Cationic polymers have received much attention as promising nonviral vectors for gene transfer. However, development of polymers with low cell toxicities and high transfection efficiencies continue to be a significant problem and a major hurdle to their success. Poly-D/L aspartate-diethylenetriamine poly(D/L Asp-DET) polymers were synthesized and evaluated as nonviral gene delivery agents. Poly(D/L Asp-DET) polymers display endosome buffering capacity. The polymers condense plasmid DNA above N:P ratios of 1 and form polyplex particles of similar to 50100 nm, with zeta potentials between neutral and +40 mV. Transmission electron microscopy shows the polyplexes to be uniform in size and shape. Polyplexes maintain the structural integrity of DNA following incubation in nucleases and also show high transfection efficiencies with minimal toxicity in both HCT-116 and PC-3 cell culture. However, it is found that these poly(D/L Asp-DET)/DNA polyplexes immediately aggregate in salt and serum conditions, making them unsuitable for use in vivo. Therefore, the polyplexes were further modified by covalent addition of polyethylene glycol (PEG). Introduction of this second step produces PEG-polyplexes of uniform size (below 100 nm), with neutral zeta potentials that are also stable in both salt and serum conditions. These results suggest poly(D/L Asp-DET) cationic polymers as potentially safe and efficient nonviral gene delivery agents. (C) 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012