to the N-terminus of phage coat protein pIII or pVIII and thus are present on the surface of the phage. Screening of peptide phage display libraries has been used in vivo to identify peptides able to target tumor vasculature [5], adipose tissue [6] and pancreatic islet cells [7]. In addition, it has been used to identify peptides able to facilitate internalization of intact, infectious phage into specific cell types such as synovial fibroblasts [8]. In vivo phage display also has been utilized to target atherosclerotic plaques [9], and to probe the heart vasculature for endothelial markers [10]. Although in vitro selection of a specific peptide sequence carrying phage resulted in increased targeting of cardiomyocytes by phage in vivo [11], it remains to be determined if the peptide can actually deliver ‘‘cargo’’ peptides or proteins of therapeutic potential to the heart. If such were indeed the case, it would open up new avenues of drug development, leading to delivery of therapeutics directly to the ischemic heart. In the current study, we utilized a combinatorial approach of cell culture and in vivo biopanning using an M13 phage peptide display library to identify peptide(s) with potential for cardiomy- ocyte transduction in vivo in a tissue specific manner. We have identified a peptide, termed Cardiac Targeting Peptide or CTP that is able to transduce cardiomyocytes specifically in culture and in vivo. This peptide could be used to deliver peptides, proteins or nucleic acid of therapeutic potential specifically to the heart. Methods Phage display A combined approach of in vitro and in vivo screening of a phage peptide display library for cardiomyocyte-specific transduction peptides was utilized. Cardiomyoblasts, H9C2 cells (ATCC, CRL- 1446), were incubated with 10 ul (161011 pfu) of a 12-mer M13 phage peptide display library (NEB, E8110S), for 6 hours at 37uC, 5% CO2. Cells were then washed extensively, trypsinized and lysed by a single freeze-thaw cycle. Recovered phage was tittered and amplified. The post-amplified phage was again tittered and administered intravenously by retro-orbital injection at a dose of 3.561011, to a female Balb/c mouse. The mice were pre-treated with intra-peritoneal injection of Chloroquine (20 mg/Kg) 24 hours prior to and on the day of the phage injection, in order to minimize intra-lysosomal destruction of internalized phage and increase the chances of recovering internalized phage. The phage was allowed to circulate for 24 hours, after which the mice were euthanized and heart and kidney tissues obtained. The rationale for this approach was based on the observation that after intravenous injection, native M13 phage had a half-life in blood of 4.5 hours [12]. Therefore we allowed the phage to circulate for ,5–6 half-lives to maximize the chance of uptake by cardiomy- ocytes and minimize contamination with non-specific phage circulating in the blood stream. To minimize the destruction of internalized phage in lysosomal compartments, the mice were pre- treated with Chloroquine, a drug known to increase the pH of lysosomal compartments and theoretically decrease intracellular destruction of phage. The collected tissues were digested with collagenase and phage recovered by a single freeze/thaw cycle. Recovered phage was then tittered, normalized by tissue weight and subsequently amplified for a second round of biopanning. A total of three in vivo biopanning rounds were performed followed by sequencing of 10 plaques. All animal studies were approved by the University of Pittsburgh Institutional Animal Care and Use Committee (protocol approval number 0804422A-1). Confocal microscopy The cardiac targeting peptide (CTP) was synthesized in the University of Pittsburgh Peptide Synthesis Facility in either 6- Identifying a Cardiac PTD carboxyfluorescene (CTP-6CF) labeled or biotinylated forms or conjugated to NBD (Nemo-binding domain), an 11-amino acid peptide (TALDWSWLQTE) which inhibits activation of the inducible NF-kB Kinase (IKK) by binding to the regulatory subunit (Nemo) of IKK. A cardiomyoblast cell line (H9C2; ATCC, CRL-1446), a mouse fibroblast cell line (NIH/3T3; ATCC, CRL-1658), a murine fibrosarcoma cell line (C57/BL6 derived fibrosarcoma MCA-205 cell line, kindly provided by P. H. Basse, Dept. of Immunology, University of Pittsburgh, Pittsburgh, PA), a cervical cancer cell line (HeLa; ATCC, CCL-2), and a human kidney tubular cell line (HK- 2; ATCC, CRL-2190) were incubated with increasing concentra- tions of CTP-6CF for 30 minutes. Cells were then washed 6 times with PBS, fixed with 2% Paraformaldehyde, and counter-stained with DRAQ5 (Molecular Probes, F1303), a nuclear stain. Cells were examined by confocal fluorescent microscopy. Luciferase assays H9C2 cells and MCA205 cells were transfected using Lipofectamine (Invitrogen, 11668-027) with a reporter plasmid expressing luciferase under an NF-kB promoter site as well as a Renilla control plasmid for normalization of transfection efficien- cies. Twenty-four hours later, cells were treated with increasing concentrations of CTP-NBD and 30-minutes later challenged with murine TNF-a, 10 ng/ml, for 3 hours. Cells were then washed, trypsinized, lysed and supernatant collected for Luciferase activity assay. Differences across groups were compared using an unpaired Student’s t-test. A two-tailed p-value of ,0.05 was considered statistically significant. In vivo imaging studies The initial in vivo targeting studies were performed using CTP- 6CF. Female Balb/C mice were injected retro-orbitally with CTP- 6CF (25 mg/Kg) and euthanized 15 minutes later. Heart cross- sections were stained for actin using phalloidin Alexa- 647 (Molecular Probes, A22287) and stained for laminin using a rabbit anti-laminin antibody followed by a goat anti-rabbit Cy3 (Jackson ImmunoResearch, 111-167-003) secondary antibody. Five non-overlapping sections were taken from each heart for quantification of green fluorescence (CTP-6CF) expressed as a percentage of total area (blue; stained for actin). A control peptide (CON; ARPLEHGSDKAT), picked from the original, unselected M13 phage library, CTP and 8-Lysine (8K, a homopolymer of lysine), a known cationic protein transduction domain, were synthesized in a biotinylated form. 200 mM of biotinylated CTP, CON and 8K, or equivalent volume of PBS, were incubated with 10 ul of Streptavidin-Alexa488 (2 ng/ml; Molecular Probes, S32354) for 2 hours at room temperature. Female Balb/c mice were intravenously (retro-orbitally) injected with peptides at a dose of 10 mg/Kg and then euthanized 30 minutes post-injection. Mice were also injected with biotinylated CTP conjugated to Streptavidin-Alexa488 at a dose of 10 mg/Kg and euthanized after varying circulation times to allow for tracking studies to be performed. Post-euthanasia heart, liver, lung, spleen, kidney, skeletal muscle and brain were harvested for cryosection- ing followed by confocal microscopy. Sections were cross-stained with DRAQ5, a nuclear stain. For confocal microscopy, laser intensities/gains were set using negative control (PBS injected) heart tissue to minimize background fluorescence. Once the laser intensity for FITC was set using the control hearts from PBS- injected mice, it was kept constant across all subsequent imaging. Also serial scanning was performed to prevent ‘‘bleed-through’’ from one laser wavelength to another. PLoS ONE | www.plosone.org 2 August 2010 | Volume 5 | Issue 8 | e12252