ω-3不饱和脂肪酸功能性纳米载体的设计及逆转肝癌多药耐药作用研究

Design of reversing multidrug resistance drug for liver cancer based on omega-3 unsaturated fatty acid nanocarriers

目的设计二十二碳六烯酸(DHA)为代表的ω-3不饱和脂肪酸功能性纳米载体,探究其协同阿霉素(DOX)给药对肝癌多药耐药(MDR)的影响及其作用机制。方法以DHA和DOX为油相,通过高压乳化法制备DOX纳米粒。将DOX或DOX纳米粒与HepG2细胞或HepG2/ADM细胞共培养后,分为HepG2/ADM组、HepG2+DOX组、HepG2+DOX纳米粒组、HepG2/ADM+DOX组、HepG2/ADM+DOX纳米粒组。采用CCK-8法检测并计算细胞存活率,倒置荧光显微镜观察细胞摄取,分光光度法检测细胞内DOX的药物浓度,Western blot法检测各实验组MDR相关蛋白(MRP)、肺耐药相关蛋白(LRP)、乳腺癌抗药性蛋白(BCRP)、凋亡相关蛋白(Bcl-2)的表达水平。结果与HepG2/ADM组比较,HepG2+DOX组、HepG2+DOX纳米粒组、HepG2/ADM+DOX组、HepG2/ADM+DOX纳米粒组细胞存活率均明显为低(均P<0.01);与HepG2/ADM+DOX组比较,HepG2/ADM+DOX纳米粒组细胞存活率低(P<0.05)。随着药物作用时间的延长,细胞药物摄取能力增强。HepG2/ADM+DOX纳米粒组细胞药物摄取能力最强,HepG2+DOX纳米粒组次之,HepG2+DOX组最弱。随着药物作用时间的延长,HepG2与HepG2/ADM细胞内DOX浓度均持续增长;HepG2+DOX纳米粒组与HepG2+DOX组、HepG2/ADM+DOX纳米粒组与HepG2/ADM+DOX组相比,细胞内DOX浓度高且增长速率较快,但差异均无统计学意义(均P>0.05)。与HepG2/ADM组相比,HepG2+DOX组、HepG2+DOX纳米粒组和HepG2/ADM+DOX纳米粒组细胞中MRP、LRP、BCRP与Bcl-2蛋白表达水平均明显为低(均P<0.05)。结论基于ω-3不饱和脂肪酸功能性纳米载体的DOX可以在一定程度上逆转肝癌MDR。

Objective To design reversing multidrug resistance drug for liver cancer based on ω-3 unsaturated fatty acid nanocarriers and to test its effects.Methods Using docosahexaenoic acid(DHA)and doxorubicin(DOX)as the oil phase,DOX nanoparticles were prepared by high-pressure emulsification.Human hepatocellular carcinoma HepG2 cells and drug-resistant HepG2/ADM cells were co-cultured with DOX or DOX nanoparticles,respectively.The cell proliferation was determined by CCK-8 method,cell uptake was observed by inverted fluorescence microscope,intracellular DOX drug concentration was detected by spectrophotometry.The expression levels of multidrug resistance-related protein(MRP),lung resistance-related protein(LRP),breast cancer resistance protein(BCRP)and apoptosis-related protein(Bcl-2)were detected by Western blot.Results Compared with the HepG2/ADM group,the survival rate of HepG2 and HepG2/ADM cells treated with DOX or DOX nanoparticles was significantly decreased(P<0.01),and DOX nanoparticles had a more significant inhibitory effect on HepG2 and HepG2/ADMcell proliferation.The HepG2/ADM+DOX nanoparticle group had the strongest drug uptake capacity,followed by the HepG2+DOX nanoparticle group,and the HepG2+DOX group was the weakest.Compared with DOX group,the intracellular drug concentration of the DOX nanoparticle group tended to be higher,but the difference was not significant(P>0.05).Western blot results showed that compared with HepG2/ADM group,the expression levels of MRP,LR,BCRP and Bcl-2 protein in HepG2+DOX group and HepG2+DOX nanoparticle group were significantly reduced(P<0.01),in HepG2/ADM+DOX nanoparticle group,only MRP and LRP protein expression levels decreased significantly(P<0.05),in HepG2/ADM+DOX group cells,MRP,LRP,BCRP,and Bcl-2 showed only a downward trend(P>0.05).Conclusion The prepared DOX nanoparticles can reverse multidrug resistance of liver cancer cells to a certain extent.