(478a) Efficient Polyethylenimine-Mediated Gene Delivery Proceeds Via Caveolae -Mediated Uptake In HeLa Cells | AIChE

(478a) Efficient Polyethylenimine-Mediated Gene Delivery Proceeds Via Caveolae -Mediated Uptake In HeLa Cells

Authors 

Pack, D. W. - Presenter, University of Illinois Urbana Champaign
Gabrielson, N. - Presenter, University of Illinois


Cancer gene therapy will require cell-specific targeting to ensure that diseased cells are treated while healthy cells are untouched. To direct genes to particular cells, peptides, proteins and small molecule ligands have been attached to the surface of gene delivery complexes. Although such targeting methods have met with success in generating particles that are capable of specific cellular interactions, little attention has been given to the effect such ligands can have on subsequent intracellular processing. For example, transferrin and folic acid are common ligands used for drug and gene delivery targeting due to the overexpression of their respective receptors on many cancer cells. Transferrin is internalized by clathrin-coated pits while folic acid is internalized by caveolae ? flask-like invaginations of the cell membrane. Endocytosis via clathrin-coated pits follows the ?traditional? model of intracellular processing ? trafficking through endosomes to lysosomes with concomitant acidification of the endocytic vesicles. Caveolae are believed to be directed towards neutral pH vesicles termed caveosomes. The difference in pH microenvironment may affect cytoplasmic release of the vectors, especially for so-called ?proton-sponge? materials.

Folic acid and human transferrin were conjugated to 25-kDa branched polyethylenimine (PEI) to yield folic acid-targeted PEI (PEI-Fol) and transferrin-targeted PEI (PEI-Tf). Caveolae inhibitors (genistein, methyl-β-cyclodextrin) inhibited uptake of PEI-Fol in HeLa cells. Likewise, clathrin inhibitors (chlorpromazine, amantadine) inhibited PEI-Tf uptake. Thus, it appears that the folic acid- and transferrin-targeted complexes are predominantly endocytosed by caveolae and clathrin, respectively. When folic acid- and transferrin-targeted polyplexes were used to transfect cells in the presence of the same drugs, inhibitors of caveolae-mediated endocytosis drastically reduced gene expression while inhibitors of clathrin-mediated endocytosis had no effect. This suggests that successful gene delivery proceeds via a caveolae-mediated process, which avoids acidic environments and lysosomes. In addition, clathrin inhibition by chlorpromazine increased gene delivery by unmodified PEI by 1.8-fold and transferrin-targeted PEI by 2.7-fold. These results suggest that the caveolin-specific pathway, as opposed to the clathrin-pathway, is effective for gene delivery in HeLa cells and suggest that the conventional understanding of PEI's proton-sponge mechanism may be flawed. Most importantly, we demonstrate that the uptake mechanism and subsequent endocytic processing are important design parameters for gene delivery materials.