新合成材料PLGA-STPGS制备的大黄素纳米粒诱导HepG2细胞凋亡
Apoptosis of HepG2 cells in vitro induced by emodin-loaded nanoparticles using a newly synthesized PLGA-STPGS as carrier

摘要：目的 合成高分子材料乙交酯丙交酯共聚物-琥珀酰基维生素E聚乙二醇1000琥珀酸酯(PLGA-STPGS)，并以其为载体制备大黄素PLGA-STPGS纳米粒(EPTN)，考察其体外诱导人肝癌细胞HepG2的凋亡效果。方法 用市售材料PLGA和TPGS为原料，采用酯化反应合成高分子材料PLGA-STPGS，并用红外光谱、氢核磁共振、凝胶渗透层析、差示扫描量热法分别对其进行表征；以PLGA-STPGS为载体，采用乳化溶剂挥发法制备EPTN和大黄素？？PLGA？？纳米粒(EPN)并测定二者的粒径、载药量和包封率；分别使用流式细胞仪检测Annexin V-FITC/PI试剂盒和荧光显微镜观察TUNEL试剂盒处理的HepG2细胞在体外与EPTN和EPN孵育24、72h后的凋亡效果。结果 成功合成高分子材料PLGA-STPGS，数均分子量和分散度分别为25347和1.42；24、72h时EPTN和EPN诱导HepG2细胞的凋亡率分别为47.2%、62.4%和41.6%、52.1%；荧光显微镜观察到EPTN组比EPN组有更多呈绿色荧光的细胞核。结论 成功合成高分子材料PLGA-STPGS，以其为载体制备的EPTN能明显诱导HepG2细胞凋亡，而材料本身对细胞无明显毒性。
ABSTRACT: Objective To prepare emodin (EMO)-loaded PLGA-STPGS nanoparticles (EPTN) and evaluate the cellular apoptosis of EPTN in the human liver cancer cell line HepG2 in vitro after synthesis and characterization of a new copolymer of polylactide-co-glycolide-succinyl-D-α-tocopheryl polyethylene glycol 1000 succinate (PLGA-STPGS). Methods PLGA-STPGS was synthesized by esterfication method using PLGA and TPGS purchased from the market, and characterized using the fourier transform infrared spectrophotometer (FTIR), 1H nuclear magnetic resonance 1H-NMR), gel permeation chromatography (GPC), and differential scanning calorimetry (DSC) analysis. EPTN and EMO-loaded PLGA nanoparticles (EPN) were prepared by an emulsification solvent evaporation method; their particle size, drug loading, and entrapment efficiency were determined. Annexin V-FITC/PI Apoptosis Kit with a fluorescence-activated cell sorter quantitatively and TUNEL Apoptosis Kit under a fluorescence inversion microscope (FIM) qualitatively were used to detect the in vitro apoptotic effect on HepG2 cells induced by EPTN and EPN after incubation for 24 and 72 hours. Results PLGA-STPGS was successfully synthesized as a new material and further confirmed by the FTIR, 1H-NMR, DSC, and GPC analysis. The number average molecular weight (Mn) and polydispersity of the PLGA-STPG random copolymer was 25347 and 1.42. The apoptosis ratios of HepG2 cells incubated with EPTN and EPN for 24 and 72 hours were 54.2%, 62.4% and 41.6%, 52.1%, respectively. Compared with the EMO solution (EMS) group, most of the cellular nuclei in the NP groups were fluorescently labelled in green with TUNEL, and the EPTN group showed the highest level of nuclear green fluorescence observed using FIM. Conclusion PLGA-STPGS was successfully synthesized, and EPTN prepared with PLGA-STPGS as carrier exhibited a stronger effect on the apoptosis of HepG2 cells. The results also indicated that PLGA-STPGS had no obvious toxicity to HepG2 cells