肿瘤靶向型联合基因治疗系统的制备及生物医学应用

Two series of nonviral combined gene therapy systems, presented tumor targeting ability, anti-tumor activity and bio-safety, will be prepared in this study. System 1: Avidin will be used as matrix, and various biotinylated cell-penetrating peptides (CPPs) obtained from solid-phase synthesis step will be coupled to avidin surface, which attributed to the avidin-biotin bridge. Then, two therapeutic genes with different anti-tumor activities will be complexed with CPP-avidin bioconjugates via electrostatic interaction. After that, biotinylated transferrin used as tumor targeted ligand will cover the complexes, and finally a series of tumor targeted combined gene therapy systems will be formed. System 2: By using avidin-biotin bridge, various biotinylated CPPs/therapeutic gene complexes, biotinylated TRAIL and biotinylated transferrin will be coupled to avidin surface, to obtain a series of tumor targeted therapeutic gene/TRAIL combined therapy systems. Plasmid pDsRed2-N1-p53 and VEGF siRNA will be used as therapeutic genes. Characteristics of these obtained biotinylated CPPs and combined gene therapy systems will be evaluated in terms of agarose gel electrophoresis, SEM, particle size and ζ-potential measurements. Their in vitro cell viability and transfection ability will be investigated in different cell lines. Apoptotic morphology will be observed using confocal microscopy, and the expression of protein in transfected cells will be evaluated by western blotting and RT-PCR. Optimize the w/w ratio of biotinylated CPPs, therapeutic gene, biotinylated TRAIL and biotinylated transferrin, select the best for further animal trails. In addition, the in vivo cytotoxicities and tumor inhibition effects of these systems were investigated in tumor-bearing nude mice preliminarily. This study provides an alternative and unique strategy to assemble tumor targeted combined gene therapy systems, which will be promising new treatments for cancer therapy and clinic applications.

制备两类不同系列的具有肿瘤靶向功能、抗肿瘤功效以及良好生物安全性的非病毒联合基因治疗系统。体系1：以亲和素为基体，通过生物素-亲和素的特异性作用，将固相合成的生物素化细胞穿膜肽偶联到亲和素表面，并与两种治疗基因通过静电作用组装得到复合物，最后在复合物表面引入生物素化人转铁蛋白作为肿瘤靶向基团，得到具有肿瘤靶向功能的双基因联合治疗系统；体系2：将生物素化穿膜肽/治疗基因复合物、肿瘤坏死因子相关凋亡诱导配体(TRAIL)以及人转铁蛋白同时偶联到亲和素表面，得到肿瘤靶向型治疗基因/TRAIL联合治疗系统。选用p53抑癌基因和抑制肿瘤血管生成的小分子干扰RNA作为治疗基因。研究这两类系统的体外细胞毒性、转染效率、肿瘤特异性以及杀瘤能力。优化穿膜肽、治疗基因、凋亡因子和靶向基团的用量比例，筛选出效果显著的体系进行动物实验。建立荷人癌细胞移植瘤的裸鼠模型，研究各系统在动物体内的毒性反应及抗肿瘤效果。

本项目设计并合成了一系列“智能”的、可用于肿瘤治疗的、具有协同治疗潜力的多功能药物/基因运载系统。以主客体作用力为驱动力，利用层层自组装技术合成了具有核-壳结构的微胶囊。该微胶囊内部的空腔可包裹大分子药物，囊膜上可负载小分子药物，从而实现了两种药物的独立装载。肿瘤组织过度表达的基质金属蛋白酶会破坏微胶囊的膜结构，释放大分子药物，而紫外光照则会促使膜上小分子药物的释放，在合适的刺激条件下能实现药物可控的独立释放，达到协同治疗效果。无外界刺激下，微胶囊不会被细胞摄取，毒副作用低。突破了传统复合载药体系同时释放种药物的限制，实现了多种药物在不同条件刺激下的分别释放。设计了一种由硼酸酯调控的新型酸敏感纳米胶束作为基因载体。体外细胞实验和体内实验结果均表明，与转染黄金标准相比，该纳米胶束在多种细胞系中均展现出了“超快速”和高效的转染能力，并且能在很短的转染时间内将大部分DNA运送入核。这是因为可逆的硼酸酯键会在溶酶体的酸性微环境中发生断裂，使胶束水解散开，快速释放DNA，使之快速入核，且具有一定的普适性。此外，该纳米胶束还表现出了相当优异的血清耐受性，具有良好的临床应用前景。