双前药共组装纳米靶向递药系统用于化疗联合免疫治疗肺癌的研究

The combination of cisplatin and cytotoxic agents used in the treatment of lung cancer can not effective improve the antitumor efficacy, and reduce toxicity and drug resistance of cisplatin. Metformin shows synergistic anti-tumor effect on chemotherapy and immunotherapy by activating AMPK-mTOR pathway. It has been used in clinical trials with cisplatin. However, the combination efficacy is limited due to the unsynchronous pharmacokinetic behavior of cisplatin and metformin used as free drug molecular form in clinical trails. Therefore, The co-delivery in vivo is a key scientific issue for combination therapy of cisplatin and metformin. Dual-prodrug targeted co-delivery technology was employed in our group, hyaluronic acid-cisplatin (HA-CDDP) prodrug and polystyrene-polymetformin (PMet) prodrug were synthesized. The dual-prodrug co-assembling nano targeted drug delivery system was developed by electrostatic binding based on negative charged HA-CDDP prodrug and positive charged PMet prodrug. In our prelimary study, the dual-prodrug co-assembling nano targeted drug delivery system showed targeted co-delivery property in vivo, intracellular enzyme-mediated co-releasing of cisplatin and metformin, and improved synergistic antitumor therapy in cell level. The in vivo pharmacokinetic and tissue distribution behavior, mechanism and effect of intracellular targeted co-delivery and enzyme-mediated co-releasing, and anti-tumor pharmacodynamics of dual-prodrug co-assembling nano targeted drug delivery system were studied, for illustrating the efficacy improvement of dual-prodrug nano drug delivery system on combination therapy of cisplatin and metformin. The action pattern and molecular mechanism of prodrug nano drug delivery system on chemoimmunotherpy was illustrated. Our study aimed to develop a dual-prodrug co-assembling nano-targeted drug delivery technology for chemoimmunotherapy on lung cancer.

临床上顺铂联合细胞毒药物治疗肺癌不能有效提高疗效、降低顺铂毒性和耐药性。二甲双胍通过激活AMPK-mTOR通路，发挥化疗和免疫抗肿瘤双效应，已联合顺铂用于临床试验，但临床以药物小分子形式用药，药动学过程不同步，联用功效有限。因此，体内共递送是实现两药联合化疗拟解决的关键科学问题。本课题组采用双前药靶向共递送技术，合成透明质酸-顺铂和聚苯乙烯-聚二甲双胍前药，并静电结合共组装这两种不同电荷性质的前药分子，构建双前药共组装纳米靶向递药系统，前期已初步探明该系统在体内长效共递药，并在胞内酶解释放顺铂和二甲双胍，在细胞水平提高了两药协同抗肿瘤效应。本研究通过体内药动学及组织分布行为、胞内共递药及酶解释药机制和效应、抗肿瘤药效学研究，阐明双前药递送系统提高联用药物化疗-免疫协同抗肿瘤功效这一科学问题，并探明其化疗-免疫抗肿瘤的作用规律和分子机制，为肺癌提供一项化疗联合免疫治疗的双前药靶向递送新技术。

实现联用药物同步共递送至肿瘤细胞并有效释放是肿瘤联合化疗的关键科学问题。本课题肺癌一线化疗药物顺铂（Cisplatin，CDDP）和新型抗癌药物二甲双胍（Metformin，MET）的共递送、共释放问题，构建了具有CD44受体靶向性的透明质酸-顺铂前药（HA-CDDP）和具有自组装特性的聚苯乙烯-二甲双胍（PMet）前药聚合物，通过静电结合方式将两种前药分子共组装形成HA-CDDP/PMet双前药共组装纳米粒，从而提高其抗肿瘤协同作用。本课题采用靶向前药偶联技术和可逆加成-断裂链转移（RAFT）聚合法合成HA-CDDP 和PMet前药，通过静电结合方式将两种前药分子共组装形成HA-CDDP/PMet双前药纳米粒；并通过制剂毒性研究，阐明共载药胶束对癌细胞的增殖抑制作用和诱导凋亡作用；考察共载药纳米粒的组织分布，评价胶束同步联用药物的组织分布作用；重点研究共载药胶束体内外抗肿瘤药效和探究HA-CDDP/PMet纳米粒的化疗联合免疫协同抗肿瘤的分子机制，综合评价胶束的共载药和靶向同步递送策略提高两药化疗-免疫联合治疗肺癌的功效。.研究结果表明，HA-CDDP/PMet纳米粒在模拟生理环境和模拟肿瘤细胞外液中均稳定，进入模拟内涵体的微酸性环境时内触发释药行为，可实现胶束携载药物在肿瘤细胞内高效共释放，达到协同抗肿瘤功效。体外细胞增殖抑制实验结果表显示，HA-CDDP/PMet纳米粒在LLC细胞中具有最强的抗肿瘤活性，表明纳米粒中化学键合的CDDP可有效释放于肿瘤细胞，联合MET协同抗肿瘤。考察纳米粒在荷瘤小鼠各组织中的分布情况发现，HA-CDDP/PMet纳米粒中的CDDP在肿瘤中的蓄积量显著提高。以荷瘤小鼠为模型考察纳米粒的抗肿瘤药效学，HA-CDDP/PMet纳米粒组肿瘤体积最小，肿瘤细胞坏死面积最大，提示前药载体裂解的CDDP可与共递送的Met产生联合免疫协同抗肿瘤作用，并进一步证实了双前药共组装纳米粒策略的有效性。.综上所示，本课题通过分子、细胞和动物水平评价，阐明肿瘤靶向共载药胶束同步递送联合化疗药物CDDP和Met，提高其协同抗肿瘤的功效及机制，为肿瘤治疗提供一种联合化疗新策略，也为两药化疗-免疫联合治疗肺癌的共递送平台的构建提供指导和借鉴。