淋巴血液多靶点纳米药物复合体系的构建及其时序响应性递药的调控研究

Tumor is the major disease threatening people's lives and health in our country, tumor metastasis has become the primary cause to death. Chemotherapy is extensively applied for cancer therapy, drug delivery of which deserves to be improved. Since the anti-cancer drug delivery is a sequential and integral process including series of continuous events, requiring specific technology and carrier material which could tempo-sequentially modulate the transport and release of drugs. Due to the peculiar structure and function, nano-complex offers an efficient opportunity to improve the drug transport and release efficacy. This project targets to establish a nano-complex combining targeting and temporal-sequences responsible abilities together, in response to the temporal sequential of drug delivery and tumor specific micro-environment. By means of the optimization of nano-complex's functional ingredients and structure, the drug delivery would be modulated tempo-sequentially and automatically by the nanocarrier. The strengthening efficacy of the transport towards lymph and blood as well as the intracellular release of drugs is expected to improve the cancer therapy over both initial and metastatic sides. The project will also bring theoretical and experimental basis for nanomedicine design and preparation as well as clinical therapy.

肿瘤是威胁我国国民生命与健康的头号疾病，肿瘤细胞转移是肿瘤患者死亡的主要原因。化疗是治疗肿瘤的最主要手段，但抗肿瘤药物递送是一系列连续的完整时序性事件，需要研究能够时序性调控药物输送和释放的载体材料或技术，与其相适应。纳米复合载体所独有的结构和功能，为提高药物输送和释放效率提供了有效的途径。本课题拟结合药物递送过程的时序性和肿瘤组织独特的微环境，设计构建靶向时序响应型纳米复合体系，通过对载体材料的功能组分和结构的优化，实现载体对药物递送的自发时序性调控，提高药物在淋巴和血液中的输送效率和在细胞质内的释放效率，兼顾原发和转移肿瘤的控制，改善肿瘤治疗效果，为纳米药物载体设计制备和临床治疗提供理论依据和实验基础。

本项目利用天然多糖，成功合成了羧甲基壳聚糖（CMCS）及巯基化修饰的壳聚糖（TCS）；采用简单的离子交联、二硫键交联的双交联方法制备了具有pH/还原双响应的时序响应性纳米载体CMCS-TCS NPs，所得纳米粒粒径约为126 nm，表面电位为-27.5 mV，制备的纳米载体呈球形，粒径分布均一；CMCS-TCS NPs对小鼠成纤维细胞（L929）及人乳腺癌细胞（MCF-7）均未表现出细胞毒性；以盐酸阿霉素（DOX）为模型药物制备载药纳米颗粒（DOX/CMCS-TCS NPs），药物包封率和包载率分别为4.2%与70.7%，在中性生理条件下具有良好的稳定性，在弱酸性环境中出现聚集，但纳米粒由TCS形成的骨架结构并不会受到影响，在高水平的GSH环境中，导致TCS链间及链内二硫键被还原而解开，纳米粒TCS骨架解体，纳米粒整体结构解体；酸性pH及还原环境对DOX/CMCS-TCS NPs的药物释放具有明显促进作用，pH 5.5及10 mM GSH同时作用下释药量最大，5h可释放93.2%；DOX/CMCS-TCS NPs能够促进纳米粒被MCF-7细胞摄取，高效逃逸溶酶体，并时序性响应胞内转运过程，最终在胞质或细胞核内解体，实现药物在细胞核富集；体外抗肿瘤实验证明，DOX/CMCS-TCS NPs与MCF-7细胞共培养48 h后，其IC50与游离DOX组相当，为0.6 μg/mL，特别是药物浓度较高时（3-27 μg/mL），表现出更高的癌细胞增殖抑制作用。该研究结果为肿瘤化疗药物递送提供了一种简单、便捷、高效的智能纳米载体制备方法，并为改善肿瘤化疗效果提供了实验和理论依据。.本项目发现TCS在碱性条件下具有成胶性能，并随pH的升高或浓度的增大或取代度的增加而增强。其成胶机制为，一方面，高pH导致TCS原分子内二硫键水解形成硫醇盐离子，通过巯基/二硫键置换反应，形成新的分子间二硫键，导致TCS链间交联；另一方面，TCS在pH高于其解离常数6.3时，氨基基团不带电荷，疏水作用增强，壳聚糖链发生缠绕和凝结，壳聚糖链的缠绕与二硫键的重排的协同作用使得TCS在碱性条件下成胶。TCS具有良好的血液相容性，无细胞毒性，在心血管支架表面形成的涂层在模拟体液中降解缓慢，放置2个月仍保持良好形态；与裸支架和壳聚糖涂层支架相比，TCS涂层支架可显著促进人皮静脉内皮细胞贴附和增殖，该结果为TCS作