Gene transfer by means of lipo- and polyplexes: role of clathrin and caveolae-mediated endocytosis.
In this paper we address the contribution of different endocytic pathways to the intracellular uptake and processing of differently sized latex particles and of plasmid DNA complexes by means of fluorescence microscopy and FACS analysis. By using a number of specific inhibitors of either clathrin-dependent or caveolae-dependent endocytosis we were able to discriminate between these two pathways. Latex particles smaller than 200 nm were internalized exclusively by clathrin-mediated endocytosis, whereas larger particles entered the cells via a caveolae-dependent pathway.The route of uptake of plasmid DNA complexes appears strongly dependent on the nature of the complexes. Thus, lipoplexes containing the cationic lipid DOTAP, were exclusively internalized by a clathrin-dependent mechanism, while polyplexes prepared from the cationic polymer polyethyleneimine (PEI) were internalized in roughly equal proportions by both pathways. Upon incubation of cells with lipoplexes containing the luciferase gene abundant luciferase expression was observed, which was effectively blocked by inhibitors of clathrin-dependent endocytosis but not by inhibitors of the caveolae-dependent uptake mechanism. By contrast, luciferase transfection of the cells with polyplexes was unaffected by inhibition of clathrin-mediated endocytosis, but was nearly completely blocked by inhibitors interfering with the caveolae pathway. The results are discussed with respect to possible differences in the mechanism by which plasmid DNA is released from lipoplexes and polyplexes into the cytosol and to the role of size in the uptake and processing of the complexes. Our data suggest that improvement of non-viral gene transfection could very much benefit from controlling particle size, which would allow targeting of particle internalization via a non-degradative pathway, involving caveolae-mediated endocytosis.
Rejman
Joanna
J
Institute for Experimental Treatment of Cystic Fibrosis, San Raffaele Scientific Institute, Via Olgettina 58, 20132, Milano, Italy. rejman.joanna@hsr.it
Conese
Massimo
M
Hoekstra
Dick
D
D021941:Caveolae / Q000502:physiology*; D002460:Cell Line; D002966:Clathrin / Q000502:physiology*; D004705:Endocytosis / Q000502:physiology*; D018014:Gene Transfer Techniques; D006801:Humans; D008081:Liposomes; D010316:Particle Size
D002966:Clathrin; D008081:Liposomes