Chitosan was partially converted to its chlorohydrin derivative by the reaction with epichlohydrin, which was subsequently reacted with varying amounts of lPEI(2.5kD) to obtain a series of chitosan-lPEI(2.5kD) copolymers (CP). These copolymers were then characterized and evaluated in terms of transfection efficiency (in vitro and in vivo), cell viability, DNA release and buffering capacity. The CP-4 copolymer (the best among the CP series) showed enhanced transfection (~ 2 – 24 folds) in comparison with chitosan, lPEI(2.5kD), bPEI(25kD) and Lipofectamine in HEK293, HeLa and CHO cells. The buffering capacity (in the pH range of 3 – 7.5), as shown by confocal microscopy, and DNA-release capability of the CP copolymers, was found to be significantly enhanced over chitosan. Intravenous administration of CP-4/DNA polyplex in mice followed by the reporter gene analysis showed the highest gene expression in spleen. Collectively, these results demonstrate the potential of CP-4 copolymer as a safe and efficient nonviral vector. From the Clinical EditorChitosan –PEI (2.5kD) copolymers (CP) were characterized and their transfection efficiency, DNA release and buffering capacity were studied. The CP-4 copolymer significantly enhanced buffering capacity and provided the highest gene expression levels. The method may be used to enhance DNA transfection.