A new synthesis of galactose-poly(ethylene glycol)-polyethylenimine for gene delivery to hepatocytes.

A synthesis method of conjugating polyethylenimine (PEI) derivatives with terminally galactose-grafted poly(ethylene glycol) (PEG) was developed by using a bifunctional PEG derivative containing both an alpha-vinyl sulfone (VS) and an omega-N-hydroxysuccinimidyl (NHS) ester groups (VS-PEG-NHS). VS-PEG-NHS is commonly used as a crosslinker to modify proteins with ligands by first coupling amine groups to the NHS ester, followed by coupling sulfhydryl groups to the VS ester, because the reaction of VS groups with amine groups of proteins is suppressed below pH 8. However, the 1H-NMR determination of the conjugated products of branched PEI (M(w)=25 kDa) with VS-PEG-NHS at pH 6.0-8.0 indicated that the VS groups were completely bound to the amine groups of PEI as well as the NHS groups. At pH 7.0, all VS groups reacted with the primary, secondary, or tertiary amine groups of PEI in 2 h. Such different reaction behaviors would be due to a higher density of amine groups of PEI as compared with those of proteins. In contrast, the reactions with a small molecular monoamine, such as p-aminophenyl beta-D-galactopyranoside, showed that the NHS groups selectively coupled with the amine groups, and the VS groups remained completely intact. The NHS groups of VS-PEG-NHS were selectively conjugated to amine groups of p-aminophenyl beta-D-galactopyranoside (VS-PEG-Gal). Then, the VS groups of Gal-PEG unit were completely conjugated with the primary, secondary, or tertiary amine groups of PEI. Thus, the use of only two reaction steps could conveniently carry out the conjugation of terminally galactose-grafted PEG to 1 and 5 mol.% of amine functions in PEI. The gel retardation assay of the complexes between Gal-PEG-PEI and plasmid DNA indicated that these polymeric gene carriers possess the potent ability to condense plasmid DNA electrostatically as well as PEI. The transfection efficiency with 1% Gal-PEG-PEI in human hepatocyte-derived cell lines (HepG2), a model of parenchymal cells in liver (hepatocytes), was superior to that of PEI at their corresponding optimal ratios of polymer to plasmid DNA. In HepG2 cells, luciferase activity with 1% Gal-PEG-PEI at an N/P ratio of 20 was 2.1-fold greater than that of PEI at an N/P ratio of 5. In mouse fibroblasts (NIH3T3) that have no ASGP receptors, the transfection efficiency with 1% Gal-PEG-PEI drastically decreased to 1/40 of that with PEI. These data indicate that a new synthesis method can produce polyethylenimine derivatives with terminally galactose-grafted poly(ethylene glycol) for specific gene targeting to the liver.