目的 构建并优化一种心肌靶向肽CTP（APWHLSSQYSRT）修饰的丹参酮II_A胶束（cardiac-targeting peptide CTP-modified tanshinone II_A micelles，CTP-Ms/TS II_A），并系统评价其理化特性及体外心肌保护作用。方法 采用薄膜水化法制备CTP-Ms/TS II_A，以包封率为核心指标，运用Box-Behnken设计-响应面法（Box-Behnken design-response surface method，BBD-RSM）优化处方参数［聚乙烯己内酰胺-聚乙酸乙烯酯-聚乙二醇接枝共聚物（Soluplus）质量浓度、维生素E琥珀酸酯聚乙二醇1000（TPGS₁₀₀₀）质量浓度、水化温度］。利用激光粒度仪和透射电子显微镜（transmission electron microscopy，TEM）表征CTP-Ms/TS II_A的粒径、ζ电位及形貌；以芘为探针测定其临界胶束浓度（critical micelle concentration，CMC），并考察制剂的储藏和血清稳定性。通过荧光显微镜与流式细胞术分析心肌细胞（H9c2）对CTP-Ms/TS II_A的摄取效率，并在异丙肾上腺素诱导的H9c2细胞损伤模型中，采用DCFH-DA和JC-1荧光探针、活死细胞染色及CCK-8检测CTP-Ms/TS II_A对活性氧水平、线粒体功能及细胞存活率的调控作用。结果 获得CTP-Ms/TS II_A最佳制备工艺参数：处方体积5 mL，Soluplus质量浓度12 g/L，TPGS₁₀₀₀质量浓度5 g/L，水化温度40 ℃。据此制备的CTP-Ms/TS II_A（膜材含丹参酮II_A、Soluplus、TPGS₁₀₀₀、DSPE-PEG₂₀₀₀、DSPE-PEG₂₀₀₀-CTP），包封率达（90.64±1.01）%（n＝3）。CTP-Ms/TS II_A粒径为（89.30±0.76）nm，ζ电位为（−0.033±0.150）mV；CMC为42.8 mg/L，稳定性良好。与非靶向胶束相比，CTP修饰显著增强了H9c2细胞的摄取效率，并在清除活性氧、恢复线粒体膜电位及改善细胞存活率方面表现出更优效果。结论 成功制备了理化性质稳定的CTP-Ms/TS II_A，并通过体外实验验证了其具有心肌靶向与保护潜能；为丹参酮II_A用于心血管疾病防治提供了新的实验基础与潜在的递送方案。

Objective To construct and optimize tanshinone II_A (TS II_A)-loaded micelles modified with a cardiomyocyte-targeting peptide CTP (APWHLSSQYSRT) (CTP-Ms/TS II_A), optimize their formulation, and evaluate their physicochemical properties and cardioprotective effects in vitro. Methods CTP-Ms/TS II_A were prepared using the film hydration method. A Box-Behnken response surface design was applied to optimize formulation parameters (Soluplus concentration, TPGS₁₀₀₀ concentration, hydration temperature) with encapsulation efficiency as the evaluation index. Particle size, ζ potential, and morphology of micelles were characterized by dynamic light scattering and transmission electron microscopy (TEM). Critical micelle concentration (CMC) was determined using pyrene as a probe, and storage and serum stability were assessed. Cellular uptake was investigated in H9c2 cardiomyocytes by flow cytometry and fluorescence microscopy. In an isoproterenol-induced H9c2 injury model, intracellular reactive oxygen species (ROS), mitochondrial membrane potential, cell viability, and apoptosis were evaluated by DCFH-DA, JC-1, calcein/PI staining, and CCK-8 assays. Results The optimal preparation parameters were determined as: formulation volume 5 mL, Soluplus concentration 12 g/L, TPGS₁₀₀₀ concentration 5 g/L, and hydration temperature 40 ℃. The CTP-Ms/TS II_A prepared under these conditions (with membrane materials including TS II_A, Soluplus, TPGS₁₀₀₀, DSPE-PEG₂₀₀₀, and DSPE-PEG₂₀₀₀-CTP) achieved a high EE of (90.64 ± 1.01)% (n＝3). The micelles exhibited a particle size of (89.30 ± 0.76) nm, a ζ potential of (−0.033 ± 0.150) mV, and a critical micelle concentration of 42.8 μg/mL, with good stability. Compared with non-targeted micelles, CTP-Ms/TS II_A exhibited significantly enhanced uptake in H9c2 cells, more effective ROS scavenging, restoration of mitochondrial membrane potential, and improved cell survival. Conclusion CTP-Ms/TS II_A with stable physicochemical properties were successfully prepared, and their cardiac-targeting and cardioprotective potential were verified by in vitro experiments. This work provides a new experimental basis and potential delivery strategy for the application of TS II_A in the prevention and treatment of cardiovascular diseases.

R283.6

2023年辽宁省科技计划联合计划项目（2023JH2/101700227）； 2022年国家自然科学基金青年基金项目（82104841）