Neutrophils were previously shown to digest oxidized carbon nanotubes through a myeloperoxidase (MPO)-dependent mechanism, and graphene oxide (GO) was found to undergo degradation when incubated with purified MPO, but there are no studies to date showing degradation of GO by neutrophils. Here we produced endotoxin-free GO by a modified Hummers' method and asked whether primary human neutrophils stimulated to produce neutrophil extracellular traps or activated to undergo degranulation are capable of digesting GO. Biodegradation was assessed using a range of techniques including Raman spectroscopy, transmission electron microscopy, atomic force microscopy, and mass spectrometry. GO sheets of differing lateral dimensions were effectively degraded by neutrophils. As the degradation products could have toxicological implications, we also evaluated the impact of degraded GO on the bronchial epithelial cell line BEAS-2B. MPO-degraded GO was found to be non-cytotoxic and did not elicit any DNA damage. Taken together, these studies have shown that neutrophils can digest GO and that the biodegraded GO is non-toxic for human lung cells.

Understanding the biomolecular interactions between graphene and human immune cells is a prerequisite for its utilization as a diagnostic or therapeutic tool. To characterize the complex interactions between graphene and immune cells, we propose an integrative analytical pipeline encompassing the evaluation of molecular and cellular parameters. Herein, we use single-cell mass cytometry to dissect the effects of graphene oxide (GO) and GO functionalized with amino groups (GONH2) on 15 immune cell populations, interrogating 30 markers at the single-cell level. Next, the integration of single-cell mass cytometry with genome-wide transcriptome analysis shows that the amine groups reduce the perturbations caused by GO on cell metabolism and increase biocompatibility. Moreover, GONH2 polarizes T-cell and monocyte activation toward a T helper-1/M1 immune response. This study describes an innovative approach for the analysis of the effects of nanomaterials on distinct immune cells, laying the foundation for the incorporation of single-cell mass cytometry on the experimental pipeline.

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.