SARS-CoV-2 inhibition in human airway epithelial cells using a mucoadhesive, amphiphilic chitosan that may serve as an anti-viral nasal spray
Description
There are currently no cures for coronavirus infections, making the prevention of infections the only course open at the present time. The COVID-19 pandemic has been difficult to prevent, as the infection is spread by respiratory droplets and thus effective, scalable and safe preventive interventions are urgently needed. We hypothesise that preventing viral entry into mammalian nasal epithelial cells may be one way to limit the spread of COVID-19. Here we show that N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ), a positively charged polymer that has been through an extensive Good Laboratory Practice toxicology screen, is able to reduce the infectivity of SARS-COV-2 in A549ACE2+ and Vero E6 cells with a log removal value of -3 to -4 at a concentration of 10 – 100 g/ mL (p < 0.05 compared to untreated controls) and to limit infectivity in human airway epithelial cells at a concentration of 500 g/ mL (p < 0.05 compared to untreated controls). GCPQ is currently being developed as a pharmaceutical excipient in nasal and ocular formulations. GCPQ’s electrostatic binding to the virus, preventing viral entry into the host cells, is the most likely mechanism of viral inhibition. Radiolabelled GCPQ studies in mice show that at a dose of 10 mg/ kg, GCPQ has a long residence time in mouse nares, with 13.1% of the injected dose identified from SPECT/CT in the nares, 24 hours after nasal dosing. With a no observed adverse effect level of 18 mg/ kg in rats, following a 28-day repeat dose study, clinical testing of this polymer, as a COVID-19 prophylactic is warranted.
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The cytotoxicity of compounds was assessed by incubating confluent monolayers of Vero E6 and A549ACE2+ cells with a range of GCPQ compound concentrations. The XTT assay was carried out 48 hours later, according to the manufacturer’s protocol. The ability of each compound to inhibit the virus replication was determined by infecting confluent Vero E6 and A549ACE2+ monolayers with the SARS-CoV-2 virus at 400 TCID50/mL in the presence of test compounds or phosphate buffered saline - PBS (TCID50 = 50% Tissue Culture Infectious Dose). Mock controls (cell lysate without the virus) and medium (supplemented DMEM – please see above) controls were included. Virus replication inhibition in HAE was evaluated by infecting MucilAir™ (Epithelics Sarl, Switzerland) with SARS-CoV-2 virus at 5000 TCID50/mL in the presence of GCPQa or PBS. GCPQa diluted in PBS was added to the apical side of the insert (200 μg/ml or 500 μg/ml) and incubated at 37°C for 30 minutes before the infection. GCPQ (molecular weight = 10 kDa, mole% palmitoyl groups = 16 and mole% quaternary ammonium groups = 13) was radiolabelled using a two stage strategy: first an acylating reagent [N-succinimidyl-3[4-hydroxyphenyl]propionate - the Bolton and Hunter reagent (BH)] was initially covalently coupled to GCPQ and then the GCPQ-BH complex was iodinated with 125I. A Male Balb/C mouse weighing 25 g (Charles River, UK), allowed free access to standard rodent chow and water, was intranasally administered radiolabelled GCPQ-BH (10 mg/kg, 1.2 MBq) by using a pipette to place 5uL of the radiolabelled material into the mouse nares and allowing the mouse to sniff in the dose. At various time points after the administration of the radiolabelled GCPQ-BH, animals were anaesthetised using isofluorane (1-2% in oxygen), maintained at 37 ºC and submitted for NanoSPECT/CT analysis (Mediso, USA). SPECT/CT scans of the mouse head at 30 min, 2h 30 min and 24 h after nasal administration were acquired using a NanoSPECT/CT scanner (Mediso, Hungary). The mouse was anaesthetised using isofluorane (1-2% in oxygen) and maintained at 37ºC.