Identification of a peptide sequence traverses small intestinal mucosa by in vivo phage display

Abstract

The oral administration represents one of the most attractive and acceptable routes for the delivery of therapeutics. The oral delivery of drugs would be able to avoid the pain and discomfort associated with injections. However, protein drugs administrated by oral route have low permeability across the plasma membranes of intestinal epithelial cells. We identified a peptide sequence that traverses small intestinal mucosa by in vivo phage display method. First of all, we conducted three rounds of biopanning, and phage DNA was prepared and sequenced after selecting the phage plaques randomly. We identified 89 candidate-peptide sequences from the selected phage genome, and we aligned the peptide sequence by Clustal X. Peptide sequences with high appearing frequency were re-tested to ascertain their efficiency of trans-mucosal ability. We tested the transmucosal efficiency of candidate phages included IT1, IT2 and IT4, and IT2 phage, CQSHDTSIC-encoding phage, showed the highest efficiency to the small intestinal mucosa. Binding efficiency of IT2 phage to small intestinal mucosa was tested, and there was definite difference in titers between IT2 phage and wild-type M13 phage (control). Competition assay using synthetic IT2 peptide ligand was conducted, however, titers of IT2 phage bound in mucosa tissue showed no competition with increasing concentration of synthetic IT2 peptide ligand. We inferred that the IT2 peptide could be passed through the intestinal mucosa without a specific binding counterpart such as endocytotic receptors. When we performed ex vivo binding assay in other organs (liver, spleen, lung and kidney), IT2 phage also showed binding affinity to liver and kidney. We determined localization of IT2 phage in intestinal mucosa tissue, and IT2 phage were high localized in lamina propria in contrast with wild-type M13 phage (control). Consequently, we identified a peptide traverses small intestinal mucosa tissue, and IT2 peptide, CQSHDTSIC, would be available for delivery of macromolecule into systemic circulation through the convenient oral route.