目的 制备去甲斑蝥素（3-丙羧基）三苯基溴化膦（TPP）-聚乙二醇-b-聚己内脂（PEG-PCL）纳米胶束，研究其体外释放、细胞内转运及促肝肿瘤细胞凋亡作用。方法 采用薄膜水化法制备去甲斑蝥素TPP-PEG-PCL纳米胶束，测定粒径、Zeta电位及显微电镜形态分析，同时对胶束进行稳定性、体外释放、药代动力学和临界胶束浓度测定；以香豆素-6作为荧光探针，评价TPP-PEG-PCL纳米胶束在肝肿瘤细胞内的摄取、溶酶体逃逸及线粒体靶向功能；采用给药剂量等同条件下，评价去甲斑蝥素TPP-PEG-PCL纳米胶束促肝肿瘤细胞凋亡效果。结果 去甲斑蝥素TPP-PEG-PCL纳米胶束粒径为（16.8±0.2）nm，Zeta电位为（14.3±0.2）mV，透射电镜图片该纳米胶束呈规则圆球型；荧光试验结果显示，TPP-PEG-PCL纳米胶束可以促进药物的细胞摄取、逃逸溶酶体的捕获，最终靶向聚集在线粒体部位；细胞存活率和Hoechst染色结果显示去甲斑蝥素TPP-PEG-PCL纳米胶束具有很好的促肝肿瘤细胞凋亡作用，去甲斑蝥素TPP-PEG-PCL纳米胶束可以明显降低线粒体膜电位、提高细胞内活性氧（ROS）水平、增加促凋亡蛋白Bcl-2、减少抗凋亡Bax蛋白的表达，这些促凋亡相关的实验结果均明显优于去甲斑蝥素PEG-PCL纳米胶束和去甲斑蝥素，具有统计学意义。结论 去甲斑蝥素TPP-PEG-PCL纳米胶束具有良好的肝肿瘤细胞线粒体靶向性和促肿瘤细胞凋亡作用，为一种潜在高效靶向肿瘤细胞线粒体的载药系统。

Objective To prepare norcantharidin TPP-PEG-PCL nanomicelles and study its release in vitro, intracellular transport and promoting effect on hepatoma cell apoptosis. Methods Thin film hydration method was used to prepare norcantharidin TPP-PEG-PCL nanomicelles, and the particle size, electric potential and microscopic electron microscopy morphological analysis were measured. At the same time, the nanomicelles were evaluated for stability, in vitro release, pharmacokinetics and critical micelle concentration. Coumarin-6 was used as a fluorescent probe to evaluate the uptake of TPP-PEG-PCL nanomicelles in liver tumor cells, lysosomal escape and mitochondrial targeting function; Under the same dosage conditions, the effect of norcantharidin TPP-PEG-PCL nanomicelles on promoting apoptosis of liver tumor cells was evaluated. Results The cantharidin TPP-PEG-PCL nanomicelles had a particle size of (16.8 ±0.2) nm, a Zeta potential of (14.3 ±0.2) mV, and transmission electron microscopy images showed that nanomicelles had a regular spherical shape. The fluorescence test results showed that TPP-PEG-PCL nanomicelles can promote the cellular uptake of drugs, escape lysosomal capture, and finally target aggregation at the mitochondrial site; Cell survival rate and Hoechst staining results showed that cantharidin TPP-PEG-PCL nanomicelles had a good effect on promoting apoptosis of liver tumor cells. Norcantharidin TPP-PEG-PCL nanomicelles can significantly reduce mitochondrial membrane potential, increase intracellular ROS levels, increase pro-apoptotic protein Bcl-2, and reduce resistance. The expression of apoptotic proteins Bax and these pro-apoptotic related experimental results are significantly better than those of norcantharidin PEG-PCL nanomicelles and norcantharidin, which have statistical significance. Conclusion Norcantharidin TPP-PEG-PCL nanomicelles have good liver tumor cell mitochondrial targeting and promote tumor cell apoptosis, and it is a potentially effective drug delivery system for targeting tumor cell mitochondria.

R283.6

成都市卫生健康发展基金资助项目（2018ZC002）