Development of polymeric gene delivery carriers: PEGylated copolymers of l-lysine and l-phenylalanine

Block copolymers consisting of poly(ethylene glycol) (PEG) and poly(amino acid)-based random copolymers were successfully synthesized by the ring opening polymerization of the N-carboxy anhydrides (NCA) of l-lysine and l-phenylalanine. The synthesized copolymers had a molecular weight of around 30,000 and contained l-lysine and l-phenylalanine residues with molar ratios of 10/0, 9/1, 8/2, 7/3 and 6/4. The complex formation of the copolymer and pCMV-luc plasmid DNA was confirmed by the gel retardation assay and zeta potential measurement. Complete neutralization was achieved at an N/P ratio of more than 1.0 and the size of the complex was determined to be around 150 nm by dynamic light scattering. The cytotoxicity and transfection efficiency were tested on the HEK 293T cell line. The synthesized copolymers displayed negligible cytotoxicity, resulting in a cell viability of more than 95%, while those of the poly(l-lysine) (PLL) and poly(ethylenimine) (PEI) homopolymer were around 65 and 55%, respectively, under comparable conditions. The introduction of the hydrophilic PEG is believed to reduce the toxicity of the copolymer, due to its enhanced biocompatibility, and to impart improved stability to the complex under physiological conditions. The transfection efficiency at the optimized charge ratio of 7 was dramatically improved as the molar content of the l-phenylalanine residues in the copolymers increased and reached a maximum value at an l-phenylalanine content of 30 mol%. The transfection efficiency of the PEGK7/plasmid DNA complex was around 80 times higher than that of PLL, despite the presence of neutral PEG as a block segment.