Binding and uptake of cationic lipid : pDNA complexes by polarized airway epithelial cells

To better understand the barriers associated with cationic lipid-mediated g ene transfer to polarized epithelial cells, Fischer rat thyroid (FRT) cells and polarized normal human bronchial epithelial (NHBE) cells grown on filt er supports at an air-liquid interface were used to study the binding and u ptake of cationic lipid:plasmid DNA (pDNA) complexes. The efficiencies of b inding and uptake of cationic lipid:pDNA complexes by these cell systems we re monitored using fluorescence microscopy of fluorescently tagged lipid or pDNA probes. Fluorescent probe bound to the cell surface was differentiate d from internalized probe by adding trypan blue, which quenched the fluores cence of bound but not internalized probes. For proliferating cells, bindin g and internalization of the cationic Lipid:pDNA complexes were determined to be efficient. In contrast, little binding or internalization of the comp lexes was observed using polarized epithelial cells. However, after aspirat ing a small area of cells from the filter support, virtually all of the cel ls adjoining this newly formed edge bound and internalized the cationic lip id:pDNA complexes. To determine if their uptake in edge cells was related t o the ability of the complexes to access the basolateral membranes of these cells, the binding and uptake of complexes was monitored in polarized NHBE cells that had been pretreated with EGTA or Ca2+-free media, strategies kn own to disrupt tight junctions. Cells treated in this manner bound and inte rnalized cationic lipid:pDNA complexes efficiently and also expressed signi ficant levels of transgene product. Control cells with intact tight junctio ns neither bound complexes nor expressed significant transgene product. The se data confirm and extend earlier observations that the polarized apical m embranes of airway epithelial cells are resistant to transfection by lipid: pDNA complexes. Further, in contrast to previous studies that have shown th e entry step of complexes is not an important barrier for COS and HeLa cell s, binding and entry of complexes in polarized NHBE cells appear to be rate limiting. These findings suggest that strategies designed to open the tigh t junctions of polarized epithelial cells mag improve gene delivery to thes e cells for diseases such as cystic fibrosis (CF).