Study of the toxicity and the antimicrobial activity of different forms of ZnO nanoparticles: ZnO nanoparticles linked to graphene, pristine ZnO nanoparticles and ZnO nanoparticles doped with Mn Natalia Fernández-Pampín1 , Rocío Barros1, Sonia Martel Martín1 , Olavo Cardozo2,3, Andreas Stingl2, Patricia Farias2,4 , Alejandra García-Gómez5, Elisa Peña5 , Carlos Rumbo1, Juan Antonio Tamayo-Ramos 1 1 International Research Centre in Critical Raw Materials-ICCRAM, University of Burgos, Plaza Misael Bañuelos s/n, 09001, Burgos, Spain 2 PHORNANO Holding GmbH, Kleinengersdorferstrasse 24, 2100 Korneuburg, Austria 3 Post-Graduate Program on Electrical Engineering, Federal University of Pernambuco, Cidade Universitaria, Recife 50670-901, Brazil 4 Post-Graduate Program on Material Sciences, Federal University of Pernambuco, Cidade Universitaria, Recife 50670-901, Brazil 5 Gnanomat S.L., Campus Cantoblanco, Madrid Science Park, c/ Faraday 7, 28049, Madrid, Spain Contacts: nfpampin@ubu.es, crumbo@ubu.es, ja.tamayoramos@gmail.com ¡ Zinc oxide nanoparticles (ZnO NPs) have a multiple applications due to their special physico-chemical and antimicrobial properties. Moreover, they are inexpensive and less cytotoxic than others metal nanoparticles. The increasing use of the ZnO NPs in different sectors has raised concerns about their potential risks for the workers, consumers and environment. The posible toxic effects of different zinc oxide nanomaterials (G-ZnO, ZnO and ZnO:Mn) on the respiratory system and their irritant potential were evaluated. The antimicrobial activity of G-ZnO, ZnO and ZnO:Mn was analysed against Gram positive (methicillin-resistant S. aureus and vancomycin- resistant E. faecium) and Gram negative (A. baumannii and P. aeruginosa) bacteria. by reconstructed 3D human epidermal Materials • Graphene-zinc oxide nanomaterial (G-ZnO), ZnO and ZnO:Mn NPs Biological models A549 cells Reconstructed human epidermis (RhE) Gram positive and Gram negative bacteria Assays The effect of different concentrations of G-ZnO NPs on the A549 cell viability was evaluted by the MTT and neutral red (NR) assays, as well as the production of reactive oxigen species (ROS). On the other hand, the potential irritant of ZnO and ZnO:Mn NPs was determined using a RhE model. Moreover, the antimicrobial properties of the different ZnO NPs were studied in Gram positive and Gram negative bacteria by the determination of the minimun inhibitory concentration (MIC) following the CLSI guidelines. ¡ A549 cells MTT NR ROS RhE model S. aureus E. faecium A. baumannii P. aeruginosa G-ZnO ˃ 256 ˃ 256 ˃ 256 ˃ 256 ZnO ˃ 256 ˃ 256 ˃ 256 ˃ 256 ZnO:Mn ˃ 256 ˃ 256 ˃ 256 ˃ 256 Minimum inhibitory concentration (µg mL-1) #514 ¡ Introduction • The results obtained in the MTT assay showed that the G-ZnO produced a reduction of the cell viability after 24 h of treatment, while this reduction was less stronger in the NR assays. • G-ZnO NM caused a increased in the production of ROS in the studied exposure conditions. • According to the EU and GHS classification (Category 2 or Category 1), an irritant is predicted if the mean relative tissue viability of three individual tissues exposed to the test substance is reduced below 50% of the mean viability of the negative controls. Following this criteria, ZnO and ZnO:Mn NPs can be considered as non-irritant in the conditions tested. • None of the different ZnO NPs showed antimicrobial properties in the selected conditions, being their MICs ˃ 256 µg mL-1. Materials and Methods Results Conclusions