In this study, we investigated different types of polyethyleneimine (PEI) and their block copolymers with N-isopropylacrylamide (NIPA) as temperature-sensitive polycationic non-viral vectors for transfection of HeLa cells in cell culture media. First carboxyl-terminated poly(NIPA) was synthesized and then copolymerized with PEIs branched or linear and with two different molecular weights (2 and 25 kDa). Addition of PEI units to the poly(NIPA) chains increased the LCST values up to body temperature. Zeta potentials of the copolymers were significantly lower than the corresponding PEI homopolymers. A green fluorescent protein expressing plasmid was used as a model. Complexes of this plasmid both with PEIs and their copolymers were formed. The zeta potentials of these complexes were between - 3.1 and + 21.3. Higher values were observed for the complexes prepared with branched and higher molecular weight PEIs. Copolymerization caused a profound decrease in the positive charges. Particle sizes of the complexes were in the range of 190-1235 nm. Using high polymer/plasmid ratios caused aggregation. The smallest complexes were obtained with the copolymer prepared with branched PEI with 25-kDa molecular weight. Copolymers were able to squeeze plasmid DNA more at the body temperature. Cytotoxicity was observed with PEIs especially with the branched higher molecular weights. Copolymerization reduced the cytotoxicity. The best in vitro DNA uptake efficiency (70%) was achieved with the complex prepared with poly(NIPA)/PE125B. However, poly(NIPA)/PE125L was the most successful vector for an effective gene expression without any significant toxicity. (C) 2004 Elsevier B.V. All rights reserved.