Feldman, Kyle S.

Background: Cell-penetrating peptides cross cell membrane barriers while carrying cargoes in a functional form. Our work identified two novel lung-targeting peptides, S7A and R11A. Here, we present studies on biodistribution, the cell types targeted, and an in vitro proof of application. Methods: Studies were performed in human bronchial epithelial cells (HBECs) with and without various endocytic inhibitors, and coincubation with fluorescently labeled transferrin or endocytic markers. Cyclic R11A (cR11A) was conjugated to siRNA duplexes and anti-viral activity against SARS-CoV-2 was tested. Biodistribution studies were performed by injecting wild-type mice with fluorescently labeled peptides, and various circulation times were allowed for, as well as cross-staining of lung sections or isolated single cells with various cellular markers, followed by fluorescence-activated cell sorting or confocal microscopy. Results: cR11A showed peak uptake in 15 min, with the highest uptake in airway epithelial type II (ATII) cells, followed by p63+ basal cells and ionocytes. Cyclization increased transduction efficiencies ~100-fold. Endocytosis studies showed a decrease in peptide uptake by pre-treatment with Pitstop2 but not Amiloride or Nystatin. Endocytic marker Lamp1 showed colocalization at the earliest time point, with the escape of the peptide from endocytic vesicles later. cR11A conjugated to ant-spike and anti-envelop proteins showed anti-viral effects with an EC90 of 0.6 μM and 1.0 µM, respectively. Conclusions: We have identified a novel peptide, cR11A, that targets ATII, basal cells, and ionocytes, the cyclization of which increased transduction efficiency in vitro and in vivo. The uptake mechanism appears to be via clathrin-mediated endocytosis with escape from endocytic vesicles. cR11A can act as a vector to deliver anti-viral siRNA to epithelial cells.

Targeted delivery of therapeutics specifically to cardiomyocytes would open up new frontiers for common conditions like heart failure. Our prior work using a phage display methodology identified a 12-amino-acid-long peptide that selectively targets cardiomyocytes after an intravenous injection in as little as 5 min and was hence termed a cardiac-targeting peptide (CTP: APWHLSSQYSRT). CTP has been used to deliver imaging agents, small drug molecules, photosensitizing nanoparticles, exosomes, and even miRNA to cardiomyocytes. As a natural extension to the development of CTP as a clinically viable cardiac vector, we now present toxicity studies performed with the peptide. In vitro viability studies were performed in a human left ventricular myocyte cell line with 10 µM of Cyanine-5.5-labeled CTP (CTP-Cy5.5). In vitro ion channel profiles were completed for CTP followed by extensive studies in stably transfected cell lines for several GPCR-coupled receptors. Positive data for GPCR-coupled receptors were interrogated further with RT-qPCRs performed on mouse heart tissue. In vivo studies consisted of pre- and post-blood pressure monitoring acutely after a single CTP (10 mg/Kg) injection. Further in vivo toxicity studies consisted of injecting CTP (150 µg/Kg) in 60, 6-week-old, wild-type CD1, male/female mice (1:1), with cohorts of mice euthanized on days 0, 1, 2, 7, and 14 with inhalational CO2, followed by blood collection via cardiac puncture, complete blood count analysis, metabolic profiling, and finally, liver, renal, and thyroid studies. Lastly, mouse cardiac MRI was performed immediately before and after CTP (150 µg/Kg) injection to assess changes in cardiac size or function. Human left ventricular cardiomyocytes showed no decrease in viability after a 30 min incubation with CTP-Cy5.5. No significant activation or inhibition of any of seventy-eight protein channels was observed other than OPRM1 and COX2 at the highest tested concentration, neither of which were expressed in mouse heart tissue as assessed using RT-qPCR. CTP (10 mg/Kg) injections led to no change in blood pressure. Blood counts and chemistries showed no evidence of significant hematological, hepatic, or renal toxicities. Lastly, there was no difference in cardiac function, size, or mass acutely in response to CTP injections. Our studies with CTP showed no activation or inhibition of GPCR-associated receptors in vitro. We found no signals indicative of toxicity in vivo. Most importantly, cardiac functions remained unchanged acutely in response to CTP uptake. Further studies using good laboratory practices are needed with prolonged, chronic administration of CTP conjugated to a specific cargo of choice before human studies can be contemplated.