲノム編集DDS製剤を開発し、NASH及びNASH肝がんの発症・進展の新規病態機序の解明及び治療効果を検証する。FLCN-FNIP複合体の分子動力学シミュレーション、オリジナル化合物による独自の創薬システム、安全で高効率なドラッグデリバリーシステムを組合せるとともに、ライソゾーム栄養感知機構を標的とした新規治療薬の開発を進める。本研究は栄養感知メカニズムを軸にした包括的かつ独創性の高い研究であり、NASH患者の予後改善に繋がる新たな治療法の開発が期待できる。
日本学術振興会, 基盤研究(B), 熊本大学, 23K27589

in vivo好中球エンジニアリングを可能とする核酸搭載脂質ナノ粒子
科学研究費助成事業
2023年06月30日 - 2025年03月31日
佐藤 悠介
日本学術振興会, 挑戦的研究(萌芽), 北海道大学, 23K18554

組織選択的に核酸送達可能な高分子ー脂質ハイブリッドナノ粒子の戦略的創出
科学研究費助成事業 挑戦的研究(萌芽)
2022年06月30日 - 2024年03月31日
磯野 拓也, 原島 秀吉, 佐藤 悠介, 佐藤 敏文
日本学術振興会, 挑戦的研究(萌芽), 北海道大学, 22K19907

律速段階の解明に基づいたウイルスを凌駕する革新的医薬分子送達システムの創製
科学研究費助成事業 基盤研究(A)
2019年04月 - 2023年03月
原島 秀吉, 佐藤 悠介, 中村 孝司, 山田 勇磨
１ siRNA送達（佐藤）：siRNAは細胞質中でリン酸化酵素Clp1により5’末端のリン酸化修飾を受け、RISCを形成し、標的mRNAのサイレンシングを引き起こす。我々の予備検討結果から、siRNAの多くが上述の一般的な経路をたどっていない可能性が強く示唆された。そこでブラックボックスとなっているsiRNAの細胞内運命を①5’リン酸化・RISC形成速度、②細胞質内・核内局在、の観点から解明する。
２ pDNA送達（原島）：転写(TC)/翻訳(TL)過程に大きな差が認められたナノDDSにおいて、TCとTLのどちらの寄与が大きいかを、mRNAを定量することにより識別する。 ①TLの寄与が大きい場合、ナノDDSのエンドソーム脱出時にエンドソーム内物質が漏出したり、あるいはpDNA導入に伴う各種細胞内センサーの刺激によって、導入細胞におけるTL活性が低下する、という仮説を検証する。
３ がん免疫ナノ療法（中村）：患者個人の腫瘍関連微小環境の免疫状態を把握することは治療戦略の決定に不可欠である。そこで、免疫状態が異なる担がんマウスモデルから腫瘍組織、所属リンパ節、脾臓を採取し、遺伝子発現解析を行うことで腫瘍関連微小環境の免疫状態情報を得る。文献情報からヒトで確認されている腫瘍関連微小環境の免疫状態をカテゴリー化し、それらを反映する担がんマウスモデルを5種類以上選定する。 ４ Mt送達（山田）：核酸・遺伝子をコアとするナノ粒子を形成し、細胞膜とMt膜 を突破するために、脂質膜コーティングを施す。脂質膜表面には細胞導入素子およびミトコンドリア移行性アプタマー(負電荷)を修飾したDual ligand MITO-Porterを基盤骨格とする。また、細胞内での流動性の向上を目指し、ポリエチレングリコール（PEG）などの親水性ポリマーの修飾や、微小な粒子径も検討する。
日本学術振興会, 基盤研究(A), 北海道大学, 研究分担者, 19H01170

肝線維化制御を目指した新規治療標的の同定とそのメカニズム解明
科学研究費助成事業 基盤研究(B)
2019年04月 - 2022年03月
田中 靖人, 佐藤 悠介, 大石 久史
わが国で肝細胞癌の発症要因として最も多いC型肝炎ウイルス（HCV）感染症の治療は飛躍的に進歩し、ウイルス排除が可能となった。しかし、HCV排除後の肝発癌が問題となっており、そのリスク因子である肝線維化の制御は重要な課題である。
本研究では新たな治療法の開発を目指して、①HCV排除後の肝発癌に関連するリスク因子である、Tolloid-like 1（TLL1）による新たな肝線維化メカニズムの解明、②肝細胞特異的ヒトTLL1高発現マウス系統の樹立と肝臓特異的マウスTll1欠損マウスの作製および解析、③コラーゲン遺伝子発現を制御するマイクロRNA-X（miR-X）標的分子の同定とその制御機構の解明、④マイクロRNA（miRNA）送達脂質ナノ粒子の実用化を目指した製剤検討、を行い、新規の肝線維化・肝発癌メカニズムやマイクロRNAによる直接的・間接的な標的分子の制御を明らかにする。
令和元（2019）年度は、miR-Xが細胞内の主要なシグナル伝達経路のリン酸化を抑制することを明らかにした。肝細胞特異的hTLL1高発現マウス系統を樹立し、TLL1の肝発癌における影響を検討した。また、mTll1 floxマウスとmTll1 Tgマウスも作製した。
miRNA導入については、siRNAをモデル核酸としてマイクロ流体デバイスiLiNPによるsiRNA搭載脂質ナノ粒子（LNP）製造時の諸条件を検討した結果、肝臓への核酸送達に適したLNPの製造条件を見出した。
日本学術振興会, 基盤研究(B), 名古屋市立大学, 研究分担者, 19H03640

組織浸透力に優れたsiRNA搭載極小脂質ナノ粒子による新規がん治療
科学研究費助成事業 挑戦的研究(萌芽)
2018年06月 - 2020年03月
佐藤 悠介
小さな脂質ナノ粒子は優れた組織浸透性と効率的な核酸送達が期待されることから、その開発は重要である。一方で、粒子径の減少に伴う粒子の物理科学的安定性の低下や薬物送達活性の減少がその応用可能性を大きく制限している。本研究では、活性低下メカニズムに基づいて、pH感受性カチオン性脂質の疎水性足場構造による影響に着目し、極小粒子径と活性を両立するsiRNA搭載脂質ナノ粒子の開発とメカニズム解析を行った。
日本学術振興会, 挑戦的研究(萌芽), 北海道大学, 研究代表者, 18K19889

In vivoゲノム編集による肝疾患治療の実現を目指した脂質ナノ粒子の創生　　　　　　　　　　　　　　　
科学研究費補助金(若手研究(A))
2017年04月 - 2020年03月
佐藤悠介
文部科学省, 研究代表者, 競争的資金

Ｂ型肝炎根絶を目指した安全な新規免疫療法の開発
科学研究費助成事業 基盤研究(B)
2016年04月 - 2019年03月
田中 靖人, 岸 裕幸, 五十川 正記, 佐藤 悠介
B型肝炎ウイルス(HBV)持続感染は、肝硬変・肝細胞癌への進展やHBV再活性化の問題があり、その根絶が求められている。抗ウイルス効果を持つ抗ヒトCD40抗体の作製と最適化を検討した。HBV特異的T細胞受容体発現TgマウスのCD8+T細胞を、肝臓内でHBVを複製するHBV-Tgマウスに養子移入後、抗CD40抗体で処理した。早期処理ではT細胞の機能性が回復しHBV-mRNA量が低下したが、後期処理ではHBV-mRNA量は不変であった。また、HBs抗原に特異的なB細胞応答の解析システムとしてELISPOT assayを作成し、ヒトの血液においてもHBV特異的なB細胞応答の評価が可能となった。
日本学術振興会, 基盤研究(B), 名古屋市立大学, 連携研究者, 16H05288

腫瘍原発巣および微小転移巣への革新的な核酸送達ナノシステムの創製
科学研究費補助金(若手研究(B))
2015年 - 2016年
佐藤 悠介
ナノ粒子によるがん組織への核酸送達においては、ナノ粒子ががん組織に到達後に組織深部まで浸透できずに標的のがん細胞へ到達できないというがん組織内動態の制約が大きな問題となっている。本研究では上記問題を解決するため、脂質ナノ粒子の粒子径を小さく高精度に制御するための新規脂質様材料の開発および粒子化手法の検討を行った。加えて、脂質ナノ粒子の小型化に伴って核酸導入効率が低下するという問題を解決するため、その原因の解明を行った。
文部科学省, 若手研究(B), 北海道大学, 研究代表者, 競争的資金, 15K20831

肝臓中ｍｉｃｒｏＲＮＡの発現変動が薬物代謝酵素活性に及ぼす影響
科学研究費助成事業 挑戦的萌芽研究
2012年04月 - 2015年03月
中島 美紀, 木村 健人, 小田 祐輝, 佐藤 悠介, 原島 秀吉
ヒト初代培養肝細胞にリファンピシンを処置することにより40種類のmiRNAの発現が2倍以上に変化し、それらが標的遺伝子の発現変化をもたらしている可能性が示された。マウスにmiRNAに対するアンチセンスオリゴを尾静注し、肝臓中の発現をノックダウンすると、薬物代謝酵素活性が変化したことから、miRNAが薬物代謝能の制御に重要な役割を担っていることをin vivoで明らかにした。
日本学術振興会, 挑戦的萌芽研究, 金沢大学, 連携研究者, 24659074

ヌクレアーゼ抵抗性ヌクレオシドを搭載した細胞標的化機能性一本鎖核酸の創出
科学研究費助成事業 基盤研究(A)
2011年04月 - 2015年03月
松田 彰, 佐藤 浩輔, 佐藤 悠介
核酸医薬の細胞・組織標的化を目的にした。(1)肝臓で高発現しているmicroRNA-122に対するアンチセンス分子をヌクレアーゼ抵抗性ヌクレオシドで合成し、肝臓標的化能に優れるpH応答性カチオン性脂質から作製したリポソームに搭載しマウスに静脈注射した。その結果、microRNA-122で抑制されていたmRNAs量が回復し、血中コレステロールの低下が約２週間持続した。(2)エピジェネティックスで重要なDNA CpG配列の選択的メチル化酵素阻害剤を開発し、前立腺ガンに高発現しているPSMAを標的とするリガンドを結合した。この阻害剤はPSMA発現前立腺ガン細胞選択的に増殖抑制活性を示した。
日本学術振興会, 基盤研究(A), 北海道大学, 連携研究者, 23249008

肝細胞標的siRNA送達システムの創製：体内動態と細胞内動態の真の両立
科学研究費補助金(研究活動スタート支援)
2014年 - 2015年
佐藤 悠介
静脈内投与によってshort interfering RNA（siRNA）を肝臓へ効率的に導入するためには、siRNAキャリアの体内動態と細胞内動態の両過程の効率を最大化させる必要がある。脂質ナノキャリアの体内動態を向上するためにはPEG等の親水性高分子修飾が有用であるが、PEG修飾はキャリアの細胞内動態を著しく悪化させる（PEGのジレンマ）。そこで、血中ではPEGを安定的に保持し、酸性エンドソーム内では速やかに脱離させることで両過程の最大化を試みた。
pH低下に応答して切断するリンカーとして無水マレイン酸誘導体（CDM）を基盤とし、様々な分子量のPEGを結合させたCDM-PEG誘導体を合成した。CDMは第一級アミンと反応して共有結合を形成するため、初めにモデル化合物としてグリシンを用い、CDM-PEGの脱離能を評価した。その結果、いずれの誘導体も血中pHにおける安定性とpH6.0における速やかな脱離能を示し、目的通りの反応性を示すことが確認された。
第一級アミン含有脂質を合成し、それを含む脂質ナノキャリアのCDM-PEG修飾を試みた結果、添加量依存的な修飾量の増加が確認された。また、CDM-PEGの修飾に伴って脂質ナノキャリアの表面電荷が中性に変化したことから、CDM-PEG修飾はキャリアの体内動態を改善することが示唆された。実際に、脂質ナノ粒子の静脈内投与後の肝臓内局在はCDM-PEG修飾によって顕著に変化した。また、キャリアからの脱離について評価したところ、CDM-PEGはpH6.0において10分以内に完全に脱離した。
以上より、効率的なsiRNA送達に必要な構成物質を開発したと共に、体内動態・細胞内動態を両立するためのCDMリンカーを介したPEG修飾および酸性環境下における速やかな脱離能を確認することに成功した。
文部科学省, 研究活動スタート支援, 北海道大学, 研究代表者, 競争的資金, 26893001

生体環境に応答するナノシステムによるがん標的型miRNA送達とがん治療への応用
科学研究費助成事業 特別研究員奨励費
2011年 - 2013年
佐藤 悠介
本研究の目的は、RNA干渉を誘起する機能性核酸miRNAを末梢の固形がん組織に送達可能なリポソーム型キャリアを構築することである。miRNAは短鎖二本鎖RNAであり、その構造や物性は、RNA干渉を誘起する人工核酸のsiRNAと同等であると考えられる。そこで、比較的安価なsiRNAをmiRNAの代用とした。これまでに、優れた核酸導入活性を有する新規pH応答性脂質YSK05を開発し、肝臓において高い遺伝子ノックダウン活性を有するキャリア(YSK05キャリア)の構築に成功した。YSK05キャリアをがん組織に応用したところ、遺伝子ノックダウンは認められたものの、キャリアのがん組織内浸透性が乏しく、その効率は60%程度で飽和した。がん組織内浸透性の乏しさは物理的バリアに起因すると考え、キャリアの小型化を試み、50nmから25nm程度への小型化に成功した。がん組織における遺伝子ノックダウン効率を上昇させるためには、キャリアの核酸導入活性の向上と、がん組織内分布の改善が重要であると考えられるため、今年度はそれらに着目して研究を行った。まず、YSK05よりも優れた核酸導入活性を有する脂質の開発を試みた結果、新規pH応答性脂質YSK13の合成に成功した。YSK13を含むキャリア(YSK13キャリア)はYSK05キャリアと比較して肝臓における活性が約4倍高いことが明らかとなった。これまでの検討により、肝臓における活性とがん組織における活性は正に相関することが明らかとなっており、YSK13キャリアはがん組織において優れた活性を示すと考えられる。また、25nmの小型キャリアのがん組織内分布を評価したところ、均一的な分布を示した。また、標的がん細胞への取り込み量の増加も認められた。
日本学術振興会, 特別研究員奨励費, 北海道大学, 研究代表者, 11J05557