基于Ca-P成型非对称脂质双层异性双药纳米粒弱化基质渗透屏障并高效接触深层肿瘤细胞的技术研究

Deformed vascular system, a large number of dense stromal cells and high interstitial fluid pressure are the major issues for agents to overcome the stromal permeability barriers and be delivered to the deep tumor cells. The popular strategy for improving drug distribution in tumor includes the nanocarriers targeting to tumor microenvironment. The key scientific problems needed to be solved are the delivery system for drug pairs with extreme opposite solubility according to clinical regimen, the promoted invasion to stroma through abnormal blood vessel, increased drug exposure to tumor cells in the deeper parts with the lower pH and hypoxia. Our research group found that gemcitabine was highly sensitive to CAFs, which would kill CAFs nearby and then pioneer the road for nanodrug which would across the epithelial wall to permeate in the interstitial substance. Therefore, based on the first-line chemotherapy for breast cancer, taking the new Ca-P precipitation-made nanoparticles with asymmetric lipid bilayers into consideration, the efficient encapsulation of gemcitabine and paclitaxel with opposite solubility and the endosome escape are supposed to be realized. With the active targeting assistance of transmembrane iRGD cyclopeptide, we are assumed to evaluate the dual permeation effect through remolding microenvironment of tumor cells and targeting effect of the drugs themselves. With all the data we would get, it is possible to predict and guide the redistribution and absorption of nanocarriers in deeper layer of tumor, provide a universal and systematic research mode for the calcium phosphate (Ca-P) nanoparticles loaded with drug pairs of opposite solubility and complete the pharmaceutical theory of the targeting drug-delivery system for anti-cancer agents.

异形血管系统、密集基质细胞及间质流体高压是药物克服基质渗透屏障并靶向深层肿瘤细胞的主要障碍，研究肿瘤微环境靶向纳米载体是促进癌变组织药物吸收的热点。能否依据临床方案构建异性双药系统、促进药物经异形脉管浸润间质、增大低pH低氧部位细胞的暴露率是亟待解决的关键科学问题。本课题组研究发现吉西他滨对CAFs高度敏感，可优先致附近CAFs死亡，为纳米药物跨血管壁后的间质渗透开拓道路。因此本研究根据乳腺癌一线化疗方案，以Ca-P沉淀为内核构建新型非对称脂质双层纳米粒，实现溶解性相异的吉西他滨、紫杉醇联用高效包封、内涵体逃逸，并通过穿膜iRGD环肽主动靶向，从肿瘤微环境重塑、药物自身细胞靶向规律出发，对所构建纳米载体的双重促透作用进行评价及机理研究，以期预测及指导设计纳米载体的肿瘤深层再分布吸收，为Ca-P沉淀构建异性双药纳米载体提供通用性系统性的研究模式，进一步完善抗肿瘤靶向给药系统的制剂学理论。

本课题组研究发现吉西他滨对肿瘤相关成纤维细胞（tumor-associated fibroblasts，TAFs）高度敏感，可优先致附近TAFs死亡，促进纳米粒渗透进入细胞间质。以一线乳腺癌化疗药物为模型，以Ca-P沉淀为内核构建新型非对称脂质双层纳米粒，实现吉西他滨单磷酸盐（GMP）与紫杉醇（PTX）联用，并通过穿膜肽主动靶向得到P/G-NPs。P/G-NPs阳性纳米粒呈球形，粒径分布均匀，GMP与PTX可实现按比例包封（8:1，m/m）、释放，包封率为93.6%（GMP）、98.7（PTX）。与未修饰纳米粒相比，RGD环肽修饰P/G-NPs细胞摄取明显增加约5倍，且具有良好的长循环及肿瘤组织靶向性。药效学结果显示，P/G-NPs可明显抑制肿瘤生长（16mg/kg GMP和2mg/kg PTX，i.v.），在治疗期间对裸鼠体重及主要脏器无明显毒副作用。与游离药物联用组相比，P/G-NPs组肿瘤内细胞凋亡增加约43.6%（TUNEL法），可有效调控Bcl-xL、Bcl-2、PARP-1蛋白的表达，具有良好的抗肿瘤作用。P/G-NPs为Ca-P沉淀构建异性双药纳米载体提供通用性系统性的研究模式，进一步完善抗肿瘤靶向给药系统的制剂学理论。