Fluid phase endocytosis contributes to transfection of DNA by PEI-25.
The understanding of internalization pathways of lipo- or polyplexes is crucial for engineering successful reagents for nonviral gene transfection. A known inhibitor of fluid phase endocytosis (FPE), rottlerin, was used to quantify the contribution of this pathway by flow cytometric and fluorescence assays. Rottlerin was shown to be a specific inhibitor of transfection by polyethylene imine (PEI-25)/DNA complexes, leading to a decrease in the amount of transfected HeLa and CHO-K1 cells and a decrease in the expression of enhanced green fluorescent protein (EGFP) reporter gene by up to 50%. Experiments using fluorescently labeled polyplexes result in a decrease of uptake by up to 40%. Additionally, rottlerin does not cross-inhibit clathrin- and caveolin-mediated endocytotic pathways of internalization, consistent with direct uptake inhibition by rottlerin. Nonspecific effects as a result of toxicity were ruled out by control experiments at concentrations where rottlerin inhibition was specific. These findings suggest that for CHO-K1 and HeLa cells, internalization of PEI-25/DNA complexes by FPE plays a decisive role in gene transfection. The establishment of an additional pathway that is independent of clathrin- and caveolin-mediated endocytotic uptake may have an impact on the design of future reagents of nonviral gene therapy and investigations of the uptake pathways and intracellular trafficking involved.
Hufnagel
Hansjörg
H
Department of Biochemistry, University of Cambridge, Cambridge, UK.
Hakim
Parvez
P
Lima
Aline
A
Hollfelder
Florian
F
D000098:Acetophenones / Q000494:pharmacology; D000818:Animals; D001578:Benzopyrans / Q000494:pharmacology; D016466:CHO Cells; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007097:Imines / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D011095:Polyethylenes / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
D000098:Acetophenones; D001578:Benzopyrans; D004791:Enzyme Inhibitors; D007097:Imines; D011095:Polyethylenes; C485184:enhanced green fluorescent protein; C505405:poly(ethylene imine); D049452:Green Fluorescent Proteins; D004247:DNA; C085746:rottlerin