In this study, an influenza hemagglutinin-derived peptide (HAP) was covalently linked to urethane-based cationic polymers (polyurethanes; PUs) to further improve their transfection efficiency. N-succinimidyl 3-(2-pyridylditio)propionate (SPDP) was used as a coupling agent. The peptide-conjugated PUs (with 2-4 peptides conjugated per polymer chain) resulted in conjugated PU/DNA complexes with greatly increased transfection efficiency in comparison with PU/DNA complexes made with PUs without HAP. This increased transfection efficiency came from the assistance of the cationic PUs in overcoming the cell membrane barrier and the ability of the conjugated viral peptide to destabilize the endolysosome membrane before degradation of the complexes. Furthermore, the modification of the cationic PUs through the introduction of poly-L-arginine (PA) into their side chains increased their capability for condensation with DNA, resulting in the smaller-sized complexes observed and contributing to their passage of DNA delivery. In vitro transfection and cytotoxicity data showed that the PU grafted with 1 degrees amine, PA, and HAP (i.e., PU-PA-1-HAP) exhibited substantially higher transfection activity than the PUs grafted without HAP, without PA or without HAP/PA (i.e., PU-PA-1, PU-1-HAP PU-1). Most of the cells were viable (90% cell survival) after transfection with cationic-PUs and HAP-conjugated PUs below a polymer/DNA ratio of 400/1 (w/w). Moreover, the results of the present study demonstrated that the effectiveness of PU-PA-1-HAP in DNA delivery is due to the HAP conjugation, which not only helps the formed complexes to escape from endolysosomes, but also opens an alternative pathway (via clathrin as opposed to via caveolae) in transfection. Therefore, the increased capability of the complexes to gain cellular entry causes their increased transfection efficiency. In contrast, PU without HAP (i.e. PU-PA-1) can only deliver DNA into cells through the caveolae-mediated pathway. (C) 2015 Elsevier B.V. All rights reserved.