Interactions of graphene oxide and graphene nanoplatelets with the in vitro Caco-2/HT29 model of intestinal barrier

Abstract

Carbon-based nanomaterials are being increasingly used, demanding strong information to support their safety in terms of human health. As ingestion is one of the most important exposure routes in humans, we have determined their potential risk by using an in vitro model simulating the human intestinal barrier and evaluated the effects of both graphene oxide (GO) and graphene nanoplatelets (GNPs). A coculture of differentiated Caco-2/HT29 cells presenting inherent intestinal epithelium characteristics (i.e. mucus secretion, brush border, tight junctions, etc.) were treated with GO or GNPs for 24 h. Different endpoints such as viability, membrane integrity, NPs localization, cytokines secretion, and genotoxic damage were evaluated to have a wide view of their potentially harmful effects. No cytotoxic effects were observed in the cells that constitute the barrier model. In the same way, no adverse effects were detected neither in the integrity of the barrier (TEER) nor in its permeability (LY). Nevertheless, a different bio-adhesion and biodistribution behavior was observed for GO and GNPs by confocal microscopy analysis, with a more relevant uptake of GNPs. No oxidative damage induction was detected, either by the DCFH-DA assay or the FPG enzyme in the comet assay. Conversely, both GO and GNPs were able to induce DNA breaks, as observed in the comet assay. Finally, low levels of anti-inflammatory cytokines were detected, suggesting a weak anti-inflammatory response. Our results show the moderate/severe risk posed by GO/GNPs exposures, given the observed genotoxic effects, suggesting that more extensive genotoxic evaluations must be done to properly assess the genotoxic hazard of these nanomaterials. Carbon-based nanomaterials are being increasingly used, due to their growing relevance in many fields. Their large surface area and their excellent electric conductivity offer many physicochemical advantages that are exploited in many industrial fields, including biomedical applications 1. Nevertheless, as it occurs with other nanomaterials, its wide use supposes an increased hazard for human health 2. One of the most recently discovered carbon-based nanomaterials is graphene. Since its first description in 2004 3 , different modifications have been done in their single-atom-thick sheet of planar sp 2-bound carbons, creating numerous graphene-based nanomaterials. According to the EU Graphene Flagship project, these materials can be classified according to three different characteristics: (i) the number of graphene layers, (ii) the average lateral size, and (iii) the carbon/oxygen ratio 4,5. Although a large amount of literature associated with graphene-based nanomaterials has been generated, only a few of them focus on toxicity aspects 2,6. Hence, this paper focuses on graphene oxide (GO) and graphene nanoplatelets (GNPs) exposure risk, two graphene-based nanomaterials that have shown great potential for biomedical and polymeric composites applications 7,8. GO is a highly-oxidized form of graphene, containing different ...

Ex Vivo Human Colon Tissue Exposure to Pristine Graphene Activates Genes Involved in the Binding, Adhesion and Proliferation of Epithelial Cells

“…Binding or adhesion of graphene to the surface of colonic cells was detected at 2 h and sustained a gene expression response for key genes including KRAS, TERT, SPP1, BIRC5, and TGFB1 . In our study, the binding or adhesion of graphene to epithelial or immune cells could have led to the downstream modulation of molecular and catalytic activity, as shown in an earlier study [ 39 ]. Furthermore, hydrophobic materials are more likely to adhere to the lipid bilayers of cell membranes [ 40 , 41 ].…”

Genotoxicity of Nanomaterials: Advanced In Vitro Models and High Throughput Methods for Human Hazard Assessment—A Review

“…High-aspect-ratio MWCNTs were found to be more toxic than the low-aspect-ratio MWCNTs [ 83 ]. Studies on interactions of graphene oxide and graphene nanoplatelets with the in vitro intestinal barrier model resulted in no oxidative damage induction [ 87 ]. Whereas MWCNTs induced significant DNA damage [ 88 , 89 ], an increase in 8 nitroguanine [ 88 , 90 ] and an increase in micronucleused cells [ 88 , 91 ].…”

A review on nanotoxicity and nanogenotoxicity of different shapes of nanomaterials

“…T A B L E 1 (Continued) (Alaraby et al, 2015a;Alaraby et al, 2020;Demir, Vales, Kaya, Creus, & Marcos, 2011;Demir, Burguru, et al, 2013;Demir et al, 2013aDemir et al, , 2013bDemir, Creus, & Marcos, 2014;Demir, Akça, et al, 2015;Demir et al, , 2020Demir & Castranova, 2016Demir & Marcos, 2018a, 2018bDomenech, Hernández, Demir, Marcos, & Cortés, 2020;Habas et al, 2018;Huang et al, 2013;Tokgun et al, 2015;. Although several studies focused on toxicity and genotoxicity of NMs in the literature (Alaraby, Annangi, Marcos, & Hernández, 2016;Kermanizadeh et al, 2016), we attempt to assess toxicity and genotoxicity caused by different shapes of NMs (NRs, NWs, and NSs).…”

“…Over recent decades, considerable research has been carried out employing in vivo and in vitro model organisms to determine potential effects of various NPs, with little or no specific attention to shape of the particles (Alaraby et al, 2015a; Alaraby et al, 2020; Alaraby, Demir, Hernández, & Marcos, 2015; Demir, Vales, Kaya, Creus, & Marcos, 2011; Demir, Burguru, et al, 2013; Demir et al, 2013a, 2013b; Demir, Creus, & Marcos, 2014; Demir, Kaya, & Kaya, 2014; Demir et al, 2014; Demir, Akça, et al, 2015; Demir, Aksakal, et al, 2015; Demir et al, 2017, 2020; Demir & Castranova, 2016, 2017; Demir & Marcos, 2018a, 2018b; Domenech, Hernández, Demir, Marcos, & Cortés, 2020; Habas et al, 2018; Huang et al, 2013; Tokgun et al, 2015; Vales, Demir, Kaya, Creus, & Marcos, 2013).…”