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 (VPPHPMTYSCQY), 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.