Cell-penetrating peptides (CPPs), also known as protein transduction domains, were first identified 25 years ago. They are small, ~6-30 amino acid long, synthetic, or naturally occurring peptides, able to carry a variety of cargoes across the cellular membranes in an intact, functional form. These cargoes can range from other small peptides, full-length proteins, nucleic acids including RNA and DNA, nanoparticles, and viral particles as well as radioisotopes and other fluorescent probes for imaging purposes. However, this ability to enter all cell types indiscriminately, and even cross the blood-brain barrier, hinders their development into viable vectors. Hence, researchers have adopted various strategies ranging from pH activatable cargoes to using phage display to identify tissue-specific CPPs. Use of this phage display strategy has led to an ever-expanding number of tissue-specific CPPs. Using phage display, we identified a 12-amino acid, non-naturally occurring peptide that targets the heart with peak uptake at 15 min after a peripheral intravenous injection, that we termed Cardiac Targeting Peptide (CTP). In this chapter, we use CTP as an example to describe techniques for validation of cell-specific transduction as well as provide details on a technology to identify binding partner(s) for these ever-increasing plethora of tissue-specific peptides. Given the myriad cargoes CTP can deliver, as well as rapid uptake after an intravenous injection, it can be applied to deliver radioisotopes, miRNA, siRNA, peptides, and proteins of therapeutic potential for acute cardiac conditions like myocardial infarction, where the window of opportunity for salvaging at-risk myocardium is limited to 6 hrs.
Keywords: Cardiac targeting peptide; Cell-penetrating peptides; Phage display; Protein transduction domains; TriCEPS.