Gene Transfer by Means of Lipo- and Polyplexes: Role of Clathrin and Caveolae-Mediated Endocytosis

Abstract

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.

Topology of octaarginines (R8) or IRQ ligand on liposomes affects the contribution of macropinocytosis- and caveolae-mediated cellular uptake

“…Evidently, the actual size of caveolae is very small (∼70 nm) [23] for accommodating an IRQ-modified carrier, which may be as large as 200 nm. The results of the present study are consistent with prior studies showing that large latex beads preferentially enter via caveolar endocytosis, although the mechanism is unclear [24,25]. However, other studies showed that particles with sizes exceeding 200 nm were internalized via macropinocytosis rather than caveolar endocytosis [26].…”

Lysosomal capturing of cytoplasmic injected nanoparticles by autophagy: An additional barrier to non viral gene delivery

“…As naked nucleic acids are negatively charged, they are complexed with cationic polymers or cationic liposomes to increase the cellular delivery. These complexes can take different endocytic pathways to enter the cells, dependent on their size and surface characteristics (11)(12)(13)(14). When nanoparticles fail to escape the endosomal compartment, eventual delivery to (and degradation in) the lysosomes will occur.…”

Lysine-functionalized nanodiamonds as gene carriers: development of stable colloidal dispersion for in vitro cellular uptake studies and siRNA delivery application

“…There are previous studies indicating that protein adsorption increases the overall cellular uptake of hard NPs like polystyrene. 71 It is speculated that the seruminduced increase in the particle size of diamoplexes can switch the most prominent clathrin-mediated endocytosis of NDs to claveoli-mediated endocytosis, 72 which is responsible for the uptake of complexes sized ~500 nm or above. 73,74 Additionally, albumin, the main component of FBS, can also facilitate cellular uptake of diamoplexes.…”