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Development of PIG micelle vector system for gene delivery

用于基因传递的 PIG 胶束载体系统的开发

基本信息

批准号：
14571361
负责人：
HOSHI Kazuto
金额：
$ 2.11万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14571361/
关键词：
gene delivery polymer PIC micelle DNA fluorescence resonance energy transfer confocal microscopy block copolymer 遺伝子ベクター

项目摘要

1) By the investigation into the intracellular mechanisms under confocal microscopy using fluorescence resonance energy transfer (FRET), it was revealed that fast endosomal escape and subsequent smooth disintegration of LPEI/pDNA in the cytoplasm are likely to be determining factors for the excellent transfection efficiency of this polyplex system.2) By the investigation from a physicochemical viewpoint to get insight into the structural feature of the PIC micellar vector system, PIG micelle took virus-comparable size (B100 nm) with a serum-tolerable property. The PEG-PLL (12-48)/pDNA micelles showed a gene expression comparable to the lipofection toward cultured 293 cells.3) Based on the observation of the relationship among the PIG micelle structure, physicochemnical properties, and the biological functions, new block copolymers were newly synthesized from PEG-poly(□-benzyl L-aspartate) block copolyiner (PEGPBLA) through aminolysis reaction. They have characteristics of possessing two distinct amino groups with different pKa in a side chain, i.e. one for counterparts with nucleic acids through electrostatic intcraction and the other for the buffering capacity in acidic environments such as endosomes. The transfections using these copolymers revealed their excellent efficiencies even without any endosomolytic agents such as chioroquine. It is indicated that this system has a promising feasibility for in vivo therapeutic applications owing to the proton sponge effect as well as the sufficient serum tolerability.

1) 通过使用荧光共振能量转移 (FRET) 的共聚焦显微镜对细胞内机制进行研究，发现 LPEI/pDNA 在细胞质中的快速内体逃逸和随后在细胞质中的顺利崩解可能是该复合物系统优异转染效率的决定因素。 2) 通过从物理化学角度进行研究，深入了解其结构特征在 PIC 胶束载体系统中，PIG 胶束具有与病毒相当的大小（B100 nm），并且具有血清耐受性。 PEG-PLL(12-48)/pDNA胶束表现出与培养293细胞的脂转染相当的基因表达。3)基于对PIG胶束结构、理化性质和生物学功能之间关系的观察，通过PEG-聚(□-L-天冬氨酸苄基)嵌段共聚物(PEGPBLA)新合成了新的嵌段共聚物。氨解反应。它们的特点是在侧链上具有两个不同pKa的不同氨基，即一个通过静电吸引与核酸对应，另一个则在酸性环境（例如内体）中具有缓冲能力。即使没有任何内体溶解剂（例如氯喹），使用这些共聚物的转染也显示出优异的效率。这表明由于质子海绵效应以及足够的血清耐受性，该系统在体内治疗应用中具有良好的可行性。