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

Cellulose nanofibrils (CNFs) have emerged as sustainable options for a wide range of applications. However, the high aspect ratio and biopersistence of CNFs raise concerns about potential health effects. Here, we evaluated the in vivo pulmonary and systemic toxicity of unmodified (U-CNF), carboxymethylated (C-CNF), and TEMPO (2,2,6,6-tetramethyl-piperidin-1-oxyl)-oxidized (T-CNF) CNFs, fibrillated in the same way and administered to mice by repeated (3×) pharyngeal aspiration (14, 28, and 56 µg/mouse/aspiration). Toxic effects were assessed up to 90 days after the last administration. Some mice were treated with T-CNF samples spiked with lipopolysaccharide (LPS; 0.02-50 ng/mouse/aspiration) to assess the role of endotoxin contamination. The CNFs induced an acute inflammatory reaction that subsided within 90 days, except for T-CNF. At 90 days post-administration, an increased DNA damage was observed in bronchoalveolar lavage and hepatic cells after exposure to T-CNF and C-CNF, respectively. Besides, LPS contamination dose-dependently increased the hepatic genotoxic effects of T-CNF.

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

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.

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

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.

Genotoxicity and cellular uptake of nanosized and fine copper oxide particles in human bronchial epithelial cells in vitro.

We studied the genotoxicity and cellular uptake of nanosized (<50 nm) and fine (<10 µm) copper oxide (CuO) particles in vitro in human bronchial epithelial (BEAS-2B) cells. In addition, the effect of dispersing the particles using bovine serum albumin (BSA) on DNA damage induction was investigated. DNA damage was assessed by the alkaline comet (single cell gel electrophoresis) assay after 3-h, 6-h and 24-h exposures. The cytokinesis-block micronucleus assay was applied to study chromosome damage. Both fine- and nanosized CuO particles induced a dose-dependent increase in DNA damage at all timepoints tested. However, nanosized CuO damaged DNA at lower doses and higher levels compared with fine CuO. Dispersing the nanoparticles in the presence of BSA (0.6 mg/mL) resulted in a small and inconsistent decrease in DNA damage compared with dispersions in serum-free cell culture medium only. CuO nanoparticles induced a clear dose-dependent increase in micronucleated cells at doses that strongly increased cytostasis and were markedly cytotoxic at 24 and 48 h. Fine CuO showed a slight induction of micronuclei. Hyperspectral microscopy indicated a substantial cellular uptake of both types of particles after a 3-h exposure to a dose of 20 µg/cm2. The number of particles internalized by the cells was higher for nanosized than fine CuO, as quantified by the frequency of spectral matches in the total cell area and by the number of spectrally matched visible particles or agglomerates per cell. The particle uptake was limited by particle size. The stronger genotoxic activity of nanosized than fine CuO particles is likely to derive from the higher cellular uptake and more effective intracellular dissolution of nanoparticles.

In vivo toxicological evaluation of polymer brush engineered nanoceria: impact of brush charge.

Nanoceria has a broad variety of industrial and pharmacological applications due to its antioxidant activity. Nanoceria can be modified by surface coating with polyelectrolyte brushes. Brushes can increase the surface charge of nanoceria, providing greater aqueous stability while reducing agglomeration. However, surface-coating also behaves as a barrier around nanoceria, affecting its redox equilibrium and, hence, its biological and toxicological properties. In the present study, we examined whether bare nanoceria (CeO2; 80-150 nm) and nanoceria modified by surface polymer brush, using negatively charged polyacrylic acid (CeO2@PAA) and positively charged poly (2-(methacryloyloxy)ethyl-trimethyl-ammonium chloride (CeO2@PMETAC), could induce systemic toxicity. As CeO2 has limited colloidal stability, which might result in vascular occlusion, intraperitoneal injection was used instead of intravenous administration. C57Bl/6 mice were four times injected with three different doses of each nanoceria-based sample (corresponding to 1.8, 5.3 and 16 mg Ce/kg BW/administration) for a total period of 14 days. CeO2@PMETAC induced a significant dose-dependent neutrophilia. Histopathological evaluation showed inflammatory processes in the capsule of liver, kidney, and spleen of animals at all doses of CeO2@PMETAC, and with the highest dose of CeO2@PAA and CeO2. However, none of the nanoceria-based samples tested increased the level of DNA damage or micronuclei in blood cells, even though Ce was detected by inductively coupled plasma mass spectrometry analyses in the bone marrow. Only CeO2@PMETAC induced the presence of megakaryocytes in the spleen. A higher accumulation of Ce in mononuclear phagocyte system organs (liver, spleen and bone marrow) was observed after CeO2@PMETAC treatment compared with CeO2@PAA and CeO2.