Molecular Architecture Governs Cytotoxicity and Gene Transfection Efficacy of Polyethylenimine Based Nanoplexes in Mammalian Cell Lines

Polyethylenimine (pEI) is a potent cationic transfection agent that is commonly used in conjunction with other inorganic and organometallics formulations. However, the toxicity that is intrinsic to pEI constitutes a major obstacle for both in vitro and in vivo applications. In this study, the roles of pEI molecular architectures and effects of nitrogen/phosphate (N/P) molar ratio on the cytotoxic potential and in vitro transfection performance were elucidated utilizing linear (L-pEI) and branched (B-pEI) 25 kDa pEI. Both L-pEI and B-pEI were able to self-assemble with the plasmid DNA to form positively charged nanoplexes having an average hydrodynamic diameter of 100–150 nm. In vitro cytotoxicity and transfection performance were assessed with three model mammalian cell lines namely, L929, COS-7 and HeLa cells. Transfection profiles over a wide range of N/P ratio exemplify a similar trend across three different cell lines, with B-pEI nanoplexes consistently showing maximum transgene expression of enhanced green fluorescent protein at an earlier N/P ratio compared to L-pEI nanoplexes. However, high transfection activity was maintained over a wider N/P ratio for L-pEI nanoplexes. While the absolute size of the nanoplexes exhibited no discernible effect on the transfection performance, cytotoxicity of free un-bound B-pEI was identified as the dictating factor responsible for the observed global cellular response. Collectively, our findings provided critical insights into the role of pEI molecular architecture in cellular transfection and are expected to lay the foundation for the rational design and development of advanced pEI-based inorganic and organometallic gene delivery systems.