D. Deshpande et al., ENHANCED CELLULAR UPTAKE OF OLIGONUCLEOTIDES BY EGF RECEPTOR-MEDIATEDENDOCYTOSIS IN A549 CELLS, Pharmaceutical research, 13(1), 1996, pp. 57-61
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 epithe
lial cancer cells. To overcome the problem of endosomal entrappment as
sociated 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 (P
L) was synthesized and complexed with 5' fluorescently-labeled oligonu
cleotide. Cellular uptake of the complex in presence or absence of the
fusogenic peptides was monitored fluorometrically. Microscopic studie
s were performed to visualize the intracellular distribution of the ol
igonucleotide. Results. Cells treated with the complex exhibited intra
cellular fluorescence intensity significantly enhanced over free oligo
nucleotide-treated controls. The uptake of the complex was shown to oc
cur via the EGF receptor-mediated pathway. Fluorescence microscopic st
udies revealed cellular internalization of the complex, however, the c
omplex appeared to be accumulated in endocytic vesicles. Exposure of t
he cells to complex in presence of HA2 peptide and polymyxin B resulte
d in a more diffused intracellular fluorescence pattern and a correspo
nding increase in fluorescence intensity. These results are consistent
with the known fusion and destabilizing activities of the peptides. C
onclusions. Since EGF receptors are overexpressed in many cancer cell
types, the EGF-PL conjugate may potentially be used as an effective an
d selective delivery system to enhance uptake of oligonucleotides into
cancer cells.