Antiviral Activity of Graphene Oxide–Silver Nanocomposites Against Murine Betacoronavirus

Joanna Cymerys,1,* Michalina Bartak,1,* Anna Słońska,1 Agata Lange,2 Sławomir Jaworski,2 Marcin Chodkowski,3 Agnieszka Ostrowska,2 Mateusz Wierzbicki,2 Ewa Sawosz,2 Marcin W Bańbura1 1Department of Preclinical Sciences, Institute of Veterinary Medicine, Warsaw University of Life Sciences, Warsaw, Poland; 2Department of Nanobiotechnology, Institute of Biology, Warsaw University of Life Sciences, Warsaw, Poland; 3Military Institute of Hygiene and Epidemiology, Warsaw, Poland*These authors contributed equally to this workCorrespondence: Joanna Cymerys, Department of Preclinical Sciences, Institute of Veterinary Medicine, Warsaw University of Life Sciences, 02-786, Warsaw, Poland, Email [email protected]: The high infectivity of coronaviruses has led to increased interest in developing new strategies to prevent virus spread. Silver nanoparticles (AgNPs) and graphene oxide (GO) have attracted much attention in the antiviral field. We investigated the potential antiviral activity of GO and AgNPs combined in the nanocomposite GO-Ag against murine betacoronavirus MHV using an in vitro model.Methods: GO, AgNPs, and GO-Ag characterization (size distribution, zeta potential, TEM visualization, FT-IR, and EDX analysis) and XTT assay were performed. The antiviral activity of GO-Ag nanocomposites was evaluated by RT-qPCR and TCID50 assays. The results were compared with free AgNPs and pure GO. Cell growth and morphology of MHV-infected hepatocytes treated with GO-Ag composites were analyzed by JuLI™Br. Immunofluorescence was used to visualize the cell receptor used by MHV. Ultrastructural SEM analysis was performed to examine cell morphology after MHV infection and GO-Ag composite treatment.Results: A significant reduction in virus titer was observed for all nanocomposites tested, ranging from 3.2 to 7.3 log10 TCID50. The highest titer reduction was obtained for GO 5 μg/mL - Ag 25 μg/mL in the post-treatment method. These results were confirmed by RT-qPCR analysis. The results indicate that GO-Ag nanocomposites exhibited better antiviral activity compared to AgNPs and GO. Moreover, the attachment of AgNPs to the GO flake platform reduced their cytotoxicity. In addition, the GO-Ag composite modulates the distribution of the Ceacam1 cell receptor and can modulate cell morphology.Conclusion: Graphene oxide sheets act as a stabilizing agent, inhibiting the accumulation of AgNPs and reducing their cellular toxicity. The GO-Ag composite can physically bind and inhibit murine betacoronavirus from entering cells. Furthermore, the constant presence of GO-Ag can inhibit MHV replication and significantly limit its extracellular release. In conclusion, GO-Ag shows promise as an antiviral coating on solid surfaces to minimize virus transmission and spread. Keywords: graphene oxide, silver nanoparticles, GO-Ag composite, antiviral, coronavirus, MHV