Optimization of heart-targeting agents for therapeutic delivery in cardiomyocytes

Introduction Cardiovascular diseases represent the major cause of death in western industrialized countries. In particular, 50% of heart failure patients die within 5-years. Although usual treatments exist like β-blockers, they impact the quality of life due to side effects and off-targets. Thus, targeting specifically cardiomyocytes for drug delivery would certainly represent an improvement over classical ways of administration. Interestingly, two peptides were previously described to exhibit a preferential cardiac tropism (i.e. CTP: APWHLSSQYSRT and PCM: WLSEAGPVVTVRALRGTGSW). However, CTP and PCM peptides efficacy in terms of cell penetration remains low and to our knowledge no optimization has been reported. Objective The aim of this work was to optimize original CTP and PCM peptides and to characterize their respective uptake efficacy and cytotoxicity in cardiomyocytes. Methods Over 80 different CTP- and PCM-derived peptides coupled with 6-carboxyfluoresceine (6-CF) were developed. All constructs were assessed for cellular uptake in neonatal rat ventricular cardiomyocytes (NRVC). Cellular uptake efficacy and corresponding viability were quantified by flow cytometry as percentage of 6-CF-positive cells. High content imaging was performed to localize 6-CF-peptides in NRVC. Results Two of the derived peptides, 6-CF-CTP1 and 6-CF-PCM2, exhibited, respectively, a 6.5- and 5.5-fold increase over original 6-CF-CTP and 6-CF-PCM sequences in live cellular uptake. None of the original or derived 6-CF-CTP and 6-CF-PCM-peptides exhibited cytotoxicity from 1 to 50 μM. 6-CF peptides are distributed in NRVC cytoplasm. We also performed 4 °C incubations which did not prevent the NRVC uptake, excluding an energy-driven process. Conclusion This study displays potential for optimization of the CTP or PCM peptide sequences to increase their cellular uptake efficacy without cytotoxicity. It raises the potential use of heart targeting peptides for cardiac drug delivery.