Identification of a peptide ligand facilitating percutaneous delivery of macromolecular cargo via inner root sheath in hair follicles by phage display combined with quantitative real-time PCR as a validation tool

Abstract

To identify transdermal peptide ligands, an in vivo phage display was conducted by percutaneous administration of a phage-peptide library through the abdominal skin of rats, and the skin-traversing phages were recovered from the systemic circulation after three round of biopannings. Among total 182 peptide sequences consisting of seven amino acids which were identified from the third round phages, four peptide sequences (ST1-ST4) showing high rank in abundance were selected as transdermal peptide candidates, then their transdermal abilities were further validated simultaneously by phage quantification from blood using quantitative real-time PCR (qRT-PCR). While ST1 showed the highest appearance frequency after the third round of biopanning, the most efficient transdermal activity was observed with ST3 (AFTPAKT) by qRT-PCR. The ST3-encoding phage delivered across intact epidermis was detected in blood during test period up to 240 min. Fluorescent microscopy analysis further revealed specific localization of the ST3-encoding phage on the inner root sheath surrounding the hair follicles after its percutaneous administration. Thus, it is inferred that the hair follicles would be the transdermal route for ST3 which enables its access into systemic circulation. The overall results suggest that the ST3 peptide could be potentially utilized as an efficient transdermal carrier to deliver macromolecular therapeutics into body. It is also expected that qRT-PCR could be adopted as a reliable validation tool after phage display screening in the aspects of its accuracy, promptness and economic feasibility.