胞外胞内双重响应的多功能协同增强传递基因载体的构建与评价

The development of safe and efficient gene delivery vectors is a prerequisite for the success of gene therapy. A series of extracellular and intracellular dual sensitive multi-functional synergistic delivery vectors will be synthesized. First, PEG is grafted to carboxymethyl chitosan using benzoic-imine bonds, and then different peptides with different functions is coupled to PEGylated carboxymethyl chitosan. PEG could increase colloidal stability of the complexes in blood circulation. Once the complexes reach tumor sites with a low pH value, the PEG chain will be removed from the complexes via acid-hydrolysis of hydrazone linkages. Then the exposed aspargine-glycine-arginine (NGR) targeting groups can realize cell targeting. The combination of NGR target ligand-mediated selectivity and cell-penetrating peptide mediated effective transmembrane function may enhance targeting selectivity and uptake of tumor cells. The disulfide crosslinking is readily reduced in the cytosol to achieve the facilitated release of nucleic acids from complexes. The multi-functional synergistic delivery nonviral gene vectors developed in this study have advantages include good biocompatibility, high stability in blood circulation, tumor-targeting property, cell-penetrating ability, and extracelluar and intracellular dual sensitivity. The structure of complexes, delivery process and transfection ability of the complexes will be investigated systematically by adjusting the of polymer structure such as grafting ratio of peptide and PEG. A clear relationship between structure and transfection efficiency will be elucidate, which provide new ideas of the development of efficient nonviral gene vectors with good biocompatibility for future research.

发展安全高效的基因载体是成功实施基因治疗的首要问题。拟设计胞外胞内双重响应多功能协同增强传递的基因载体-PEG化羧甲基壳聚糖串接具一定数目连续精氨酸残基与天冬酰胺-甘氨酸-精氨酸序列的多肽。据此设计的载体因PEG保护在体循环中稳定；在到达肿瘤部位后随着腙键在酸性环境下水解，复合物脱PEG保护，增强其靶向性；经NGR肽主动靶向作用及跨膜肽介导使复合物易于进入肿瘤细胞；胞内通过二硫键还原响应，实现DNA与载体的有效分离。由此构建具有良好生物相容性、体循环稳定、靶向性、跨膜性、胞外胞内双重响应多功能协同增强传递的高效低毒基因载体。通过调控大分子结构如改变侧链多肽的接枝率及多肽精氨酸残基的数目、PEG的接枝率等，系统、深入地研究该多功能载体与DNA的组装结构、传递过程以及体内体外的传递及治疗效果。在此基础上，阐明该类载体结构与基因传递效率之间的构效关系，为进一步研究高效低毒非病毒载体提供理论依据。

恶性肿瘤严重威胁人类健康，发展安全高效的基因及药物载体是成功实施肿瘤治疗的首要问题。本课题利用具有良好生物可降解性的天然多糖为大分子骨架，通过化学修饰引入二硫键等连接基，制备了一系列的多糖基基因载体。通过设计并引入具有跨膜功能的多肽，赋予多糖基基因载体多功能协同增强基因传递，提高基因转染的效率。研究了该多功能载体与DNA 的组装结构、传递过程以及体内外的传递及治疗效果。结果表明所制备的多糖基基因载体具有较低的细胞毒性和较高的基因转染能力，具有潜在应用价值。另外，考虑到智能给药系统在肿瘤疾病的治疗中具有重要意义，本项目还以天然多糖为大分子骨架，设计和制备了一系列高分子前药，研究了高分子前药自组装以及高分子前药在肿瘤细胞环境下的药物智能响应释放。这些研究工作为进一步发展基因与药物的共载体提供理论依据。