Endocytosis and intracellular processing of bioreducible polyamidoamine - gene complexes in cells of the retinal pigment epithelium.

摘要

In this work, we studied for the first time in vitro endocytosis and intracellular processing of gene complexes composed of a bioreducible polyamidoamine CBA ABOL and plasmid DNA, in cells of the retinal pigment epithelium (RPE), the latter being an interesting target for ocular gene therapy. These polymer based gene complexes or 'polyplexes' are non-toxic and very effective in transfecting ARPE-19 cells. We found that cationic CBA ABOL DNA polyplexes attach to cell surface proteoglycans and get subsequently internalized via a phagocytosis-like mechanism, as well as Flotillin dependent endocytosis. While a fraction of these complexes is able to escape out of the endosomes, it seems that the largest fraction of gene complexes is recognized by the innate defence mechanism, resembling autophagy, shielding the polyplexes for further endosomal escape and directing them to the lysosomes for degradation. By getting a detailed insight into the uptake and subsequent intracellular processing of these polyplexes, we want to provide the knowledge necessary for a further rational optimization of the design and composition of this polymer carrier.