基于中医“扶正固本”与逐级靶向递药的“里应外合”抗肿瘤效应和机制研究

Immune cells are damaged remarkably in treatment of chemotherapy due to the nonselective distribution of chemotherapeutic drugs, and antitumor effect by the autoimmune system is seriously reduced after conventional chemotherapy. Hence, the objective of this study is to obtain an enhanced antitumor effect via increasing selectivity of antitumor drugs and enhancing immune functions simultaneously. In the study, a novel targeting molecule is synthesized with matrix metalloproteinase (MMP) sensitive cell-penetrating peptide as a linker, and a new kind of cascade targeting liposome is prepared subsequently. Formed by oligomeric hyaluronic acid, the hydration shell on the liposomal surface prevents the liposome being quickly cleared in the blood circulation. Once arriving at tumor tissues, the PD-L1 inhibitor could be released by the overexpressed MMP in tumor microenvironment, and the cell-penetrating peptide residues are exposed after dissociation by hyaluronidase, thus further enhancing the tumor cell uptake. In the process of treatment, the body immune function is sufficiently enhanced, and the ability to “clean up” tumor cells is fully achieved by “strengthening and consolidating body resistance”. Then, the drugs in the liposome are cascade released, tumor immune escape is inhibited and active targeting tumor cells is acquired by the released agents. Finally, the synergetic antitumor effect of drug therapy and autoimmune therapy is reached to obtain better overall therapeutic effect. This study would further improve the incorporation between traditional Chinese medicine and modern targeting drug delivery technology and expand the tumor treatments with traditional Chinese medicine.

肿瘤化疗过程中，由于化疗药物体内选择性差，在杀伤肿瘤细胞时会损伤免疫细胞，严重影响自身免疫系统抗肿瘤作用。因此，本研究旨在通过提高药物递送选择性和增强免疫功能两方面改善肿瘤治疗效果。研究拟利用基质金属蛋白酶敏感的穿膜肽为连接臂合成一种级联靶向分子，并构建全新的逐级靶向脂质体。该脂质体表面由寡聚透明质酸形成一层水化膜，可避免其被快速清除，到达肿瘤微环境后，在基质金属蛋白酶和透明质酸酶的作用下，释放出PD-L1抑制剂，并脱去表面水化膜而暴露出穿膜肽残基，增加药物摄取率。治疗中，首先利用中医“扶正固本”增强机体免疫功能，提高机体自身对肿瘤细胞的“清理”能力，然后利用逐级靶向脂质体的释药选择性，依次发挥抑制肿瘤细胞免疫逃逸和肿瘤细胞主动靶向的作用，最终实现药物治疗与自身免疫相互促进的治疗策略，提高治疗效果。本课题可完善中医药理论与现代制剂技术的深入融合，并拓展中医药在抗肿瘤方面的应用。

本课题首先以出膏率和含糖量综合评分为指标，采用星点设计-效应面法优选“扶正固本”方提取工艺。为在保持汤剂原有药效基础上方便用药，将“扶正固本”方制成颗粒剂。确定干燥方式、辅料、制粒筛号等成型工艺后，为评价扶正固本颗粒质量标准，建立炙甘草、黄芪、五味子TLC鉴别方法和人参Rg1的HPLC测定方法，结果显示扶正固本颗粒制备工艺良好。建立C57BL/6荷瘤小鼠模型，考察“扶正固本”方对免疫器官、脾淋巴细胞及肿瘤浸润性淋巴细胞影响，结果显示扶正固本颗粒可有效抑制荷瘤小鼠肿瘤生长，增强机体免疫功能。采用星点设计-效应面法优选异长春花碱脂质体处方，将成功合成的功能性载体材料DSPE-PEG2000-TATmmp-S7911对脂质体修饰。采用含量测定、细胞抑制及摄取实验考察MMP酶与透明质酸酶的降解对多功能靶向脂质体的影响，结果表明多功能靶向脂质体具有良好逐级释放能力及靶向性。构建多功能靶向异长春花碱脂质体，分别从粒径、粒径分布、Zeta电位、形态学、包封率、体外释放度和稳定性等方面考察各脂质体理化性质。多功能靶向异长春花碱脂质体粒径均匀，包封率较高，物理稳定性良好。构建CTL细胞与肿瘤细胞共培养模型，用扶正固本颗粒与多功能靶向异长春花碱脂质体联合给药方式，考察T淋巴细胞杀伤活性、INF-γ及IL-2表达水平以及FAS、FASL、caspase-3等蛋白表达。结果显示，联合给药明显提高免疫作用同时促进细胞凋亡。以SD大鼠为动物模型，通过HPLC法评价联合给药前后不同脂质体在SD大鼠中代谢动力学参数，结果显示联合给药能有效提高生物利用度。组织分布表明：药物包封入脂质体后，可延长其在体内的循环时间，多功能靶向脂质体可增强脂质体的肿瘤组织靶向性和积蓄。分别从生存曲线和HE染色等方面考察联合给药前后不同制剂对荷瘤小鼠药效学，研究表明联合给药一定程度上提高荷瘤小鼠生存时间，增加肿瘤的抑瘤率，诱导肿瘤细胞凋亡，提高联合给药对荷瘤小鼠的治疗作用。