Identification of an internalising peptide in differentiated Calu-3 cells by phage display technology; application to gene delivery to the airways
| Autor: | Hans E. Junginger, Tom J.M. Molenaar, Gerrit Borchard, Bogdan I. Florea, T. J. C. Van Berkel, Ingrid N. Michon, Ilze Bot, E.A.L. Biessen, Johan Kuiper |
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| Rok vydání: | 2004 |
| Předmět: |
Streptavidin
Phage display Pharmaceutical Science Biotin Respiratory Mucosa Gene delivery Biology Ligands Transfection law.invention Bacteriophage chemistry.chemical_compound law Peptide Library Cell Line Tumor Humans Polyethyleneimine Microscopy Confocal Gene Transfer Techniques DNA biology.organism_classification Molecular biology In vitro chemistry Biotinylation Recombinant DNA Peptides |
| Zdroj: | Journal of drug targeting. 11(7) |
| ISSN: | 1061-186X |
| Popis: | 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 (P0.05) 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. |
| Databáze: | OpenAIRE |
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