Gal-PEG-PEI/psiRNA肝靶向性纳米基因载体对人胎肝细胞系L-02细胞的转染效率检测

Transfection efficiency of galactosylated poly(ethylene glycol)-graft-polyethylenimine as hepatocyte-targeting psiRNA carrier in human fetus liver cells L-02 cells

目的:检测半乳糖-聚乙二醇-聚乙烯亚胺/小干扰RNA质粒(Gal-PEG-PEI/psiRNA)肝靶向性纳米基因载体对人胎肝细胞系L-02细胞的转染效率。方法:合成Gal-PEG-PEI/psiRNA纳米复合物,采用纳米粒径仪测定复合物的粒径和zeta电位,用不同Gal-PEG-PEI氨基与psiRNA磷酸基比例(N/P)的Gal-PEG-PEI/psiRNA纳米复合物转染L-02细胞,以Lipofectamine2000、非靶向性载体PEG-PEI/psiRNA和裸psiRNA转染为对照组,48 h后用流式细胞仪测定转染效率。转染前加入1 mg半乳糖观察其半乳糖竞争拮抗结果。结果:Gal-PEG-PEI/psiRNA纳米复合物的粒径随N/P增大而减小,N/P≥1/15时达最小粒径,约为80 nm。Gal-PEG-PEI/psiRNA纳米基因载体的转染率为(20.4±0.9)%,明显高于非肝靶向性载体PEG-PEI/psiRNA(P<0.01)及裸psiRNA(P<0.01),但低于Lipofectamine 2000(P<0.05);加入竞争性拮抗剂半乳糖后Gal-PEG-PEI/psiRNA纳米基因载体的转染率下降至(4.8±2.1)%,PEG-PEI/psiRNA介导的转染率则不受影响。结论:Gal-PEG-PEI/psiRNA纳米基因载体可明显提高人胎肝细胞系L-02细胞的转染效率,且具有良好的肝细胞靶向性。

Objective：To evaluate the transfection efficiency of galactosylated poly（ethylene glycol）-graft-polyethylenimine（Gal-PEG-PEI） as a nonviral gene carrier of hepatocyte-targeting plasmid siRNA（psiRNA） in human fetus liver cells L-02 cells.Methods：The characteristics of the Gal-PEG-PEI/psiRNA nanoparticles were measured by dynamic light scattering.The transfection experiments were performed with the Gal-PEG-PEI/psiRNA using GFP as the reporter gene in L-02 cells and the transfection efficiency was evaluated by flow cytometry after 48 h.The competition assay was carried out to confirm the uptake of Gal-PEG-PEI /psiRNA by ASGP-R in L-02 cells by adding 1mg galactose into the transfection media.Results：The results indicated that the particle sizes decreased with increasing charge ratios of Gal-PEG-PEI to psiRNA and had a minimum value around 80 nm at the charge ratio of 15.The transfection efficiency of Gal-PEG-PEI was higher than that of PEG-PEI or naked psiRNA in L-02 cells.When galactose was added,the transfection efficiency of Gal-PEG-PEI was drastically decreased from（20.4±0.9）% to（4.84±2.12）%.Conclusion：The Gal-PEG-PEI nanospheres display perfect hepatocyte-targeting ability and can be potentially used as a nonviral gene carrier in liver diease therapy.