Design of hepatocyte-targeted gene transfer vector and its in vitro transfer of tumor-suppressor p53 gene

Abstract

In this paper, a highly efficient gene transfer vector with hepatocyte-targeted function, galactosylated poly(L-succinimide)-g-polyethylenimine-g-lactobionic acid (PSI-g-PEI-g-LA), was synthesized by conjugating lactobionic acid (LA) to biodegradable cationic PSI-g-PEI polymers. The physicochemical properties of PSI-g-PEI-g-LA including buffer capability, plasmid DNA (pDNA) binding ability, cytotoxicity, zeta potential and complex size were explored. Dynamic light scattering (DLS) reveals that PSI-g-PEI-g-LA can compactly condense pDNA into nano-sized particles with a hydrodynamic diameter of 95–175 nm. PSI-g-PEI-g-LA exhibits much higher cell-biocompatibility compared to PSI-g-PEI and PEI. The hepatocyte-targeted function was demonstrated by comparative studies on three polymeric vectors including PSI-g-PEI-g-LA, PSI-g-PEI and PEI25k. The transfection efficiency was evaluated in two different cell lines including asialoglyco protein receptor (ASGP-R) bearing HepG2 cells and ASGP-R-lacking HeLa cells. The in vitro transfection tests using different reporter genes indicate that PSI-g-PEI-g-LA displays higher transfection activity in HepG2 cells due to the specific interaction between LA segments and its ASGP-R on HepG2 cells compared with PSI-g-PEI and PEI. In contrast, the three polymers exhibit similar transfection activity in HeLa cells. PSI-g-PEI-g-LA displays better serum-tolerant transfection ability. Based on these analyses, PSI-g-PEI-g-LA was used as the vector to mediate the transfection of a tumor-suppressor gene p53. By means of western blotting analysis, flow cytometry and confocal laser scanning microscopy techniques, considerably high p53 gene expression and consequently strong p53-inducing apoptosis of HepG2 cells are distinctly observed.