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*2HCl (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 N/P 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 intra-cross-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.
Gosselin
M A
MA
College of Pharmacy, Division of Pharmaceutics and Pharmaceutical Chemistry, The Ohio State University, Columbus, Ohio 43210, USA.
Guo
W
W
Lee
R J
RJ
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry; D018014:Gene Transfer Techniques; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008970:Molecular Weight; D010957:Plasmids / Q000737:chemistry; D011094:Polyethyleneimine / Q000737:chemistry*; D014162:Transfection / Q000379:methods
D003432:Cross-Linking Reagents; D011094:Polyethyleneimine; D004247:DNA; D008156:Luciferases