基于修饰型siRNA的基因和药物共递送纳米载体的构建及研究

Study on gene and drug co-delivery is now one of the hot topics in the cancer therapy research area. In this study, using aptamer technology, a novel gene/drug co-delivery system could be constructed with reasonable genes and drugs co-loading, controlled release and simple preparation process. Due to the aptamer with continuous -CG- nucleotide sequence could combine with antitumor drug doxorubicin (DOX), this aptamer could be used to modify siRNA in order to obtain drug loaded siRNA. Thereby, after drug loading, the modified siRNA could also be called co-loaded siRNA (siRNA-DOX). Thus, to construct reasonable siRNA delivery system was actually to construct gene and drug co-delivery system. The gene and drug co-loaded PEI/siRNA-DOX nanoparticles were obtained by condensing siRNA-DOX with positively charged PEI. NGR oligopeptides which can specifically target to the over-expressed CD13 antigen on tumor neovascular endothelial cells were linked to peglated Carboxymethyl Chitosan (CMCS) to obtain novel pH-sensitive targeting material CMCS-PEG-NGR. The negative charged CMCS-PEG-NGR could self-assemble on the PEI/siRNA-DOX nanoparticles. That is to say, the expected multifunctional gene and drug co-delivery nanocarriers were formed with PEI/siRNA-DOX core and CMCS-PEG-NGR shell. CMCS-PEG-NGR could enhance the stability of the nanoparticles, reduce toxicity of PEI, and make nanoparticles active celluar targeting and long circulation. This novel multifunctional gene and drug co-delivery nanocarriers were designed to be simple and ingenious, possess many good properties such as less influential factors, controlled gene and drug co-loading and release. Therefore, positive results would be expected by exploring the evaluation and anti-tumor synergy studies of this drug and gene co-delivery system. Furthermore, the output of this project is expected to establish a solid foundation for the further studies and utilizations on gene and drug co-delivery strategy.

基因和药物共给药是目前肿瘤治疗研究前沿。本课题拟利用适配体技术构建基因和药物合理共载、释放可控、制备过程简单的共递送系统。连续-CG-碱基序列的适配体可结合抗癌药物多柔比星（DOX），利用此适配体修饰治疗siRNA可获得具有载药链的修饰型siRNA，修饰型siRNA载药后即为双载siRNA（siRNA-DOX），设计合理的siRNA给药系统即为基因和药物共给药系统。以PEI压缩siRNA-DOX获得双载PEI/siRNA-DOX纳米粒；靶向CD13的NGR修饰羧甲基壳聚糖(CMCS)得到pH敏感靶向材料CMCS-PEG-NGR，自组装到PEI/siRNA-DOX纳米粒最外层，可提高该纳米粒的稳定性、降低PEI毒性，并获得靶向和长循环性质。此共载系统设计简单巧妙，影响因素少，基因和药物的共载与释放可控。通过该共递送载体的性质评价和抗肿瘤功效研究，为基因和药物共给药深入研究及未来应用奠定基础。

癌症是威胁人类健康的重大安全隐患，全球致死率高达13%，且近年来其发病率和死亡率呈明显上升的趋势。研制安全有效的癌症治疗手段是当前全人类面临的重大挑战。目前研究发现，化疗与基因治疗联合治疗，是提高肿瘤化疗效果、减轻毒副作用的一种常用治疗手段。将化疗药物与基因装载于同一载体中，有利于实现两者生物药剂学性质同步化，确保化疗药物与基因以合适比例递送至同一靶细胞。化疗与基因治疗联合治疗的研究成为癌症联合治疗研究领域的热点和难点。而实现基因和药物共给药的挑战之一就是共递送系统的设计与开发。本课题利用富含CGA重复序列的寡聚核苷酸可以与抗肿瘤药物多柔比星特异结合的性质，采用一步组装法设计合成新型多功能药物/基因共递送纳米载体。取得了以下主要研究成果和进展：1、成功合成新型多功能材料。所合成载体组装材料均具备载体构建所需功能，为进一步研究多功能一步组装纳米载体奠定基础；2、成功通过一步组装方法构建多功能药物/基因共递送纳米载体；3、成功验证多功能药物/基因共递送纳米载体的共递送效果、体外抗肿瘤效果、体外基因转染效果；4、探索了多功能药物/基因共递送纳米载体的细胞摄取、细胞内药物释放、入胞动力学及入胞机制等；5、成功完成了多功能药物/基因共递送纳米载体的体内分布、体内安全性和体内抗肿瘤效果评价。该载体体外基因转染效果好，抗肿瘤活性高，具有良好的应用前景。上述工作在合成和组装新型多功能基因/药物共递送载体，提高抗肿瘤活性方面取得一定进展，为基因/药物共递送载体系统的进一步研究奠定基础，为肿瘤联合治疗提供了一种新思路、新工具和新手段。上述相关研究共计发表研究论文5篇，其中4篇SCI，申请中国发明专利1项，另有1篇论文处于修改阶段。