Fluid Phase Endocytosis Contributes to Transfection of DNA by PEI-25

• Published in: Molecular therapy 2009-08, Vol.17 (8), p.1411-1417
• Main Authors: Hufnagel, Hansjörg, Hakim, Parvez, Lima, Aline, Hollfelder, Florian
• Format: Article
• Language: English
• Subjects: Acetophenones - pharmacology; Animals; Benzopyrans - pharmacology; Biochemistry; Cell Survival; CHO Cells; Cricetinae; Cricetulus; DNA - chemistry; DNA - metabolism; Endocytosis - drug effects; Endocytosis - physiology; Enzyme Inhibitors - pharmacology; Experiments; Flow Cytometry; Genetic engineering; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; HeLa Cells; Humans; Imines - chemistry; Imines - metabolism; Kinases; Mice; Original; Polyethylene; Polyethylenes - chemistry; Polyethylenes - metabolism; Proteins; Ratios; Reagents; Toxicity; Transfection - methods
• ISSN: 1525-0016; 1525-0024
• DOI: 10.1038/mt.2009.121

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.