In vivo phage display to identify M cell-targeting ligands

Purpose. The purpose of this study was to use in vivo phage display screening technology to identify novel lead peptides that target delivery to M cells and to follicle-associated epithelium (FAE) of the intestine. Methods. Phage display libraries were screened in vivo within the gastrointestinal tract of a rat model by successive screenings across four cycles of selection. Results. Following four cycles of in vivo screening, we identified 30 unique peptide sequences that bound to Peyer's patch tissue, human Caco-2, and rat IEC-6 epithelial cells. Two of the lead targeting peptides, peptides P8 (LETTCASLCYPS) and P25 (VPPHPM-TYSCQY), were shown to bind to receptors on the surface of human intestinal tissue. The L-form, D-form, retro-inverted D-form, and selective Cys-to-Ala site-directed mutants of peptides P8 and P25 were also shown to retain binding to Caco-2 cell membranes when immobilized on the surface of a model particulate. Finally, the D-peptide analog of peptide P8 (yqcsytmphppv) enhanced the delivery of polystyrene particles to M cells in vivo in a mouse model, and these particles were delivered into Peyer's patch tissue, as determined by confocal microscopy. Conclusions. In summary, we have identified novel ligands that target M cells and Peyer's patch tissue, and thus may have utility in the targeted oral delivery of vaccines and vaccine carrier systems to the mucosal immune system within the gastrointestinal tract.