Hepatocyte-targeted psiRNA delivery mediated by galactosylated poly(ethylene glycol)-graft-polyethylenimine in vitro

Gene silencing in liver disease could be achieved by delivering siRNA with nonviral vectors. However, the transfection efficiency of plasmid siRNA (psiRNA) applied through this approach in hepatocytes is generally low. Based on the fact that the asialoglycoprotein receptors present on hepatocytes can recognize galactose, we synthesized galactosylated poly(ethylene glycol)-graft-polyethylenimine (Gal-PEG-PEI) as a nonviral psiRNA carrier for hepatocyte targeting. Our results indicate that 0.2% (molar percentage) of amine groups of PEI was conjugated with PEG having galactose on its distal end. Increasing the molar ratios of Gal-PEG-PEI to psiRNA in complexation led to a decrease in particle size but an increase in zeta potential of complexes. The transfection efficiency of nanocomplexes, that is, Gal-PEG-PEI/psiRNA, in HepG2 cell line depends on the N/P value, which reflects the molar ratio of Gal-PEG-PEI to psiRNA in the complex. The highest transfection efficiency was 37.34%, which was obtained at N/P 8. At the same N/P value, the transfection efficiency with the nontargeting PEG-PEI/psiRNA or Lipofectamine 2000/psiRNA was much lower. The transfection efficiency of Gal-PEG-PEI/psiRNA dropped to 3.60% from 37.34% after an excessive amount of free galactose was added into the medium for HepG2 cell incubation. By contrast, the similar phenomenon was observed neither when using PEG-PEI or Lipofectamine 2000 as a delivery vector nor in human embryonic kidney 293 cell line lacking ASGR. Real-time PCR analysis and western blot assay demonstrate that the knockdown of HLA-E gene expression by psiRNA/Gal-PEG-PEI (N/P 8) can reach about 60% in HepG2 cells after a 48-h transfection.