Effect of endocytosis inhibitors on gene transfection efficiency with Chitosan/Poly-L-arginine/DNA Complex in HeLa cells

Abstract

Keywords: polyarginine; gene transfer; chitosan; endocytosis; inhibitor

Objectives: To investigate the effect of endocytosis inhibitors on gene transfection efficiency of poly-L-arginie/DNA/chitosan (PLA/DNA/CS) complexes in human cervical carcinoma cells (HeLa cells).

Methods: Chitosan salt (CS; chitosan HCl, MW of 45 kDa and 87% degree of deacetylation) combined with poly-L-arginine (PLA, MW of > 70 kDa) was formulated. The ternary complexes (PLA, DNA and CS) at various weight ratios were formulated and characterized by gel electrophoresis. Their particle sizes and charges were measured using Zetasizer Nano ZS. The cell viability was evaluated by MTT assay. The effect of endocytosis inhibitors (ammonium chloride, filipin III, chlorpromazine, nocodazole, genistein and wortmannin) on enhanced green fluorescent protein (EGFP) gene delivery efficiency in human cervical carcinoma cells (HeLa cells) was then analysed. Polyethylenimine (PEI, 25 kDa) complexed with DNA (PEI/DNA) at the weight ratio of 1 were used as a positive control.

Results: The ternary complexes (PLA/DNA/CS) were completely formed at the weight ratio above 0.5. The mean size and zeta potential of these complexes were in the range of 200-260 nm and 30-35 mV, respectively. For cytotoxicity studies with MTT assay, the ternary complexes at various weight ratios showed an average cell viability of over 80%. However, the transfection efficiency of the ternary complexes was lower than that of PEI/DNA. When pre-incubated cells with each inhibitor for 30 min prior PLA/DNA/CS complexes treatment, both ammonium chloride and filipin III significantly increased gene transfection efficiency. On the contrary, other inhibitors caused the decrease in transfection efficiency. The results demonstrated the possibility of using an association of ammonium chloride or filipin III with PLA/CS as an attractive improved DNA delivery potential in vitro.

Conclusion: Ammonium chloride and filipin III might be preferable for the enhancement of gene transfer, while chlorpromazine, nocodazole, genistein and wortmannin may reduce the gene transfer with their endocytosis inhibitory properties.