Enhanced cellular uptake of oligonucleotides by EGF receptor-mediated endocytosis in A549 cells

Purpose. The goal of this study was to investigate the feasibility of utilizing epidermal growth factor (EGF) receptor-mediated endocytosis to enhance cellular uptake and targeting of oligonucleotides in epithelial cancer cells. To overcome the problem of endosomal entrappment associated with receptor-mediated delivery, we also examined the effects of two fusogenic peptides, polymyxin B and influenza HA2 peptide, for their capability to promote cytoplasmic delivery of oligonucleotides. Methods. A molecular conjugate consisting of EGF and poly-L-lysine (PL) was synthesized and complexed with 5' fluorescently-labeled oligonucleotide. Cellular uptake of the complex in presence or absence of the fusogenic peptides was monitored fluorometrically. Microscopic studies were performed to visualize the intracellular distribution of the oligonucleotide. Results. Cells treated with the complex exhibited intracellular fluorescence intensity significantly enhanced over free oligonucleotide-treated controls. The uptake of the complex was shown to occur via the EGF receptor-mediated pathway. Fluorescence microscopic studies revealed cellular internalization of the complex, however, the complex appeared to be accumulated in endocytic vesicles. Exposure of the cells to complex in presence of HA2 peptide and polymyxin B resulted in a more diffused intracellular fluorescence pattern and a corresponding increase in fluorescence intensity. These results are consistent with the known fusion and destabilizing activities of the peptides. Conclusions. Since EGF receptors are overexpressed in many cancer cell types, the EGF-PL conjugate may potentially be used as an effective and selective delivery system to enhance uptake of oligonucleotides into cancer cells.