Abstract
The polarized architecture of epithelium presents a barrier to therapeutic drug/gene carriers, which is mainly due to a limited (apical) internalization of the carrier systems. The bacterium Pseudomonas aeruginosa invades epithelial cells by inducing production of apical phosphatidylinositol-3, 4, 5-triphosphate (PIP3), which results in the recruitment of basolateral receptors to the apical membrane. Since basolateral receptors are known receptors for gene delivery vectors, apical PIP3 may improve the internalization of such vectors into epithelial cells. PIP3 and nucleic acids were complexed by the cationic polymer polyethylenimine (PEI), forming PEI/PIP3 polyplexes. PEI/PIP3 polyplexes showed enhanced internalization compared to PEI polyplexes in polarized MDCK cells, while basolateral receptors were found to redistribute and colocalize with PEI/PIP3 polyplexes at the apical membrane. Following their uptake via endocytosis, PEI/PIP3 polyplexes showed efficient endosomal escape. The effectiveness of the PIP3-containing delivery system to generate a physiological effect was demonstrated by an essentially complete knock down of GFP expression in 30% of GFP-expressing MDCK cells following anti-GFP siRNA delivery. Here, we demonstrate that polyplexes can be successfully modified to mimic epithelial entry mechanisms used by Pseudomonas aeruginosa. These findings encourage the development of pathogen-inspired drug delivery systems to improve drug/gene delivery into and across tissue barriers.
Highlights
In mammalian cells, phosphoinositides play a key role in determining cell polarity
PIP3 is localized at the basolateral plasma membrane and excluded from the apical plasma membrane, while PIP2 is enriched at the apical membrane
The polarized distribution of PIP3 was verified in polarized MDCK cells that were stably transfected with the PIP3 sensor GFP-pleckstrin homology (PH)-Akt, i.e., a GFP fusion protein of the PIP3-binding pleckstrin-homology domain of Akt[7]
Summary
Phosphoinositides play a key role in determining cell polarity. Phosphatidylinositol-4, 5-bisphosphate (PIP2) primarily localizes to the apical surface, whereas phosphatidylinositol-3, 4, 5-trisphosphate (PIP3) is found at the basolateral membrane[6]. Ternary complexes of PEI, DNA and poly (α -glutamic acid) or heparin have previously been made to reduce the overall positive charge of the complexes in order to avoid the undesired interaction with negatively charged serum proteins, which may lead to recognition and clearance by the reticuloendothelial system[16,17]. It is investigated whether PIP3-containing PEI polyplexes induce the recruitment of basolateral receptors to the apical cell surface in MDCK cells. PEI polyplexes with and without PIP3 are compared for their cellular binding and uptake, intracellular trafficking, endosomal escape, and transfection efficiency
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