Identifying a Cardiac PTD Figure 2. CTP specifically transduces cardiomyocytes in culture. Confocal micrographs of H9C2, 3T3, MCA-205, HeLa and HK-2 cells incubated with increasing concentrations (50 uM, 100 uM and 200 uM) of CTP-6CF for 30 minutes, followed by multiple washings and cross-staining with a nuclear stain, DRAQ5 (206) (a). A higher (406) confocal micrograph of H9C2 cells is shown demonstrating pattern of transduction (b); CTP-6CF - green, Nuclei – blue. Scale bars represent 100 uM. doi:10.1371/journal.pone.0012252.g002 brain (Fig. 5) or spleen (data not shown). The only other organs with uptake were a small percentage of lung capillaries as well as limited transduction of endothelial cells of the glomerular capillaries in the cortex of the kidneys (Fig. 5). These results demonstrate the specificity of CTP transduction in vivo. To examine the biodistribution of CTP-biotin-SA488 over time, mice were euthanized at different time points following intrave- nous injection. Even with the large CTP-biotin-SA488 complex, efficient transduction of the heart was seen at 15 minutes, mainly confined to the sub-epicardial region of the heart. At 30 minutes this became more diffuse and by 120 minutes there was almost no fluorescence seen in the heart (Fig. 6). Over these three time points, the fluorescence gradually increased in the kidney glomerular capillaries (Fig. 6, center column), suggesting that this might be the mode of excretion of this peptide or at least the fluorescence after peptide breakdown. Figure 3. CTP functionally delivers the IKK/NF-kB inhibitory peptide NBD to cardiomyocytes. H9C2 cells (a) and MCA205 (b) cells, were co-transfected with a NF-kB dependent luciferase reporter plasmid and a control renilla-expression plasmid. Twenty-four hours post-transfection, cells were stimulated with TNF-a (10 ng/ml) or pre-treated with increasing concentrations of CTP-NBD (50 uM, 100 uM, 200 uM and 400 uM) for 30 minutes followed by TNF-a stimulation for three hours. Cells were then washed, lysed and luciferase activity measured and normalized to renilla activity. (N = 4 in each group; error bars represent one standard deviation). doi:10.1371/journal.pone.0012252.g003 PLoS ONE | www.plosone.org 5 August 2010 | Volume 5 | Issue 8 | e12252