川芎嗪PEG-PE纳米胶束的体外评价、细胞摄取及抗心肌细胞凋亡研究

In vitro Assessment, Cellular Uptake and Anti-cardiomyocyte Apoptosis of Tetramethylpyrazine PEG-PE Micelles

关键词:  PEG-PE纳米胶束  川芎嗪  细胞摄取  细胞凋亡

Key words:  PEG-PE micelles  tetramethylpyrazine  cellular uptake  cell apoptosis

摘要:

目的 制备川芎嗪PEG-PE纳米胶束，并评价该纳米胶束的细胞摄取和抗心肌细胞凋亡效果。方法 采用薄膜水化法制备川芎嗪PEG-PE纳米胶束，并进行表征。采用体外释药、细胞摄取和细胞凋亡试验对该载药系统进行评价。结果 川芎嗪PEG-PE纳米胶束粒径为（15.8±0.9） nm，Zeta电势为-（20.5±0.4） mV，载药量为（5.7±0.3）%，包封率为（87.2±5.4）%。电镜结果表明川芎嗪PEG-PE纳米胶束呈形态规则的圆球型结构；采用芘测定法测定PEG-PE纳米胶束的临界胶束浓度约为5.3 μg·mL^-1；细胞摄取试验结果表明，PEG-PE纳米胶束可以增强药物的细胞摄取量，细胞外残留量减少；川芎嗪PEG-PE纳米胶束在10%胎牛血清DMEM培养基稳定性良好，采用异丙肾上腺素诱导心肌细胞凋亡，Hoechst染色提示凋亡心肌细胞出现了大量形态学改变，而川芎嗪PEG-PE纳米胶束可以明显减少凋亡细胞和促凋亡Caspase-3活性、抑制促凋亡蛋白Bax表达，提高抗凋亡蛋白Bcl-2表达，均显著优于川芎嗪（P<0.01）。结论 川芎嗪PEG-PE纳米胶束具有粒径小，载药量高，释药缓慢等优势，可很大程度上提高川芎嗪的心肌细胞摄取量，增强药物的抗心肌细胞凋亡作用。

Abstract:

OBJECTIVE To prepare tetramethylpyrazine PEG-PE(TMP-PEG-PE) micelles, and to evaluate the cellular uptake and anti-cardiomyocyte apoptosis of the nanomicelle. METHODS TMP-PEG-PE micelles were prepared by thin film hydration method and characterized. The drug delivery system was evaluated by in vitro release, cell uptake and apoptosis assays. RESULTS The particle size of TMP-PEG-PE micelles was (15.8±0.9)nm, the zeta potential was -(20.5±0.4)mV, the drug loading was (5.7±0.3)% and the encapsulation efficiency was (87.2±5.4)%. The results of electron microscopy showed that the TMP-PEG-PE micelles were in the shape of a regular spherical structure. The critical micelle concentration of PEG-PE micelles was determined by Pyrene assay to be about 5.3 μg·mL^-1. The results of cell uptake assay showed that PEG-PE nanomicelles could enhance the cellular uptake of the drug and reduce the extracellular residual amount. TMP-PEG-PE micelles had good stability in 10% fetal bovine serum DMEM medium. Isoproterenol was used to induce myocardial apoptosis, Hoechst staining indicated that there were a lot of morphological changes in apoptotic cardiomyocytes. TMP-PEG-PE micelles could significantly reduce the pro-apoptotic caspase-3 activity, inhibit the expression of the pro-apoptotic protein Bax, and increase the expression of anti-apoptotic protein Bcl-2, which were significantly better than the test results of TMP(P<0.01). CONCLUSION TMP-PEG-PE micelles have the advantages of small particle size, high drug loading and slow drug release, which can greatly improve the myocardial cell uptake of TMP and enhance its anti-cardiomyocyte apoptosis effect.