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Nanofibrillated cellulose (NFC) is a renewable nanomaterial that has beneficial uses in various applications such as packaging materials and paper. Like carbon nanotubes (CNT), NFCs have high aspect ratio and favorable mechanical properties. The aspect ratio also rises a concern whether NFC could pose a health risk and induce pathologies, similar to those triggered by multi-walled CNT. In this study, we explored the immunomodulatory properties of four NFCs in vitro and in vivo, and compared the results with data on bulk-sized cellulose fibrils and rigid multi-walled CNT (rCNT). Two of the NFCs were non-functionalized and two were carboxymethylated or carboxylated. We investigated the production of pro-inflammatory cytokines in differentiated THP-1 cells, and studied the pulmonary effects and biopersistence of the materials in mice. Our results demonstrate that one of the non-functionalized NFCs tested reduced cell viability and triggered pro-inflammatory reactions in vitro. In contrast, all cellulose materials induced innate immunity response in vivo 24 h after oropharyngeal aspiration, and the non-functionalized NFCs additionally caused features of Th2-type inflammation. Modest immune reactions were also seen after 28 days, however, the effects were markedly attenuated as compared with the ones after 24 h. Cellulose materials were not cleared within 1 month, as demonstrated by their presence in the exposed lungs. All effects of NFC were modest as compared with those induced by rCNT. NFC-induced responses were similar or exceeded those triggered by bulk-sized cellulose. These data provide new information about the biodurability and pulmonary effects of different NFCs; this knowledge can be useful in the risk assessment of cellulose materials.

Nanofibrillated cellulose (NFC) is a sustainable and renewable nanomaterial, with diverse potential applications in the paper and medical industries. As NFC consists of long fibres of high aspect ratio, we examined here whether TEMPO-(2,2,6,6-tetramethyl-piperidin-1-oxyl) oxidised NFC (length 300-1000nm, thickness 10-25nm), administrated by a single pharyngeal aspiration, could be genotoxic to mice, locally in the lungs or systemically in the bone marrow. Female C57Bl/6 mice were treated with four different doses of NFC (10, 40, 80 and 200 µg/mouse), and samples were collected 24h later. DNA damage was assessed by the comet assay in bronchoalveolar lavage (BAL) and lung cells, and chromosome damage by the bone marrow erythrocyte micronucleus assay. Inflammation was evaluated by BAL cell counts and analysis of cytokines and histopathological alterations in the lungs. A significant induction of DNA damage was observed at the two lower doses of NFC in lung cells, whereas no increase was seen in BAL cells. No effect was detected in the bone marrow micronucleus assay, either. NFC increased the recruitment of inflammatory cells to the lungs, together with a dose-dependent increase in mRNA expression of tumour necrosis factor α, interleukins 1β and 6, and chemokine (C-X-C motif) ligand 5, although there was no effect on the levels of the respective proteins. The histological analysis showed a dose-related accumulation of NFC in the bronchi, the alveoli and some in the cytoplasm of macrophages. In addition, neutrophilic accumulation in the alveolar lung space was observed with increasing dose. Our findings showed that NFC administered by pharyngeal aspiration caused an acute inflammatory response and DNA damage in the lungs, but no systemic genotoxic effect in the bone marrow. The present experimental design did not, however, allow us to determine whether the responses were transient or could persist for a longer time.

Wood-derived nanofibrillated cellulose (NFC) has emerged as a sustainable material with a wide range of applications and increasing presence in the market. Surface charges are introduced during the preparation of NFC to facilitate the defibrillation process, which may also alter the toxicological properties of NFC. In the present study, we examined the in vitro toxicity of NFCs with five surface chemistries: nonfunctionalized, carboxymethylated, phosphorylated, sulfoethylated, and hydroxypropyltrimethylammonium-substituted. The NFC samples were characterized for surface functional group density, surface charge, and fiber morphology. Fibril aggregates predominated in the nonfunctionalized NFC, while individual nanofibrils were observed in the functionalized NFCs. Differences in surface group density among the functionalized NFCs were reflected in the fiber thickness of these samples. In human bronchial epithelial (BEAS-2B) cells, all NFCs showed low cytotoxicity (CellTiter-GloVR luminescent cell viability assay) which never exceeded 10% at any exposure time. None of the NFCs induced genotoxic effects, as evaluated by the alkaline comet assay and the cytokinesis-block micronucleus assay. The nonfunctionalized and carboxymethylated NFCs were able to increase intracellular reactive oxygen species (ROS) formation (chloromethyl derivative of 2′,7′-dichlorodihydrofluorescein diacetate assay). However, ROS induction did not result in increased DNA or chromosome damage.

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