可触发释药的聚合物胶束给药系统构建及靶向异质性肿瘤细胞的研究

The main objectives of the proposal are to design and study a novel polymeric micellar drug delivery systems with triggered release mechanism for targeting heterogeneous cancer cells. Two kinds of polymeric micelles responding to tumor interstitial pH and endosome (or lysosome)pH respectively were constructed using temperature/pH responsive copolymer of poly(L-histidine)-poly(lactic acid-co-glycolic acid)-PEG-poly (lactic acid-co-glycolic acid)-poly (L-histidine),folate conjugated Pluronic 127 and Pluronic P85 (or Pluronic L61).The physical mixed micelles were used for targeting delivery of resveratrol and doxorubicin in combination. The tumor interstitial pH responsive micelles were designed to release resveratrol and Pluronic P85 (or Pluronic L61) for killing cancer stem cells under the synergistic effect of inducing apoptosis from resveratrol and overcoming multi-drug resistance from Pluronic P85 (or Pluronic L61). The endosome (or lysosome)pH responsive micelles were designed for targeting cancer cells via folate mediated endocytosis approach. The micelles would respond to the acidic pH in the endosome (or lysosome)and local hyperthemia, resulting burst release of the incoporated doxorubicin.The polymeric micellar drug delivery systems for targeting cancer stem cells and cancer cells simultaneously are of great creativity and have not been reported so far. The study of the project will help to improve the design of tumor targeting drug delivery systems. More importantly, the study will reveal the validity of synergistic effect of overcoming multi-drug resistance and inducing apoptosis pathways in targeting cancer stem cells.

本项目设计一种新型可触发释药的同步靶向异质性肿瘤细胞的聚合物胶束给药系统。构建该系统的基本思路是将温度/pH双敏聚组氨酸-聚乳酸羟基乙酸-聚乙二醇-聚乳酸羟基乙酸-聚组氨酸、叶酸修饰的普朗尼克F127和多靶点耐药逆转剂普朗尼克P85（或普朗尼克L61）分别组成肿瘤间质pH和肿瘤细胞内涵体（或溶酶体）pH触发释药的两种胶束，用于靶向递送白藜芦醇和阿霉素。肿瘤间质pH触发释药胶束通过响应肿瘤间质弱酸性环境释放白藜芦醇和逆转剂普朗尼克，协同诱导细胞凋亡和多靶点逆转耐药两种机制靶向肿瘤干细胞；肿瘤内涵体（或溶酶体）pH触发释药胶束通过叶酸配体介导实现肿瘤细胞的主动靶向，响应弱酸性的内涵体或溶酶体环境和局部热疗（40oC-43oC）加速释放阿霉素。本项目设计的胶束给药系统具有创新性，国内外未见报道。项目的研究对于完善抗肿瘤药物纳米靶向给药系统的设计及探索多途径联合靶向肿瘤干细胞，具有很好的科学意义。

肿瘤细胞异质性导致的多药耐药是临床上化疗失败的主要原因之一。本研究合成了系列pH敏感聚合物（PHis-b-PLA-b-PEG-b-PLA-b-PHis和PEG-b-PLA-b-PHis），应用PHis-b-PLA-b-PEG-b-PLA-b-PHis与叶酸修饰的普朗尼克F127（folate-Pluronic® F127），构建了新型叶酸介导的内涵体pH触发释药混合胶束（pHendoSM-P85/f/DOX）；应用PEG-b-PLA-b-PHis聚合物与普朗尼克L61（Pluronic ®L61）构建内涵体pH触发释药胶束。两种胶束可利用内涵体的弱酸性环境，pHendoSM-P85/f/DOX可实现肿瘤细胞内递送Pluronic® P85分子和抗肿瘤药物阿霉素（DOX），PEG-b-PLA-b-PHis胶束可实现肿瘤细胞内递送Pluronic ®L61、DOX和白藜芦醇，逆转细胞异质性所致的多药耐药现象。开环聚合法分别合成了系列pH敏感共聚物，并对共聚物结构进行表征。考察混合胶束在不同pH条件下的体外释药行为，当PHis-b-PLA-b-PEG-b-PLA-b-PHis和Pluronic® F127的质量百分比为8/2时，该胶束具有内涵体pH敏感性。分别以耐阿霉素人乳腺癌细胞MCF-7/ADR和采用MCF-7人乳腺癌细胞为模型，对两种pH触发释药胶束逆转肿瘤MDR的机制进行了研究。pH触发释药混合胶束通过靶向叶酸介导的主动靶向作用，将药物运送至耐药肿瘤部位和肿瘤细胞内部，通过PHis的pH敏感性和“质子海绵作用”，可实现药物在耐药肿瘤细胞内的有效释放，同时也可以实现Pluronic® P85分子在耐药肿瘤细胞内的有效释放，发挥Pluronic® P85分子逆转肿瘤MDR的作用，通过耗竭细胞内ATP和降低线粒体膜电势等促进耐药细胞的凋亡。PEG-b-PLA-b-PHis胶束通过细胞内协同递送Pluronic ®L61和白藜芦醇，杀伤肿瘤干细胞。荷瘤裸小鼠为模型的活体成像研究结果表明，pH触发释药胶束组能显著增加示踪染料DIR在肿瘤部位的蓄积，有效地降低DIR在心脏部位的蓄积，提示pH触发释药胶束能减少药物的心脏毒性。与对照组相比，pH触发释药胶束组能显著延长DOX在血中的滞留时间。pH触发释药胶束组具有明显地控制肿瘤生长和转移的作用，显著延长荷瘤小鼠的生存周期。