Adsorption of lactate dehydrogenase enzyme on carbon nanotubes: how to get accurate results for the cytotoxicity of these nanomaterials.

Carbon nanotube (CNT) cytotoxicity is frequently investigated using in vitro classical toxicology assays. However, these cellular tests, usually based on the use of colorimetric or fluorimetric dyes, were designed for chemicals and may not be suitable for nanosized materials. Indeed, because of their unique physicochemical properties CNT can interfere with the assays and bias the results. To get accurate data and draw reliable conclusions, these artifacts should be carefully taken into account. The aim of this study was to evaluate qualitatively and quantitatively the interferences occurring between CNT and the commonly used lactate dehydrogenase (LDH) assay. Experiments under cell-free conditions were performed, and it was clearly demonstrated that artifacts occurred. They were due to the intrinsic absorbance of CNT on one hand and the adsorption of LDH at the CNT surface on the other hand. The adsorption of LDH on CNT was modeled and was found to fit the Langmuir model. The K(ads) and n(eq) constants were defined, allowing the correction of results obtained from cellular experiments to get more accurate data and lead to proper conclusions on the cytotoxicity of CNT.

Toxicity of boehmite nanoparticles: impact of the ultrafine fraction and of the agglomerates size on cytotoxicity and pro-inflammatory response.

Boehmite (γ-AlOOH) nanoparticles (NPs) are used in a wide range of industrial applications. However, little is known about their potential toxicity. This study aimed at a better understanding of the relationship between the physico-chemical properties of these NPs and their in vitro biological activity. After an extensive physico-chemical characterization, the cytotoxicity, pro-inflammatory response and oxidative stress induced by a bulk industrial powder and its ultrafine fraction were assessed using RAW264.7 macrophages. Although the bulk powder did not trigger a significant biological activity, pro-inflammatory response was highly enhanced with the ultrafine fraction. This observation was confirmed with boehmite NPs synthesized at the laboratory scale, with well-defined and tightly controlled physico-chemical features: toxicity was increased when NPs were dispersed. In conclusion, the agglomerates size of boehmite NPs has a major impact on their toxicity, highlighting the need to study not only raw industrial powders containing NPs but also the ultrafine fractions representative of respirable particles.

In vitro toxicity of carbon nanotubes, nano-graphite and carbon black, similar impacts of acid functionalization.

Carbon nanotubes (CNT) and nano-graphite (NG) are graphene-based nanomaterials which share exceptional physicochemical properties, but whose health impacts are unfortunately still not well understood. On the other hand, carbon black (CB) is a conventional and widely studied material. The comparison of these three carbon-based nanomaterials is thus of great interest to improve our understanding of their toxicity. An acid functionalization was carried out on CNT, NG and CB so that, after a thorough characterization, their impacts on RAW 264.7 macrophages could be compared for a similar surface chemistry (15 to 120 µg·mL(-1) nanomaterials, 90-min to 24-h contact). Functionalized nanomaterials triggered a weak cytotoxicity similar to the pristine nanomaterials. Acid functionalization increased the pro-inflammatory response except for CB which did not trigger any TNF-α production before or after functionalization, and seemed to strongly decrease the oxidative stress. The toxicological impact of acid functionalization appeared thus to follow a similar trend whatever the carbon-based nanomaterial. At equivalent dose expressed in surface and equivalent surface chemistry, the toxicological responses from murine macrophages to NG were higher than for CNT and CB. It seemed to correspond to the hypothesis of a platelet and fiber paradigm.

In vitro cellular responses to silicon carbide particles manufactured through the Acheson process: impact of physico-chemical features on pro-inflammatory and pro-oxidative effects.

Silicon carbide (SiC) an industrial-scale product manufactured through the Acheson process, is largely employed in various applications. Its toxicity has been poorly investigated. Our study aims at characterizing the physico-chemical features and the in vitro impact on biological activity of five manufactured SiC powders: two coarse powders (SiC C1/C2), two fine powders (SiC F1/F2) and a powder rich in iron impurities (SiC I). RAW 264.7 macrophages were exposed to the different SiC particles and the cellular responses were evaluated. Contrary to what happens with silica, no SiC cytotoxicity was observed but pro-oxidative and pro-inflammatory responses of variable intensity were evidenced. Oxidative stress (H2O2 production) appeared related to SiC particle size, while iron level regulated pro-inflammatory response (TNFα production). To investigate the impact of surface reactivity on the biological responses, coarse SiC C1 and fine SiC F1 powders were submitted to different thermal treatments (650-1400 °C) in order to alter the oxidation state of the particle surface. At 1400 °C a decrease in TNFα production and an increase in HO·, COO(·-) radicals production were observed in correlation with the formation of a surface layer of crystalline silica. Finally, a strong correlation was observed between surface oxidation state and in vitro toxicity.

New insight into artifactual phenomena during in vitro toxicity assessment of engineered nanoparticles: study of TNF-α adsorption on alumina oxide nanoparticle.

Biomolecules can be adsorbed on nanoparticles (NPs) and degraded during in vitro toxicity assays. These artifactual phenomena could lead to misinterpretation of biological activity, such as false-negative results. To avoid possible underestimation of cytokine release after contact between NP and cells, we propose a methodology to account for these artifactual phenomena and lead to accurate measurements. We focused on the pro-inflammatory cytokine tumor necrosis factor TNF-α. We studied well-characterized boehmite engineered NP [aluminum oxide hydroxide, AlO(OH)]. The rate of TNF-α degradation and its adsorption (on boehmite and on the walls of wells) were determined in cell-free conditions by adding a known TNF-α concentration (1500 pg/ml) under various experimental conditions. After a 24-h incubation, we quantified that 7 wt.% of the initial TNF-α was degraded over time, 6 wt.% adsorbed on the walls of 96-well plates, and 13 wt.% adsorbed on the boehmite surface. Finally, boehmite NP were incubated with murine macrophages (RAW 264.7 cell line). The release of TNF-α was assessed for boehmite NP and the experimental data were corrected considering the artifactual phenomena, which accounted for about 20-30% of the total.