Identifying a Cardiac PTD Figure 8. CTP specifically transduces cardiac tissue in contrast to a cationic transduction domain. Confocal analysis of heart, kidney, liver and spleen (a–d) 30 minutes following intravenous injection of CTP+SA488, 8K+SA488 or RAN+SA488 (10 mg/Kg body weight) conjugates. N = 2 in each group; scale bars represent 100 uM. doi:10.1371/journal.pone.0012252.g008 atherosclerotic plaques [9] as well as heart endothelial cells [10]. This approach also has been used in cell culture with adherent primary cardiomyocytes to isolate a 20-mer peptide with a homology to tenascin-X [11], an extracellular matrix protein. The phage displaying this peptide was found to be associated with cardiomyocytes isolated from mice treated with it in vivo. However, although it was preferentially associated with cardiomyocytes, it could still be isolated from lung tissue. Furthermore, it was unclear whether this 20-mer peptide was able to function as a cardiac transduction domain and transduce heart tissue in vivo independent of the phage carrying it. In our screening approach, we combined a combination of cell culture and in vivo screening to identify a peptide able to be internalized into cardiac tissue. The first cycle was performed on cardiomyocytes as a screening approach to limit the population of non-specific phage from the initial phage library. All subsequent cycles were in vivo with intravenous injection in mice followed by a prolonged circulation time of 24 hours. Using this approach we identified a peptide that is able to deliver fluorescently labeled Streptavidin, a ,60 kDa complex, to cardiac cells in vivo without transduction of liver, spleen, skeletal muscle or brain, with minimal uptake by lung and glomerular capillaries. A BLAST search in the NCBI data base did not reveal homology to any known, naturally occurring proteins. Interestingly two separate groups of investigators, using an in vitro screening of a phage display library approach, have identified the exact same sequence as CTP, and shown it to have high affinity for binding to apatite-based, bone-like minerals [14] and two specific sulfated carbohydrates [15]. However, it is unclear how the ability of CTP to interact with these cellular components PLoS ONE | www.plosone.org 10 August 2010 | Volume 5 | Issue 8 | e12252