肿瘤干细胞化学治疗的纳米给药系统设计与评价

Regulating signal pathway of nanoparticle delivery system is considered to be one of the most potential ways to treat cancer stem cells. In this project, we introduced two types of delivery systems: glycolipid and lipid nanoparticles, which display excellent uptake ability, escaping capacity from endosome, and blocking the delivery system from being identified by drug-resistant associated transporters. The delivery system was formed by regulating the ratio of two types of glycolipid nanomaterial, providing the ability of targeting different sites in cells and different releasing rates. Furthermore, we will examine the correlation between the modification of the amino groups, responsive ability of intracellular redox potential and the specific site, rate of drug release. Choose different liquid-lipid ratios of lipid nanoparticles to investigate the mechanism of how the intracellular physical form of drugs impact on drug release. Also, we will illustrate the relevance between physiochemical property, spatial structure and rate of intracellular drug release. Moreover, experiments will be conducted including establishing animal models of cancer stem cells, body distribution, intracellular dynamic drug release and combination therapy against tumor cells and cancer stem cells. Inherent regularity of formulation design of nanoparticle delivery systems, cytotoxicity and drug efficacy will be clarified. This study will provide new ideas, strategies and approaches for efficient cancer stem cell therapy of nanoparticle delivery system, and enlarge and develop the cancer therapeutical theory.

纳米给药系统的信号通路调控，被认为是肿瘤干细胞有效治疗的主要路径之一。本项目采用具有高效跨细胞膜、从溶酶体中逃逸、屏蔽耐药转运蛋白识别的糖脂及脂质二类纳米载体。通过纳米载体中不同类型糖脂比例的控制，形成细胞内靶向不同部位、具有不同释放能力的糖脂纳米给药系统，研究纳米给药系统的氨基取代度、细胞内还原性环境响应能力与细胞内药物释放位点、释放速率之间的相关性。选择不同液体脂质比例的纳米结构脂质载体，研究纳米给药系统中药物物理形态促进细胞内药物释放机理。阐明纳米给药系统的理化性质、空间结构与细胞内药物释放速率之间的内在联系。通过肿瘤干细胞球、肿瘤干细胞模型动物的体内动态释放、细胞内药物动态释放、及针对肿瘤细胞及肿瘤干细胞的联合治疗研究，探明纳米给药系统的处方设计、细胞药效与药物疗效之间的内在规律性，为纳米给药系统的肿瘤干细胞高效治疗，提供新思路、新策略和新方法，丰富和发展肿瘤治疗理论

肿瘤干细胞可能是肿瘤难以治愈的主因。本项目针对肿瘤干细胞特性，设计制备糖脂纳米载体CSOSA，以阿霉素（DOX）为模型药物，构建纳米给药系统。结果显示，肿瘤干细胞球模型中，糖脂纳米给药系统CSOSA/DOX具有较强的摄取和渗透能力。酸性磷酸酶试验显示，糖脂纳米给药系统抑制细胞球生长IC50值为1.07μg/mL，其抑制作用是盐酸阿霉素阳性对照的2.47倍。模型动物多周期抗肿瘤药效研究显示，糖脂纳米给药系统抑制肿瘤生长的药效显著优于对照制剂。为进一步探索肿瘤干细胞与耐药的相关性，采用模拟临床的三周期给药方案，选择MCF-7乳腺癌模型小鼠，评价纳米给药系统抗肿瘤药效。三周期治疗结束后，CSOSA/DOX、阳性对照及阴性对照组的肿瘤干细胞比例，分别为10.62%、69.36%及10.95%，显示化疗药物导致肿瘤干细胞富集，糖脂纳米给药系统可同时清除肿瘤细胞及肿瘤干细胞，有效抑制肿瘤生长及复发。采用高剂量脉冲法及低剂量连续刺激法，分别以CSOSA/DOX和盐酸阿霉素诱导MCF-7细胞发生耐药。结果表明，CSOSA/DOX的体外高剂量脉冲刺激及低剂量连续刺激，均不导致肿瘤细胞耐药，而盐酸阿霉素诱导对照组在连续刺激条件下均产生耐药。采用适配体修饰脂质纳米载体A15-PEG-SLN、肝靶向脂质载体A54-PEG-SLN、脂质结构载体NLC和基因递送脂质载体SLN，分别以奥沙利铂（OXA）、盐霉素（SAL）、紫杉醇（PTX）和miR-200c为模型药物，构建纳米给药系统。结果显示，A15-PEG-SLN/SAL抑制肝肿瘤干细胞IC50值为0.69±0.015μg/mL，A54-PEG-SLN/OXA抗肝癌IC50值为16.0±1.2μg/mL。A54-PEG-SLN/OXA与A15-PEG-SLN/SAL序贯给药，模型动物抗肿瘤药效为游离药物对照组的2.41倍。基因递送脂质载体SLN可递释miR-200c至肿瘤细胞，沉默靶蛋白TUBB3的表达，逆转肿瘤干细胞对紫杉醇的耐药。SLN/miR-200c复合物与NLC/PTX纳米粒序贯给药，可将NLC/PTX对乳腺癌干细胞的抑制作用提高7.35倍。.本项目阐明了纳米给药系统的处方设计与药物疗效之间的内在规律性，揭示了多周期化疗诱导肿瘤耐药的原因，探索了纳米给药系统的肿瘤干细胞高效治疗新思路，为纳米给药系统的肿瘤高效安全治疗提供理论基础。