Robbins1* Abstract Identification of a Cardiac Specific Protein Transduction Domain by In Vivo Biopanning Using a M13 Phage Peptide Display Library in Mice Maliha Zahid1, Brett E. Phillips2, Sean M. Albers3, Nick Giannoukakis2, Simon C. Watkins3, Paul D. 1 Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America, 2 Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America, 3 Center for Biologic Imaging, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America Background: A peptide able to transduce cardiac tissue specifically, delivering cargoes to the heart, would be of significant therapeutic potential for delivery of small molecules, proteins and nucleic acids. In order to identify peptide(s) able to transduce heart tissue, biopanning was performed in cell culture and in vivo with a M13 phage peptide display library. Methods and Results: A cardiomyoblast cell line, H9C2, was incubated with a M13 phage 12 amino acid peptide display library. Internalized phage was recovered, amplified and then subjected to a total of three rounds of in vivo biopanning where infectious phage was isolated from cardiac tissue following intravenous injection. After the third round, 60% of sequenced plaques carried the peptide sequence APWHLSSQYSRT, termed cardiac targeting peptide (CTP). We demonstrate that CTP was able to transduce cardiomyocytes functionally in culture in a concentration and cell-type dependent manner. Mice injected with CTP showed significant transduction of heart tissue with minimal uptake by lung and kidney capillaries, and no uptake in liver, skeletal muscle, spleen or brain. The level of heart transduction by CTP also was greater than with a cationic transduction domain. Conclusions: Biopanning using a peptide phage display library identified a peptide able to transduce heart tissue in vivo efficiently and specifically. CTP could be used to deliver therapeutic peptides, proteins and nucleic acid specifically to the heart. Citation: Zahid M, Phillips BE, Albers SM, Giannoukakis N, Watkins SC, et al. (2010) Identification of a Cardiac Specific Protein Transduction Domain by In Vivo Biopanning Using a M13 Phage Peptide Display Library in Mice. PLoS ONE 5(8): e12252. doi:10.1371/journal.pone.0012252 Editor: Anil Kumar Tyagi, University of Delhi, India Received April 9, 2010; Accepted July 9, 2010; Published August 17, 2010 Copyright: 2010 Zahid et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by DOD grants 17-03-1-0488 and 17-03-0142 to P.D.R. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: probb@pitt.edu Introduction Ischemic heart disease and occlusive coronary artery disease continue to be the number one killer in the developed world. There are an estimated 500,000 acute ST-elevation myocardial infarctions (MI) in the US alone each year [1], and this is becoming an increasingly significant problem in the developing world [2]. Current approaches for management of an acutely occluded coronary artery leading to an MI consist of anti-platelet and anti-thrombotic strategies with intervention aimed at opening the infarct-related artery in a timely fashion. Although this approach is able to protect cardiomyocytes from necrosis, with resulting decrease in morbidity and mortality, it necessitates exposing the heart to post-ischemic reperfusion injury. Limiting this reperfusion injury and decreasing apoptosis would ultimately lead to greater myocardial salvage and prevention of development of heart failure. Numerous animal studies have identified biological agents able to ameliorate this ischemia-reperfusion injury and reduce the ultimate infarct size [3,4]. However, further development of these approaches is hindered by the inability to deliver the biologic agents to the myocardium in a tissue-specific, efficient and rapid manner. A protein transduction peptide specific for the heart would be able to deliver biologic agents in a timely fashion to the heart when given at the time of reperfusion for an infarction. Protein transduction domains (PTD) are small cationic peptides that can cross cellular membranes, and are able to transport large, biologically active molecules into mammalian cells in culture as well as in vivo. The limitation of PTDs is the non-specific transduction of all tissue types with some tissues, such as liver and kidney, taking up the PTD much more avidly than heart tissue. Thus there is a need to identify peptides able to target cardiac tissue specifically for delivery of biologics of therapeutic potential. Screening approaches using peptide phage display libraries are effective for identifying peptides able to bind to specific ligand targets as well as identifying peptides with novel properties. Phage display uses filamentous bacteriophage, such as M13, that are able to replicate in E. coli. The proteins or peptides to be displayed are fused PLoS ONE | www.plosone.org 1 August 2010 | Volume 5 | Issue 8 | e12252