具有刚性疏水主链的阳离子聚噻吩衍生物作为核酸类药物载体的研究

Nucleic acid-based therapeutics has gained great attention in the treatment of human diseases, ranging from genetic disorders to viral infections and cancer. However, a lack of safe and efficient delivery system is still the bottleneck in nucleic acid therapy. One limitation of cationic polymers as non-viral gene vectors is that they must escape from endosome to avoid acid degradation of cargos, with cellular internalization by clathrin-mediated endocytosis. To solve this problem, we proposed in this project that polythiophene derivatives with hydrophobic and rigid rod-like backbones have potential plasma membrane permeability, and will improve the intracellular delivery of nucleic acid by internalization via a caveolae-mediated endocytosis or a membrane penetration pathway. In detail, polythiophene derivatives with a variety of cationic groups and adjustable hydrophobic/hydrophilic side chains are designed and screened for siRNA vehicles for gene silencing, and for DNA vectors for gene transfection in vitro. Their endocytosis mechanism (membrane penetration or caveolae-mediated pathway) and intracellular fates will be investigated through flow cytometry and confocal laser scanning microscopy. Finally, the best polythiophene vectors will be applied as DNA vaccine adjunct in vivo, to enhance the specific antibody and cellular immune responses.

基于核酸药物的疗法已经在治疗基因缺陷、病毒感染和肿瘤等疾病领域引起广泛关注，但其发展仍受限于缺乏安全、有效的核酸载体。阳离子聚合物作为非病毒类载体的局限之一是内吞后必须从内涵体逃逸以避免遭受溶酶体降解；因此，更有效的策略是能够避免以网格蛋白介导内吞的方式进入细胞。为此，本项目拟设计和制备一系列含有不同阳离子侧基和不同亲疏水侧链比例的阳离子聚噻吩衍生物，期望利用其疏水且刚硬性的共轭主链和可调的侧链组成结构，实现以小窝内陷介导内吞或直接穿透细胞膜的方式进入细胞，从而达到更高的核酸药物传递效率。具体地，本项目将采用流式细胞术和激光共聚焦显微镜等手段系统研究其内吞机理(膜穿透或小窝内陷介导)和在细胞内的分布、代谢过程，筛选出适合于传递DNA进行基因转染或传递siRNA实现基因沉默的最佳载体，最后将优选的聚噻吩载体用作DNA疫苗辅剂，并对特异性抗体和细胞性免疫的增强作用进行研究。

基于核酸药物的疗法已经在治疗基因缺陷、病毒感染和抗肿瘤领域引起广泛关注，但其发展仍受限于缺乏安全、有效的核酸载体。为此，本项目设计了一系列含有刚性主链的阳离子聚噻吩材料作为载体，期望其疏水且刚硬的共轭主链的膜穿透力能协助药物进入细胞，从而实现更高的核酸传递效率。具体研究成果如下：（1）成功合成了阳离子聚噻吩衍生物，研究发现控制材料的亲疏水性和正电荷密度可调控其自组装行为和与核酸药物的相互作用；（2）细胞实验筛选得到了适合于传递DNA进行基因转染的聚噻吩载体，能够担载并传输核酸药物进入细胞，实现高效基因转染的同时保持较低的细胞毒性；（3）研究过程中新合成的几种聚氨基酸共聚物药物载体和前药纳米粒已在老鼠模型上实现了增强的抗肿瘤治疗作用。综上，在本项目的资助下，得到了一系列的聚噻吩衍生物和聚氨基酸类药物载体，实现了高效的基因转染和抗肿瘤治疗效果, 该研究成果有望为高分子纳米药物的研发和应用提供有益参考。本项目完成了预期的研究任务，并发表学术论文9篇，申请中国发明专利1项。