The pH (low) insertion peptide (pHLIP) family enables targeting of cells in tissues with low extracellular pH. Here, we show that ischemic myocardium is targeted, potentially opening a new route to diagnosis and therapy. The experiments were performed using two murine ischemia models: regional ischemia induced by coronary artery occlusion and global low-flow ischemia in isolated hearts. In both models, pH-sensitive pHLIPs [wild type (WT) and Var7] or WT-pHLIP-coated liposomes bind ischemic but not normal regions of myocardium, whereas pH-insensitive, kVar7, and liposomes coated with PEG showed no preference. pHLIP did not influence either the mechanical or the electrical activity of ischemic myocardium. In contrast to other known targeting strategies, the pHLIP-based binding does not require severe myocardial damage. Thus, pHLIP could be used for delivery of pharmaceutical agents or imaging probes to the myocardial regions undergoing brief restrictions of blood supply that do not induce irreversible changes in myocytes.acidity | cardiac | fluorescence imaging | targeted delivery T he pH (low) insertion peptide (pHLIP) enables targeting of cells in tissues with low extracellular pH (1). In this publication, we show that ischemic myocardial tissues are targeted, potentially opening new avenues for diagnosis and therapy. The pHLIP family consists of water-soluble polypeptides originally derived from the bacteriorhodopsin C helix, which is triggered by low pH to insert and fold across a membrane to form a stable transmembrane α helix as a consequence of titrating two carboxyl groups in the transmembrane (TM) helix to render them uncharged (2). In solution, at pH values above 7.0-7.2, a typical pHLIP partitions as a mainly unstructured peptide to the surface of a lipid bilayer, inserting when the pH drops from neutral or high to slightly acidic (7.0-6.5 and less). The insertion process is unidirectional and rapid (<2 min in lipid vesicles) (3-5). Variations of the pHLIP sequence can be used to change the time required for insertion across and exit from the membrane, ranging from milliseconds to minutes (6). The apparent pK of insertion across a liposomal bilayer membrane is 6.0 for the original WT-pHLIP sequence (2). pHLIP has been shown to locate imaging agents in tumors and arthritic inflammation (7-11), and to accomplish pH-selective cytoplasmic delivery of peptide nucleic acids, fluorescent dyes, and anticancer agents (3, 12, 13). Low pH is associated with a number of important pathological states including myocardial ischemia. According to the American Heart Association (14), âŒ9 million Americans are estimated to experience angina pectoris, and a similar incidence of angina has been estimated for most European countries (15). It has been demonstrated that extracellular pH is lowered in ischemic heart models (16, 17) and clinically in patients during pacing-induced ischemia in coronary artery disease (18). To follow targeting by pHLIP, fluorescent dyes and fluorescently labeled liposomes coated with pHLIP were u...