Abstract
The interest in graphene and its translation into commercial products has been expanding at a high pace. Based on previously described pulmonary safety concerns for carbon nanomaterials, there is a great need to define parameters guiding interactions between graphene-based materials and the pulmonary system. The aim of the present study was to determine the importance of two critical parameters: lateral dimensions of the material and coating with proteins in relation to each other and their pulmonary impact. Endotoxin-free materials with distinct lateral dimensions, s-GO (50-200 nm) and l-GO (5-15 μm), were produced and thoroughly characterized. Exploiting intrinsic fluorescence of graphene oxide (GO) and using confocal live-cell imaging, the behavior of the cells in response to the material was visualized in real time. Although BEAS-2B cells internalized GO efficiently, l-GO was linked to higher plasma membrane interactions correlated with elevated reactive oxygen species (ROS) levels, pro-inflammatory response, and greater cytotoxicity, in agreement with the oxidative stress paradigm. For both GO types, the presence of serum alleviated lipid peroxidation of plasma membrane and decreased intracellular ROS levels. However, protein coating was not enough to entirely mitigate toxicity and inflammatory response induced by l-GO. In vitro results were validated in vivo, as l-GO was more prone to induce pulmonary granulomatous response in mice compared to s-GO. In conclusion, the lateral dimension of GO played a more important role than serum protein coating in determining biological responses to the material. It was also demonstrated that time-lapse imaging of live cells interacting with label-free GO sheets can be used as a tool to assess GO-induced cytotoxicity.
Highlights
The interest in graphene and its translation into commercial products have been expanding during the past few years
atomic force microscopy (AFM) showed that the average thickness for both materials was approximately 1−2 nm (Figure S1a), indicating that both graphene oxide (GO) samples were mainly composed of monolayer sheets
Protein coating of GO was shown to have a secondary role in comparison to the effect of lateral dimensions in inducing oxidative-stress-mediated cellular responses
Summary
The interest in graphene and its translation into commercial products have been expanding during the past few years. Hazard assessment studies have indicated that structure− function relationships should be based on specific physicochemical properties of the studied nanomaterial Both intrinsic (e.g., physicochemical properties) and acquired (e.g., aggregation state in local milieu or secondary coating with components from the cell culture medium, blood, or different organs) features need to be taken into consideration before obtaining a clear understanding of the parameters that could lead to adverse effects.[7] In addition, the oxidative stress paradigm has been shown to successfully explain and predict the outcomes of the cellular response to many nanomaterials.[8−10] According to this paradigm, cellular response to nanomaterials is governed by induction of oxidative stress, which can either be counterbalanced by the activity of antioxidative enzymes (tier 1 response), lead to subsequent activation of pro-inflammatory pathways (tier 2 response), or result in cell death in the case of excessive levels of reactive oxygen species (ROS) production (tier 3 response)
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