Identification of an internalising peptide in differentiated Calu-3 cells by phage display technology; application to gene delivery to the airways.
Differentiated, human submucosal-gland carcinoma, Calu-3 cell monolayers were used as in vitro model for the airway epithelium. Internalised phage were selected from a recombinant pComb8 phage library by repetitive cycles of bio-panning on Calu-3 monolayers, protease K degradation, cell-lysis and amplification. After four selection rounds, sequence analysis of 15 enriched phage colonies revealed two clones of 73 and 27% abundancy, named IB1 and IB2, respectively. The IB2 sequence was eliminated due to a frame shift. IB1-phage internalisation at 4 degrees C was significantly lower (P < 0.05) than at 37 degrees C, suggesting involvement of a receptor-mediated endocytosis pathway. The IB1 peptide was synthesised, biotinylated and complexed to streptavidin. IB1/streptavidin-complexes co-administrated with PEI/DNA-polyplexes, enhanced polyplex transfection efficiency, dose dependently, by 6- and 4-fold in Calu-3 cells. IB1/Alexa488-streptavidin complexes were used for confocal laser-scanning microscopy (CLSM) visualisation and showed basolateral localisation in membrane associated and internalising vesicles. This study demonstrates the potential of phage display technology for identification of internalising peptide-epitopes that can enhance gene delivery efficiency in differentiated airway epithelial cells.
Florea
B I
BI
Division of Pharmaceutical Technology, Leiden/Amsterdam Center for Drug Research, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
Molenaar
T J M
TJ
Bot
I
I
Michon
I N
IN
Kuiper
J
J
Van Berkel
T J C
TJ
Junginger
H E
HE
Biessen
E A L
EA
Borchard
G
G
D001710:Biotin; D045744:Cell Line, Tumor; D004247:DNA / Q000235:genetics; D018014:Gene Transfer Techniques; D006801:Humans; D008024:Ligands; D018613:Microscopy, Confocal; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry*; D011094:Polyethyleneimine; D020545:Respiratory Mucosa / Q000166:cytology / Q000502:physiology; D014162:Transfection
D008024:Ligands; D019151:Peptide Library; D010455:Peptides; D001710:Biotin; D011094:Polyethyleneimine; D004247:DNA