Cardiac Targeting Peptide, a Novel Cardiac Vector: Studies in Bio-Distribution, Imaging Application, and Mechanism of Transduction

Abstract

Our previous work identified a 12-amino acid peptide that targets the heart, termed cardiac targeting peptide (CTP). We now quantitatively assess the bio-distribution of CTP, show a clinical application with the imaging of the murine heart, and study its mechanisms of transduction. Bio-distribution studies of cyanine5.5-N-Hydroxysuccinimide (Cy5.5) labeled CTP were undertaken in wild-type mice. Cardiac targeting peptide was labeled with Technetium 99m (99mTc) using the chelator hydrazino-nicotinamide (HYNIC), and imaging performed using micro-single photon emission computerized tomography/computerized tomography (SPECT/CT). Human-induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMCs) were incubated with dual-labeled CTP, and imaged using confocal microscopy. TriCEPs technology was utilized to study the mechanism of transduction. Bio-distribution studies showed peak uptake of CTP at 15 min. 99mTc-HYNIC-CTP showed heart-specific uptake. Robust transduction of beating human iPSC-derived CMCs was seen. TriCEPs experiments revealed five candidate binding partners for CTP, with Kcnh5 being felt to be the most likely candidate as it showed a trend towards being competed out by siRNA knockdown. Transduction efficiency was enhanced by increasing extracellular potassium concentration, and with Quinidine, a Kcnh5 inhibitor, that blocks the channel in an open position. We demonstrate that CTP transduces the normal heart as early as 15 min. 99mTc-HYNIC-CTP targets the normal murine heart with substantially improved targeting compared with 99mTc Sestamibi. Cardiac targeting peptide’s transduction ability is not species limited and has human applicability. Cardiac targeting peptide appears to utilize Kcnh5 to gain cell entry, a phenomenon that is affected by pre-treatment with Quinidine and changes in potassium levels.

Photosensitive nanocarriers for specific delivery of cargo into cells

“…S10B,D, S.I. ), consistent with the literature 44,46 . Therefore, 8-h of NBc incubation was the optimal internalization time selected for further experiments (Fig.…”

“…S4) 32,33 . The CTP has shown to be able to transduce cardiomyocytes rapidly, specifically and efficiently with very little uptake by other cells [44][45][46] . The average dynamic diameter of the PNcs was 91.00 ± 4.23 nm, which increased by about 10 nm after the functionalization process ( Fig.…”

“…4B). This is likely a receptor-mediated cellular uptake through the cell bioreceptor-CTP interaction, whose mechanism (endocytic or non-endocytic) is still unclear 46 , but generates an intracellular occupied area of 0.83 ± 0.02% (Fig. S9, S.I.).…”

Cell Penetrating Peptides, Novel Vectors for Gene Therapy

“…Although CPPs have been used as vectors for delivery of drugs [48][49][50][51], other peptides of therapeutic potential [52][53][54][55][56][57][58][59][60], proteins [56,[61][62][63][64][65], radioisotopes [66][67][68][69], quantum dots [70,71] and photosensitizers [72,73], this review will focus on use of CPPs as vectors for nucleic acid delivery, be they genes, oligonucleotides, peptide nucleic acid conjugates, small interfering RNA (siRNA) or the newest application with DNA origami. Although there is literature on all of these applications, most interest and work done to date has been with siRNA.…”

“…Excited by these reports, we constructed a tandem peptide by adding 6-arginine to the N-terminus of an optimized version of our cardiac targeting peptide (CTP-B). Based on our prior bio-distribution studies [66], this fluorescently labeled peptide was injected intravenously into mice and allowed to circulate for 15 min. However, the conjugate peptide actually reduced cardiac uptake compared to the parent CTP-B peptide, while increasing kidney and liver uptake ( Figure 1).…”