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

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.

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.

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
研究論文（学術雑誌）, We report on a novel strategy for treating liver fibrosis through reprogramming activated Hepatic Stellate Cells (aHSCs) into quiescent Hepatic Stellate Cells (qHSCs) using siRNA-loaded lipid nanoparticles (LNPs). The in vivo screening of an array of molecularly-diverse ionizable lipids identified two candidates, CL15A6 and CL15H6, with a high siRNA delivery efficiency to aHSCs. Optimization of the composition and physico-chemical properties of the LNPs enabled the ligand-free, selective, and potent siRNA delivery to aHSCs post intravenous administration, with a median effective siRNA dose (ED50) as low as 0.08 mg/Kg. The biosafety of the LNPs was confirmed by escalating the dose to 50-fold higher than the ED50 or by chronic administration. The recruitment of the novel LNPs for the simultaneous knockdown of Hedgehog (Hh) and Transforming Growth Factor Beta 1 (TGFβ1) signaling pathways using an siRNA cocktail enabled the reversal of liver fibrosis and the restoration of the normal liver function in mice. Analysis of the key transcription factors in aHSCs suggested that the reprogramming of aHSCs into qHSCs mediated the therapeutic outcomes. The scalable ligand-free platform developed in this study as well as the novel therapeutic strategy reported herein are promising for clinical translation.

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）

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.

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, ROYAL SOC CHEMISTRY, 2021年01月
英語, 研究論文（学術雑誌）, Sub 100 nm-sized lipid nanoparticles (LNPs) have been widely used in drug delivery systems (DDSs). The size of the LNPs is an important parameter for the DDS performance, such as biodistribution and gene silencing using siRNAs. However, the LNPs prepared by the conventional preparation method show a wide size distribution. To improve the LNP size distribution, we developed a microfluidic device, named the iLiNP (TM) device, in a previous study. This device could produce LNPs in the size range of 20 to 150 nm, but the size distribution of the large-sized LNPs needs to be further improved. From the viewpoint of the LNP formation process, a homogeneous and slow rate dilution of ethanol plays an important role in improving the large-size LNP size distribution. In this study, we developed a three-dimensional, symmetrically assembled microfluidic device named the 3D-iLiNP device with the aim of precise size control of large-sized LNPs. We designed the 3D-iLiNP device using a computational fluid dynamics simulation and demonstrated that the 3D-iLiNP device can improve the LNP size distribution. The gene silencing activity of four kinds of siRNA-loaded LNPs was investigated via in vitro and in vivo experiments to elucidate the effect of the LNP size distribution. The results revealed that the LNPs with a size between 90 and 120 nm showed higher gene silencing activity than those with other sizes. The 3D-iLiNP device is expected to improve DDS performance by precisely controlling the size of LNPs.

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, 核酸ナノ医療の時代が始まり、医薬品業界ではゴールドラッシュが巻き起こっている。一方で、ゾルゲンスマに代表される超高額医療費の経済的問題は、国家予算の破綻も招きかねず、新たなジレンマに直面することが予想される。本稿では、前半ではCAR-T療法、パティシラン、ゾルゲンスマについて紹介し、革新的核酸医薬・遺伝子治療の最先端技術について考察する。後半では、我々の研究成果に基づいて、細胞内動態制御の重要性とポストEPRの時代のDDS戦略について紹介し、バイオマテリアルによってこのジレンマをいかにして突破するべきか、議論したい。(著者抄録)

The Effect of Size and Charge of Lipid Nanoparticles Prepared by Microfluidic Mixing on Their Lymph Node Transitivity and Distribution.
Takashi Nakamura, Minori Kawai, Yusuke Sato, Masatoshi Maeki, Manabu Tokeshi, Hideyoshi Harashima
Molecular pharmaceutics, 17, 3, 944, 953, 2020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）, A number of drugs that are currently on the market, as well as new candidates for drugs, are poorly water soluble. Because of this, a need exists to develop drug formulations that will permit the expanded use of such drugs. The use of liposomes and lipid nanoparticles for drug delivery has attracted attention as a technique for solubilizing molecules that are poorly water soluble, but this technique faces serious scale-up risks. In this study, we report on attempts to encapsulate Coenzyme Q10 (CoQ10) as a model of a poorly water-soluble drug in an MITO-Porter, a liposome for mitochondrial delivery using a microfluidic device (a CoQ10-MITO-Porter [μ]). The physical properties of the CoQ10-MITO-Porter [μ] including homogeneity, size, and preparation volume were compared with those for a CoQ10-MITO-Porter prepared by the ethanol dilution method (a CoQ10-MITO-Porter [ED]). In the case where a microfluidic device was used, a small-sized CoQ10-MITO-Porter was formed homogeneously, and it was possible to prepare it on a large scale. Intracellular observations using HeLa cells showed that the CoQ10-MITO-Porter [μ] was efficiently internalized by cells to reach mitochondria. These results indicate that the CoQ10-MITO-Porter [μ] represents a potential candidate for use in mitochondrial nanomedicine.

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）, The development of efficient gene delivery systems targeting the lung endothelium remains a serious challenge. This study reports on the design and optimization of a multifunctional envelope-type nanodevice (MEND) for an efficient siRNA delivery to the lung endothelium based on GALA-peptide targeting ability. The incorporation of a pH-sensitive lipid (YSK05) results in a dramatic improvement in silencing efficiency by enhancing endosomal escape, but this also causes a reduction in the lung selectivity. Contrary to the assumption that active targeting is largely dependent on the presence of a targeting ligand, the findings of the present study indicate that nanocarrier composition is critical for achieving the organ selectivity. Interestingly, helper lipids substantially mask the liver delivery resulting in optimum lung targeting. The optimized YSK05-MEND is 40-fold more efficient than a previously developed MEND, with a robust lung endothelium gene knockdown at small doses. The YSK05-MEND strongly inhibits a metastatic lung cancer model and exerts superior control over lung metastasis compared to chemotherapy or the previously developed MEND. The YSK05-MEND is well-tolerated in mice after acute or chronic administration. As far as it is known, YSK05-MEND achieves the most efficient lung endothelium gene silencing reported thus far with a median effective dose of 0.01 mg siRNA kg −1 while minimally affecting the endothelium of other organs.

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）, Lipid nanoparticles (LNPs) are one of the more promising technologies for efficiently delivering short interfering RNA (siRNA) in vivo. A pH-sensitive cationic lipid that facilitates the targeting of hepatocytes and endosomal escape, strongly influences the availability of siRNA, thus making it a key material for efficient siRNA delivery. A systematic knowledge regarding lipid structure-activity relationships would greatly facilitate the development of sophisticated pH-sensitive cationic lipids for use in siRNA-based therapeutics. The systemic derivatization of a hydrophilic head group and hydrophobic tails of YSK12-C4, a pH-sensitive cationic lipid that was developed in our laboratory, revealed that hydrophilic head significantly affected the apparent pKa of the final product, a key factor in both intrahepatic distribution and endosomal escape. The clogP value of a hydrophilic head group was found to be associated with the apparent pKa of the product. In contrast, the hydrophobic tail structure strongly affected intrahepatic distribution without depending on apparent pKa. A structure-activity relationship study enabled the selection of an adequate combination of a hydrophilic head group and hydrophobic tails and permitted a potent LNP composed of a pH-sensitive cationic lipid CL4H6 (CL4H6-LNPs) to be developed that showed efficient gene silencing activity (50% effective dose: 0.0025 mg/kg), biodegradability and was tolerated. In vivo experiments revealed that the CL4H6-LNPs showed a superior efficiency for endosomal escape, cytosolic release and the RNA-induced silencing for the complex-loading of siRNAs compared to the previously developed LNPs.

【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, ＜文献概要＞Short interfering RNA(siRNA)は,RNA干渉により任意の遺伝子発現が抑制可能な機能性核酸である。多くの難治性疾患に対する新規治療モダリティとして魅力的であるが,単独では組織移行能を示さないことから,医薬としての応用には目的細胞へ選択的かつ効率的にsiRNAを送達する技術(drug delivery system:DDS)の開発が不可欠である。これまでに脂質,ポリマーやリガンド分子コンジュゲート等の多様な分子設計・マテリアル開発がなされてきており,複数の製剤が臨床試験に進んでいる。2018年8月には世界初のsiRNA医薬品が認可され,まさにsiRNA医薬時代の幕開けとなった。本稿では,近年のsiRNAのDDS開発状況を概説するとともに,今後の課題や展望について述べる。

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）, This paper reports a methodology for precise controlling the lipid nanoparticle (LNP) size by stepwise and rapid ethanol dilution using an integrated microfluidic device with baffle mixers. The integrated microfluidic device coupling the LNP synthesis and the post-treatment regions had better size controllability of LNPs than the conventional preparation methods. Additionally, 30 nm-sized siRNA-loaded LNPs prepared by the post-treatment process using the integrated microfluidic device showed great gene-silencing activity and specific intrahepatic biodistribution. The stepwise and rapid ethanol dilution methodology using the integrated microfluidic device provides LNPs with homogeneous size distribution for improving the efficacy of nanomedicines.

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, WILEY, 2017年09月, ［査読有り］
英語, 研究論文（学術雑誌）, Most anticancer drugs are intended to function in the nuclei of cancer cells. If an anticancer drug could be delivered to mitochondria, the source of cellular energy, this organelle would be destroyed, resulting in the arrest of the energy supply and the killing of the cancer cells. To achieve such an innovative strategy, a mitochondrial drug delivery system targeted to cancer cells will be required. We recently reported on the development of a MITO-Porter, a liposome for mitochondrial delivery. In this study, we validated the utility of such a cancer therapeutic strategy by delivering anticancer drugs directly to mitochondria. We succeeded in packaging doxorubicin (DOX) as a model cargo in MITO-Porter to produce a DOX-MITO-Porter. We evaluated cellular toxicity of OS-RC-2 cell, a type of DOX-resistant cancer cell, after delivering DOX to mitochondria using the MITO-Porter system. Cell viability was decreased by the DOX-MITO-Porter treatment, while cell viability was not decreased in the case of naked DOX and a conventional DOX liposomal formulation. We also found a relationship between cellular toxicity and mitochondrial toxicity. The use of a MITO-Porter system for mitochondrial delivery of a toxic agent represents a possible therapeutic strategy for treating drug-resistant cancers. (C) 2017 American Pharmacists Association (R). Published by Elsevier Inc. All rights reserved.

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, WILEY, 2017年09月, ［査読有り］
英語, 研究論文（学術雑誌）, A alpha-helical GALA peptide (WEAALAEALAEALAEHLAEALAEALEALAA) has been found to possess dual functions: a pH-dependent inducer of endosomal escape, and a ligand that targets lung endothelium. In the present study, the flexibility of GALA was improved by modifying the edge with polyethylene glycol linker, to increase lung-targeting activity. We first investigated the uptake of the GALA-modified liposomes in which GALA was directly conjugated to the lipid (Cholesterol: GALA/Chol) or the phospholipid-PEG (GALA/PEG(2000)). The liposomes that were modified with GALA/PEG(2000) (GALA/PEG(2000)-LPs) were taken up at a higher level by human lung endothelial cells (HMVEC-L), in comparison with particles that were modified with GALA/Chol (GALA/Chol-LPs). Small-interfering RNA-encapsulating liposomal-based nanocarriers (multifunctional envelope-type nano device: MEND) that were formulated with a vitamin E-scaffold SS-cleavable pH-activated lipid-like material, namely GALA/PEG(2000)-MENDssPalmE were also modified with GALA/PEG(2000). Gene silencing activity in the lung endothelium was then evaluated against an endothelial marker; CD31. In comparison with the unmodified MENDssPalmE, GALA/PEG(2000)-MENDssPalmE exhibited a higher silencing activity in the lung. Optimization of GALA/PEG(2000)-MENDssPalmE resulted in silencing activity in the lung with an ED50 value of 0.21 mg/kg, while non-specific gene silencing in liver was marginal. Collectively, PEGylated GALA is a promising device for use in targeting the lung endothelium. (C) 2017 American Pharmacists Association (R). Published by Elsevier Inc. All rights reserved.

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, PHARMACEUTICAL SOC JAPAN, 2017年07月, ［査読有り］
英語, 研究論文（学術雑誌）, In the active targeting of a drug delivery system (DDS), the density of the ligand on the functionalized liposome determines its affinity for binding to the target. To evaluate these densities on the surface of different sized liposomes, 4 liposomes with various diameters (188, 137, 70, 40 nm) were prepared and their surfaces were modified with fluorescently labeled ligand lipid conjugates by the post-insertion method. Each liposomal mixture was fractionated into a series of fractions using size exclusion chromatography (SEC), and the resulting liposome fractions were precisely analyzed and the surface ligand densities calculated. The data collected using this methodology indicate that the density of the ligand on a particle is greatly dependent on the size of the liposome. This, in turn, indicates that smaller lip osomes (75-40nm) tend to possess higher densities. For developing active targeting systems, size and the density of the ligands are two important and independent factors that can affect the efficiency of a system as it relates to medical use.

リンパ節内樹状細胞を標的とした極小ナノキャリアシステムの開発　　　　　　　　　　　　　　　
河合 美典, 中村 孝司, 佐藤 悠介, 真栄城 正寿, 原島 秀吉
日本DDS学会学術集会プログラム予稿集, 33回, 175, 175, 日本DDS学会, 2017年06月
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）, Lipid nanoparticles (LNPs) or liposomes are the most widely used drug carriers for nanomedicines. The size of LNPs is one of the essential factors affecting drug delivery efficiency and therapeutic efficiency. Here, we demonstrated the effect of lipid concentration and mixing performance on the LNP size using microfluidic devices with the aim of understanding the LNP formation mechanism and controlling the LNP size precisely. We fabricated microfluidic devices with different depths, 11 μm and 31 μm, of their chaotic micromixer structures. According to the LNP formation behavior results, by using a low concentration of the lipid solution and the microfluidic device equipped with the 31 μm chaotic mixer structures, we were able to produce the smallest-sized LNPs yet with a narrow particle size distribution. We also evaluated the mixing rate of the microfluidic devices using a laser scanning confocal microscopy and we estimated the critical ethanol concentration for controlling the LNP size. The critical ethanol concentration range was estimated to be 60–80% ethanol. Ten nanometer-sized tuning of LNPs was achieved for the optimum residence time at the critical concentration using the microfluidic devices with chaotic mixer structures. The residence times at the critical concentration necessary to control the LNP size were 10, 15–25, and 50 ms time-scales for 30, 40, and 50 nm-sized LNPs, respectively. Finally, we proposed the LNP formation mechanism based on the determined LNP formation behavior and the critical ethanol concentration. The precise size-controlled LNPs produced by the microfluidic devices are expected to become carriers for next generation nanomedicines and they will lead to new and effective approaches for cancer treatment.

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, WILEY-V C H VERLAG GMBH, 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）, Nanomedicines promise to extend drug therapy from small molecular compounds to proteins/nucleic acids/genes. Multifunctional envelope-type nanodevices (MENDs) have been developed for delivering such molecules to the site of action. The YSK-MEND contains new types of pH-responsive cationic lipids to efficiently deliver siRNA to hepatocytes via receptor-mediated endocytosis and use in treating hepatitis C and B in model mice. The RGD ligand is introduced to target tumor endothelial cells (TEC) and RGD-MEND is able to send siRNA to TEC to regulate the function of tumor microenvironments. The MITO-Porter is also developed to target mitochondria via membrane fusion. Antisense oligo RNA in the MITO-Porter permits the knock down of mitochondrial function. Finally, the ssPalms is designed based on a new concept of pH-dependent protonation in endosomes and cleavage of SS bonds in the reducing conditions in cytosol. These new technologies promise to stimulate the use of Nanomedicines in the future.

Development of a multifunctional envelope-type nano device and its application to nanomedicine
Yusuke Sato, Takashi Nakamura, Yuma Yamada, Hideyoshi Harashima
JOURNAL OF CONTROLLED RELEASE, 244, Pt B, 194, 204, ELSEVIER SCIENCE BV, 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）, Successful nanomedicines should be based on sound drug delivery systems (DDS) the permit intracellular trafficking as well as the biodistribution of cargos to be controlled. We have been developing new types of DDS that are multifunctional envelope-type nano devices referred to as MENDs. First, we will focus the in vivo delivery of siRNA to hepatocytes using a YSK-MEND which is composed of pH-responsive cationic lipids. The YSK-MEND is capable of inducing efficient silencing activity in hepatocytes and can be used to cure mice that are infected with hepatitis C or B. The YSK-MEND can also be applied to cancer immunotherapy through the activation of immune cells by delivering different compounds such as cyclic-di-GMP, siRNA or alpha-galactosylceramide as a lipid antigen. The findings indicate that, as predicted, these compounds, when encapsulated in the YSK-MEND, can be delivered to the site of action and induced immune activation through different mechanisms. Finally, a MITO-Porter, amembrane fusion-based delivery system to mitochondria, is introduced as an organelle targeting DDS and a new strategy for cancer therapy is proposed by delivering gentamicin to mitochondria of cancer cells. These new technologies are expected to extend the therapeutic area of Nanomedicine by increasing the power of DDS, especially from the view point of controlled intracellular trafficking. (C) 2016 Elsevier B.V. All rights reserved.

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, PHARMACEUTICAL SOC JAPAN, 2016年12月, ［査読有り］
英語, 研究論文（学術雑誌）, The surface topology of ligands on liposomes is an important factor in active targeting in drug delivery systems. Accurately evaluating the density of anchors and bioactive functional ligands on a liposomal surface is critical for ensuring the efficient delivery of liposomes. For evaluating surface ligand density, it is necessary to clarify that on the ligand-modified liposomal surfaces, some anchors are attached to ligands but some are not. To distinguish between these situations, a key parameter, surface anchor density, was introduced to specify amount of total anchors on the liposomal surface. Second, the parameter reaction yield was introduced to identify the amount of ligand-attached anchors among total anchors, since the conjugation efficiency is not always the same nor 100%. Combining these independent parameters, we derived: incorporation ratio=surface anchor densityxreaction yield. The term incorporation ratio defines the surface ligand density. Since the surface anchor density represents the density of polyethylene glycol (PEG) on the surfaces in most cases, it also determines liposomal function. It is possible to accurately characterize various PEG and ligand densities and to define the surface topologies. In conclusion, this quantitative methodology can standardize the liposome preparation process and qualify the modified liposomal surfaces.

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, NATURE PUBLISHING GROUP, 2016年11月, ［査読有り］
英語, 研究論文（学術雑誌）, Gene silencing by small interfering RNA (siRNA) is useful for analyzing the functions of human immune cells. However, the transfection of siRNA to human immune cells is difficult. Here, we used a multifunctional envelope-type nanodevice (MEND) containing YSK12-C4 (YSK12-MEND) to efficiently introduce siRNA to human immune cell lines, Jurkat, THP-1, KG-1 and NK92. The YSK12-MEND was transfected to human immune cell lines at a siRNA dose range of 1-30 nM, resulting that maximum gene silencing efficiencies at the mRNA level in Jurkat, THP-1, KG-1 and NK92 were 96%, 96%, 91% and 75%, respectively. The corresponding values for Lipofectamine RNAiMAX (RNAiMAX) were 37%, 56%, 43% and 19%, respectively. The process associated with cellular uptake played a role in effective gene silencing effect of the YSK12-MEND. The small size and high non-aggregability of the YSK12MEND were advantageous for the cellular internalization of siRNA to immune cell lines. In the case of RNAiMAX, a drastic increase in particles size was observed in the medium used, which inhibited cellular uptake. The YSK12-MEND reported in herein appears to be appropriate for delivering siRNA to human immune cells, and the small particle size and non-aggregability are essential properties.

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, PHARMACEUTICAL SOC JAPAN, 2016年10月, ［査読有り］
英語, 研究論文（学術雑誌）, Lipin1, a bifunctional protein, regulates fatty acid utilization in the triglyceride biosynthesis pathway. In the current study, using a liver-specific in vivo short interfering RNA (siRNA) delivery system, we examined the pathological and physiological roles of hepatic Lipin1 in the development of insulin resistance and the maintenance of systemic energy homeostasis. Liver-specific silencing of Lipin1 expression was achieved by the systemic administration of siRNA against Lpin1 mRNA (siLpin1)-loaded lipid nanoparticles (LNPs) to wild type mice at 3-4 d intervals for 25 d. The siLpin1-treated mice showed normal blood glucose levels and insulin sensitivity, however, triglyceride (TG) levels were reduced in liver and peripheral blood of them. The knockdown of hepatic Lipin1 in mice led to marked decrease in adipose tissue mass and adipocyte diameters in epididymal and inguinal fat depots without the undesired silencing of Lipin1 in adipose tissue. In summary, we report for the first time that the down-regulation of hepatic Lipin1 expression leads to less adiposity as well as a decrease in TG level in the liver and blood circulation, without any alterations in the glucose tolerance and blood glucose levels. Our findings may provide new insights into the physiological roles of hepatic Lipin1 in systemic energy homeostasis.

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, ELSEVIER SCIENCE BV, 2016年07月, ［査読有り］
英語, 研究論文（学術雑誌）, The PEGylated liposomal (PEG-LP) Doxorubicin, PEG-LP (DOX), with a diameter of around 100 nm, accumulates in tumors via the enhanced permeability and retention (EPR) effect, and is used clinically for the treatment of several types of cancer. However, there are a number of tumor types that are resistant to DOX. We report herein on a unique anti-tumor effect of PEG-LP (DOX) in a DOX-resistant tumor xenograft model. PEG-LP (DOX) failed to suppress the growth of the DOX-resistant tumors (ex. non-small cell lung cancer, H69AR; renal cell carcinoma, OSRC-2) as observed in the xenograft model. Unexpectedly, tumor growth was suppressed in a DOX-resistant breast cancer (MDA-MB-231) xenograft model. We investigated the mechanism by which PEG-LP (DOX) responses differ in different drug resistant tumors. In hyperpermeable OSRC-2 tumors, PEG-LP was distributed to deep tumor tissues, where it delivers DOX to drug-resistant tumor cells. In contrast, extracellular matrix (ECM) molecules such as collagen, pericytes, cancer-associated fibroblasts render MDA-MB-231 tumors hypopermeable, which limits the extent of the penetration and distribution of PEG-LP, thereby enhancing the delivery of DOX to the vicinity of the tumor vasculature. Therefore, a remarkable anti-angiogenic effect with a preferential suppression in tumor growth is achieved. Based on the above findings, it appears that the response of PEG-LP (DOX) to drug-resistant tumors results from differences in the tumor microenvironment. (C) 2016 Elsevier B.V. All rights reserved.

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, ELSEVIER SCIENCE BV, 2016年05月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, Because nanoparticles with diameters less than 50 nm penetrate stromal-rich tumor tissues more efficiently, the synthesis of small-sized nanoparticles encapsulating short interfering RNA (siRNA) is important in terms of realizing novel siRNA medicine for the treatment of various cancers. Lipid nanoparticles (LNPs) are the leading systems for the delivery of siRNA in vivo. Limit size LNPs were successfully synthesized using a microfluidic mixing technique. However, the physicochemical properties and potential for in vivo siRNA delivery of the limit-size LNPs have not been examined in detail. In the present study, we prepared LNPs with different diameters from 32 to 67 nm using a microfluidic mixing devise and examined the physicochemical properties of the particles and the potential for their use in delivering siRNA in vitro and in vivo to liver. Reducing the size of the LNPs causes poor-packing and an increased surface area, which result in their instability in serum. Moreover, it was revealed that the ability of endosomal escape (cytosolic siRNA release) of the smaller LNPs is subject to inhibition by serum compared to that of larger counterparts. Taken together, an increase in packing and avoiding the adsorption of serum components are key strategies for the development of next-generation highly potent and small-sized LNPs. (C) 2016 Elsevier B.V. All rights reserved.

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, CELL PRESS, 2016年04月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, While a variety of short interfering RNA (siRNA) delivery compounds have been developed, a deep understanding of the key parameters that determine the quality of siRNA delivery are not known with certainty. Therefore, an understanding of the factors required for the efficient, selective, and safe delivery of siRNA is a great challenge for successful siRNA delivery. Herein, we report on the development of two pH-sensitive cationic lipids and their use in examining the impact of the acid dissociation constant (pK(a)) value, lipase sensitivity and the size of lipid nanoparticles on the biodistribution, and efficiency and cell specificity of gene silencing in the liver. An increase in the pK(a) value resulted in a significant change in the intrahepatic localization of siRNA and gene-silencing efficiency in hepatocytes and liver sinusoidal endothelial cells (LSECs). The sensitivity of the pH-sensitive cationic lipid to lipases was a major factor in achieving hepatocyte-specific gene silencing. Increasing the particle size can improve the LSEC specificity of gene silencing. As a consequence, we succeeded in developing both a highly efficient, hepatocyte-specific formulation, and the most efficacious LSEC-targeted formulation reported to date. These findings will facilitate the development of more sophisticated siRNA delivery systems.

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, ELSEVIER SCIENCE BV, 2016年03月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, Background & Aims: Antiviral agents including entecavir (ETV) suppress the replication of the hepatitis B virus (HBV) genome in human hepatocytes, but they do not reduce the abundance of viral proteins. The present study focused on effectively reducing viral protein levels.
Methods: We designed siRNAs (HBV-siRNA) that target consensus sequences in HBV genomes. To prevent the emergence of escaped mutant virus, we mixed three HBV-siRNAs (HBV-siRNAmix); the mixture was encapsulated in a novel pH-sensitive multifunctional envelope-type nanodevice (MEND), a hepatocyte-specific drug delivery system. Coagulation factor 7 siRNA was used to assess delivery and knockdown efficiencies of MEND/siRNA treatments in mice. The potency of MEND/HBV-siRNAmix was evaluated in primary human hepatocytes and in chimeric mice with humanized liver persistently infected with HBV.
Results: Effective knockdown of targets, efficient delivery of siRNA, and liver-specific delivery were each observed with MEND. MEND/HBV-siRNA caused efficient reduction of HBsAg and HBeAg in vitro and in vivo. However, ETV treatment did not efficiently reduce HBsAg or HBeAg when compared with a single MEND/HBV-siRNAmix treatment. Furthermore, the suppressive effects of a single dose of MEND/HBV-siRNAmix persisted for 14 days in vitro and in vivo.
Conclusion: We demonstrated that MEND/HBV-siRNA controlled HBV more efficiently than did ETV. Furthermore, the effect of a single dose of MEND/HBV-siRNA persisted for a long time. These results indicated that MEND/HBV-siRNA may be a promising novel HBV treatment that is more effective than reverse transcriptase inhibitors. (C) 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

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, ELSEVIER SCIENCE BV, 2016年03月, ［査読有り］
英語, 研究論文（学術雑誌）, Applying small interfering RNA (siRNA) to dendritic cell (DC) based therapy represents a potential candidate for cancer immunotherapy. However, delivering siRNA to DCs is a challenging issue for non-viral vectors. To date, only viral vectors have achieved efficient gene silencing in DCs. We report herein that a novel cationic lipid, YSK12-C4, when loaded in a nanoparticle with siRNA (YSK12-C4 multifunctional envelope type nano device [YSK12-MEND]), greatly facilitated gene silencing in mouse DCs. The use of the YSK12-MEND resulted in a gene silencing efficiency in excess of 90%, with a median effective dose (ED50) of 1.5 nM, whereas the maximum gene silencing efficiency of Lipofectamine RNAiMAX was less than 60% and the ED50 was 25 nM. Furthermore, suppressor of cytokine signaling 1, an immune suppressive molecule in DCs, silenced in the mouse DC by the YSK12-MEND showed a drastic enhancement in cytokine production, resulting in the significant suppression of tumor growth when it was applied to DC-based therapy against a mouse lymphoma. These results clearly indicate that YSK12-MEND overcomes the obstacle associated with non-viral vectors and can be considered to be a promising non-viral vector for siRNA delivery to DCs, thus accelerating DC-based therapies with siRNA. (C) 2016 Elsevier B.V. All rights reserved.

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, ELSEVIER SCIENCE BV, 2015年11月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, Macrophages are key contributors to various inflammatory diseases. Therefore, the development of an efficient in vivo short interference RNA (siRNA) system that can be delivered to macrophages represents a novel treatment strategy for addressing these disorders. It was recently revealed that peritoneal macrophages (PEMs) are involved in several diseases including ovarian cancer, and are now recognized as a promising drug target. We report herein on the use of pH-sensitive cationic YSK05-MENDs as siRNA carriers and on the impact of both the size of the YSK05-MENDs and their administration routes for the efficient targeting PEMs to achieve a high level of gene silencing activity. The size of the YSK05-MENDs had a dramatic effect on their specificity for PEMs when administered intravenously, but not for intraperitoneal injection. Also, significant gene silencing was achieved by an intraperitoneal administration of the YSK05-MEND at a dose in the single digit mg/kg range. To our knowledge, this is the most efficacious method for siRNA delivery for gene silencing in PEMs in vivo reported to date. These findings enabled us to investigate the complex function of PEMs through several gene silencing simultaneously. (C) 2015 Elsevier B.V. All rights reserved.

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, ELSEVIER SCIENCE BV, 2015年10月, ［査読有り］
英語, 研究論文（学術雑誌）, The delivery of small interfering RNA (siRNA) to dendritic cells (DCs) is a challenging issue for siRNA-loaded lipid nanoparticles. The cause of this difficulty is unknown. The findings reported herein indicate that the rate-limiting step in gene silencing using siRNA-loaded lipid nanoparticles in DCs, as evidenced by a quantitative analysis of each process in siRNA delivery between mouse bone marrow derived DC (BMDC) and other cell lines, was not associated with the actual delivery of siRNA. A gene silencing of only 50% was observed in BMDC, even when a high dose was used. Contrary to our expectation, the interval between cellular uptake and the delivery of siRNA to the cytosol was not responsible for the low gene silencing. Meanwhile, a drastic difference was found in the relationship between the efficiency of gene silencing and the amount of intracellular intact siRNA. This fact indicates that the processes after cytosolic delivery of siRNA, namely the intracellular pharmacodynamics (PD) of siRNA, appear to be the rate-limiting step in gene silencing in BMDC. The findings reported here demonstrate the importance of the intracellular PD of siRNA delivered to cytosol in the development of siRNA delivery systems for gene silencing in DCs. (C) 2015 Elsevier B.V. All rights reserved.

Liposomes loaded with a STING pathway ligand, cyclic di-GMP, enhance cancer immunotherapy against metastatic melanoma
Takashi Nakamura, Hiroko Miyabe, Mamoru Hyodo, Yusuke Sato, Yoshihiro Hayakawa, Hideyoshi Harashima
JOURNAL OF CONTROLLED RELEASE, 216, 149, 157, ELSEVIER SCIENCE BV, 2015年10月, ［査読有り］
英語, 研究論文（学術雑誌）, Malignant melanomas escape immunosurveillance via the loss/down-regulation of MHC-I expression. Natural killer (NK) cells have the potential to function as essential effector cells for eliminating melanomas. Cyclic di-GMP (c-di-GMP), a ligand of the stimulator of interferon genes (STING) signal pathway, can be thought of as a new class of adjuvant against cancer. However, it is yet to be tested, because technologies for delivering c-di-GMP to the cytosol are required. Herein, we report that c-di-GMP efficiently activates NK cells and induces antitumor effects against malignant melanomas when loaded in YSK05 lipid containing liposomes, by assisting in the efficient delivery of c-di-GMP to the cytosol. The intravenous administration of c-di-GMP encapsulated with-in YSK05-liposomes (c-di-GMP/YSK05-Lip) into mice efficiently induced the production of type I interferon (IFN) as well as the activation of NK cells, resulting in a significant antitumor effect in a lung metastasis mouse model using B16-F10. This antitumor effect was dominated by NK cells. The infiltration of NK cells was observed in the lungs with B16-F10 melanomas. These findings indicate that the c-di-GMP/YSK05-Lip induces MHC-I nonrestricted antitumor immunity mediated by NK cells. Consequently, c-di-GMP/YSK05-Lip represents a potentially new adjuvant system for use in immunotherapy against malignant melanomas. (C) 2015 Elsevier B.V. All rights reserved.

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, AMER CHEMICAL SOC, 2015年09月, ［査読有り］
英語, 研究論文（学術雑誌）, A lipid nanoparticle (LNP) composed of a series of SS-cleavable and pH-activated lipid-like materials (ssPalm) was previously developed as a platform of a gene delivery system. A tertiary amine and disulfide bonding were employed to destabilize the endosomal membrane and for intracellular collapse. We report herein on the development of a hepatocyte-targeting siRNA carrier by the molecular tuning of the hydrophobic scaffold, and tertiary amine structures. The gene knockdown activity against a hepatocyte-specific marker (factor VII: FVII) was improved when a more fat-soluble vitamin (vitamin E) was employed as a hydrophobic scaffold. Moreover, to allow the tertiary amines to accept protons by sensing a slight change in endosomal acidification, its structural flexibility was minimized by fixing it in a piperidine structure, and the distance between the surface of the particle to the ternary amine was increased. As a result, the pK(a) value was increased to the approximately 6.18 depending on its distance, while the pK(a) reached plateau when the tertiary amine was linked by an excess number of linear carbon chains. The pH-dependent membrane destabilization activity, as assessed by a hemolysis assay, was increased in parallel with the pK(a) value. Moreover, the gene knockdown activity was improved in parallel with hemolytic activity. Finally, further optimization of the lipid/siRNA ratio, and the use of chemically (2'-fluoro) modified siRNA synergistically improved the gene knockdown efficacy to an effective dose (ED50) of 0.035 mg/kg. The developed ssPalm represents a promising platform for use as a hepatocyte-targeting siRNA carrier.

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, SOC POLYMER SCIENCE JAPAN, 2015年
日本語, 研究論文（学術雑誌）, This comprehensive paper describes the anionic ring-opening polymerization of substituted epoxides using a phosphazene base catalyst, t-Bu-P-4, and its application for the synthesis of architecturally complex polyethers. The t-Bu-P-4-catalyzed ROP of 1,2-butylene oxide (BO) produced the poly(BO) having the predicted molecular weight and narrow polydispersity. The kinetic analysis revealed that the ROP system proceeded in a living manner. The t-Bu-P(4)catalyzed ROP was then applied to the polymerization of various epoxides having a variety of side chains, e.g. styrene oxide. The synthesis of star-shaped polyethers was achieved by the t-Bu-P-4-catalyzed ROP using polyols as an initiator. The syntheses of cyclic-, figure-eight-shaped, and tadpole-shaped polyethers were also achieved via the intramolecular click reaction of the well-defined linear precursors bearing azido and ethynyl functions at designed positions.

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）, This paper described a simple preparation method for small-size and monodispersed lipid nanoparticles (LNPs) by using microfluidic devices. The fundamental role and importance of chaotic micromixer in the microfluidic device was demonstrated. The suitable cycle number of chaotic micromixer was confirmed for precise controlling LNPs size with narrow distribution under the any flow rate conditions. In addition, LNPs containing siRNA was synthesized for evaluation of penetration efficiency via in vivo experiment. The PEGylated LNPs containing siRNA with a diameter of 30 nm could penetrate to the mouse parenchymal liver cells rather than the LNPs with a diameter of 50 nm.

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, CELL PRESS, 2015年01月, ［査読有り］, ［国際共著］
英語, 研究論文（学術雑誌）, Host innate recognition triggers key immune responses for viral elimination. The sensing mechanism of hepatitis B virus (HBV), a DNA virus, and the subsequent downstream signaling events remain to be fully clarified. Here we found that type III but not type I interferons are predominantly induced in human primary hepatocytes in response to HBV infection, through retinoic acid-inducible gene-I (RIG-I)-mediated sensing of the 5'-epsilon region of HBV pregenomic RNA. In addition, RIG-I could also counteract the interaction of HBV polymerase (P protein) with the 5'-epsilon region in an RNA-binding dependent manner, which consistently suppressed viral replication. Liposome-mediated delivery and vector-based expression of this e region-derived RNA in liver abolished the HBV replication in human hepatocyte-chimeric mice. These findings identify an innate-recognition mechanism by which RIG-I dually functions as an HBV sensor activating innate signaling and to counteract viral polymerase in human hepatocytes.

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, ELSEVIER SCIENCE BV, 2014年09月, ［査読有り］
英語, Nanomedicine is expected to be a basic technology for using nucleic acids as a drug, in which treating the cause of diseases represent the ultimate therapy. However, a sophisticated delivery system is required for efficient delivery of RNA/DNA, since these compounds need precise control of intracellular trafficking as well as biodistribution. Here we report on the use of a multifunctional envelope-type nano device (MEND) which is capable of intracellular trafficking such as endosomal escape, delivery to mitochondria, as well as active targeting to selective tissues/cells in vivo. In this review, we focused on the controlled intracellular trafficking of antigens for advanced immunotherapy, and then introduced a mitochondrial delivery system as an organelle targeting system for unmet medical needs. We also provide a successful in vivo delivery of siRNA to the liver based on a newly designed pH-responsive cationic lipid. Finally we will discuss an important role of an active targeting system using a peptide ligand to adipose vasculature. These progresses in drug delivery system will break through the barriers exist in our body, tissues and cells and open a window for future Nanomedicine. (C) 2014 Elsevier B.V. All rights reserved.

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, ELSEVIER SCIENCE BV, 2014年06月, ［査読有り］
英語, 研究論文（学術雑誌）, Cyclic dinucleotides are of importance in the field of microbiology and immunology. They function as second messengers and are thought to participate in the signal transduction of cytosolic DNA immune responses. One such dinucleotide, cyclic di-GMP (c-di-GMP), stimulates the immune system. It is thought that c-di-GMP is recognized by ATP dependent RNA helicase (DDX41) in the cytosol, forms a complex with the Stimulator of interferon genes protein (STING), triggers a signal via the tank binding kinase 1-interferon regulatory factor 3 (TBK1-IRF3) pathway and induces the production of type I interferons. Therefore c-di-GMP can be thought of as a new class of adjuvant. However, because c-di-GMP contains two phosphate groups, this prevents its use as an adjuvant because it cannot pass through the cell membrane, even though the target molecule of c-di-GMP is located in the cytoplasm.
Our group has been developing a series of liposomal drug delivery systems and recently investigated YSK05 which is a synthetic, pH sensitive lipid that has a high fusogenicity. We utilized this lipid as a carrier to transport c-di-GMP into the cytosol to then use c-di-GMP as an adjuvant. Based on screening experiments, YSK05/POPE/cholesterol = 40/25/35 was found to induce IFN-beta in Raw264.7 cells. The induction of IFN-beta from c-di-GMP liposomes was inhibited by adding BX795, a TBK1 inhibitor, indicating that the production of IFN-beta caused the activation of the STING-TBK1 pathway. C-di-GMP liposomes also showed significantly higher levels of expression of CD80, CD86 and MHC class I. The c-di-GMP/YSK05 liposome facilitated antigen specific cytotoxic T cell activity and the inhibition of tumor growth in a mouse model. These findings indicate that c-di-GMP/YSK05 liposomes could be used, not only to transfer c-di-GMP to the cytosol and induce an innate immune system but also as a platform for investigating the mechanism of immune sensing with cyclic dinucleotides in vitro and in vivo. (C) 2014 Elsevier B.V. All rights reserved.

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, ELSEVIER SCIENCE BV, 2014年04月, ［査読有り］
英語, 研究論文（学術雑誌）, A disorder in the brain endothelium is thought to be closely related to the pathophysiology of brain diseases. A method for delivering nucleic acids (i.e. short interference RNA; siRNA) to the brain endothelium should be an attractive strategy for curing brain disorders. A liposornal nanoparticle containing a proton ionizable amino lipid was recently developed as a carrier of encapsulated siRNA. The aim of this study was to evaluate the utility of apolipoprotein E (ApoE) as a targeting ligancl for mouse brain endothelial cells (MBEC4 cells). The cellular uptake of the ApoE-modified nanoparticles was gradually increased in an ApoE-density dependent mariner. Furthermore, the ApoE-modified nanoparticles were taken up via both clathrin and caveolae mediated endocytosis, thus permitting them to avoid lysosomal degradation. Finally, endogenous gene silencing in MBEC4 cells was efficiently achieved depending on the ApoE-modification. Collectively, the ApoE-modified nanoparticle is a promising carrier for delivering nucleic acids to the brain endothelium. (c) 2014 Elsevier B.V. All rights reserved.

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, NATURE PUBLISHING GROUP, 2014年04月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, The development of RNA interference (RNAi)-based therapy faces two major obstacles: selecting small interfering RNA (siRNA) sequences with strong activity, and identifying a carrier that allows efficient delivery to target organs. Additionally, conservative region at nucleotide level must be targeted for RNAi in applying to virus because hepatitis C virus (HCV) could escape from therapeutic pressure with genome mutations. In vitro preparation of Dicer-generated siRNAs targeting a conserved, highly ordered HCV 5' untranslated region are capable of inducing strong RNAi activity. By dissecting the 5'-end of an RNAi-mediated cleavage site in the HCV genome, we identified potent siRNA sequences, which we designate as Dicer-hunting siRNAs (dh-siRNAs). Furthermore, formulation of the dh-siRNAs in an optimized multifunctional envelope-type nano device inhibited ongoing infectious HCV replication in human hepatocytes in vivo. Our efforts using both identification of optimal siRNA sequences and delivery to human hepatocytes suggest therapeutic potential of siRNA for a virus.

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, NATURE PUBLISHING GROUP, 2014年03月, ［査読有り］
英語, 研究論文（学術雑誌）, Over the past decade, considerable advances have been made in the discovery of gene targets in metabolic diseases. However, in vivo studies based on molecular biological technologies such as the generation of knockout mice and the construction of short hairpin RNA vectors require considerable effort and time, which is a major limitation for in vivo functional analysis. Here, we introduce a liver-specific nonviral small interfering RNA (siRNA) delivery system into rapid and efficient characterization of hepatic gene targets in metabolic disease mice. The comparative transcriptome analysis in liver between KKAy diabetic and normal control mice demonstrated that the expression of monoacylglycerol O-acyltransferase 1 (Mogat1), an enzyme involved in triglyceride synthesis and storage, was highly elevated during the disease progression. The upregulation of Mogat1 expression in liver was also found in other genetic (db/db) and diet-induced obese mice. The silencing of hepatic Mogat1 via a liver-specific siRNA delivery system resulted in a dramatic improvement in blood glucose levels and hepatic steatosis as well as overweight with no apparent overall toxicities, indicating that hepatic Mogat1 is a promising therapeutic target for metabolic diseases. The integrated approach with transcriptomics and nonviral siRNA delivery system provides a blueprint for rapid drug discovery and development.

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）, Efficient delivery continues to be a challenge in microRNA (miRNA) therapeutics. We utilized a pH-sensitive multifunctional envelope-type nano device (MEND) containing a pH-sensitive lipid YSK05 (YSK05-MEND) to regulate liver specific miRNA-122 (miR-122). Anti-microRNA oligonucleotides including 2′-OMe and phosphorothioate modifications against miR-122 (AMO122) were encapsulated in the YSK05-MEND. Despite the lower uptake, the YSK05-MEND showed a higher activity in liver cancer cells than Lipofectamine2000 (LFN2k) due to efficient endosomal escape. Cytotoxicity was minimal at 100 nM of AMO122 in YSK05-MEND treated cells, but LFN2k showed toxicity at 50 nM. When mice were administrated with free AMO122, it was eliminated via the kidney due to its molecular weight, and lesser amounts were detected in the liver. Conversely, the YSK05-MEND delivered higher amounts of the AMO122 to the liver. Systemic administration of YSK05-MEND induced the knockdownofmiR-122andan increase in target genes inthe liver, and a subsequent reduction in plasma cholesterol at a dose of 1mgAMO/kgwhile free AMO122 showed no activity at the same dose. The effect ofAMO122 delivered by YSK05-MEND persisted for over 2 weeks. These results suggest that YSK05-MEND is a promising system for delivering AMOs to the liver.

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, DOVE MEDICAL PRESS LTD, 2014年, ［査読有り］
英語, 研究論文（学術雑誌）, An innovative drug delivery technology is urgently needed to satisfy unmet medical needs in treating various brain disorders. As a fundamental carrier for plasmid DNA or nucleic acids, we developed a liposomal nanoparticle (multifunctional envelope-type nano device [MEND]) containing a proton-ionizable amino lipid (YSK-MEND). Here we report on the impact of apolipoprotein E (ApoE) modification on the function of YSK-MEND in terms of targeting brain cells. The cellular uptake and function of YSK-MEND encapsulating short interference RNA or plasmid DNA were significantly improved as a result of ApoE modification in mouse neuron-derived cell lines (Neuro-2a and CAD). Intracerebroventricular administration of ApoE-modified YSK-MEND (ApoE/YSK-MEND) encapsulating plasmid DNA also resulted in higher transgene expression in comparison with YSK-MEND that was not modified with ApoE. Moreover, observation of fluorescence-labeled ApoE/YSK-MEND and expression of mCherry (fluorescence protein) derived from plasmid DNA indicated that this carrier might be useful for delivering and conferring transgene expression in neural stem cells and/or neural progenitor cells. Thus, this system may be a useful tool for the treatment of neurodegenerative disease.

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, ELSEVIER ACADEMIC PRESS INC, 2014年, ［査読有り］
英語, 論文集(書籍)内論文, It is anticipated that nucleic acid medicines will be in widespread use in the future, since they have the potential to cure diseases based on molecular mechanisms at the level of gene expression. However, intelligent delivery systems are required to achieve nucleic acid therapy, since they can perform their function only when they reach the intracellular site of action. We have been developing a multifunctional envelope-type nanodevice abbreviated as MEND, which consists of functional nucleic acids as a core and lipid envelope, and can control not only biodistribution but also the intracellular trafficking of nucleic acids. In this chapter, we review the development and evolution of the MEND by providing several successful examples, including the R8-MEND, the KALA-MEND, the MITO-Porter, the YSK-MEND, and the PALM.

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, OXFORD UNIV PRESS, 2013年12月, ［査読有り］
英語, 研究論文（学術雑誌）, MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression post-transcriptionally. Previous studies, which characterized miRNA function, revealed their involvement in fundamental biological processes. Importantly, miRNA expression is deregulated in many human diseases. Specific inhibition of miRNAs using chemically modified anti-miRNA oligonucleotides (AMOs) can be a potential therapeutic strategy for diseases in which a specific miRNA is overexpressed. 2'-O-Methyl (2'-OMe)-4'-thioRNA is a hybrid type of chemically modified oligonucleotide, exhibiting high binding affinity to complementary RNAs and high resistance to nuclease degradation. Here, we evaluate 2'-OMe-4'-thioribonucleosides for chemical modification on AMOs. Optimization of the modification pattern using a variety of chemically modified AMOs that are perfectly complementary to mature miR-21 revealed that the uniformly 2'-OMe-4'-thioribonucleoside modified AMO was most potent. Further investigation showed that phosphorothioate modification contributed to long-term miR-122 inhibition by the 2'-OMe-4'-thioribonucleoside-modified AMO. Moreover, systemically administrated AMOs to mouse using a liposomal delivery system, YSK05-MEND, showed delivery to the liver and efficient inhibition of miR-122 activity at a low dose in vivo.

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）, SS-cleavable proton-activated lipid-like material (ssPalm) functions as a key element in a lipid nanoparticle in which pDNA is encapsulated. The ssPalm contains dual sensing motifs that can respond to the intracellular environment
a proton-sponge unit (tertiary amines) that functions in response to an acidic environment (endosome/lysosome), and disulfide bonding that can be cleaved in a reducing environment (cytosol). © 2013 WILEY-VCH Verlag GmbH &
Co. KGaA, Weinheim.

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, ELSEVIER SCIENCE BV, 2012年11月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, Modification of liposomal siRNA carriers with polyethylene glycol, i.e., PEGylation, is a generally accepted strategy for achieving in vivo stability and delivery to tumor tissue. However, PEGylation significantly inhibits both cellular uptake and the endosomal escape process of the carriers. In a previous study, we reported on the development of a multifunctional envelope-type nano device (MEND) for siRNA delivery and peptide-based functional devices for overcoming the limitations and succeeded in the efficient delivery of siRNA to tumors. In this study, we synthesized a pH-sensitive cationic lipid, YSK05, to overcome the limitations. The YSK05-MEND had a higher ability for endosomal escape than other MENDs containing conventional cationic lipids. The PEGylated YSK05-MEND induced efficient gene silencing and overcame the limitations followed by optimization of the lipid composition. Furthermore, the intratumoral administration of the YSK05-MEND resulted in a more efficient gene silencing compared with MENDs containing conventional cationic lipids. Collectively, these data confirm that YSK05 facilitates the endosomal escape of the MEND and thereby enhances the efficacy of siRNA delivery into cytosol and gene silencing. (c) 2012 Elsevier B.V. All rights reserved.

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, ELSEVIER SCI LTD, 2011年08月, ［査読有り］
英語, 研究論文（学術雑誌）, An siRNA that specifically silences the expression of mRNA is a potential therapeutic agent for dealing with many diseases including cancer. However, the poor cellular uptake and bioavailability of siRNA remains a major obstacle to clinical development. For efficient delivery to tumor tissue, the pharmacokinetics and intracellular trafficking of siRNA must be rigorously controlled. To address this issue, we developed a liposomal siRNA carrier, a multi-functional nano device (MEND). We describe herein an approach for systemic siRNA delivery to tumors by combining the MEND system with shGALA, a fusogenic peptide. In cultured cell experiments, shGALA-modification enhanced the endosomal escape of siRNA encapsulated in a polyethylene glycol modified MEND (PEG-MEND), resulting in an 82% knockdown of the target gene. In vivo systemic administration clarified that the shGALA-modified MEND (shGALA-MEND) showed 58% gene silencing in tumor tissues at a dose of 4 mg of siRNA/kg body weight. In addition, a significant inhibition of tumor growth was observed only for the shGALA-MEND and no somatic or hepatic toxicity was observed. Given the above data, this peptide-modified delivery system, a shGALA-MEND has great potential for the systemic delivery of therapeutic siRNA aimed at cancer therapy. (C) 2011 Elsevier Ltd. All rights reserved.

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, PHARMACEUTICAL SOC JAPAN, 2010年07月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, The delivery of nucleic acids to cancer cells represents a potentially useful strategy. Previously, we developed a multifunctional envelope-type nano device (MEND) for the efficient delivery of plasmid DNA. In addition, we successfully delivered short interference RNA (siRNA) into cytoplasm using a MEND which contains siRNA particles that were produced using stearyl octaarginine (STR-R8). In the present study, to achieve further gene silencing activity compared with STR-R8, various additional polycations were screened. We used protamine and 10 different polypeptides containing random sequence of basic amino acids. The ability of these polycations to form nano particles with siRNA were evaluated by measuring the size and zeta-potential of produced nano particles, and as a consequence, 6 of the polycations were selected for further evaluation. We then prepared MENDs containing the particles. The lipid composition of the MEND consisted of dioleoylphosphatidyl ethanolamine (DOPE)/phosphatidic acid (PA) (7/2). For cellular uptake and endosomal escape, the MEND was modified with PPD (polyethylene glycol (PEG)-peptide-DOPE), STR-R8 and GALA, pH-sensitive fusogenic peptide. The resulting MEND had a diameter of 120-170 nm and a zeta-potential of 15-25 mV. The MEND was transfected into HeLa cells stably expressing luciferase and the silencing activity of the polycations was compared. Most of the polycations failed to knockdown luciferase activity. However, the polypeptide containing ornithine and tryptophan (Orn/Trp) induced a higher knockdown than STR-R8. In addition, Orn/Trp induced a silencing effect at lower doses than STR-R8, as evidenced by dose-response data. In conclusion, the findings suggest that Orn/Trp is a superior polycation to STR-R8 for siRNA delivery.

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
The last few years have witnessed a great advance in the development of nonviral systems for in vivo targeted delivery of nucleic acids. Lipid nanoparticles (LNPs) are the most promising carriers for producing clinically approved products in the future. Compared with other systems used for nonviral gene delivery, LNPs provide several advantages including higher stability, low toxicity, and greater efficiency. Additionally, systems based on LNPs can be modified with ligands and devices for controlled biodistribution and internalization into specific cells. Efforts are ongoing to improve the efficiency of lipid-based gene vectors. These efforts depend on the appropriate design of nanocarriers as well as the development of new lipids with improved gene delivery ability. Several ionizable lipids have recently been developed and have shown dramatically improved efficiency. However, enhancing the ability of nanocarriers to target specific cells in the body remains the most difficult challenge. Systemically administered LNPs can access organs in which the capillaries are characterized by the presence of fenestrations, such as the liver and spleen. The liver has received the most attention to date, although targeted delivery to the spleen has recently emerged as a promising tool for modulating the immune system. In this review, we discuss recent advances in the use of LNPs for cell-specific targeted delivery of nucleic acids. We focus mainly on targeting liver hepatocytes and spleen immune cells as excellent targets for gene therapy. We also discuss the potential of endothelial cells as an alternate approach for targeting organs with a continuous endothelium., 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］
This paper describes development of a three-dimensional (3D) baffle mixer device for precise size control of various types of lipid nanoparticles (LNPs) with high encapsulation efficiency of short interfering RNAs (siRNAs). The 3D baffle mixer device achieved more precise size control of various LNPs than that of the conventional micromixer device. In addition, the 3D baffle mixer enabled effective capturing of siRNAs into LNPs without any assistance of electrostatic interaction between lipid molecules and siRNAs. The 3D baffle mixer device is expected to become one of the key platforms for production of novel lipid-based nucleic acid nanocarriers., 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
Introduction: The discovery of RNA interference (RNAi) earned the 2006 Nobel Prize in Physiology or Medicine for its biological significance and potential for developing novel therapeutics. The small interfering RNA (siRNA) is the most promising tool for translating RNAi to clinical use. Efforts are ongoing to improve siRNA delivery through developing novel biomaterials and delivery strategies. Given time, it appears that siRNA drugs will eventually become a reality. Areas covered: The currently used approaches for siRNA delivery are discussed with a focus on siRNA therapeutics currently in clinical testing. A comparison of advantageous aspects of currently available platforms and the possibility of further optimization for increased efficiency and safety are presented. Future directions in siRNA delivery are also highlighted. Expert opinion: The recent success in the field of siRNA delivery is based mainly on developing new biomaterials with extraordinarily high activities. Notably, the introduction of ionizable lipids and novel targeting ligands represent two huge steps for realizing siRNA therapy. The currently available systems are largely directed to the liver and the new challenge is to extend their applicability for treating diseases of other organs. Active targeting to different organs is the most promising approach for developing new siRNA-based therapeutics., 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
This paper reports a lipid nanoparticles (LNP) production method and its formation behavior using microfluidic devices with baffle structures. The microfluidic devices showed great mixing efficiency at 500 μL/min, and we achieved 20 nm-sized LNPs production that chaotic micromixers were not able to produce at the same flow rate condition. Additionally, we found that the smaller-sized LNPs/siRNA prepared by baffle structures have higher penetration efficiency than that of the larger-sized LNPs, but all of them showed the gene silencing activity. The microfluidic devices with baffle structures are expected to be a practicable apparatus for DDSs application., 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］
This paper describes lipid nanoparticles (LNPs) formation behavior in microfluidic devices equipped with the staggered herringbone micromixers (SHM) in order to control the LNPs size precisely. Three types of microfluidic devices with different heights of mixer structures were fabricated to examine the effect of mixer structure on the LNPs formation behavior. We found the height of the grooved structures, the lipid concentration, and the ethanol concentration were significant factors for controlling LNPs size and its distribution., 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
The development of a carrier for the delivery of siRNA is a factor in the realization of RNA interference (RNAi) therapeutics. Modification of siRNA carriers with polyethylene glycol, <i>i.e</i>., PEGylation, is a general strategy for stabilizing a particle in the blood stream and delivering it to tissue or cells. However, it is well-known that, when a carrier is modified by PEGylation, it results in a significant inhibition of both cellular uptake and the endosomal escape process. In a previous study, we reported on the development of a multifunctional envelope-type nano device (MEND) for delivering siRNA and peptide-based functional devices for overcoming the effects conferred by PEGylation and succeeded in the delivery of siRNA to tumor tissue. In this study, we noticed that the pH-sensitive property, changing from neutral to cationic in response to a decrease in pH, could avoid the inhibition caused by PEGylation and succeeded in synthesizing a pH-sensitive cationic lipid, YSK05. The YSK05-MEND had a higher fusogenicity and potency for endosomal escape than other MENDs containing conventional cationic lipids. The PEGylated YSK05-MEND induced efficient gene silencing and avoided the inhibition of endosomal escape caused by PEGylation followed by optimization of the lipid composition. Furthermore, the intratumoral injection of the PEGylated YSK05-MEND resulted in a more efficient gene silencing compared with MENDs containing conventional cationic lipids. Thus, the YSK05-MEND is a promising siRNA carrier for avoiding the inhibition in intracellular trafficking caused by PEGylation both <i>in vitro</i> and <i>in vivo</i>.<br>, 公益社団法人 日本薬学会, 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
MARY ANN LIEBERT INC, 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
MARY ANN LIEBERT INC, 英語, 研究発表ペーパー・要旨（国際会議）

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

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

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