PEG grafting of polyethylenimine (PEI) exerts different effects on DNA transfection and siRNA-induced gene targeting efficacy

Background: Gene targeting by RNA interference (RNAi) is mediated through small interfering RNA (siRNA), which, as plasmid DNA molecules, can be delivered into cells by polyethylenimines (PEI). Grafting with poly(ethylene glycol) has been introduced previously to improve PEI biocompatibility; however, data on the effects of PEGylation have been somewhat contradictory and various PEI(-PEG) need to be evaluated independently for DNA transfection and siRNA gene targeting efficacies. Aim: We directly compare plasmid DNA transfection and siRNA-mediated gene targeting efficacies, employing a larger set of polyethylenimine-graft-poly(ethylene glycol) (PEI-g, PEG; PEI(-PEG)) with different molecular weights and degrees of PEG substitution. Method: We performed tissue culture-based bioassays on DNA transfection and siRNA-mediated targeting efficacies as well as on toxicity and cellular nucleic acid uptake, and, using sensitive assays based on radioactive labelling, physicochemically characterize the complexes regarding the degree of nucleic acid complexation and complex stabilities under various conditions. Results: In contrast to the DNA transfection efficacy, siRNA-mediated gene targeting is much less dependent on the PEGylation of PEI or on the NIP ( = PEI nitrogen/nucleic acid phosphate) ratio. A more detailed analysis reveals that, in order to define optimal NIP ratios for DNA transfection, complex toxicities and nucleic acid uptake are the most critical parameters. In contrast, at optimal NIP ratios, complex stabilities and complexation efficacies determine PEI(-PEG)/DNA transfection efficacies and the major differences between various PEI(-PEG) are observed. All these parameters are less critical for PEI(-PEG)/siRNA gene targeting efficacy. Thus, our data lead to the distinction between three PEI(-PEG) groups, which relies on the differences in transfection rather than gene targeting efficacies, and which is correlated with the molecular weights and degrees of PEG substitution. Conclusion: In contrast to PEI(-PEG)/DNA complexes, a broader panel of PEI-PEG are capable of siRNA-mediated gene targeting. Thus, PEG grafting of PEI requires a separate evaluation of siRNA and DNA complexes, which expands the portfolio of available PEI(-PEG) for the preparation of non-toxic, biocompatible siRNA delivery reagents for the induction of RNAi.