脂質・核酸複合体の動的構造と核酸送達機能

• Project Area: Molecular Science of Fluctuations toward Biological Functions
• Project/Area Number: 21107510
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Science and Engineering
• Research Institution: Nagoya Institute of Technology
• Principal Investigator: 出羽 毅久 名古屋工業大学, 工学研究科, 准教授 (70335082)
• Project Period (FY): 2009 – 2010
• Project Status: Completed (Fiscal Year 2010)
• Budget Amount: ¥7,020,000 (Direct Cost: ¥5,400,000、Indirect Cost: ¥1,620,000); Fiscal Year 2010: ¥3,510,000 (Direct Cost: ¥2,700,000、Indirect Cost: ¥810,000); Fiscal Year 2009: ¥3,510,000 (Direct Cost: ¥2,700,000、Indirect Cost: ¥810,000)
• Keywords: 遺伝子治療 / 核酸送達 / ポリアミン / 脂質 / 原子間力顕微鏡 / 形態変化 / DDS

Research Abstract

核酸医薬として有望視されているカチオン性脂質と核酸からなる複合体(Lipoplex)は揺らいだ動的分子集合体であり、細胞内でのその動的挙動が機能に関与していると考えられるが、その詳細は全く不明である。本研究では、高い遺伝子送達能をもつポリアミン脂質を合成し、Lipoplexの揺らぎをともなう構造変化を蛍光顕微鏡および原子間力顕微鏡(AFM)により詳細に観察することにより、揺らぎ(構造変化)と生体機能(遺伝子送達)との相関を明らかにする。研究成果として、高い遺伝子送達能を有する新規ポリアミンの合成に成功し、その類縁体の合成も行った。ポリアミン部位のわずかな化学構造の違いが遺伝子送達活性に影響を及ぼすことが示唆された。高い活性をもつLipoplexの構造をAFMで観測した結果、ポリアミン脂質からなる二分子膜構造の間にDNAが挿入された層状構造であることが明らかとなった。細胞内取り込み後のエンドソーム環境をモデル化した系において、LipoplexからのDNAの放出が見られ、高い活性をもつLipoplexほど速やかに構造変化を起こすことが蛍光分光法およびAFMから明らかとなった。また、蛍光ラベル化したポリアミン脂質/DNA系で蛍光共鳴エネルギー移動法により、Lipoplex内のDNAとポリアミン脂質はある程度乖離することがわかり、AFM観察の結果とよい一致を示した。さらに、RNA干渉実験から、これらの誘導体は遺伝子発現抑制作用も有することが分かった。

Research Products

• [Journal Article] Selective Assembly of Photosynthetic Antenna Proteins into a Domain-Structured Lipid Bilayer for the Construction of Artificial Photosynthetic Antenna Systems : Structural Analysis of the Assembly using Surface Plasmon Resonance and Atomic Force Microscopy (2011)
  • Author(s): Sumino, Dewa, et al.
  • Journal Title: Langmuir Volume: 27 Pages: 1092-1099
  • Peer Reviewed
• [Journal Article] Reconstitution and AFM Observation of Photosynthetic Membrane Prote in Assembly in Planar Lipid Bilayers (2011)
  • Author(s): Sumino, Dewa, et al.
  • Journal Title: e-J.Surf.Sci.Nanotech. Volume: 9 Pages: 15-20
  • Peer Reviewed
• [Journal Article] Dicetyl phosphate-tetraethylenepentamine-based Liposomes for Systemic siRNA Delivery (2011)
  • Author(s): Asai, et al.
  • Journal Title: Bioconjugate Chem. Volume: 22 Pages: 429-435
  • Peer Reviewed
• [Journal Article] Liposomal Polyamine-Dialkyl Phosphate Conjugates as Effective Gene Carriers : Chemical Structure, Morphology, and Gene Transfer activity (2010)
  • Author(s): Dewa, et al.
  • Journal Title: Bioconjugate Chem. Volume: 21 Pages: 844-852
  • Peer Reviewed
• [Journal Article] 光合成でのアンテナ系タンパク質超分子複合体の構造形成とカロテノイド色素の機能評価 (2010)
  • Author(s): 中川勝統, ら
  • Journal Title: 高分子論文集 Volume: 67 Pages: 574-583
  • Peer Reviewed
• [Journal Article] Immobilization of Porphyrin Derivatives with a Defined Distance and Orientation onto a Gold Electrode Using Synthetic Light-Harvesting α-Helix Hydrophobic Polypeptides (2010)
  • Author(s): Ochiai, et al.
  • Journal Title: Langmuir Volume: 26 Pages: 14419-14422
  • Peer Reviewed
• [Journal Article] Peroxide Decoloration of CI Acid Orange 7 Catalyzed by Manganese Chlorophyll Derivatives at the Surfaces of Micelles and Lipid Bilayers (2010)
  • Author(s): Ishigure, et al.
  • Journal Title: Langmuir Volume: 26 Pages: 7774-7782
  • Peer Reviewed
• [Journal Article] Reconstitution of the Light-harvesting 1 (LH1) Complex Using LH1-a and LH1-b Polypeptides, Separately Isolated from the Purple Photosynthetic Bacterium Rhodospirillum rubrum, Together with Bacteriochlorophyll a and All-trans Carotenoids (2009)
  • Author(s): Nakagawa, et al.
  • Journal Title: Carotenoid Science Volume: 14 Pages: 54-57
  • Peer Reviewed
• [Journal Article] Carotenoid Specificity During Reconstitution of the Light-harvesting 1 (LH1) Complexes Using LH1-polypeptides Isolated from the Purple Photosynthetic Bacterium Rhodobacter sphaeroides Together with Bacteriochlorophyll a and Carotenoids (2009)
  • Author(s): Nakagawa, et al.
  • Journal Title: Carotenoid Science Volume: 14 Pages: 58-61
  • Peer Reviewed
• [Journal Article] Reconstitution of the Light-harvesting (LH1) Complex Using Zinc-substit uted Bacteriochlorophyll a and LH1-Polypeptides Isolated from the Purple Photosynthetic Bacterium Rhodospirillum rubrum Together with all-trans Carotenoids (2009)
  • Author(s): Nakagawa, et al.
  • Journal Title: Carotenoid Science Volume: 14 Pages: 62-65
  • Peer Reviewed
• [Presentation] 光線力学的療法のための新規ナノキャリアの開発とガン細胞への導入 (2011)
  • Author(s): 二井知紀, ら
  • Organizer: 日本化学会第91春季年会
  • Place of Presentation: 神奈川大学
  • Year and Date: 2011-03-29
• [Presentation] 組み換えLH1タンパク質を用いた再構成LH1型複合体の基板上への組織化 (2011)
  • Author(s): 酒井俊亮, ら
  • Organizer: 日本化学会第91春季年会
  • Place of Presentation: 神奈川大学
  • Year and Date: 2011-03-29
• [Presentation] 光合成膜タンパク質分子集合体の構築と機構評価 (2011)
  • Author(s): 出羽毅久, ら
  • Organizer: 日本化学会第91春季年会
  • Place of Presentation: 神奈川大学
  • Year and Date: 2011-03-28
• [Presentation] Supramolecular Assembly of Bacterial Light-harvesting/reaction center complexes (LH2/LH1-RC)in Planar Lipid Bilayers : AFM Observation, Energy Transfer and Photocurrent Generation (2010)
  • Author(s): T.Dewa, et al.
  • Organizer: The 70^<th> Okazaki Conference
  • Place of Presentation: 岡崎
  • Year and Date: 2010-12-05
• [Presentation] Development of Polyamine-lipid conjugates as effective gene carriers : DNA release from lipoplexes characterized by fluorescence spectroscopy (2010)
  • Author(s): Y.Kozuma, et al.
  • Organizer: 4^<th> International Symposium on Nanomedicine (ISNM2010)
  • Place of Presentation: 岡崎
  • Year and Date: 2010-12-01
• [Presentation] Functional Analysis of Poluamine-Lipid/DNA Complex(Lipoplex) as Gene Carrier : Relationship Between Metamorphosis of Lipoplex and its Function (2010)
  • Author(s): T.Dewa, et al.
  • Organizer: The 4^<th> International Symposium : Molecular Science of Fluctuations toward Biological Functions
  • Place of Presentation: 滋賀
  • Year and Date: 2010-11-30
• [Presentation] 光合成膜タンパク質集合体の構築と機能解析 (2010)
  • Author(s): 出羽毅久, ら
  • Organizer: 第59回高分子討論会
  • Place of Presentation: 北海道大学(札幌)(依頼講演)
  • Year and Date: 2010-09-15
• [Presentation] 遺伝子導入能を有するポリアミン脂質の開発とその作用機構 (2010)
  • Author(s): 加藤清志, ら
  • Organizer: 第59回高分子討論会
  • Place of Presentation: 北海道大学(札幌)
  • Year and Date: 2010-09-15
• [Presentation] 遺伝子送達能を有するポリアミン脂質の開発とその機能評価:原子間力顕微鏡によるDNA複合体の形態観察とその作用機構 (2010)
  • Author(s): 加藤清志, ら
  • Organizer: DDSカンファレンス
  • Place of Presentation: 静岡
  • Year and Date: 2010-09-04
• [Presentation] Reconstitution of light-harvesting 1 (LH1) complexes using zinc-substituted bacteriochlorophyll a and LH1-polypeptides isolated from purple photosynthetic bacteria together with all-trans carotenoids (2010)
  • Author(s): K.Nakagawa, et al.
  • Organizer: Photosynthetic Light Harvesting, The Satellite Meeting to the 15^<th> International Congress of Photosynthesis
  • Place of Presentation: Tianjin(China)
  • Year and Date: 2010-08-20
• [Presentation] AFM Observation of Artificial Photosynthetic Antenna Protein Complexes Assembled on a Solid Membrane Support (2010)
  • Author(s): A.Sumino, et al.
  • Organizer: Zero-Carbon Energy Kyoto 2010 The 2nd International Symposium : Kyoto University Global COE Program Energy Science in the Age of Global Warming Toward CO2
  • Place of Presentation: 京都
  • Year and Date: 2010-08-19
• [Presentation] LIPOSOMAL POLYAMINE-DIALKYL PHOSPHATE CONJUGATES AS EFFECTIVE GENE CARRIERS : CHEMICAL STRUCTURE, MORPHOLOGY, AND GENE TRANSFER ACTIVITY (2010)
  • Author(s): T.Dewa, et al.
  • Organizer: International Liposome Research Days and Lipids Liposomes & Membrane Biophysics
  • Place of Presentation: Vancouver(Canada)
  • Year and Date: 2010-08-06
• [Presentation] Organization and AFM observation of photosynthetic membrane proteins assembly in planar lipid bilayer (2010)
  • Author(s): T.Dewa
  • Organizer: 13th International Conference on Non-Contact Atomic Force Microscopy, 167 Committee satellite workshop on SPM
  • Place of Presentation: 金沢(招待講演)
  • Year and Date: 2010-08-04
• [Presentation] Reconstitution and AFM observation of Photosynthetic Membrane Protein Assembly in Artificial Planar Lipid Bilayers (2010)
  • Author(s): A.Sumino, et al.
  • Organizer: 13th International Conference on Non-Contact Atomic Force Microscopy
  • Place of Presentation: 金沢
  • Year and Date: 2010-08-01
• [Presentation] 光合成膜タンパク質を含む平面脂質膜の構築と原子間力顕微鏡による直接観察 (2010)
  • Author(s): 角野歩, ら
  • Organizer: 第20回バイオ・高分子シンポジウム
  • Place of Presentation: 東京大学(駒場キャンパス)
  • Year and Date: 2010-07-28
• [Presentation] 脂質・核酸複合体の動的構造と核酸送達機能 (2010)
  • Author(s): 出羽毅久
  • Organizer: 脂質・核酸複合体の動的構造と核酸送達機能
  • Place of Presentation: 加賀
  • Year and Date: 2010-06-30
• [Presentation] 原子間力顕微鏡による生体分子集合体の構造-機能相関解明 (2010)
  • Author(s): 出羽毅久
  • Organizer: 神戸大学セミナー
  • Place of Presentation: 神戸大学(六甲)
  • Year and Date: 2010-06-24
• [Presentation] Morphological analysis of polyamine-lipid/DNA complexes as effective gene carriers (2009)
  • Author(s): Dewa, et al.
  • Organizer: The third international symposium on "molecular Science of Fluctuations toward Biological function
  • Place of Presentation: 名古屋大学
  • Year and Date: 2009-12-20
• [Presentation] Development of siRNA delivery system using novel polycation liposomes for cancer therapy (2009)
  • Author(s): Kenjo, et al.
  • Organizer: 4th International Liposome Society Conference
  • Place of Presentation: London
  • Year and Date: 2009-12-14
• [Presentation] Development of in vivo delivery system for siRNA-based cancer therapy (2009)
  • Author(s): Tsuzuku, et al.
  • Organizer: 第31回生体膜と薬物の相互作用シンポジウム
  • Place of Presentation: 大阪
  • Year and Date: 2009-11-30
• [Presentation] Liposomal Polyamine-Dialkyl Phosphate Conjugates as Effective Gene Carriers : Chemical Structure, Morphology, and Gene Transfer Activity (2009)
  • Author(s): Dewa, et al.
  • Organizer: International Symposium on Nanomedicine 2009-2
  • Place of Presentation: 岡崎
  • Year and Date: 2009-11-05
• [Presentation] Polyamine-lipid/DNA Complexes as Gene Carriers : Morphological Observation of the Complexes by Using AFM (2009)
  • Author(s): Kato, et al.
  • Organizer: International Symposium on Nanomedicine 2009-2
  • Place of Presentation: 岡崎
  • Year and Date: 2009-11-05
• [Presentation] ポリアミン脂質による遺伝子送達とDNA複合体形態の直接観察 (2009)
  • Author(s): 出羽毅久, 他
  • Organizer: 第58回 高分子討論会
  • Place of Presentation: 熊本大学
  • Year and Date: 2009-09-16
• [Presentation] ポリアミン脂質・DNA複合体の構造と核酸送達能 (2009)
  • Author(s): 出羽毅久
  • Organizer: 揺らぎと生体機能 平成21年度合同班会議
  • Place of Presentation: 熊本
  • Year and Date: 2009-09-11
• [Presentation] ポリアミン脂質-DNA複合体のAFMによる形態観察と遺伝子送達能 (2009)
  • Author(s): 出羽毅久, 他
  • Organizer: バイオ高分子シンポジウム
  • Place of Presentation: 東京大学先端研
  • Year and Date: 2009-07-29
• [Presentation] 遺伝子伝子導入能をもつ新規ポリアミン脂質の開発-DNA複合体の形態観察とその機能評価 (2009)
  • Author(s): 加藤清志, 他
  • Organizer: 第58回高分子学会年次大会
  • Place of Presentation: 神戸国際会議場
  • Year and Date: 2009-05-27