Role of Surface Chemistry in the In Vitro Lung Response to Nanofibrillated Cellulose

Abstract

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.

Keywords: genotoxicity; human bronchial epithelial cells; nanocellulose; nanofibrillated cellulose; nanotoxicity; reactive oxygen species; surface chemistry.

Figure 1

Chemical structures of the nanofibrillated cellulose (NFC) materials under study.

Figure 2

Representative TEM images of nanofibrillated cellulose (NFC) aqueous suspensions showing the morphology of the nanofibers. (a) U-NFC, (b) C-NFC, (c) H-NFC, (d) P-NFC and (e) S-NFC.

Figure 3

Examples of calcofluor staining (blue) in cells treated with U-NFC (167 µg/mL) (a), H-NFC (56 µg/mL) (b) and untreated cells (c). Counterstaining by acridine orange.

Figure 4

Cytotoxicity of the nanofibrillated cellulose (NFC) materials. The total number of living cells was counted at 24 and 48 h exposure to (a) U-NFC, (b) C-NFC, (c) H-NFC, (d) P-NFC and (e) S-NFC, and the results were normalized with the unexposed control. Results are presented as the mean ± standard error of the mean. Graphs are plotted with 95% confidence interval (colored areas). The range of 45 ± 5% living cells (corresponding to the 55 ± 5% cytotoxicity) is indicated by the grey area in each graph.

Figure 5

Induction of intracellular reactive oxygen species (ROS) by the nanofibrillated cellulose (NFC) materials. The production of ROS was assessed at 3, 6, and 24 h exposure to (a) U-NFC, (b) C-NFC, (c) H-NFC, (d) P-NFC and (e) S-NFC. Data are expressed as relative fluorescence units (RFU) and presented as the mean ± standard error of the mean. Significant linear dose-responses are plotted with 95% confidence interval (colored areas). Statistical significance in comparison with control cultures (one-way ANOVA): * p < 0.05. Statistical significance, linear regression: ^†† p < 0.01; ^††† p < 0.001; ^†††† p < 0.0001.

Figure 6

Induction of DNA damage by the nanofibrillated cellulose (NFC) materials. DNA strand breaks were assessed at 24 h exposure to (a) U-NFC, C-NFC, H-NFC, P-NFC, and S-NFC. A significant linear dose response (p = 0.0427) was induced by P-NFC (b). Data are expressed as percentage of DNA in tail and presented as the mean ± standard error of the mean. Significant linear dose-response is plotted with 95% confidence interval (colored area).

Figure 7

Induction of micronuclei by the nanofibrillated cellulose (NFC) materials. Micronucleus induction was assessed at 48 h exposure to (a) U-NFC, (b) C-NFC, (c) H-NFC, (d) P-NFC and (e) S-NFC. Purple symbols show the frequency of micronucleated cells in 2000 binucleated cells (MNCs/2000 BNCs). Blue symbols show the percentage of cytostasis. Data are presented as the mean ± standard error of the mean.