Apoptosis induced by the polyethylenimine/DNA complex in polymeric gene delivery

Abstract

Polymeric gene carriers have been developed as alternatives for viral vectors because of their excellent characteristics such as no immunogenecity, moderate transfection efficiency, no size-limit, convenience of handling, low cost, and reproducibility. High molecular weight polyethylenimine (25,000 Da, PEI) has been extensively studied for the development of efficient gene carrier. The previous report showed that there are at least two types of cytotoxicity in PEI-mediated cell transfection, which are immediate and delayed toxicities. Delayed toxicity is related to the duration of gene expression after gene delivery, since it induces continuous loss of the transfected cells. Most PEI-mediated gene delivery protocols use net cationic complexes with an excess of PEI to maintain equilibrium between the complexed and dissociated form in solution. Until now, it is not clear which is related to the delayed toxicity, the complexed or dissociated form of PEI in the complex solution. In this study, human embryonic kidney 293 cells were incubated with naked DNA, free PEI, and PEI/DNA complex for 4 hrs. Then, the cells were analyzed at 6, 24, 48, and 96 hrs after the incubation by Tunnel, MTT and luciferase assays. At 6 hrs after the incubation, apoptosis was detected in the cells incubated with the PEI/DNA complex or free PEI solution. From 24 hrs after the incubation, apoptosis was observed in the cells incubated with the PEI/DNA complex solution, but not in the cells incubated with the naked DNA or free PEI solution. This suggests that immediate toxicity may be induced by free PEI and that the delayed toxicity is mediated by PEI/DNA complex, not free PEI in the complex solution.