Toxicity Studies of Cardiac-Targeting Peptide Reveal a Robust Safety Profile.

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: APHLSSQYSRT). 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.

Cardiac-Targeting Peptide: From Discovery to Applications.

Despite significant strides in prevention, diagnosis, and treatment, cardiovascular diseases remain the number one cause of mortality in the United States, with rates climbing at an alarming rate in the developing world. Targeted delivery of therapeutics to the heart has been a lofty goal to achieve with strategies ranging from direct intra-cardiac or intra-pericardial delivery, intra-coronary infusion, to adenoviral, lentiviral, and adeno-associated viral vectors which have preference, if not complete cardio-selectivity, for cardiac tissue. Cell-penetrating peptides (CPP) are 5-30-amino-acid-long peptides that are able to breach cell membrane barriers while carrying cargoes up to several times their size, in an intact functional form. Identified nearly three decades ago, the first of these CPPs came from the HIV coat protein transactivator of transcription. Although a highly efficient CPP, its clinical utility is limited by its robust ability to cross any cell membrane barrier, including crossing the blood-brain barrier and transducing neuronal tissue non-specifically. Several strategies have been utilized to identify cell- or tissue-specific CPPs, one of which is phage display. Using this latter technique, we identified a cardiomyocyte-targeting peptide (CTP) more than a decade ago, a finding that has been corroborated by several independent labs across the world that have utilized CTP for a myriad of different purposes in pre-clinical animal models. The goal of this publication is to provide a comprehensive review of the identification, validation, and application of CTP, and outline its potential in diagnostic and therapeutic applications especially in the field of targeted RNA interference.

Assessment of Antibiotic Levels, Multi-Drug Resistant Bacteria and Genetic Biomarkers in the Waters of the Rio Grande River Between the United States-Mexico Border.

BACKGROUND: The worldwide emergence of multi-drug resistant bacteria has become a health crisis, as fewer or sometimes no antimicrobial agents are effective against these bacteria. The Rio Grande River is the natural boundary between the United States (US) and Mexico. It spans a border region between Texas, New Mexico and Mexico. Underserved populations on the Mexican side use the river for recreational purposes, while on the US side, the river is used for irrigation and as a source of drinking water. OBJECTIVES: The purpose of the present study was to evaluate the concentration of antibiotic residues, to determine the presence of genetic elements conferring antibiotic resistance and to characterize multi-drug resistant bacteria in the waters of the Rio Grande River. METHODS: Water samples were obtained from the Rio Grande River. Deoxyribonucleic acid (DNA) was extracted from both isolated bacteria and directly from the water. Amplification of selected genetic elements was accomplished by polymerase chain reaction. Identification and isolation of bacteria was performed through MicroScan autoSCAN-4. Fecal contamination was assessed by IDEXX Colilert. Antibiotic residues were determined by liquid chromatography and mass spectrometry. RESULTS: Antibiotics were found in 92% of both water and sediment samples. Antibiotic concentrations ranged from 0.38 ng/L - 742.73 ng/L and 0.39 ng/l - 66.3 ng/g dry weight in water and sediment samples, respectively. Genetic elements conferring resistance were recovered from all collection sites. Of the isolated bacteria, 91 (64.08%) were resistant to at least two synergistic antibiotic combinations and 11 (14.79%) were found to be resistant to 20 or more individual antibiotics. Fecal contamination was higher during the months of April and July. CONCLUSIONS: The 26 km segment of the Rio Grande River from Sunland Park NM to El Paso, TX and Juarez, Mexico is an area of concern due to poor water quality. The presence of multidrug resistant bacteria, antibiotics and mobile genetic elements may be a health hazard for the surrounding populations of this binational border region. Policies need to be developed for the appropriate management of the environmental natural resources in this border region. COMPETING INTERESTS: The authors declare no competing financial interests.