Efficient gene transfer using reversibly cross-linked low molecular weight polyethylenimine

Polyethylenimine (PEI) is a polycation with potential application as a nonviral vector for gene delivery. Here we show that after conjugation with homobifunctional amine reactive reducible cross-linking reagents, low molecular weight polyethylenimine efficiently mediates in vitro gene delivery to Chinese hamster ovary (CHO) cells. Two cross-linking reagents, dithiobis(succinimidylpropionate) (DSP) and dimethyl-3,3'-dithiobispropionimidate-2HCI (DTBP), were utilized based on their reactivity and chemical properties. Both reagents react with primary amines to form reducible cross-links; however, unlike DSP, the DTBP cross-linker maintains net polymer charge through amidine bond formation. PEI with a reported weight-average molecular weight (M-w) of 800 Da was reacted with either DSP or DTBP at PEI primary amine:cross-link reactive group ratios of 1:1 and 2:1. The transfection efficiencies of the resulting cross-linked products were evaluated in CHO cells using a luciferase reporter gene under a cytomegalovirus (CMV) promoter. Our results show that cross-linked polymers mediate variable levels of transfection depending on the cross-linking reagent, the extent of conjugation, and the NIP ratio. In general, we found conjugate size to be proportional to gene transfer efficiency. Using gel retardation analysis, we also evaluate the capacity of the cross-linked polymers to condense plasmid DNA before and after reduction with 45 mM dithiothreitol (DTT). DTT mediated reduction of intracross-link disulfide bonds and inhibited condensation of DNA by conjugates cross-linked with DSP at a ratio of 1:1, but had little effect on the remaining polymers. Analogous intracellular reduction of transfection complexes by reduced glutathione could facilitate uncoupling of PEI from DNA to enhance gene expression.