Lipoplex-mediated transfection of mammalian cells occurs through the cholesterol-dependent clathrin-mediated pathway of endocytosis

IS Zuhorn, Ruby Kalicharan, D Hoekstra*

*Bijbehorende auteur voor dit werk

OnderzoeksoutputAcademicpeer review

269 Citaten (Scopus)


Synthetic amphiphiles are widely used as a carrier system. However, to match transfection efficiencies as obtained for viral vectors, further insight is required into the properties of lipoplexes that dictate transfection efficiency, including the mechanism of delivery. Although endocytosis is often referred to as the pathway of lipoplex entry and transfection, its precise nature has been poorly defined. Here, we demonstrate that lipoplex-mediated transfection is inhibited by more than 80%, when plasma membrane cholesterol is depleted with methyl-beta-cyclodextrin. Cholesterol replenishment restores the transfection capacity. Investigation of the cellular distribution of lipoplexes after cholesterol depletion revealed an exclusive inhibition of internalization, whereas cell-association remained unaffected. These data strongly support the notion that complex internalization, rather than the direct translocation of plasmid across the plasma membrane, is a prerequisite for accomplishing effective lipoplex-mediated transfection. We demonstrate that internalized lipoplexes colocalize with transferrin in early endocytic compartments and that lipoplex internalization is inhibited in potassium-depleted cells and in cells overexpressing dominant negative Eps15 mutants. In conjunction with the notion that eaveolae-mediated internalization can be excluded, we conclude that efficient lipoplex-mediated transfection requires complex internalization via the cholesterol-dependent clathrin-mediated pathway of endocytosis.

Originele taal-2English
Pagina's (van-tot)18021-18028
Aantal pagina's8
TijdschriftThe Journal of Biological Chemistry
Nummer van het tijdschrift20
StatusPublished - 17-mei-2002

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