Surface functionalization and size modulate the formation of reactive oxygen species and genotoxic effects of cellulose nanofibrils

Abstract

Background Cellulose nanofibrils (CNFs) have emerged as a sustainable and environmentally friendly option for a broad range of applications. The fibrous nature and high biopersistence of CNFs call for a thorough toxicity assessment, but it is presently unclear which physico-chemical properties could play a role in determining the potential toxic response to CNF. Here, we assessed whether surface composition and size could modulate the genotoxicity of CNFs in human bronchial epithelial BEAS-2B cells. We examined three size fractions (fine, medium and coarse) of four CNFs with different surface chemistry: unmodified (U-CNF) and functionalized with 2,2,6,6-tetramethyl-piperidin-1-oxyl (TEMPO) (T-CNF), carboxymethyl (C-CNF) and epoxypropyltrimethylammonium chloride (EPTMAC) (E-CNF). In addition, the source fibre was also evaluated as a non-nanosized material. Results The presence of the surface charged groups in the functionalized CNF samples resulted in higher amounts of individual nanofibrils and less aggregation compared with the U-CNF. T-CNF was the most homogenous, in agreement with its high surface group density. However, the colloidal stability of all the CNF samples dropped when dispersed in cell culture medium, especially in the case of T-CNF. CNF was internalized by a minority of BEAS-2B cells. No remarkable cytotoxic effects were induced by any of the cellulosic materials. All cellulosic materials, except the medium fraction of U-CNF, induced a dose-dependent intracellular formation of reactive oxygen species (ROS). The fine fraction of E-CNF, which induced DNA damage (measured by the comet assay) and chromosome damage (measured by the micronucleus assay), and the coarse fraction of C-CNF, which produced chromosome damage, also showed the most effective induction of ROS in their respective size fractions. Conclusions Surface chemistry and size modulate the in vitro intracellular ROS formation and the induction of genotoxic effects by fibrillated celluloses. One cationic (fine E-CNF) and one anionic (coarse C-CNF) CNF showed primary genotoxic effects, possibly partly through ROS generation. However, the conclusions cannot be generalized to all types of CNFs, as the synthesis process and the dispersion method used for testing affect their physico-chemical properties and, hence, their toxic effects.

High aspect ratio nanomaterial-induced macrophage polarization is mediated by changes in miRNA levels

“…CNF on contrary, is not effectively internalized by alveolar macrophages in vitro as demonstrated here by immuno-TEM analysis, however exposed cells showed an increased presence of lysosomal structures. These findings are supported by previous studies showing increased presence of vacuoles in the cytoplasm of CNF exposed cells despite low or no particle uptake (24,55). Previous studies also suggest that CNF may absorb to the plasma membrane resulting in limited uptake and that its effects may involve receptormediated mechanisms (23,56,57).…”

Effect of Surface Modification on the Pulmonary and Systemic Toxicity of Cellulose Nanofibrils

“…The surface of all the used equipment was extensively cleaned through consecutive washes with deionized water, ethanol (Altia, Helsinki, Finland), sterile purified water, and endotoxin-free water (Thermo Fisher Scientific, UT, USA). Sterile centrifuge tubes (Thermo Fisher Scientific, UT, USA) and sterile and endotoxin-free water were also used to prepare the samples in aqueous media …”

“…Characterization was performed as previously described . Atomic force microscopy (AFM) was used to investigate the morphology of the CNF samples using a Dimension 5000 scanning probe microscope (Veeco, TX, USA).…”

“…Sterile centrifuge tubes (Thermo Fisher Scientific, UT, USA) and sterile and endotoxin-free water were also used to prepare the samples in aqueous media. 27 CNFs were sourced as commercially low endotoxins containing bleached sulfite birch-dissolving pulp (UPM Kymmene Oyj, Finland), whose lignin content was below 0.5%. The wood fibers were refined using a laboratory-scale PFI refiner (Hamjern Maskin AB, Norway).…”

Toxicity of metal-based nanoparticles: Challenges in the nano era

“…The surface chemistry properties of NPs can be modified by covalently coupling with functional groups like anionic, nonionic, zwitterionic, and cationic groups onto the surface (Blanco et al, 2015;Zakharova et al, 2019;Han et al, 2020). These functional groups can influence the charge density and hydrophobicity of NPs (Nel et al, 2009;Aimonen et al, 2022). Au NPs can be an appropriate example where surface chemistry modification could influence NP toxicity.…”