FULL PAPER © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 602 wileyonlinelibrary.com De Novo Design of Skin-Penetrating Peptides for Enhanced Transdermal Delivery of Peptide Drugs Stefano Menegatti, Michael Zakrewsky, Sunny Kumar, Joshua Sanchez De Oliveira, John A. Muraski, and Samir Mitragotri* DOI: 10.1002/adhm.201500634 S. Menegatti Department of Chemical and Biomolecular Engineering North Carolina State University Raleigh, NC 27695, USA M. Zakrewsky, J. S. De Oliveira, Prof. S. Mitragotri Center for Bioengineering Department of Chemical Engineering University of California Santa Barbara, CA 93106, USA E-mail: samir@engineering.ucsb.edu S. Kumar Nitto Denko Oceanside, CA 92058, USA J. Muraski Convoy Therapeutics Oro Valley, AZ 85704, USA Skin-penetrating peptides (SPPs) are attracting increasing attention as a non- invasive strategy for transdermal delivery of therapeutics. The identification of SPP sequences, however, currently performed by experimental screening of peptide libraries, is very laborious. Recent studies have shown that, to be effec- tive enhancers, SPPs must possess affinity for both skin keratin and the drug of interest. We therefore developed a computational process for generating and screening virtual libraries of disulfide-cyclic peptides against keratin and cyclo- sporine A (CsA) to identify SPPs capable of enhancing transdermal CsA delivery. The selected sequences were experimentally tested and found to bind both CsA and keratin, as determined by mass spectrometry and affinity chromatography, and enhance transdermal permeation of CsA. Four heptameric sequences that emerged as leading candidates (ACSATLQHSCG, ACSLTVNWNCG, ACTSTGRNACG, and ACSASTNHNCG) were tested and yielded CsA per- meation on par with previously identified SPP SPACE TM . An octameric peptide (ACNAHQARSTCG) yielded significantly higher delivery of CsA compared to heptameric SPPs. The safety profile of the selected sequences was also vali- dated by incubation with skin keratinocytes. This method thus represents an effective procedure for the de novo design of skin-penetrating peptides for the delivery of desired therapeutic or cosmetic agents. reduces both risk of infections and dis- comfort in patients. [1] Drug permeation into and across skin, however, still poses serious challenges, mainly related to the natural imperviousness of this tissue. [2] Among the various skin layers, the stratum corneum (SC) is particularly important in protecting underlying organs from foreign agents, such as pathogens and toxins. [3] SC comprises keratin-rich cells embedded in multiple lipid bilayers. [4] The hydropho- bicity and the densely packed structure of this layer limit the permeation of even small therapeutically active ingredients. [5] To increase drug permeation across the tissue, chemical permeation enhancers (CPEs) have been proposed, including small synthetic chemicals (azone deriva- tives, fatty acids, alcohols, esters, sulfox- ides, pyrrolidones, glycols, surfactants, and terpenes) and peptides. [6] We have discovered and extensively characterized a number of small (1000–1500 Da) skin penetrating peptides for the transdermal delivery of highly relevant drug models, such as siRNA, hyaluronic acid, and cyclo- sporine A (CsA). [7] In a recent study, we described several funda- mental aspects underlying the mechanism of skin permeation enhancement by peptides. [8] Our findings, obtained based on studies of five sequences (skin penetrating and cell entering (SPACE TM ), TD-1, poly-R, dermis localizing peptide (DLP), and LP-12) with different physicochemical properties, strongly indi- cate that skin penetrating peptides’ (SPPs) action occurs mainly through the keratin domain of the skin. This is supported by experimental observations of the structural alteration by SPPs of the proteins of the stratum corneum, as well as by affinity binding studies that indicate an affinity between SPPs and ker- atin, the most abundant skin protein. Further, the studies evi- denced a strong correlation between the strength of the ternary complex formed between keratin, a model therapeutic CsA and SPP and the ability of CsA to permeate into the skin. We there- fore contend that the noncovalent binding (affinity) of SPPs to keratin enables their migration across the skin through a pro- gressively penetrating protein binding mechanism, while the affinity for CsA enables the concurrent transport of the drug along with the SPPs. These observations suggest a simple strategy for the de novo design of SPPs for transdermal delivery of a desired drug, 1. Introduction Transdermal drug delivery is a growing area within the field of drug delivery, as it offers numerous advantages over traditional techniques, especially due to the elimination of needles, which Adv. Healthcare Mater. 2016, 5, 602–609 www.advhealthmat.de www.MaterialsViews.com