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Graphene derivatives are expected to have a great impact in a wide range of applications, among them as food packaging materials. This is one of the sources of potential human oral exposure to them. However, studies devoted to investigating their putative toxic effects at the intestinal level are underrepresented in the scientific literature. Thus, this study aimed to investigate the in vitro toxicity of reduced graphene oxide (rGO) and graphene oxide (GO) in the human intestinal Caco-2 cell line. rGO and GO were firstly characterized and later, cell viability was assessed after exposure to 0–250 µg/mL rGO/GO for 24 and 48 h. Internalization was evidenced for both materials using transmission electron microscopy. A mean effective concentration (24 h) of 176.3 ± 7.6 µg/mL for cytotoxicity was obtained for rGO, whereas GO did not induce any change at the concentration range evaluated. However, both of them altered oxidative stress biomarkers, causing increased reactive oxygen species (ROS) and depletion of the glutathione content (GSH) after exposures up to 24 h. Further studies, particularly with rGO, are required to elucidate their toxicity profile in experimental models relevant for oral exposures.

The growing interest in graphene derivatives is a result of their variety of applications in many fields. Due to their use, the oral route could be a potential way of entrance for the general population. This work assesses the biotransformation of reduced graphene oxide (rGO) after an in vitro digestion procedure (mouth, gastric, intestinal, and colon digestion), and its toxic effects in different cell models (HepG2, Caco-2, and 3D intestinal model). The characterization of rGO digestas evidenced the agglomeration of samples during the in vitro gastrointestinal (g.i.) digestion. Internalization of rGO was only evident in Caco-2 cells exposed to the colonic phase and no cellular defects were observed. Digestas of rGO did not produce remarkable cytotoxicity in any of the experimental models employed at the tested concentrations (up to 200 µg/mL), neither an inflammatory response. Undigested rGO has shown cytotoxic effects in Caco-2 cells, therefore these results suggest that the digestion process could prevent the systemic toxic effects of rGO. However, additional studies are necessary to clarify the interaction of rGO with the g.i. tract and its biocompatibility profile.

The interest of graphene materials has increased markedly in the recent years for their promising applications in many fields as food packing. These new applications have caused some concern regarding their safety for consumers since the intake of these materials may increase. In this sense, a battery of in vitro test is required before its use as a food contact material. Then, the aim of this study was to assess the potential mutagenicity and genotoxicity of graphene oxide (GO) and reduced-graphene oxide (rGO) following the recommendations of the European Food Safety Authority (EFSA). Thus, the mouse lymphoma assay (MLA) and the micronucleus test (MN) were performed in L5178YTk ± cells, and the Caco-2 cells were used for the standard and modified comet assays. The results indicated that GO (0-250 μg/mL) was not mutagenic in the MLA. However, rGO revealed mutagenic activity from 250 μg/mL and 125 μg/mL after 4h and 24h of exposure, respectively. In the MN test, negative results were obtained for both compounds at the concentrations assayed (0-250 μg/mL) for GO/rGO. Moreover, no DNA strand breaks, or oxidative DNA damage were detected in Caco-2 cells exposed to GO (0-250 μg/mL) and rGO (0-176.3 μg/mL for 24h and 0-166.5 μg/mL for 48h). Considering the mutagenic potential of rGO observed further investigation is needed to describe its toxic profile., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Taking advantage of the high functionalization capacity of poly(vinyl alcohol) (PVA), bead-free homogeneous nanofibrous mats were produced. The addition of functional groups by means of grafting strategies such as the sulfonation and the addition of nanoparticles such as graphene oxide (GO) were considered to bring new features to PVA. Two series of sulfonated and nonsulfonated composite nanofibers, with different compositions of GO, were prepared by electrospinning. The use of sulfosuccinic acid (SSA) allowed crosslinked and functionalized mats with controlled size and morphology to be obtained. The functionalization of the main chain of the PVA and the determination of the optimum composition of GO were analyzed in terms of the nanofibrous morphology, the chemical structure, the thermal properties, and conductivity. The crosslinking and the sulfonation treatment decreased the average fiber diameter of the nanofibers, which were electrical insulators regardless of the composition. The addition of small amounts of GO contributed to the retention of humidity, which significantly increased the proton conductivity. Although the single sulfonation of the polymer matrix produced a decrease in the proton conductivity, the combination of the sulfonation, the crosslinking, and the addition of GO enhanced the proton conductivity. The proposed nanofibers can be considered as good candidates for being exploited as valuable components for ionic polyelectrolyte membranes.