Synthesis of Poly(ethylene glycol)-g-Chitosan-g-Poly(ethylene imine) Co-polymer and In Vitro Study of Its Suitability as a Gene-Delivery Vector

There are two main hindrances for the application of chitosan (CS) as a gene-delivery vector: poor water solubility and low transfection efficiency. To address these problems, we modified chitosan with poly(ethylene glycol) (PEG) and poly(ethylene imine) (PEI). As previously described, PEG was grafted onto CS by a reaction between the activated PEG and CS amine. This increased the solubility of CS in neutral or basic solution. Then, monomers of PEI (i.e., aziridine) were polymerized on the CS chain of the PEG(40k)-CS(50k) co-polymer obtained in the previous step. The resulting PEG-CS-PEI (PCP) co-polymer was characterized by 1H-NMR, 13C-NMR and gel-permeation chromatography (GPC). It was found in the preliminary experiments that, amongst the series of PEG-CS-PEI co-polymers with various PEI molecular weights, PEG(40k)-CS(50k)-PEI(20k) was the most efficient one; therefore, it was chosen for the study. The PCP co-polymer showed lower cytotoxicity compared to PEI (25k) by MTT assay. Particle size and zeta potential of PCP/DNA complexes were measured by dynamic light scattering (DLS) and were shown to be predominantly affected by N/P ratios. PCP/DNA complexes at N/P ratio 20 were observed under a transmission electron microscope (TEM) as spherical particles with a mean diameter of about 50 nm. Plasmid DNA could be efficiently protected by PCP co-polymer from DNase I. The in vitro gene-transfection efficiency of PCP/pEGFP was higher than that of PEI(25k)/pEGFP and was markedly facilitated by serum.