Cyto-genotoxic effects of distinct micro/nanocelluloses in human osteoblastic and mice fibroblastic cells

The unique characteristics of micro/nanocelluloses (MNCs) have been boosting their use in a wide variety of industrial and biomedical applications. With the expected decrease of their production cost, it is likely to observe an even faster growing adoption of these materials in the coming years. With the increasing use of MNCs comes an increased likelihood of human exposure to these materials, raising concerns about potential human health effects. The number of studies on the potential adverse effects of MNCs is still scarce and sometimes inconclusive. The aim of the present study is to shed some light on the impact of human exposure to cellulose-based nanomaterials. For that, three MNCs were produced from bleached Eucalyptus kraft pulp using different pretreatments. A microfibrillated (CMF) and a nanofibrillated cellulose (CNF) were respectively obtained by enzymatic and TEMPO-mediated oxidation pretreatments followed by high-pressure homogenization. In addition, cellulose nanocrystals (CNC) were obtained by acid hydrolysis with diluted sulfuric acid. The potential cytotoxicity of the MNCs was evaluated in vitro in two mammalian cell lines (human MG-63 osteoblasts and mice V79 lung fibroblasts) by the MTT and the clonogenic assays, and the genotoxicity was assessed by the micronucleus assay. Cytotoxicity was observed by the clonogenic assay in V79 cells, particularly for CNC, but not by the MTT assay. CNF induced micronuclei in both cell lines and nucleoplasmic bridges in MG-63 cells. CMF and CNC induced micronuclei and nucleoplasmic bridges in MG-63 cells, but not in V79 cells. From the results, it was concluded that under the tested conditions all MNCs present cytotoxicity and genotoxicity, although at different concentration levels, which may be related to their physicochemical differences, to the availability for cell uptake, and to differences in the DNA damage response of the cell model.The work was funded by the Portuguese Foundation for Science and Technology (FCT/MCTES), through national funds (PIDDAC) under the project ToxApp4NanoCELFI (PTDC/SAU-PUB/32587/2017). The following funding is also acknowledged: ERDF and POR Lisboa2020 through the COMPETE 2020 POCI and PORL; POCI-01-0145- FEDER-007688 (UIDB/50025/2020-2023); NanoCell2SEC (PTDC/CTM-REF/30529/2017); ToxOmics (UIDP/00009/2020; UIDB/00009/2020); CIEPQPF (UIDB/00102/2020); European Topology Interdisciplinary Action (EUTOPIA CA17139) and Inpactus Project – innovative products and technologies from eucalyptus (Project N.º 21874 funded by Portugal 2020 through ERDF in the frame of COMPETE 2020 nº246/AXIS II/2017). Schemes created with BioRender.com.N/