Fullerenol C60(OH)40 Nanoparticles and Ectoine Protect Human Nasal Epithelial Cells Against the Cytokine Storm After Addition of the Full-Length Spike Protein from SARS-CoV-2

Malwina Sosnowska,1 Mateusz Wierzbicki,1 Barbara Nasiłowska,2 Totka Nikolaeva Bakalova,3 Klara Piotrowska,4 Barbara Strojny-Cieślak,1 Ewa Sawosz,1 Marta Kutwin1 1Department of Nanobiotechnology, Institute of Biology, Warsaw University of Life Sciences, Warsaw, Poland; 2Biomedical Engineering Center, Institute of Optoelectronics, Military University of Technology, Warsaw, Poland; 3Department of Material Science, Faculty of Mechanical Engineering, Technical University of Liberec, Liberec, Czech Republic; 4Department of Animal Nutrition, Institute of Animal Sciences, Warsaw University of Life Sciences, Warsaw, PolandCorrespondence: Malwina Sosnowska, Department of Nanobiotechnology, Institute of Biology, Warsaw University of Life Sciences, st. Ciszewskiego 8, Warsaw, 02-786, Poland, Tel +48 22 593 66 71, Email [email protected] and Objective: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the nasal cavity, penetrates the nasal epithelial cells through the interaction of its spike protein with the host cell receptor angiotensin-converting enzyme 2 (ACE2) and then triggers a cytokine storm. We aimed to assess the biocompatibility of fullerenol nanoparticles C60(OH)40 and ectoine, and to document their effect on the protection of primary human nasal epithelial cells (HNEpCs) against the effects of interaction with the fragment of virus - spike protein. This preliminary research is the first step towards the construction of a intranasal medical device with a protective, mechanical function against SARS-CoV-2 similar to that of personal protective equipment (eg masks).Methods: We used HNEpCs and the full-length spike protein from SARS-CoV-2 to mimic the first stage of virus infection. We assessed cell viability with the XTT assay and a spectrophotometer. May–Grünwald Giemsa and periodic acid–Schiff staining served to evaluate HNEpC morphology. We assessed reactive oxygen species (ROS) production by using 2′,7′-dichlorofluorescin diacetate and commercial kit. Finally, we employed reverse transcription polymerase chain reaction, Western blotting and confocal microscopy to determine the expression of angiotensin-converting enzyme 2 (ACE2) and inflammatory cytokines.Results: There was normal morphology and unchanged viability of HNEpCs after incubation with 10 mg/L C60(OH)40, 0.2% ectoine or their composite for 24 h. The spike protein exerted cytotoxicity via ROS production. Preincubation with the composite protected HNEpCs against the interaction between the spike protein and the host membrane and prevented the production of key cytokines characteristic of severe coronavirus disease 2019, including interleukin 6 and 8, monocyte chemotactic protein 1 and 2, tissue inhibitor of metalloproteinases 2 and macrophage colony-stimulating factor.Conclusion: In the future, the combination of fullerenol and ectoine may be used to prevent viral infections as an intranasal medical device for people with reduced immunity and damaged mucous membrane. Keywords: ACE2, cytokine storm, ectoine, nasal epithelium, polyhydroxylated fullerene, spike