Biotinylated CON or CTP peptides were labeled with neutravidin-conjugated fluospheres (Molecular Probes, F8770) with an overnight incubation at 4uC. These fluospheres are 40 nm in diameter and fluoresce at an excitation wavelength of 605 nm, allowing for in vivo bead tracking. Female Balb/c mice received intracardiac injections of fluospheres-labeled CON peptide, CTP peptide or control PBS incubated fluospheres alone. Mice were anesthetized with isoflurane delivered by the XGI-8 Gas Anesthesia System (Xenogen). Initial isoflurane concentration was set to 2.5% and was reduced to 1.5% once the animals were anesthetized. Mice were then imaged at 30, 60, 120, and 180 minutes post-injection with the IVIS Lumina (Caliper Life Sciences Inc.). All mouse studies were approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh (protocol approval number 0804422A-1). Results Identification of a cardiac specific transduction peptide by biopanning of a M13 phage display peptide library In order to identify a peptide able to preferentially transduce cardiac tissue in vivo, a screening protocol using a 12 amino acid peptide M13 peptide phage display library was utilized. The first cycle of screening of the phage peptide display library for cardiomyocytes specific transduction peptides was performed on a rat cardiomyocyte cell line, H9C2, in culture. The H9C2 cells were incubated with the M13 phage peptide display library then washed extensively and possibly internalized phage recovered following trypsinization and lysis by freeze-thaw. For each of the subsequent three rounds, the phage were injected intravenously and mice euthanized 24 hours post-injection. The hearts and kidneys were isolated, enyzmatically digested and associated phage recovered. The isolated phage were quantified and expressed as number of phage per gram of tissue weight. Following each round of in vivo screening, there was a steady increase in the ratio of phage recovered from the heart relative to the kidneys, suggesting enrichment of phage targeting the heart (Fig. 1). After the third round of in vivo screening, 10 plaques were selected and sequenced. Six of the 10 phage contained the identical nucleic acid sequence of gcgccgtggcatctttcgtcgcagtattctcgtact, corresponding to the peptide APWHLSS- QYSRT, termed cardiac targeting peptide (CTP). A BLAST search in the NCBI database revealed that this sequence shared no homology with known naturally occurring peptides or proteins. Confocal microscopy analysis demonstrates preferential targeting of cardiomyoblasts In order to examine the ability of CTP to transduce cardiomyocytes preferentially in a dose-dependent manner, fluorescent confocal microscopy was performed using the peptide coupled to 6-carboxy- flouroscene (6-CF). H9C2, 3T3, MCA-205, HeLa and HK-2 cells were incubated with increasing concentrations of CTP-6-CF, washed, fixed and counterstained with DRAQ5, a nuclear stain. As shown in figure 2, significant internalization of CTP-6-CF was observed in H9C2 cells compared to relatively minor internalization by 3T3, MCA-205 and HeLa cells at high concentrations, with no appreciable uptake by HK-2 cells. These results, performed by confocal analysis, demonstrate both the specificity of transduction by CTP as well as that the peptide is internalized, and not simply binding to the cell membrane. Inhibition of IKK/NF-kB signal transduction by a CTP-NBD fusion peptide demonstrates functional delivery to cardiomyoblasts To confirm functional transduction of H9C2 cells by CTP, the ability to deliver a peptide, NBD, able to block activation of the Identifying a Cardiac PTD Figure 1. Enrichment of cardiac specific phage by multiple rounds of biopanning. After a single, screening cycle of phage incubated with H9C2 cells, recovered phage was amplified, tittered and injected intravenously into Balb/c mice. After a circulation time of 24 hours, mice were euthanized, heart and kidney dissected, digested with collagenase II, cells lysed and recovered phage tittered. Recovered phage was amplified, re-tittered and injected for subsequent round of biopanning. Phage recovered from heart versus kidney from each cycle of in vivo phage display was normalized by gram of tissue weight and expressed as a ratio of heart to kidney. doi:10.1371/journal.pone.0012252.g001 IKK/NF-kB transduction pathway, was examined. H9C2 and MCA205 cells were transfected with a plasmid expressing the luciferase marker under the control of a NF-kB-dependent promoter. Twenty-four hours post-transfection, cells were pre- treated with the CTP-NBD fusion peptide for 30 minutes, followed by stimulation with murine TNF-a for three hours. TNF-a treatment alone caused an increase in NF-kB transcriptional activity, which was inhibited by pre-treating the H9C2 cells with increasing concentrations of CTP-NBD, in a dose-dependent fashion (Fig. 3-a). In contrast, experiments performed using MCA205 cells did not show any inhibition of TNFa mediated NF-kB activation (Fig. 3b). CTP transduces cardiac tissue in vivo To demonstrate transduction of heart tissue in vivo, CTP-6CF or the biotinylated forms of CTP, RAN and 8K peptides coupled to Streptavidin-Alexa 488 (SA488) were injected intravenously (retro- orbitally). Mice were euthanized at varying time points and heart and multiple other organs harvested for confocal microscopy. Confocal microscopy of heart tissue from mice injected with CTP- 6CF showed rapid (15 minutes) transduction of heart tissue (Fig. 4). Staining for actin and laminin showed co-localization of CTP-6CF fluorescence (green; Figure 4a) with actin (blue; Figure 4b), but not laminin (red; Figure 4c). These co-localization studies strongly suggest that CTP is internalized into cardiac cells in vivo, similar to the cell culture experiments. Quantification of transduction, using Metamorph software, revealed that approximately 15% of the total heart was being transduced by CTP following intravenous injection (Fig. 4e). Injection of the CTP-biotin-SA488 complex showed rapid, efficient and specific transduction of heart tissue at 30 minutes in a diffuse pattern compared to Streptavidin-Alexa 488 alone. There was no appreciable transduction seen of liver, skeletal muscle, PLoS ONE | www.plosone.org 3 August 2010 | Volume 5 | Issue 8 | e12252