Background: Polyethylenimine (PEI) polyplexes mediate efficient gene transfer only at high +/- charge ratios at which free noncomplexed PEI is present. The excess of PEI gives polyplexes a positive surface charge that plays a role in polyplex binding on the cell membrane. Although positively charged PEI polyplexes are known to interact with anionic cell-surface glycosaminoglycans (GAGs), the exact role of free PEI in such interactions is unclear.
Methods: Chinese hamster ovary wild-type cells and mutants lacking cell-surface GAGs were transfected with marker genes using PEI polyplexes with and without free PEI. The total amount of cell-associated plasmid DNA (pDNA) delivered by polyplexes was determined by quantitative real-time PCR and transgene expression was determined using β-galactosidase and luciferase assays.
Results: Transfection activity of polyplexes without free PEI in cells expressing cell-surface GAGs was low even though pDNA was delivered to cells. In the absence of cell-surface GAGs, polyplexes without free PEI had high transfection efficacy. This indicates that the cell-surface GAGs inhibit transfection by purified polyplexes. PEI polyplexes with free carrier mediated transfection in both normal and GAG-deficient cells because free PEI overcomes the inhibitory effect of cell-surface GAGs on transfection. The intracellular elimination of pDNA was faster in the presence of GAGs and, despite improved transfection, free PEI reduced pDNA association with the cells.
Conclusions: Free PEI is essential for minimizing the undesirable binding of polyplexes to cell-surface GAGs that have a negative impact on transfection. The same mechanism may be important in transfections with other polyplexes that require high charge ratios for transfection.
Copyright © 2011 John Wiley & Sons, Ltd.