Galactose-PEI-DNA complexes for targeted gene delivery: degree of substitution affects complex size and transfection efficiency

Complexes of galactosylated polyethylenimines (gal-PEI) with DNA have been proposed for gene delivery to hepatocytes. We synthesized gal-PEI with a broad range of degrees of substitution (DS) ranging from 3.5 to 31% of all PEI amino groups by reductive amination to determine physico-chemical and biological properties with respect to the DS. Gel retardation assay for herring testes DNA-polymer polyplexes showed that increasing DS compromised DNA complexation and especially condensation. Using photon correlation spectroscopy, gal-PEI complexes formed with plasmid DNA were found to increase in size with increasing galactosylation (156+/-7 nm for 0%, 486+/-76 nm for 3.5%, 467+/-86 nm for 9.7% and 652+/-123 nm for 31% DS). Zeta potentials decreased in inverse proportion to DS (0%: 30+/-3 mV, 3.5%: 22+/-2 mV, 9.7%: 15+/-1 mV, 31%: -26+/-3.5 mV) suggesting a shielding effect by carbohydrate coupling. Cytotoxicity of gal-PEI was found to decrease with increasing galactosylation (MTT and LDH assay), no toxicity was detectable for polyplexes with plasmid DNA (LDH assay). The transfection efficiency of a reporter gene complexed with gal-PEI in a hepatocyte cell culture model (HepG2) expressing the asialoglycoprotein receptor was slightly but not significantly increased for galactosylated PEIs, at a nitrogen to phosphate (N/P) ratio of 2 and strongly reduced at higher N/P ratios, compatible with only a minor targeting efficiency, strongly affected by DS. In NIH-3T3 mouse fibroblasts, increasing the DS led to a decreased transfection efficiency for all N/P ratios. Our study highlights the necessity of careful optimization of polyplex composition for active gene targeting. (C) 2002 Elsevier Science B.V. All rights reserved.