研究者データベース

中村 孝司(ナカムラ タカシ)
薬学研究院 医療薬学部門 医療薬学分野
助教

基本情報

所属

  • 薬学研究院 医療薬学部門 医療薬学分野

職名

  • 助教

学位

  • 博士(生命科学)(北海道大学)

論文上での記載著者名

  • Takashi Nakamura
  • 中村 孝司

J-Global ID

研究キーワード

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

研究分野

  • ライフサイエンス / 薬系化学、創薬科学
  • ライフサイエンス / 薬系分析、物理化学
  • ライフサイエンス / 薬系分析、物理化学
  • ライフサイエンス / 生体材料学
  • ライフサイエンス / 生体医工学

職歴

  • 2012年11月 - 2017年10月 国立薬品食品衛生研究所 薬品部 協力研究員
  • 2011年01月 北海道大学大学院薬学研究院 助教
  • 2010年04月 - 2010年12月 北海道大学大学院薬学研究院 博士研究員
  • 2007年04月 - 2010年03月 日本学術振興会 特別研究員(DC1)

学歴

  • 2007年04月 - 2010年03月   北海道大学   大学院生命科学院   生命科学専攻博士後期課程
  • 2005年04月 - 2007年03月   北海道大学   大学院薬学研究科   医療薬学専攻修士課程

所属学協会

  • 日本核酸医薬学会   日本がん免疫学会   

研究活動情報

論文

  • Takashi Nakamura, Koharu Yamada, Yusuke Sato, Hideyoshi Harashima
    International journal of pharmaceutics 587 119652 - 119652 2020年07月17日 [査読有り][通常論文]
     
    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.
  • Takashi Nakamura, Hideyoshi Harashima
    Advanced drug delivery reviews 2020年06月06日 [査読有り][通常論文]
     
    It is generally known that the lymph nodes (LNs) are important tissues in cancer immunotherapy. Therefore, delivering immune functional compounds to LNs is a useful strategy for enhancing cancer immunotherapy. Lipid-based nanocarriers have been widely used as delivery systems that target LNs, but lipid nanoparticle (LNP) technology has recently attracted increased interest. High levels of nucleic acids can be efficiently loaded in LNPs, they can be used to actively deliver nucleic acids into the cytoplasm, and they can be produced on an industrial scale. The use of microfluidic devices has been particularly valuable for producing small-sized LNPs, thus paving the way for successful LN targeting. In the review, we focus on the potential of LNP technology for targeting LNs.
  • Hideyuki Masuda, Takashi Nakamura, Hideyoshi Harashima
    Journal of pharmaceutical sciences 109 6 1943 - 1950 2020年06月 [査読有り][通常論文]
     
    Interest has developed in the bacillus Calmette-Guerin (BCG) cell wall skeleton (BCG-CWS) as a noninfectious adjuvant. Although BCG-CWS readily undergoes aggregation, in a previous study, we applied it to cancer immunotherapy via intravenous administration by encapsulating the BCG-CWS into nanoparticles (CWS-NPs). The CWS-NPs were taken up by major histocompatibility complex (MHC) class II+ (MHC-II+) cells and induced antigen-specific cytotoxic T lymphocyte (CTL) activity. However, the nature of the contribution of MHC-II+ cells to the CTL response continues to be unclear. In this study, we investigated the relationship between the distribution of CWS-NPs in the spleen and CTL activity. The main MHC-II+ cells that internalized the CWS-NPs were B cells. Decreasing the level of polyethylene glycol modification increased the uptake of CWS-NPs by B cells, leading to an increased CTL activity. A comparison of CWS-NPs with different uptake efficiencies into dendritic cells and B cells suggested that the DCs with internalized CWS-NPs may contribute to CTL activation compared with B cells. We succeeded in enhancing CTL activity by the CWS-NPs, and the findings reported herein should provide important information regarding target cells for the development of CWS-NP.
  • Mio Maeta, Naoya Miura, Hiroki Tanaka, Takashi Nakamura, Ryo Kawanishi, Yoshifumi Nishikawa, Kenichi Asano, Masato Tanaka, Shinya Tamagawa, Yuta Nakai, Kota Tange, Hiroki Yoshioka, Hideyoshi Harashima, Hidetaka Akita
    Molecular pharmaceutics 17 4 1237 - 1247 2020年04月06日 [査読有り][通常論文]
     
    DNA vaccinations are promising strategies for treating diseases that require cellular immunity (i.e., cancer and protozoan infection). Here, we report on the use of a liposomal nanocarrier (lipid nanoparticles (LNPs)) composed of an SS-cleavable and pH-activated lipidlike material (ssPalm) as an in vivo DNA vaccine. After subcutaneous administration, the LNPs containing an ssPalmE, an ssPalm with vitamin E scaffolds, elicited a higher gene expression activity in comparison with the other LNPs composed of the ssPalms with different hydrophobic scaffolds. Immunization with the ssPalmE-LNPs encapsulating plasmid DNA that encodes ovalbumin (OVA, a model tumor antigen) or profilin (TgPF, a potent antigen of Toxoplasma gondii) induced substantial antitumor or antiprotozoan effects, respectively. Flow cytometry analysis of the cells that had taken up the LNPs in draining lymph nodes (dLNs) showed that the ssPalmE-LNPs were largely taken up by macrophages and a small number of dendritic cells. We found that the transient deletion of CD169+ macrophages, a subpopulation of macrophages that play a key role in cancer immunity, unexpectedly enhanced the activity of the DNA vaccine. These data suggest that the ssPalmE-LNPs are effective DNA vaccine carriers, and a strategy for avoiding their being trapped by CD169+ macrophages will be a promising approach for developing next-generation DNA vaccines.
  • 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][通常論文]
     
    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.
  • Nakamura T, Yamada Y, Sato Y, Khalil IA, Harashima H
    Biomaterials 218 119329  2019年10月 [査読有り][通常論文]
  • Endo R, Nakamura T, Kawakami K, Sato Y, Harashima H
    Scientific reports 9 1 11335  2019年08月 [査読有り][通常論文]
  • Masuda H, Nakamura T, Noma Y, Harashima H
    Molecular pharmaceutics 15 12 5762 - 5771 2018年12月 [査読有り][通常論文]
  • Kawai M, Nakamura T, Miura N, Maeta M, Tanaka H, Ueda K, Higashi K, Moribe K, Tange K, Nakai Y, Yoshioka H, Harashima H, Akita H
    Nanomedicine : nanotechnology, biology, and medicine 2018年08月 [査読有り][通常論文]
  • Nakamura T, Yamada K, Fujiwara Y, Sato Y, Harashima H
    Molecular pharmaceutics 15 6 2142 - 2150 2018年06月 [査読有り][通常論文]
  • Kajimoto K, Katsumi T, Nakamura T, Kataoka M, Harashima H
    Journal of the American Oil Chemists Society 95 1 101 - 109 2018年 [査読有り][通常論文]
  • Nakamura T
    Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan 138 12 1443 - 1449 2018年 [査読有り][通常論文]
  • Nakamura T, Harashima H
    Therapeutic delivery 8 11 987 - 1000 2017年11月 [査読有り][通常論文]
  • Naoya Miura, Hidetaka Akita, Naho Tateshita, Takashi Nakamura, Hideyoshi Harashima
    MOLECULAR THERAPY 25 4 1003 - 1013 2017年04月 [査読有り][通常論文]
     
    For a successful anti-cancer vaccine, antigen presentation on the major histocompatibility complex (MHC) class I is a requirement. To accomplish this, an antigen must be delivered to the cytoplasm by overcoming the endosome/lysosome. We previously reported that a lipid nanoparticle modified with a KALA peptide (WEAKLAKALAKALAKHLAKALAICALICA), an alpha-helical cationic peptide, permits the encapsulated pDNA to be efficiently delivered to the cytoplasm in bone marrow derived dendritic cells (BMDCs). Herein, we report on the use of KALA-modified liposomes as an antigen carrier, in an attempt to induce potent antigen-specific cellular immunity. The subcutaneous injection of KALA-modified ovalbumin (OVA)-encapsulating liposomes (KALA-OVA-LPs) elicited a much more potent OVA-specific cytotoxic T lymphocyte activity and anti-tumor effect in comparison with particles that were modified with octa-arginine (R8), a cell-penetrating peptide (R8-OVA-LPs). In addition, the numbers of OVA-specific CD8(+) T cells were increased by immunization the KALAOVA-LPs. The treatment of BMDCs with KALA-OVA-LPs induced a substantial MHC class I antigen presentation. Furthermore, the acidic pH-dependent membrane destabilization activity of KALA-OVA-LPs strongly suggests that they are able to escape from endosomes/lysosomes and thereby deliver their cargos to the cytoplasm. Collectively, the KALAmodified liposome is a potential antigen delivery platform for use as a protein vaccine.
  • Takashi Nakamura, Yosuke Noma, Yu Sakurai, Hideyoshi Harashima
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 40 2 234 - 237 2017年02月 [査読有り][通常論文]
     
    Intravesical drug delivery by cationic liposomes (Cat-LPs) represents a potent nanotechnology for enhancing therapeutic effects against bladder disorders. However, preventing the aggregation of Cat-LPs in urine poses a significant barrier. We report on an examination of the effect of modifying liposomes with polyethylene glycol (PEG) lipids to prevent Cat-LPs from aggregating in human urine. Although Cat-LPs underwent significant aggregation in human urine, introducing 5 mol% of PEG2k lipid or 2 mol% of PEG5k lipid completely inhibited the aggregation of the Cat-LPs. When 2 mol% of PEG2k lipids were introduced, the lipid structures of 1,2-distearoly-sn-glycero-3-phosphoethanolamine (DSPE) and 1,2-distearoyl-sn-glycerol (DSG) greatly prevented aggregation compared with cholesterol. By contrast, when Cat-LPs, after incubation in urine, were exposed to bladder cancer cells, only introducing cholesteryl-PEG into the Cat-LPs showed a significant enhancement in cellular uptake. These results offer the potential for incorporating cholesteryl-PEG into Cat-LPs for achieving both stability in urine and effective cellular uptake.
  • Yusuke Sato, Yu Sakurai, Kazuaki Kajimoto, Takashi Nakamura, Yuma Yamada, Hidetaka Akita, Hideyoshi Harashima
    MACROMOLECULAR BIOSCIENCE 17 1 2017年01月 [査読有り][通常論文]
     
    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.
  • Yusuke Sato, Takashi Nakamura, Yuma Yamada, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 244 Pt B 194 - 204 2016年12月 [査読有り][通常論文]
     
    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.
  • Takashi Nakamura, Moeka Kuroi, Yuki Fujiwara, Shota Warashina, Yusuke Sato, Hideyoshi Harashima
    SCIENTIFIC REPORTS 6 37849  2016年11月 [査読有り][通常論文]
     
    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.
  • Takashi Nakamura
    YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 136 11 1477 - 1484 2016年11月 [査読有り][通常論文]
     
    Delivery systems are a powerful technology for enhancing the effect of cancer immunotherapy. We have been in the process of developing lipid-based delivery systems for controlling the physical properties and dynamics of immunofunctional molecules such as antigens and adjuvants. The lipid nanoparticulation of these molecules improves their physical properties, resulting in a good water dispensability, greater stability, and small size. The cell wall skeleton of bacille Calmette-Guerin (BCG-CWS) could be used to replace live BCG as a drug for treating bladder cancer, but problems associated with the physical properties of BCG-CWS have prevented its use. To overcome such problems, we developed a novel packaging method that permits BCG-CWS to be encapsulated into lipid nanoparticles, which induce antitumor responses against bladder cancer. Lipid nanoparticulation also improves the intracellular trafficking and biodistribution of immunofunctional molecules. Cyclic di-GMP (c-di-GMP) is an adjuvant that is recognized by the cytosolic sensor. However, c-di-GMP cannot pass through the cell membrane. We encapsulated c-di-GMP into lipid nanoparticles containing a pH-responsive lipid that was developed in our laboratory and achieved efficient cytosolic delivery and the induction of antitumor immunity. Furthermore, we are attempting to control the functions of immune cells by RNA interference. We have recently succeeded in the efficient delivery of small interfering RNA into mouse dendritic cells (DCs), which led to the enhancement of antitumor activity of DCs. In this review, our recent efforts regarding cancer immunotherapy using lipid-based nanoparticles are reviewed.
  • Shota Warashina, Takashi Nakamura, Yusuke Sato, Yuki Fujiwara, Mamoru Hyodo, Hiroto Hatakeyama, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 225 183 - 191 2016年03月 [査読有り][通常論文]
     
    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.
  • 小暮 健太朗
    Biomat. Sci. 4 No.3 439 - 447 2016年02月23日 [査読有り][通常論文]
     
    Membrane fusion is a rational strategy for crossing intracellular membranes that present barriers to liposomal nanocarrier-mediated delivery of plasmid DNA into the nucleus of non-dividing cells, such as dendritic cells. Based on this strategy, we previously developed nanocarriers consisting of a nucleic acid core particle coated with four lipid membranes [Akita, et al., Biomaterials, 2009, 30, 2940-2949]. However, including the endosomal membrane and two nuclear membranes, cells possess three intracellular membranous barriers. Thus, after entering the nucleus, nanoparticles coated with four membranes would still have one lipid membrane remaining, and could impede cargo delivery. Until now, coating a core particle with an odd number of lipid membranes was challenging. To produce nanocarriers with an odd number of lipid membranes, we developed a novel coating method involving lipid nano-discs, also known as bicelles, as a material for packaging DNA in a carrier with an odd number of lipid membranes. In this procedure, bicelles fuse to form an outer coating that resembles a patchwork quilt, which allows the preparation of nanoparticles coated with only three lipid membranes. Moreover, the transfection activity of dendritic cells with these three-membrane nanoparticles was higher than that for nanoparticles coated with four lipid membranes. In summary, we developed novel nanoparticles coated with an odd number of lipid membranes using the novel "patchwork-packaging method" to deliver plasmid DNA into the nucleus via membrane fusion.
  • Asako Yamada, Asako Mitsueda, Mahadi Hasan, Miho Ueda, Susumu Hama, Shota Warashina, Takashi Nakamura, Hideyoshi Harashima, Kentaro Kogure
    BIOMATERIALS SCIENCE 4 3 439 - 447 2016年 [査読有り][通常論文]
     
    Membrane fusion is a rational strategy for crossing intracellular membranes that present barriers to liposomal nanocarrier-mediated delivery of plasmid DNA into the nucleus of non-dividing cells, such as dendritic cells. Based on this strategy, we previously developed nanocarriers consisting of a nucleic acid core particle coated with four lipid membranes [Akita, et al., Biomaterials, 2009, 30, 2940-2949]. However, including the endosomal membrane and two nuclear membranes, cells possess three intracellular membranous barriers. Thus, after entering the nucleus, nanoparticles coated with four membranes would still have one lipid membrane remaining, and could impede cargo delivery. Until now, coating a core particle with an odd number of lipid membranes was challenging. To produce nanocarriers with an odd number of lipid membranes, we developed a novel coating method involving lipid nano-discs, also known as bicelles, as a material for packaging DNA in a carrier with an odd number of lipid membranes. In this procedure, bicelles fuse to form an outer coating that resembles a patchwork quilt, which allows the preparation of nanoparticles coated with only three lipid membranes. Moreover, the transfection activity of dendritic cells with these three-membrane nanoparticles was higher than that for nanoparticles coated with four lipid membranes. In summary, we developed novel nanoparticles coated with an odd number of lipid membranes using the novel "patchwork-packaging method" to deliver plasmid DNA into the nucleus via membrane fusion.
  • Abdelmegeed H, Nakamura T, Harashima H
    Journal of pharmaceutical sciences 105 1 250 - 256 2016年01月 [査読有り][通常論文]
     
    Alpha-galactosylceramide (GC) represents a potentially new class of adjuvant because GC strongly induces interferon (IFN) gamma production from natural killer T (NKT) cells, leading to the induction of strong antitumor immunity. Interleukin (IL)-12 is another stimulating signal that induces IFN-gamma production by NKT cells. We report herein on an investigation of the effect of recombinant IL-12 on NKT cell activation, when used in combination with GC-loaded octaarginine modified liposomes (GC-Lip). IFN-gamma production from splenocytes simulated with GC-Lip was dose dependently enhanced in the presence of IL-12 in vitro. In contrast, IFN-gamma production in vivo was enhanced at a low dose of IL-12. Enhanced IFN-gamma production was observed in the case of low doses (0.5 mu g and 2.5 mu g) of GC-Lip but not a high dose (5 mu g), that is, the IL-12 combination enhanced NKT cell activation at a 10-fold lower GC dose. The use of the above combination also enhanced the expansion of the NKT cell population. These findings indicate that in vivo IFN-gamma production is inversely correlated with the dose of IL-12 during dual signal stimulation of NKT cells via both GC-Lip and IL-12, indicating that the dose of GC-Lip can be reduced without weakening NKT cell activation. (C) 2016 American Pharmacists Association (R). Published by Elsevier Inc. All rights reserved.
  • Takashi Nakamura, Yuki Fujiwara, Shota Warashina, Hideyoshi Harashima
    INTERNATIONAL JOURNAL OF PHARMACEUTICS 494 1 271 - 277 2015年10月 [査読有り][通常論文]
     
    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.
  • Takashi Nakamura, Hiroko Miyabe, Mamoru Hyodo, Yusuke Sato, Yoshihiro Hayakawa, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 216 149 - 157 2015年10月 [査読有り][通常論文]
     
    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.
  • Nakamura T, Kuroi M, Harashima H
    Molecular pharmaceutics 12 8 2791 - 2799 2015年08月 [査読有り][通常論文]
     
    Alpha-galactosylceramide (GC), a lipid antigen present on CD1d molecules, is a unique adjuvant that enables a strong antitumor effect to be induced via activation of natural killer T cells. We previously reported that a liposomal formulation of GC significantly enhanced GC presentation via CD1d and antitumor immunity. However, the influence of the intracellular fate of liposomes controlled by the lipid composition on GC presentation using GC-loaded liposomes (GC-Lip) remains unclear. In this study, we prepared a GC-Lip formulation by incorporating dioleoyl-phosphatidylethanolamine (DOPE)/cholesterol, egg phosphatidylcholine (EPC)/cholesterol, and distearoyl phosphocholine (DSPC)/cholesterol, and investigated the relationship between the intracellular trafficking of GC-Lip and GC presentation in antigen-presenting cells. When GC-Lip was prepared using DOPE, a fusogenic lipid, the endosomal escape of liposomes was enhanced, resulting in a decrease in GC presentation of CD1d, compared to the EPC based GC-Lip (EPC/GC-Lip). The stability of liposomes in endosomes/lysosomes had no influence on GC presentation. The DSPC based GC-Lip (DSPC/GC-Lip) induced GC presentation without any detectable degradation in liposomal structure, although the EPC/GC-Lip induced GC presentation with degradation of liposomal structure. The efficiency of GC presentation between EPC/GC-Lip and DSPC/GC-Lip was comparable. These GC presentations that were independent of the degradation of liposomes were dominated by saposins, sphingolipid activator proteins. Our findings reveal that GC presentation on CD1d from the fluid liposomes involves the action of saposins, regardless of whether liposome degradation occurs. This insight can be of use in terms of developing GC-Lip formulation for efficient GC presentation.
  • Naoya Miura, Sharif M. Shaheen, Hidetaka Akita, Takashi Nakamura, Hideyoshi Harashima
    NUCLEIC ACIDS RESEARCH 43 3 1317 - 1331 2015年02月 [査読有り][通常論文]
     
    Technologies that delivery antigen-encoded plasmid DNA (pDNA) to antigen presenting cell and their immune-activation are required for the success of DNA vaccines. Here we report on an artificial nanoparticle that can achieve these; a multifunctional envelope-type nanodevice modified with KALA, a peptide that forms alpha-helical structure at physiological pH (KALA-MEND). KALA modification and the removal of the CpG-motifs from the pDNA synergistically boosted transfection efficacy. In parallel, transfection with the KALA-MEND enhances the production of multiple cytokines and chemokines and co-stimulatory molecules via the Toll-like receptor 9-independent manner. Endosome-fusogenic lipid envelops and a long length of pDNA are essential for this immune stimulation. Furthermore, cytoplasmic dsDNA sensors that are related to the STING/TBK1 pathway and inflammasome are involved in IFN-beta and IL-1 beta production, respectively. Consequently, the robust induction of antigen-specific cytotoxic T-lymphoma activity and the resulting prophylactic and therapeutic anti-tumor effect was observed in mice that had been immunized with bone marrow-derived dendritic cells ex vivo transfected with antigen-encoding pDNA. Collectively, the KALA-MEND possesses dual functions; gene transfection system and immune-stimulative adjuvant, those are both necessary for the successful DNA vaccine.
  • Takashi Nakamura, Masafumi Fukiage, Yoshiteru Suzuki, Ikuya Yano, Jun Miyazaki, Hiroyuki Nishiyama, Hideyuki Akaza, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 196 161 - 167 2014年12月 [査読有り][通常論文]
     
    We previously reported on the development of a water soluble formulation of the cell wall skeleton of BCG (BCG-CWS), a major immune active center of BCG, by encapsulating it into a nanoparticle (CWS-NP). The CWS-NP allowed us to clarify the machinery associated with the BCG mediated anti-bladder tumor effect, especially the roles of bladder cancer cells and dendritic cells (DCs) in the initial step, which remains poorly understood. We show herein that the internalization of BCG-CWS by bladder cancer cells, but not DCs, is indispensable for the induction of an antitumor effect against bladder cancer. Tumor growth was significantly inhibited in mice that had been inoculated with mouse bladder cancer (MBT-2) cells containing internalized BCG-CWS. On the other hand, the internalization of BCG-CWS by DCs had only a minor effect on inducing an antitumor effect against MBT-2 tumors. This was clarified for the first time by using the CWS-NP. This finding provides insights into our understanding of the role of bladder cancer cells and DCs in BCG therapy against bladder cancer. (C) 2014 Elsevier B.V. All rights reserved.
  • Kazuaki Kajimoto, Yusuke Sato, Takashi Nakamura, Yuma Yamada, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 190 593 - 606 2014年09月 [査読有り][通常論文]
     
    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.
  • Takashi Nakamura, Kouhei Ono, Yoshiteru Suzuki, Rumiko Moriguchi, Kentaro Kogure, Hideyoshi Harashima
    MOLECULAR PHARMACEUTICS 11 8 2787 - 2795 2014年08月 [査読有り][通常論文]
     
    Exogenous antigen proteolysis by proteasomes and amino peptidases is essential for the production of mature major histocompatibility complex class 1 (MHC-I) peptides to induce cross-presentation. We report here that when liposomes are modified with octaarginine (R8-Lip), a type of cell-penetrating peptide, the production of the mature MIC-I peptide is enhanced by promoting the C-terminal trimming of the antigen peptide. The efficiency of cross-presentation of ovalbumin (OVA) using the R8-Lip was dramatically higher than that by octalysine modified liposomes (K8-Lip) in mouse bone-marrow derived dendritic cells (BMDCs), although the physical characters of both liposomes were comparable. In this study, we investigated the mechanism responsible for the enhancement in cross-presentation by R8-Lip. Although the efficiencies of cellular uptake, endosomal escape, proteolysis of OVA and DC maturation between the two systems were essentially the same, an analysis of peptide trimming to SIINFEKL (mature MHC-I peptide of OVA) by using R8-Lip and K8-Lip encapsulating peptides of various length dearly indicates that the use of R8-Lip enhances the efficiency of the C-terminal cleavage of antigen-derived peptides. This finding provides a new strategy for achieving efficient cross-presentation by using R8 peptide and arginine-rich peptides. Moreover, this result may contribute to the development of a new paradigm regarding the machinery associated with antigen peptide production.
  • Hiroko Miyabe, Mamoru Hyodo, Takashi Nakamura, Yusuke Sato, Yoshihiro Hayakawa, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 184 20 - 27 2014年06月 [査読有り][通常論文]
     
    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.
  • Yuki Hattori, Daisuke Morita, Nagatoshi Fujiwara, Daiki Mori, Takashi Nakamura, Hideyoshi Harashima, Sho Yamasaki, Masahiko Sugita
    JOURNAL OF BIOLOGICAL CHEMISTRY 289 22 15405 - 15412 2014年05月 [査読有り][通常論文]
     
    Background: A host receptor has not yet been identified for glycerol monomycolate (GroMM), an immunostimulatory lipid of mycobacteria. Results: GroMM recognition occurred in cell transfectants expressing human, but not mouse Mincle. Human Mincle transgenic mice acquired the ability to respond to GroMM. Conclusion: GroMM is a ligand for human Mincle. Significance: The molecular basis underlying the innate immune recognition of GroMM has been elucidated. An array of lipidic compounds that constitute the cell wall of mycobacteria is recognized by host receptors. Examples include trehalose dimycolate (TDM), which is a major surface-exposed glycolipid of mycobacteria, that interacts with the macrophage inducible C-type lectin, Mincle, and exerts its highly potent adjuvant functions. Recent evidence has suggested that glycerol monomycolate (GroMM), another mycolate-containing lipid species produced by mycobacteria, can stimulate innate immune cells; however, its specific host receptors have yet to be identified. We here demonstrated that cell transfectants expressing human Mincle (hMincle) reacted to both TDM and GroMM, while those expressing mouse Mincle (mMincle) only reacted to TDM and failed to recognize GroMM. Studies using domain swap chimeras confirmed that the ectodomain of hMincle, but not that of mMincle, interacted with GroMM, and site-directed mutagenesis analyses revealed that short stretches of amino acid residues at positions 174-176 and 195-196 were involved in GroMM recognition. To further substantiate the differential recognition of GroMM by hMincle and mMincle, hMincle transgenic/mMincle knock-out mice (i.e. hMincle(+) mice) were established and compared with non-transgenic mice (i.e. mMincle(+) mice). We showed that macrophages derived from hMincle(+) mice were activated by GroMM and produced inflammatory cytokines, whereas those derived from mMincle(+) mice did not exhibit any reactivity to GroMM. Furthermore, local inflammatory responses were elicited in the GroMM-injected skin of hMincle(+), but not mMincle(+) mice. These results demonstrated that GroMM is a unique ligand for hMincle that is not recognized by mMincle.
  • Takashi Nakamura, Masafumi Fukiage, Megumi Higuchi, Akihiro Nakaya, Ikuya Yano, Jun Miyazaki, Hiroyuki Nishiyama, Hideyuki Akaza, Toshihiro Ito, Hiroyuki Hosokawa, Toshinori Nakayama, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 176 44 - 53 2014年02月 [査読有り][通常論文]
     
    The Mycobacterium bovis Bacille Calmette-Guerin cell wall skeleton (BCG-CWS) could be used to replace live BCG as a bladder cancer drug. However, because BCG-CWS is poorly soluble, has a strong-negative charge, very high molecular weight and heterogeneity in size of tens of mu m, it cannot be used in such an application. We report herein on the development of a novel packaging method that permits BCG-CWS to be encapsulated into 166 nm-sized lipid particles. The BCG-CWS encapsulated nano particle (CWS-NP) has a high uniformity and can be easily dispersed. Thus, it has the potential for use as a packaging method that would advance the scope of applications of BCG-CWS as a bladder cancer drug. In a functional evaluation, CWS-NP was efficiently taken up by mouse bladder tumor (MBT-2) cells in vitro and inhibited tumor growth in mice bearing MBT-2 tumors. Moreover, intravesically administered CWS-NP showed significant antitumor effects in a rat model with naturally developed bladder cancer. An enhancement in Th1 differentiation by CWS-NP was also confirmed in human T cells. In conclusion, CWS-NP represents a promising delivery system for BCG-CWS for clinical development as a potent bladder cancer drug. (C) 2013 Elsevier B. V. All rights reserved.
  • Yusuke Sato, Takashi Nakamura, Yuma Yamada, Hidetaka Akita, Hideyoshi Harashima
    NONVIRAL VECTORS FOR GENE THERAPY LIPID- AND POLYMER-BASED GENE TRANSFER 88 139 - 204 2014年 [査読有り][通常論文]
     
    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.
  • Morita D, Miyamoto A, Hattori Y, Komori T, Nakamura T, Igarashi T, Harashima H, Sugita M
    Biochemical and biophysical research communications 441 1 108 - 113 2013年11月 [査読有り][通常論文]
  • Hidetaka Akita, Soichiro Ishii, Naoya Miura, Sharif Mohammad Shaheen, Yasuhiro Hayashi, Takashi Nakamura, Noritada Kaji, Yoshinobu Baba, Hideyoshi Harashima
    BIOMATERIALS 34 35 8979 - 8990 2013年11月 [査読有り][通常論文]
     
    Technologies for the transfection of antigen-encoding genes into the dendritic cells, and subsequent immune-activation are both prerequisites for a successful DNA vaccine. We herein report on the density-dependent enhancement of transgene expression by the simple modification by stearyl-conjugated KALA, an alpha-helical peptide (STR-KALA), onto a lipid envelope-type nanoparticle (the R8-MEND, an octaarginine-modified multifunctional envelope-type nano device). The enhanced transgene expression in the KALA-modified R8-MEND (R8/KALA-MEND) cannot be explained by cellular uptake and nuclear delivery efficacy. Thus, the post-nuclear delivery process (i.e. transcription), but not intracellular trafficking processes attributed the enhanced transfection efficacy. Microarray analyses revealed that transfection with the R8/KALA-MEND resulted in a greater perturbation in host genes expression in comparison with the R8-MEND and that this effect was time-dependent. Further pathway analyses in the category of transcription-related genes and a gene ontology analysis indicated that the R8/KALA-MEND stimulated the expression of transcription factors that are closely related to immune-activation (i.e. NF-kB and STAT). Inhibition of the transfection efficacy by blockage of the STAT pathways revealed that the enhanced transcription activity is the result of immune-stimulation. Collectively, the R8/KALA-MEND mounts a "switch-on" function that triggers signal transduction forward to the immune-stimulation analogous to an adjuvant, and consequently elicits active transcription. (C) 2013 Elsevier Ltd. All rights reserved.
  • Nakamura T, Yamazaki D, Yamauchi J, Harashima H
    Journal of controlled release : official journal of the Controlled Release Society 171 2 216 - 224 2013年10月 [査読有り][通常論文]
  • Kuraya D, Watanabe M, Koshizuka Y, Ogura M, Yoshida T, Asahi Y, Kamachi H, Nakamura T, Harashima H, Ozaki M, Umezawa K, Matsushita M, Yamashita K, Todo S
    Transplantation 96 5 445 - 453 2013年09月 [査読有り][通常論文]
  • Morita D, Hattori Y, Nakamura T, Igarashi T, Harashima H, Sugita M
    Infection and immunity 81 1 311 - 316 2013年01月 [査読有り][通常論文]
  • Takashi Nakamura, Rumiko Moriguchi, Kentaro Kogure, Hideyoshi Harashima
    INTERNATIONAL JOURNAL OF PHARMACEUTICS 441 1-2 476 - 481 2013年01月 [査読有り][通常論文]
     
    In a previous study, we reported on the efficient delivery of an antigen to the cytosol and a specific-antigen presentation on MHC class I in dendritic cells by rationally controlling the intracellular trafficking of ovalbumin (OVA), a model antigen, with stearylated octaarginine-modified liposomes (R8-Lip/OVA). However, no significant difference in antitumor effects against E.G7-OVA, OVA expressed lymphoma, was observed between R8-Lip/OVA and an electrostatic complex of R8 and OVA (R8/OVA-Com). In this study, we hypothesized that use of adjuvants clarified the difference in immune responses between R8-Lip/OVA and R8/OVA-Com, and selected polyinosine-polycytidylic acid (polyI:C) as an adjuvant. Cytotoxic T lymphocyte (CTL) activity of the polyI:C and OVA encapsulated R8-Lip (R8-Lip/PIC/OVA) was drastically enhanced compared to R8-Lip/OVA and complete Freund's adjuvant with OVA. Moreover, the incorporation of polyI:C clearly was critical for the difference in antitumor effects and CTL activities between R8-Lip/OVA and R8/OVA-Com. These findings suggest that the carriers that are incorporated polyI:C has a great influence on the induction of cellular immunity in vivo. (C) 2012 Elsevier B. V. All rights reserved.
  • Miyazaki J, Kawai K, Kojima T, Oikawa T, Joraku A, Shimazui T, Nakaya A, Yano I, Nakamura T, Harashima H, Akaza H
    BJU international 108 9 1520 - 1526 2011年11月 [査読有り][通常論文]
     
    OBJECTIVE To conduct a preclinical evaluation of the ability of natural killer cells to cytolyze bladder cancer cells that were modified to show enhanced expression of natural-killer group 2, member D (NKG2D) ligands by R8-liposome-bacillus Calmette-Guein (BCG)-cell wall skeleton (CWS) treatment. MATERIALS AND METHODS The T24 cells and RT-112 cells were co-cultured with R8-liposome-BCG-CWS and BCG for 2, 4, or 6 h, and then the surface expression of NKG2D ligands was analyzed using TaqMan real-time quantitative RT-PCR. Peripheral blood mononuclear cells were obtained with a conventional preparation kit, and then lymphokine-activated killer (LAK) cells were generated from these purified peripheral blood mononuclear cells via interleukin-2 stimulation. The anti-tumour effect of LAK cells against untreated and R8-liposome-BCG-CWS co-cultured with cells of the human bladder cancer cell lines T24 and RT-112 was analyzed using the cytotoxic WST-8 assay method at 4 h of culture at various effector/target (E : T) ratios. RESULTS Major histocompatibility complex class I-related chain B (MICB) expression was increased approximate to 1.5-fold on T24 cells and RT-112 cells with BCG. UL-16-binding protein (ULBP) 1 expression was also increased approximate to 1.5-fold on T24 cells and RT-112 cells with BCG. R8-liposome-BCG-CWS increased the surface expression of MICB 2.2-fold on T24 cells but did not increase it significantly on RT-112 cells. ULBP1 expression was increased approximate to 2.2-fold on RT-112 cells, although no differences were observed between the expression of ULBP2 and 3 with R8-liposome-BCG-CWS. T24 cells that were co-cultured with R8-liposome-BCG-CWS showed an approximate to 1.3-fold increase in sensitivity to cytolysis by LAK cells at an E : T ratio of 4 and RT-112 cells showed an approximate to 1.4-fold increase at an E : T ratio of 2. CONCLUSIONS In the present study, the induction of surface NKG2D ligands by R8-liposome-BCG-CWS rendered cancer cells more susceptible to cytolysis by LAK cells. T24 cells and RT-112 cells, even when cultured singly in the absence of immune cells, can directly respond to R8-liposome-BCG-CWS. The results obtained in the present study may therefore indicate a novel adoptive immunotherapy against bladder cancers.
  • Sharif M Shaheen, Hidetaka Akita, Takashi Nakamura, Shota Takayama, Shiroh Futaki, Atsushi Yamashita, Ryo Katoono, Nobuhiko Yui, Hideyoshi Harashima
    Biomaterials 32 26 6342 - 6350 2011年09月 [査読有り][通常論文]
     
    DNA vaccines are a new-generation vaccines that elicit an immunological response against a wide-variety of antigens with frequent mutations. However, an effective non-viral vector for genetically engineered DNA to dendritic cells is yet to be developed. We previously reported that an octaarginine (R8)-modified tetra-lamellar multi-functional envelope-type nano device (R8-T-MEND) increases transfection efficiency in dendritic cell cultures (JAWS II). The critical structural elements of the R8-T-MEND are a DNA-polycation condensed core coated with two nuclear membrane-fusogenic inner envelopes, and two endosome-fusogenic outer envelopes. While the gene expression was drastically enhanced by R8-T-MEND, antigen presentation using an epitope-encoding plasmid DNA remains an obstacle for future non-viral vectors in DNA vaccinations. In the present study, we upgraded the function of R8-T-MEND by improving the membrane-fusion processes with endosome- and nuclear membranes by incorporating the KALA peptide, and by reducing the charge ratio (+/-), in an attempt to accelerate intra-nuclear decondensation. The resulting KALA-modified T-MEND (R8/KALA-T-MEND) showed an approximately 20-fold highe
  • Sharif M. Shaheen, Hidetaka Akita, Takashi Nakamura, Shota Takayama, Shiroh Futaki, Atsushi Yamashita, Ryo Katoono, Nobuhiko Yui, Hideyoshi Harashima
    BIOMATERIALS 32 26 6342 - 6350 2011年09月 [査読有り][通常論文]
     
    DNA vaccines are a new-generation vaccines that elicit an immunological response against a wide-variety of antigens with frequent mutations. However, an effective non-viral vector for genetically engineered DNA to dendritic cells is yet to be developed. We previously reported that an octaarginine (R8)-modified tetra-lamellar multi-functional envelope-type nano device (R8-T-MEND) increases transfection efficiency in dendritic cell cultures (JAWS II). The critical structural elements of the R8-T-MEND are a DNA-polycation condensed core coated with two nuclear membrane-fusogenic inner envelopes, and two endosome-fusogenic outer envelopes. While the gene expression was drastically enhanced by R8-T-MEND, antigen presentation using an epitope-encoding plasmid DNA remains an obstacle for future non-viral vectors in DNA vaccinations. In the present study, we upgraded the function of R8-T-MEND by improving the membrane-fusion processes with endosome- and nuclear membranes by incorporating the KALA peptide, and by reducing the charge ratio (+/-), in an attempt to accelerate intra-nuclear decondensation. The resulting KALA-modified T-MEND (R8/KALA-T-MEND) showed an approximately 20-fold higher transgene expression compared with the conventional R8-T-MEND in JAWS II, and exceeded that of Lipofectamine PLUS, a commercially available transfection reagent. Furthermore, significant antigen presentation of a specific epitope (SIINFEKL) was observed for the R8/KALA-T-MEND but was not detected for the conventional T-MEND or Lipofectamine PLUS when an ovalbumin (OVA)-encoding plasmid DNA was transfected. It thus appears that the R8/KALA-T-MEND has the potential for use as a vector in DNA vaccinations. (C) 2011 Elsevier Ltd. All rights reserved.
  • Shota Warashina, Takashi Nakamura, Hideyoshi Harashima
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 34 8 1348 - 1351 2011年08月 [査読有り][通常論文]
     
    In a previous report, we described the development of lipid envelope-type nanoparticles (MEND) modified with octaarginine (R8) and a pH-sensitive fusogenic peptide (GALA) for delivering short interference RNA (siRNA) to mouse dendritic cells (DCs). A20 was recently reported to be a negative regulator of the toll-like receptor and the tumor necrosis factor receptor signaling pathways. Although A20 would be expected to be a useful target for boosting the effects of adjuvants in DC immunotherapy, limited information is available regarding the use of A20-silenced DC by an original non-viral vector. In this study, we loaded anti-A20 siRNA into a MEND and investigated the gene knockdown activity in DC and the immunological functions of A20-silenced DC. The use of a MEND resulted in a significant A20 knockdown effect, and the A20-silenced DC resulted in an enhanced production of proinflammatory molecules, after lipopolysaccharide (LPS) stimulation. The expression of co-stimulatory molecules by LPS stimulation was also increased in the A20-silenced DC. The findings reported herein show that a MEND loaded with anti-A20 siRNA is a potent non-viral vector that has the ability to enhance the adjuvant effect of LPS in DC.
  • The Therapeutic Effects of R8-Liposome-BCG-CWS on BBN-Induced Rat Urinary Bladder Carcinoma
    Jun Miyazaki, Hiroyuki Nishiyama, Ikuya Yano, Akihiro Nakaya, Hideyasu Kohama, Koji Kawai, Akira Joraku, Takashi Nakamura, Hideyoshi Harashima, Hideyuki Akaza
    ANTICANCER RESEARCH 31 6 2065 - 2071 2011年06月 [査読有り][通常論文]
     
    Background: The present gold standard for bladder cancer is Mycobacterium bovis bacillus Calmette-Guerin (BCG) immunotherapy, but serious side-effects are common. We previously reported that C3H/HeN mice vaccinated with a mixture of MBT-2 cells and artificial BCG, octaarginine-modified liposomes incorporating the cell wall of BCG (R8-liposome-BCG-CW), significantly inhibited growth of R8-liposome-BCG-CW pretreated MBT-2 cells. Our aim was to determine if a non-live bacterial agent could be as efficacious as live BCG in a model of bladder cancer. We investigated the suppressive effect of liposome-incorporating cell wall skeleton (BCG-CWS) on N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced urinary bladder carcinogenesis in rats. Materials and Methods: F344 rats were fed with BBN and sodium ascorbate for 8 weeks, after which all rats were confirmed to have excreted atypical epithelial cells in the urine. Rats were administered BCG-CW(1.0 mg/rat) or R8-liposome-BCG-CWS (0.1 or 1.0 mg/rat) intravesically once/week for 8 weeks from week 28 to 35 of the experimental protocol. Results: Rats receiving R8-liposome-BCG-CWS intravesically showed significantly inhibited numbers of tumors, especially those of simple hyperplasia, in comparison with the control rats. Conclusion: R8-liposome-BCG-CWS administration had inhibitory effects on rat bladder carcinogenesis. These results may indicate a novel adoptive immunotherapy against bladder cancers.
  • Hattori Y, Matsunaga I, Komori T, Urakawa T, Nakamura T, Fujiwara N, Hiromatsu K, Harashima H, Sugita M
    Biochemical and biophysical research communications 409 2 304 - 307 2011年06月 [査読有り][通常論文]
  • Komori T, Nakamura T, Matsunaga I, Morita D, Hattori Y, Kuwata H, Fujiwara N, Hiromatsu K, Harashima H, Sugita M
    The Journal of biological chemistry 286 19 16800 - 16806 2011年05月 [査読有り][通常論文]
  • Kaoru Kigasawa, Kazuaki Kajimoto, Takashi Nakamura, Susumu Hama, Kiyoshi Kanamura, Hideyoshi Harashima, Kentaro Kogure
    JOURNAL OF CONTROLLED RELEASE 150 3 256 - 265 2011年03月 [査読有り][通常論文]
     
    Oligodeoxynucleotides containing unmethylated cytosine-phosphate-guanosine motifs (CpG-ODN) possess immunostimulatory effects and potential antitumor activity. Since the skin is an easily available site of administration of CpG-ODN due to its accessibility and the presence of abundant antigen presenting cells, it is expected that the application of CpG-ODN to the skin would induce systemic immune response and antitumor activity. However, it is difficult to deliver hydrophilic macromolecules including CpG-ODN through the skin. We have previously demonstrated that small interfering RNA (siRNA) was efficiently delivered into rat epidermis by iontophoresis. In this report, we investigate the effect of transdermal iontophoretic delivery of CpG-ODN on the induction of immune responses and antitumor activity against B16F1 melanoma in mice. Iontophoresis promoted CpG-ODN delivery into the epidermis and dermis. Furthermore, iontophoretic delivery of CpG-ODN to the skin induced the expression of proinflammatory and Th1-type cytokines in the skin and draining lymph node. Finally, transdermal iontophoretic delivery of CpG-ODN led to antitumor activity against B16F1 melanoma. Interestingly, the CpG-ODN administration site is not restricted to the tumor area. In conclusion, CpG-ODN delivered transdermally induced potent antitumor activity, and our system is expected to serve as a simple and noninvasive approach for cancer immunotherapy. (C) 2011 Elsevier B.V. All rights reserved.
  • Hidetaka Akita, Kentaro Kogure, Rumiko Moriguchi, Yoshio Nakamura, Tomoko Higashi, Takashi Nakamura, Satoshi Serada, Minoru Fujimoto, Tetsuji Naka, Shiroh Futaki, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 143 3 311 - 317 2010年05月 [査読有り][通常論文]
     
    We previously developed octaarginine (R8)-modified lipid envelope-type nanoparticles for siRNA delivery (R8-MEND). Herein, we report on their ex vivo siRNA delivery to primary mouse bone marrow-derived dendritic cells (BMDCs) for potential use as a cancer vaccine. Quantitative imaging analysis of the intracellular trafficking of siRNA revealed that the dissociation process, as well as the rate of endosomal escape limits the siRNA efficiency of the prototype R8-MEND, prepared by the hydration method (R8-MENDhydo). Successful endosomal escape was achieved by using a pH-dependent fusogenic peptide (GALA) modified on a lipid mixture that was optimized for endosomal fusion. Furthermore, a modified protocol for the preparation of nanoparticles, mixing the 5iRNA/STR-R8 complex and small unilamellar vesicles (R8/GALA-MENDSUV), results in a more homogenous, smaller particle size, and results in a more efficient intracellular dissociation. Gene knockdown of the suppressor of cytokine signaling 1 (SOCS1), a negative-feedback regulator of the immune response in BMDCs resulted in an enhanced phosphorylation of STAT1, and the production of proinflammatory cytokines. Moreover, SOCS1-silenced BMDCs were more potent in suppressing tumor growth. Collectively, these results show that siRNA loaded in R8/GALA-MENDSUV efficiently suppresses endogenous gene expression and consequently enhances dendritic cell-based vaccine potency in vivo. (C) 2010 Elsevier B.V. All rights reserved.
  • Atthachai Homhuan, Kentaro Kogure, Takashi Nakamura, Nilabh Shastri, Hideyoshi Harashima
    JOURNAL OF CONTROLLED RELEASE 136 1 79 - 85 2009年05月 [査読有り][通常論文]
     
    To improve uptake and cross-presentation of exogenous antigens (Ag) by dendritic cells (DCs), octaarginine-modified liposomes (R8-Lip) were used as a novel strategy for protein-Ag transduction. Immature DCs endocytose macromolecules efficiently. While mature DCs lose their ability to capture Ag, but have an increased capacity for T-cell activation. Thus Ag-transduction has been performed mostly in immature DCs. In the present study, R8-Lip were efficiently taken up by both immature and mature DCs. DCs transduced after maturation were highly efficient at cross-presentation of Ag and induced higher cytotoxic T-lymphocytes (CTL) activity than were DCs transduced before maturation. The mechanism of Ag presentation involved the escape of R8-Lip from endosomes to cytosol, which require the acidic environment. The Ag released was then processed by a proteasome-dependent pathway. This novel transduction approach is clinically applicable, easy to perform, and has more practical advantages than current protein transduction methods. (C) 2009 Elsevier B.V. All rights reserved.
  • Takashi Nakamura, Rumiko Moriguchi, Kentaro Kogure, Nilabh Shastri, Hideyoshi Harashima
    MOLECULAR THERAPY 16 8 1507 - 1514 2008年08月 [査読有り][通常論文]
     
    Recently, much attention has been paid to cell-penetrating peptides (CPPs) as an antigen-delivery tool for presentation through the major histocompatibility complex class I (MHC-I) pathway. However, escape of CPPs from the endosome is inefficient and therefore a bottleneck for antigen delivery. Previously, we showed the importance of topological control of octaarginine (R8) peptides on the liposome surface for regulating cellular uptake as well as intracellular trafficking, especially endosomal escape. In this study, we hypothesized that efficient MHC-I presentation could be achieved by controlled intracellular trafficking of antigen encapsulated in R8-modified liposomes (R8-Lip). The mechanism of uptake of both R8-Lip and cationic liposomes was shown to be by macropinocytosis in dendritic cells. However, confocal laser scanning microscopy (CLSM) revealed that R8-Lip are able to release significantly more antigen to the cytosol than are cationic liposomes. Processing of the antigens delivered by R8-Lip was shown to be proteasome-dependent, which is consistent with selective antigen presentation by R8-Lip via MHC-I. According to antigen-presentation analysis, R8-Lip can induce significantly higher MHC-I presentation at lower doses than either soluble ovalbumin ( OVA) or OVA in pH-sensitive or cationic liposomes. Moreover, R8-Lip showed an efficient antitumor effect in vivo. Therefore, R8-Lip is a promising new carrier for MHC-I-specific antigen presentation.
  • Takashi Nakamura, Rumiko Moriguchi, Kentaro Kogure, Arisa Minoura, Tomoya Masuda, Hidetaka Akita, Kazunori Kato, Hirofumi Hamada, Shiroh Futaki, Hideyoshi Harashima
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 29 6 1290 - 1293 2006年06月 [査読有り][通常論文]
     
    In this study, we developed novel double-membranous non-viral gene delivery system modified with SV-40 T antigen-derived nuclear localization signal (NLS-DMEND) for delivery of luciferase plasmid DNA to nucleus of non-dividing mouse bone marrow-derived dendritic cells (BMDC). Intracellular trafficking and gene expression of NLS-DMEND in the BMDC were evaluated. Condensed DNA was observed in the nucleus by confocal laser scanning microscopy, and the NLS-DMEND induced significant luciferase activity in the BMDC. It was suggested that the condensed DNA particle transferred into nucleus via energy dependent manner, since the nuclear transfer was inhibited by metabolic inhibitors. In conclusion, condensed plasmid DNA was delivered into the nucleus of non-dividing BMDC by NLS-DMEND.
  • Shiroh Futaki, Yumi Masui, Ikuhiko Nakase, Yukio Sugiura, Takashi Nakamura, Kentaro Kogure, Hideyoshi Harashima
    Harashima 7 1450 - 1458 2005年 [査読有り][通常論文]

その他活動・業績

  • BCG-CWS搭載ナノ粒子を用いた膀胱がん免疫療法剤の開発
    中村 孝司, 吹上 雅文, 鈴木 嘉晃, 矢野 郁也, 宮崎 淳, 西山 博之, 赤座 英之, 中山 俊憲, 原島 秀吉 日本薬学会年会要旨集 135年会 (4) 84 -84 2015年03月 [査読無し][通常論文]
  • BCG-CWS搭載ナノ粒子を基盤とした膀胱癌治療剤の開発
    中村 孝司, 吹上 雅文, 中谷 彰洋, 矢野 郁也, 宮崎 淳, 西山 博之, 赤座 英之, 伊藤 俊宏, 細川 裕之, 中山 俊憲, 原島 秀吉 泌尿器外科 27 (3) 338 -339 2014年03月 [査読無し][通常論文]
  • 常樂 晃, 宮崎 淳, 中谷 彰洋, 中村 孝司, 河合 弘二, 矢野 郁也, 原島 秀吉, 赤座 英之 日本泌尿器科學會雜誌 102 (2) 2011年03月20日 [査読無し][通常論文]

受賞

  • 2019年07月 日本DDS学会 奨励賞
     
    受賞者: 中村 孝司
  • 2018年03月 日本薬学会 奨励賞
     
    受賞者: 中村 孝司
  • 2016年05月 日本薬剤学会 奨励賞
     
    受賞者: 中村孝司
  • 2015年05月 日本薬学会北海道支部 奨励賞
     
    受賞者: 中村孝司
  • 2012年05月 日本薬剤学会 日本薬剤学会第27年会 最優秀発表者賞
     
    受賞者: 中村孝司
  • 2005年03月 日本薬剤学会 日本薬剤学会創立20周年記念大会 最優秀発表者賞
     
    受賞者: 中村孝司

共同研究・競争的資金等の研究課題

  • PD-1抗体への獲得抵抗性を攻略する記憶NK細胞誘導型ナノがん免疫療法の開発
    日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2020年04月 -2023年03月 
    代表者 : 中村 孝司
  • 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
    日本学術振興会:科学研究費助成事業 基盤研究(A)
    研究期間 : 2019年04月 -2023年03月 
    代表者 : 原島 秀吉, 佐藤 悠介, 中村 孝司, 山田 勇磨
  • 腫瘍微小環境のナノDDS関連免疫ステータス解析に基づいたナノがん免疫療法の開発
    日本学術振興会:科学研究費助成事業 挑戦的研究(萌芽)
    研究期間 : 2018年06月 -2021年03月 
    代表者 : 中村 孝司
     
    本研究では、ドラッグデリバリーシステム(DDS)の体内動態/細胞内動態に関連する特有の免疫ステータスパラメータや変動を明らかにし、腫瘍微小環境のDDS関連免疫ステータスに基づいたDDS設計戦略を立案、現行のがん免疫療法抵抗性の難治性がんを克服するための新規がん免疫療法の開発を目指す。 まずは、研究に使用する担がんマウスモデルを選定する必要がある。そこで本年度はマウス腫瘍微小環境の免疫ステータス解析とそのフェノタイプ分類を行った。実験方針として、異なる種類のがん細胞をマウスに移植し、形成された腫瘍内の免疫ステータスを解析することで、どのようなタイプの腫瘍であるかを分類する。 5種類のマウスがん細胞をそれぞれマウス皮下に移植し、腫瘍体積が400-500ミリ立方メートルに成長した時点で腫瘍を回収した。腫瘍からRNAを抽出後、RT-qPCR法により遺伝子発現を解析した。解析対象の遺伝子はがん免疫応答に関連する遺伝子(免疫細胞のマーカー、免疫チェックポイント分子、サイトカイン、抑制性細胞マーカー、免疫抑制因子など)を文献情報から約20種類を選定した。その結果、活性化したT細胞などの免疫細胞の浸潤が起こっている腫瘍、免疫細胞の浸潤が少ない腫瘍、免疫細胞の機能を抑制する分子の発現が亢進している腫瘍、抑制性の細胞が多く浸潤している腫瘍に分類することができた。この結果は、ヒトの腫瘍で形成される免疫ステータスに類似したモデルを選定できる可能性を示している。
  • 腫瘍リンパ節標的型クラスターナノDDSによる複合がん免疫療法
    文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 2017年04月 -2020年03月 
    代表者 : 中村孝司
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2015年04月 -2018年03月 
    代表者 : 杉田 昌彦, 中村 孝司
     
    結核は依然としてグローバルな感染症であり、現行唯一の抗結核ワクチンであるBCGワクチンに代わる新たな抗結核ワクチンの開発は、社会的急務と言える。研究代表者は結核菌脂質を標的とした新しい免疫応答の研究を展開し、その成果をもとに抗結核脂質ワクチンの可能性を追究してきた。本研究において、抗結核脂質ワクチン候補を絞り込み、その生合成経路や宿主体内で誘起される免疫応答についてサルやモルモットを用いた研究を展開し、今後につながる基礎知見を得た。
  • c-di-GMP搭載脂質ナノ粒子と抗PD-1抗体による複合的がん免疫療法
    文部科学省:科学研究費補助金(挑戦的萌芽研)
    研究期間 : 2016年04月 -2018年03月 
    代表者 : 中村孝司
  • 文部科学省:科学研究費補助金(若手研究(A))
    研究期間 : 2014年04月 -2017年03月 
    代表者 : 中村 孝司
  • 文部科学省:科学研究費補助金(挑戦的萌芽研究)
    研究期間 : 2014年04月 -2016年03月 
    代表者 : 中村 孝司
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2012年04月 -2015年03月 
    代表者 : 杉田 昌彦, 五十嵐 樹彦, 中村 孝司, 原島 秀吉
     
    世界人口の約3分の1が結核菌に感染しており、現行唯一の抗結核ワクチンであるBCGに代わる新たなワクチン開発が社会的急務である。研究代表者は結核菌脂質に対する免疫応答について、第一線の研究を進めてきた。本研究ではこの知見を活用し、脂質をベースにした新しいコンセプトの抗結核ワクチン開発に向けて、その研究基盤の確立を目指した研究を進めた。その結果、モルモットにおいて結核を制御する新しい脂質ワクチンの原型を確立できた。またサルを用いた実験から脂質に対する免疫応答を効率的に誘導できるプロトコールを見いだした。以上から抗結核脂質ワクチン開発の基盤を確立できた。
  • 文部科学省:科学研究費補助金(若手研究(B))
    研究期間 : 2012年04月 -2014年03月 
    代表者 : 中村 孝司
  • 細胞内生態解析に基づいた樹状細胞選択的DNAワクチンの開発
    日本学術振興会:科学研究費助成事業 特別研究員奨励費
    研究期間 : 2007年 -2009年 
    代表者 : 中村 孝司
     
    前年度は、タンパク質抗原を用いた樹状細胞へのデリバリーを行い、抗原提示と抗腫瘍効果の評価を行った。本年度では、DNAワクチンの前段階としてsiRNAを樹状細胞に導入し、機能を示すキャリアを見出した。 R8/GALA-DMENDを用いた樹状細胞におけるSOCS1のノックダウン DNAワクチンの前段階としてsiRNAを樹状細胞に導入し、その効果を調べた。当研究室で開発されたR8/GALA-DMENDはエンドソーム脱出のための機能性素子であるGALAと膜枚数の制御により、培養細胞で高いRNAi効果を示すことが明らかになっている。本年度では、樹状細胞においてサイトカインシグナルの抑制因子であるSOCS1を標的とした R8/GALA-DMENDの機能評価を行った。siRNAを封入したR8/GALA-DMENDを樹状細胞に導入した結果、未処理の樹状細胞と比較して87.5%の発現抑制が認められた。またSOCS1をノックダウンした樹状細胞ではTNF-αやIL-6産生が増加した。さらにSOCS1をノックダウンした樹状細胞をマウスに投与した後、癌細胞をマウスに移植し、その増殖を観察した。その結果、コントロールsiRNAを用いた場合と比較して著しい腫瘍増殖抑制が認められた。以上のことから、R8/GALA-DMENDは樹状細胞においても十分なRNAi効果を誘導できるキャリアであることが明らかになった(Akita H et al.J.Control.Release)。 本年度の成果によりR8/GALA-DMENDが樹状細胞への核酸デリバリーシステムとして有用であることが示された。故にR8/GALA-DMENDはDNAワクチンのデリバリーシステムとしての応用が期待される。

教育活動情報

主要な担当授業

  • 生命科学研究
    開講年度 : 2018年
    課程区分 : 修士課程
    開講学部 : 生命科学院
  • 大学院共通授業科目(一般科目):自然科学・応用科学
    開講年度 : 2018年
    課程区分 : 修士課程
    開講学部 : 大学院共通科目
  • 生命科学実習
    開講年度 : 2018年
    課程区分 : 修士課程
    開講学部 : 生命科学院
  • 基礎実習
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 薬学部
  • 生命科学論文講読Ⅰ
    開講年度 : 2018年
    課程区分 : 修士課程
    開講学部 : 生命科学院
  • 薬剤学実習
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 薬学部
    キーワード : 消化管吸収、pH分配仮説、コンパートメントモデル、尿中排泄、粒子径測定
  • 生命科学論文講読Ⅱ
    開講年度 : 2018年
    課程区分 : 修士課程
    開講学部 : 生命科学院
  • 薬剤学Ⅳ
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 薬学部
    キーワード : コントロールドリリース、プロドラッグ、ターゲティング、ナノメディシン、ゲノム創薬、細胞内動態制御


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