Structure-function relationships of gene delivery vectors in a limited polycation library

A library of 13 polylysine-graft-imidazoleacetic acid conjugates was synthesized to examine the collective effects of polymer molecular weight, side chain substitution, and DNA:polymer ratio on cytotoxicity, transfection efficiency, and polycation-DNA interaction. In general, the relationships between the physicochemical characteristics and the gene transfer capabilities of these polycations appear nonlinear. The in vitro cytotoxicity of these polymers decreased, while total protein expression increased, with decreasing molecular weight and increasing imidazole content. Flow cytometry experiments indicated, however, that an increase in marker gene expression does not always correlate with the total number of cells transfected, even when similar polymer structures are used for transfection. The maximum level of luciferase gene expression was mediated by transfection with a low molecular weight, high imidazole content (9400 M, 95 mol% imidazole) polymer. The extent of DNA condensation, as determined by ethidium bromide fluorescence quenching, also decreased with decreasing polymer molecular weight and increasing imidazole content. Relative binding affinity between DNA and the polycations, measured via competitive binding in the presence of a synthetic polyanion, decreased with decreasing polymer molecular weight; however, the relative affinity also appeared to increase with increasing imidazole, suggesting that electrostatic contributions are not solely responsible for DNA-polycation binding interactions. This limited library and corresponding structure/function analysis forms the foundation upon which larger, more comprehensive polycationic libraries can be designed and evaluated to further understand how polycation transfection reagents function. (c) 2004 Elsevier B.V. All rights reserved.