丹参酮ⅡA-甘草酸自组装纳米胶束的制备及体外抗脑胶质瘤评价

  目的  将丹参酮ⅡA(Tanshinone ⅡA, Tan ⅡA)封装于甘草酸(Glycyrrhizic acid, GL)中自组装得到胶束, 考察其制备工艺并对产物进行表征, 同时进行体外抗胶质瘤细胞活性评价。  方法  采用薄膜分散法制备载丹参酮ⅡA-甘草酸自组装胶束(Tan ⅡA-GL/Micelle, TGM)。测定TGM的粒径、多分散系数以及Zeta电位；透射电子显微镜观察TGM的形态特征；高效液相色谱法测定TGM的包封率；透析袋法考察TGM的药物释放情况。倒置荧光显微镜考察TGM体外跨血脑屏障(Blood-brain barrier, BBB)的能力; 定量考察细胞摄取的具体机制; MTT法考察游离Tan ⅡA、游离GL和TGM分别对鼠源性脑胶质瘤细胞GL261的细胞毒性; 流式细胞仪检测游离Tan ⅡA、游离GL及TGM对GL261的细胞凋亡作用。  结果  薄膜分散法是制备载丹参酮ⅡA-甘草酸自组装胶束的最佳工艺方法; TGM粒径约为(121.87±5.85)nm, Tan ⅡA包封率为(88.54±14.27)%, 呈类球形形态, 制剂均一稳定; 可在5 h左右释放包载药物。TGM具有良好的跨BBB能力; 脑胶质瘤细胞摄取自组装胶束的机制为通过网格蛋白介导的胞吞来进行制剂的摄取; 对于脑胶质瘤细胞GL261具有良好的细胞毒性; 细胞凋亡实验结果表明, TGM相较于Free TanⅡA、Free GL组显著提高细胞凋亡水平, 促细胞凋亡率分别由12.28%、13.32%上升至31.16%。  结论  GL与Tan IIA通过自组装形成了纳米胶束，有效克服TanⅡA生物利用度低、水溶性差等缺点。TGM可有效跨越BBB，通过网格蛋白介导的胞吞途径被脑胶质瘤细胞摄取，具有良好的细胞毒性与促细胞凋亡能力。 

Preparation and in vitro Evalution of Glycyrrhizic Acid Self-Assembled Micelles Loading Tanshinone ⅡA

  OBJECTIVE  Encapsulate tanshinone ⅡA (Tan ⅡA) in glycyrrhizic acid (GL) self-assembled micelles, to investigate the preparation process and characterization, and evaluate the anti-glioma cell activity in vitro.  METHODS  Thin film dispersion method was used to prepare self-assembled micelles loaded with tanshinone ⅡA glycyrrhizic acid (TGM, Tan ⅡA-GL/Micelle). The particle size, polydispersity index (PDI) and Zeta potential of TGM were measured. The typical morphological characteristics of TGM were observed by transmission electron microscopy (TEM). HPLC was employed to determine the encapsulation rate of TGM, then the drug release of TGM under different pH conditions was investigated by dialysis bag method. The in vitro blood-brain barrier (BBB)-crossing ability of TGM was inspected by inverted fluorescence microscopy. Quantitative cell uptake experiment to investigate the specific mechanism of cell uptake. The cytotoxicity of free Tan ⅡA, GL and TGM on GL261 cells was investigated by MTT assay. The apoptotic effects of free Tan ⅡA, GL and TGM on GL261 cells were detected by flow cytometry.  RESULTS  Thin film dispersion method was the best method to prepare self-assembled micelles loaded with tanshinone ⅡA glycyrrhizic acid. The particle size of TGM was about (121.87±5.85) nm, and the encapsulation efficiency of Tan ⅡA was (88.54±14.27)%, which was almost spherical in shape and the preparation was uniform and stable. TGM was capable of effective BBB-penetrating and the mechanism of uptake of self-assembled micelles by glioma cells was the uptake of preparations by clathrin-mediated endocytosis. TGM enhanced GL261 cell apoptosis from 12.28% (Free Tan ⅡA) and 13.32% (Free GL) to 31.16% (TGM), providing a promising potential to the GBM treatment.  CONCLUSION  The TGM nano-micelles are self-assembled from GL and Tan IIA, which effectively overcomes low bioavailability and poor water solubility of Tan IIA. TGM can effectively cross the blood-brain barrier and be taken up by glioma cells through clathrin-mediated endocytosis, with good cytotoxicity and pro-apoptotic ability.