黄芪甲苷PEG-PE纳米胶束的制备、细胞内分布及抗心肌细胞凋亡的研究

Preparation,Intracellular Distribution and Anti-cardiomyocyte Apoptosis of AstragalosideⅣPEG-PE Nanomicelles

目的研究黄芪甲苷聚乙二醇衍生化磷脂酰乙醇胺(PEG-PE)纳米胶束的制备、细胞内分布及抗心肌细胞凋亡。方法采用薄膜水化法制备黄芪甲苷PEG-PE(AST-Ⅳ@PEG-PE)纳米胶束,并进行表征观察;采用稳定性实验和体外释药对载药系统进行评价;以香豆素-6作为荧光探针,评价PEG-PE纳米胶束的细胞摄取及细胞内分布;采用10μmol·L^(-1)异丙肾上腺素诱导H9c2心肌细胞凋亡,将等剂量(50μmol·L^(-1))黄芪甲苷和黄芪甲苷PEG-PE纳米胶束在造模前预处理2 h,测定caspase 3活性、活性氧(reactive oxygen species,ROS)水平,Bcl-2和BAX蛋白的表达水平。结果黄芪甲苷PEG-PE纳米胶束具有粒径小、稳定性良好、释药缓慢等特点;荧光实验表明,PEG-PE纳米胶束可以促进药物的细胞摄取,入胞后,还可将药物聚集在线粒体周围,具有良好的线粒体趋向性;细胞凋亡实验结果表明,黄芪甲苷PEG-PE纳米胶束可以明显降低caspase 3蛋白活性、Bax蛋白表达、ROS水平(P<0.05),显著提高Bcl-2蛋白的表达(P<0.05),这些结果均与等剂量的黄芪甲苷存在显著性差异。结论AST-Ⅳ@PEG-PE纳米胶束可以很大程度上提高黄芪甲苷的心肌细胞摄取量,并聚集在线粒体周围,增强药物抗心肌细胞凋亡作用。

OBJECTIVE To investigate the preparation of astragalosideⅣPEG-PE nanomicelles,its intracellular distribution and the study of its anti-apoptosis of cardiomyocytes.METHODS AstragalosideⅣPEG-PE(AST-Ⅳ@PEG-PE)nanomicelles were prepared by thin film hydration method and characterized and observed.The drug delivery system was evaluated by stability test and in vitro release.Coumarin-6 as a fluorescent probe was used to evaluate the uptake and distribution of PEG-PE micelles in cells.The model of H9c2 cardiomyocyte apoptosis was induced by 10μmol·L^(-1) isoproterenol.The same dose(50μmol·L^(-1))of astragalosideⅣand astragalosideⅣPEG-PE micelles were pretreated for 2 h before modeling.The caspase 3 activity,ROS level,Bcl-2 and BAX protein expression level were measured.RESULTS AstragalosideⅣPEG-PE nanomicelles had excellent characteristics such as small particle size,good stability and slow drug release.Fluorescence tests showed that PEG-PE nanomicelles could promote the cellular uptake of drugs.After entering the cells,they can also gather the drugs around mitochondria,which has a good mitochondrial tendency.The results of cell apoptosis test showed that astragalosideⅣPEG-PE nanomicelles can significantly reduce caspase 3 protein activity,Bax protein expression,and ROS levels(P<0.05),and significantly increase Bcl-2 protein expression(P<0.05).CONCLUSION These results are significantly better than that of the astragalosideⅣgroup.AstragalosideⅣPEG-PE can greatly increase the myocardial uptake of astragalosideⅣ,aggregate around the mitochondria and enhance the anti-cardiomyocyte apoptosis effect of the drug.