胞内外阶梯式触发的钙磷复合脂质药物基因共递送系统对三阴性乳腺癌的治疗研究

The project aims to resolve recurrence, metastasis and drug resistance of triple negative breast cancer, as well as the “PEG dilemma” in long-circulating nano-drug delivery system. A PTX-miR-124 co-delivery nano system of multi-level tumor targeting and anti-tumor capability was designed. A material of mPEG-Hydrazone bond-HA- Disulfide bond-Phospholipids was designed and synthesized. The material was prepared into stepped cleavable long-circulating calcium phosphate composed lipid delivery system response to tumor microenvironment loading PTX in the lipid layer and miR-124 composed with calcium phosphate by electric charge..The NPs depends on the long-circulating capacity of PEG and stabilizing effect of calcium phosphate in the cycle. The hydrazone bond is broken and PEG layer get off in the acidic environment after the NPs get into tumor tissue through EPR effect. Oligo-HA is exposed to target to CD44 on the surface of tumor cells and mediated endocytosis. The disulfide bond is broken by the glutathione in tumor cells and oligo-HA is released to bind with resistance-associated proteins antagonizing endogenous HA. The oligo-HA also increases tumor cell adhesion and inhibits metastasis. At last, the calcium phosphate composed lipid NPs collapse, releasing PTX and miR-124 to produce synergistic antitumor effect.

针对三阴性乳腺癌易耐药和术后复发、转移的现状，本项目设计一种胞内外阶梯式触发的药物基因共载系统，多机制联合作用提高纳米制剂的靶向性，抑制肿瘤生长和转移。合成mPEG-腙键-透明质酸(HA)-二硫键-磷脂，制备长循环脂质体，包裹抗肿瘤药物紫杉醇(PTX)；同时钙磷与miR-124静电结合，包裹在脂质体内，形成钙磷复合的可裂解长循环脂质给药系统。.给药系统在血循环中依赖于PEG的长循环效应和钙磷对脂质体的稳定作用；到达肿瘤部位时，经由EPR效应进入肿瘤部位，在酸性环境下腙键断裂，PEG脱落，克服PEG的摄取抑制缺点；暴露出的寡聚HA靶向于肿瘤细胞表面的CD44而被摄取；在肿瘤细胞内谷胱甘肽的作用下，二硫键断裂，寡聚HA脱落，拮抗内源性HA，与耐药相关蛋白结合从而克服耐药性；并增加肿瘤细胞粘附性，抑制其转移；钙磷复合脂质纳米粒在溶酶体内解离，释放出PTX和miR-124，发挥抗肿瘤协同作用。

本项目利用肿瘤微环境特点设计一种胞内外阶梯式触发的CaP复合的脂质纳米系统，共载PTX和miR-124，协同发挥抗肿瘤作用。共得到了mPEG-HA 比例1:1、2:1、4:1的三种mPEG-HA-PC材料。经pH和谷胱甘肽敏感性、抑制肿瘤细胞侵袭迁移能力和安全性筛选，选择mPEG-HA 2:1的mPEG-HA-PC作为功能性载体材料制备纳米系统。.经正交设计和单因素实验，确定了共载PTX和miR-124的CaP复合脂质纳米系统的最佳制备工艺，并对纳米系统进行了表征。.共载PTX和miR-124的CaP复合脂质纳米粒能够增加MDA-MB-231细胞对PTX和miR-124的摄取；抑制其侵袭和迁移能力；并能够直接抑制MDA-MB-231细胞的生长。.分别建立了人乳腺癌MDA-MB-231细胞的裸鼠皮下瘤和非转移瘤模型，分别进行了药效试验。共载PTX和miR-124的CaP复合脂质纳米粒能够抑制MDA-MB-231细胞裸鼠皮下移植瘤的生长，并对正常组织器官无明显毒性，效果强于PTX和miR-124溶液以及对应的单载纳米粒；能够抑制MDA-MB-231细胞在裸鼠体内的肺转移，并抑制转移灶的生长，促进其凋亡，对正常肺组织无明显毒性，侧面证实了其肿瘤靶向性；并对重要器官心、脾、肾无明显毒性。.共载PTX和miR-124的CaP复合脂质纳米粒可下调caspase3/9、PARP和AKT蛋白的表达，对BCL-1和2的表达影响相反；上调BECN1、BCL、CDK4、Ets-1、Snail-2基因，提示其通过PARP/AKT通路抑制肿瘤细胞的生长的侵袭；促进细胞凋亡；并可能与自噬有关。.PTX和miR-124之间具有协同作用，并且部分蛋白和基因的表达差异在PTX和miR-124经复合纳米粒包载后效果显著提高，提示纳米系统通过PTX、miR-124和mPEG-HA-PC三方的共同作用发挥抗肿瘤效果，可为临床治疗提供新的思路。