HER2抗体介导的pH敏感光交联聚合物胶束递药系统的构建及抗肿瘤研究

To solve the problems that existing micelles are not stable enough in the body and/or release slowly in targeted cells, we aim to build a multifunctional polymer micelle drug delivery system, with functions of stability in systemic circulation, active tumor targeting and pH-responsive intracellular release of drug, in order to enhance tumor targeting efficiency of the micelles and achieve a rapid release of anti-cancer drugs in targeted cells. The micelles were prepared by PEG-PAC-PTMBPEC and Trastuzumab-PEG-PTMBPE self-assembled in the water and then cross-linked by UV irradiation. The PAC is polycarbonate which contains photo-cross-linkable acrylic groups and the PTMBPEC is polycarbonate whose side chain contains degradable acid-sensitive acetal groups. Trastuzumab is HER2 targeting antibody. This smart micelles have following characteristics: (1) by cross-linking, the micelles have a high degree of stability so they can stay longer in the body circulation, and thus increase their passive targeting efficiency; (2) modified by the monoclonal antibody, the micelles can be specifically endocytosed by targeted cells; (3) the micelles can be hydrolysed rapidly in the acidic environment of the endosomes and quickly release the drug. The antibody-mediated pH-sensitive cross-linked micelles are expected to achieve efficient and targeted delivery of anti-cancer drugs to tumors, and provide theoretical and practical basis for the design of intelligent delivery system.

针对现有胶束存在体内不够稳定和/或在靶细胞内释药缓慢的问题，本项目旨在构建集体循环稳定、肿瘤靶向识别及细胞内涵体pH响应释药等多功能的胶束递药系统，以期提高胶束的肿瘤靶向效率，同时实现抗癌药物在靶细胞内的快速释放。该胶束由PEG-PAC-PTMBPEC和Trastuzumab-PEG-PTMBPEC在水中自组装后光照交联制备得到，其中PAC为含可光交联丙烯酸基团的聚碳酸酯、PTMBPEC为侧链含酸敏感可降解缩醛基团的聚碳酸酯，Trastuzumab为靶向HER2抗体。该智能胶束具有以下特点：（1）通过交联，胶束具有高度的稳定性，可在体内滞留更长时间，增加其被动靶向效率；（2）经单抗修饰的胶束可被靶细胞特异性内吞；（3）胶束在细胞内涵体酸性环境中快速水解并迅速释放药物。本项目设计的抗体介导的pH敏感交联胶束有望实现乳腺癌的高效靶向药物递送，同时为智能递送系统的设计提供理论及实验依据。

本项目研究旨在构建集肿瘤细胞靶向识别、体循环稳定、细胞内环境刺激响应释药等功能于一体的聚合物胶束药物递送系统，以期提高胶束的肿瘤靶向效率，同时实现抗癌药物在靶细胞内的快速释放。本项目执行过程中，主要围绕3个研究主题展开。.研究主题1，通过加成反应合成DSPE-PEG2000-GE11，利用薄膜分散-后插入法制备GE11修饰阿霉素脂质体（GE11-LP/DOX），通过DLS、TEM和HPLC对脂质体的粒径、电位、形貌和包封率进行表征。通过MTT试验、荧光显微镜及流式细胞仪等筛选GE11多肽的最佳密度，探索GE11修饰脂质体的內吞机理。利用近红外活体成像仪动态观察脂质体在肿瘤组织的聚集过程。结果表明，GE11配体密度对肿瘤摄取效率、脂质体细胞毒性及脂质体体内肿瘤富集均有较大影响。为进一步改善肿瘤细胞对纳米载体的摄取效率，尝试了双配体共修饰策略。特异性配体叶酸（FA）用长链PEG连接，非特异性配体（TAT肽）用短链PEG连接，将两个不同功能的配体整合到一个纳米载体。采用相同的制备工艺及表征手段制备并考察了不同FA/TAT肽组合比例的双靶脂质体。结果表明，FA/TAT肽共修饰脂质体的摄取量约为FA单配体修饰脂质体的2倍，并在动物肿瘤模型中展现了最优的肿瘤富集及最长的肿瘤滞留时间。.研究主题2，通过开环聚合反应合成了共聚物PEG-PTMBPEC，通过酰胺反应将cRGDyK肽连接到PEG-PTMBPEC末端，采用透析法制备含有不同cRGDyK肽密度的阿霉素载药聚合物胶束。结果显示，cRGDyK修饰的聚合物胶束不仅能较好地响应pH释药，还能被肿瘤细胞及新生血管表皮细胞摄取。动物试验表明，胶束治疗组小鼠能耐受15 mg/kg的阿霉素剂量，较非靶向胶束表现出更强的抑瘤效果。.研究主题3，为解决快速释药与载体稳定性的矛盾，选用具有三维立体结构、单一粒径的球形聚合物大分子PAMAM作为载体。通过还原敏感的-S-S-键将PEG连接到PAMAM表面，通过疏水作用将阿霉素载入PAMAM分子的疏水内核。结果显示，合成的PAMAM-S-S-PEG聚合物具有极低的溶血性及良好的血浆稳定性，在低pH或高还原环境中，药物均能快速从聚合物载体中释放。最重要的是，对比与DOX溶液，载药胶束显示了更好的抑瘤效果及更低的毒性。.上述研究为项目预定目标的实现奠定了基础，同时本研究的开展为肿瘤治疗提供了新途径。.