Nanomaterials can enter into the body through different routes, and inhalation, dermal adsorption, and ingestion are the most likely routes of unintended exposure in occupational or environmental settings. (96,97) Dermal injection is pertinent in the particular case of tattoo pigments, some of which are nanosized. (98) Parenteral administration and primarily intravenous injection is relevant for intentional exposure to nanomaterials designed for specific medical applications. Nanomaterials may subsequently travel through the body and reach sites beyond their initial portal of entry, as discussed above. However, at some point, the materials manifest their biological (or toxicological) effects at the level of specific target organs. (99) The potential adverse outcomes of nanomaterial exposure have been extensively investigated over the past decade, with particular attention to common classes of nanomaterials including metal and metal oxide nanoparticles and carbon-based materials, especially carbon nanotubes. These studies have informed us on specific material features that contribute to toxicity, for instance, particle dissolution and the release of toxic metal ions in the case of certain metal nanoparticles, or the high aspect ratio and fiber-like dimensions in the case of long and rigid carbon nanotubes. (6) Hazard assessment of GBMs has been lagging behind, but in the past 5 years or more, the potential toxicity of GBMs has been explored in a systematic fashion bothand, in the EU-funded Graphene Flagship project and elsewhere. The following sections provide an overview of the toxicological impact of GBMs on the immune system, our primary defense against foreign intrusion, as well as the pulmonary, dermal, cardiovascular, gastrointestinal, reproductive, and central nervous systems, following which we will discuss the environmental impact of GBMs.

Immune Interactions of Graphene-Based Materials

in vivo. Hence, large GO was able to generate inflammatory responses significantly higher than those of small GO in mice after intraperitoneal injection.i.e., macrophage-like cell lines versus primary cells, as primary macrophages are far more efficient in terms of phagocytosis. It thus appears that inflammasome activation leading to IL-1β secretion may transpire for a range of carbon-based nanomaterials including carbon nanotubes as well as spherical particles and flat materials such as GO The immune system consists of complex molecular and cellular networks that protect our body from infections and other exogenous materials while maintaining tolerance to self-components. In the development of new materials, it is fundamental to assess their impact on the immune system in order to understand if the presence of such materials can be tackled, eventually leading to their elimination, or to clarify if the persistence of the materials provokes chronic diseases. (100) Macrophages are key cells of the innate immune system tasked with eliminating exogenous and endogenous materials. Therefore, it is important to know if GBMs affect the viability and/or activation of macrophages. (101) One of the first studies to address this question compared the effect of GO of different sizes on human and murine primary macrophages. (102) The three GO materials tested displayed a dose-dependent cytotoxic effect. GO of smaller lateral size (130 and 270 nm) were internalized to a higher extent in comparison to large GO (∼1320 nm), leading to significant effects on cell viability and cell activation (supporting text and Figure S2 ). In addition, a specific interaction of GO sheets with the cell membrane was noted whereby GO sheets adopted an arrangement parallel to the cell surface (designated as the cell “masking” effect). (102) Another study based on two GO materials of different sizes (350 and 2000 nm) showed opposite results. (103) Both GO materials had no effect on cell viability and were internalized by cells in an energy-dependent process but showed different intracellular locations. In addition, except for IL-10, the release of all other cytokines–chemokines including IL-6, IL-12, TNF-α, MCP-1, and IFN-γ significantly increased after 2 days in cells exposed to large GO, whereas a weak increase was measured for small GO. (103) The GO materials used in these two studies were obtained by the same Hummers’ method, and the only difference that might explain the contrasting effects is the number of layers (single-layer GO in the former study and few-layer GO in the latter study). In another recent study, large GO showed a stronger adsorption onto the plasma membrane of macrophages when compared to small GO, and this elicited more robust interaction of Toll-like receptors (TLRs) and more potent activation of the NF-κB pathway. (104) Large GO was also shown to promote M1 polarization, associated with enhanced production of inflammatory cytokines and recruitment of immune cells. These size-dependent responses to GO were also evidenced. Hence, large GO was able to generate inflammatory responses significantly higher than those of small GO in mice after intraperitoneal injection. (104) Naturally, careful characterization of the materials is crucial. In particular, endotoxin content must be controlled in any studies using immune-competent cells. Recent work in the Graphene Flagship has focused on establishing a protocol for sterile production of GO according to Hummers’ method. (26) Using this protocol, endotoxin-free GO of differing lateral dimensions (50–300 nm and 10–40 μm, respectively, thickness 1–2 nm) was produced, and cytotoxicity assessment as well as cytokine profiling was performed using primary human macrophages. (39) These studies showed that small and large GO sheets were readily internalized by macrophages without any toxicity ( Figure 3 ). Furthermore, GO did not trigger the production of pro-inflammatory TNF-α in this model. (39) However, GO was found to elicit caspase-dependent IL-1β expression, a hallmark of inflammasome activation, in LPS-primed macrophages. Moreover, a specific role of the inflammasome sensor, NLRP3, in GO-induced IL-1β secretion was demonstrated. In contrast to the above-mentioned study, (104) the effects were independent of the lateral dimensions of GO. These differences could be ascribed to differences between the cell models,, macrophage-like cell linesprimary cells, as primary macrophages are far more efficient in terms of phagocytosis. It thus appears that inflammasome activation leading to IL-1β secretion may transpire for a range of carbon-based nanomaterials including carbon nanotubes as well as spherical particles and flat materials such as GO (105−109) and other exogenous materials, (100) indicating that the inflammasome functions as a universal “sensor” for xenobiotic agents. It is of interest to note that the commonly used adjuvant alum also triggers NLRP3-dependent release of IL-1β in macrophages. (110) Thus, in analogy, the immunomodulatory effects of GBMs could perhaps be harnessed for biomedical uses. (111)

Figure 3 Figure 3. Macrophage uptake of GO. Primary human monocyte-derived macrophages readily ingest GO without ultrastructural signs of acute toxicity. Macrophages were incubated for 3 h with or without small or large GO (50 μg/mL). TEM images (scale bar: 2 μm) show (a) control cells, (b) cells exposed to GO-S, and (c) cells exposed to GO-L. Internalized GO can be seen in panels (b,c). Higher-magnification micrographs (scale bar: 1 μm) show (d) control cells, (e) cells exposed to GO-S, and (f,g) cells exposed to GO-L. The asterisk in panel (e) indicates GO sheets that are undergoing internalization. The asterisk in panel (f) shows a large aggregation of GO inside the cell, whereas the image in panel (g) shows the presence of GO sheets at the plasma membrane of the cell as well as GO internalized within the cell. The asterisk marks a mitochondrion, for comparison. Finally, at higher magnification (scale bar: 200 nm), the micrographs in panels (h,i) show internalized GO-S and GO-L, respectively. Reprinted with permission from ref (39). Copyright 2018 Wiley-VCH Verlag GmbH & Co.

et al. examined a panel of GO materials prepared by a modified Hummers’ method comprising pristine, rGO, and hydrated GO (hGO) in which quantitative assessment of the hydroxyl, carboxyl, epoxy, and carbon radical contents was used to study the impact on epithelial cells and macrophages as well as in the murine lung. In a recent study, Liexamined a panel of GO materials prepared by a modified Hummers’ method comprising pristine, rGO, and hydrated GO (hGO) in which quantitative assessment of the hydroxyl, carboxyl, epoxy, and carbon radical contents was used to study the impact on epithelial cells and macrophages as well as in the murine lung. (112) The authors could show that hGO, which exhibited the highest carbon radical density, triggered cell death in THP-1 and BEAS-2B cells with attendant lipid peroxidation of the cell membrane, albeit at relatively high concentrations (up to 200 μg/mL). The authors also demonstrated that hGO was more prone than the other materials to trigger lung inflammation, accompanied by lipid peroxidation in alveolar macrophages. (112) Thus, carbon radical content plays an important role for toxicity of GO.

et al. found that small GO (350 nm) induced the formation of small vacuoles in RAW264.7 cells without causing apparent cell death.et al. prepared GO using Hummers’ method and measured the amount of the model protein, bovine serum albumin (BSA), adsorbed to GO. They found that the loading capacity was, respectively, ∼9-fold and ∼1.8-fold higher than that of BSA to multiwalled and single-walled carbon nanotubes.et al. prepared a series of GO derivatives including aminated GO (GO-NH 2 ), poly(acrylamide)-functionalized GO (GO-PAM), poly(acrylic acid)-functionalized GO (GO-PAA), and poly(ethylene glycol)-functionalized GO (GO-PEG) and compared their toxicity with that of pristine GO. In addition to primary macrophages, the effects of GO were studied on macrophage-like cell lines. Chenfound that small GO (350 nm) induced the formation of small vacuoles in RAW264.7 cells without causing apparent cell death. (113) Increasing the GO concentration triggered the formation of more vacuoles and significant cell death. In addition, GO treatment provoked TLR signaling and triggered consequent cytokine responses. Molecular analysis identified that TLR4 and TLR9 and their downstream signaling mediators MyD88, TRAF6, and NFkB played critical roles in the GO-induced inflammatory responses. (113) This was confirmed in a subsequent study, in which necrotic cell death was shown to be mediated by activation of TLR4. (114) In contrast, large GO (average lateral dimension ∼1 μm) did not activate TLR2 or TLR4 reporter cell lines, whereas single-walled carbon nanotubes (with or without a protein corona) activated TLR signaling with subsequent chemokine release. (115) Protein adsorption biocorona formation is reminiscent of the process of opsonization whereby microorganisms or apoptotic cells are “tagged” for phagocytosis with antibodies, complement factors, or other soluble proteins. (116,117) The impact of protein adsorption or biocorona formation on cell interactions of GBMs has been explored in a few studies using human cell lines. Huprepared GO using Hummers’ method and measured the amount of the model protein, bovine serum albumin (BSA), adsorbed to GO. They found that the loading capacity was, respectively, ∼9-fold and ∼1.8-fold higher than that of BSA to multiwalled and single-walled carbon nanotubes. (118) The data suggested that GO possessed an exceptionally high adsorption capacity arising from the 2D structure that provides a very high surface-to-volume ratio. In addition, GO possesses many surface defects that could serve as binding sites for proteins, and this could contribute to the observed differences in protein adsorption ability of GO and carbon nanotubes. In another study, protein adsorption was confirmed using experimental and theoretical approaches, and the authors proposed that protein-coated GO sheets lack the capacity for destructive membrane interactions due to the increase in the thickness of the GO sheets and reduction of the available surface area of GO, instead exposing largely hydrophilic surfaces that may lead to more benign interactions with membrane phospholipids. (119) To improve the biocompatibility of pristine GO, Xuprepared a series of GO derivatives including aminated GO (GO-NH), poly(acrylamide)-functionalized GO (GO-PAM), poly(acrylic acid)-functionalized GO (GO-PAA), and poly(ethylene glycol)-functionalized GO (GO-PEG) and compared their toxicity with that of pristine GO. (120) Among these GO derivatives, GO-PEG and GO-PAA induced less toxicity than pristine GO, and GO-PAA was the most biocompatible material. The differences in biocompatibility were suggested to be due to the differential compositions of the protein corona, formed on their surfaces that determine their cell interactions and pro-inflammatory effects. (120) In another recent study, coating of GO with complement factor H afforded almost complete protection (>90% reduction) against complement activation, suggesting that a “stealth” effect can be achieved through purposeful biocorona formation. (121) By contrast, coating of GO with serum albumins achieved moderate protection (∼40% reduction), whereas immunoglobulin G amplified complement activation by several-fold.

v β 8 is involved in initiating signal transduction related to the membrane binding of PEGylated GO.i.e., the morphology and density of PEG chains on the surface of GO) should be carefully evaluated, and endotoxin contamination should be excluded. PEGylation was shown in several studies to reduce the cytotoxic effects of GO on macrophages. (122−124) However, a recent report suggested that PEGylation of small GO flakes (single-layer, ∼200 nm in lateral size) resulted in the stimulation of a potent cytokine response, despite not being internalized by macrophages. (125) The authors performed extensive molecular dynamics simulations of pristine and PEGylated GO in the presence of lipid membranes. PEGylated GO appeared to preferentially adsorb onto and partially insert into cell membranes, thereby amplifying the interactions with stimulatory surface receptors. The authors also put forward the hypothesis that the integrin αis involved in initiating signal transduction related to the membrane binding of PEGylated GO. (125) Overall, these results are surprising as they suggest that PEGylation does not lead to passivation but, instead, might lead to macrophage activation. Clearly, not only the characteristics of the parent material (GO) but also the surface modification (, the morphology and density of PEG chains on the surface of GO) should be carefully evaluated, and endotoxin contamination should be excluded.

et al. performed studies using the rat NR8383 alveolar macrophage cell line as a model to predict the pulmonary toxicity of 18 different inorganic nanomaterials including graphite nanoplatelets and distinguish active from passive nanomaterials.in vitro passive materials. Neutrophils are among the first cells to be recruited in the airways upon pulmonary exposure to GBMs and also play a key role in inflammation in many other tissues. Interestingly, a recent study has shown that when GO sheets interact with isolated human neutrophils, this triggers a dose-dependent loss of cell viability and size-dependent formation of neutrophil extracellular traps (NETs). Macrophages belong to the front line of the innate immune defense against pathogens or foreign materials. (111) Most studies on macrophages have been performed using macrophage-like cell lines or monocyte-derived macrophages. However, alveolar macrophages are likely one of the first cell types, along with epithelial cells, to interact with GBMs, reaching the lungs after pulmonary exposure. Studies on alveolar macrophages are scarce, but Weimannperformed studies using the rat NR8383 alveolar macrophage cell line as a model to predict the pulmonary toxicity of 18 different inorganic nanomaterials including graphite nanoplatelets and distinguish active from passive nanomaterials. (126) Graphite nanoplatelets (<30 μm flakes) were classified aspassive materials. Neutrophils are among the first cells to be recruited in the airways upon pulmonary exposure to GBMs and also play a key role in inflammation in many other tissues. Interestingly, a recent study has shown that when GO sheets interact with isolated human neutrophils, this triggers a dose-dependent loss of cell viability and size-dependent formation of neutrophil extracellular traps (NETs). (127) NETs consist of nuclear chromatin decorated with granule proteins such as neutrophil elastase (NE) and myeloperoxidase (MPO), and these structures are normally deployed by neutrophils for extracellular destruction of pathogens. In the latter study, the effects of GO were attributed to cholesterol oxidation in the plasma membrane, as evidenced by time-of-flight secondary ion mass spectrometry (ToF-SIMS) of exposed cells. (127) The latter study underscores the importance of direct membrane interactions of 2D materials and implies that immune cells may respond to such materials in a manner that is comparable with immune responses to bacteria and fungi.

et al. investigated the interactions of graphene and FLG microsheets with macrophages and other cell types and with model lipid membranes by combining molecular dynamics simulations with confocal fluorescence imaging and electron microscopic imaging. In contrast to GO, there are relatively fewer studies on graphene and its effects on the immune system. As graphene is too hydrophobic to obtain homogeneous dispersions in aqueous solutions, it is necessary to use appropriate biocompatible surfactants or coating molecules. In a recent study, Graphene Flaghsip scientists discovered that FLG obtained by solvent-free ball-milling treatment of graphite in the presence of melamine, subsequently dispersed in cell culture medium, is able to specifically kill monocytes while preserving the viability of macrophages. (128) The capacity of FLG to trigger monocyte cell death was exploited to selectively kill monocytoid cancer cells isolated from patients affected by myelomonocytic leukemia. One of the most biocompatible surfactants used to disperse nanomaterials is pluronic F108. It has been found that exposure of macrophages to graphene in 1% pluronic decreases cell viability in a dose-dependent manner. This graphene significantly stimulated the secretion of Th1/Th2 cytokines and chemokines, and the morphology of naïve macrophages was altered, with reduced capacity to adhere to the extracellular matrix and attenuated phagocytic capacity. (129) The same type of material, again dispersed in 1% pluronic, can induce cytotoxic effects with dissipation of the mitochondrial membrane potential and increase of intracellular reactive oxygen species (ROS), resulting in apoptosis. (130) Graphene or graphene that had undergone a direct oxidative process to introduce oxygenated species on its surface was also tested upon dispersion in physiological medium. Both materials do not cause any premature immune cell activation or suppression up to 75 μg/mL after 72 h of incubation. Macrophages showed relatively high intracellular uptake of oxidized, hydrophilic graphene compared to the hydrophobic graphene, which was found to be mainly retained on the cell surface and induced ROS-mediated apoptosis above 50 μg/mL. (131) When graphite is only partially exfoliated, a material in the micrometer lateral size range composed of multilayers of stacked graphene sheets (incorrectly called nanographite) can be isolated. Further treatment with strong acid generates oxidized (micro)graphite. Both types of (micro)graphite were shown to trigger a weak cytotoxicity with dose-dependent pro-inflammatory cytokine release. (132) Liinvestigated the interactions of graphene and FLG microsheets with macrophages and other cell types and with model lipid membranes by combining molecular dynamics simulations with confocal fluorescence imaging and electron microscopic imaging. (133) The imaging experiments suggested edge-first uptake of FLG into cells. The authors speculated that the ability of large graphene microsheets to penetrate and enter cells, documented experimentally and through simulations, may lead to cytoskeletal disruption, impaired cell motility, compromised epithelial barrier function, or other “geometric and steric effects”. (133) However, the lack of cell viability tests precluded any quantification of possible cellular damage.

et al. investigated the effects on human immune cells of two types of thoroughly characterized GO sheets, differing in their lateral size distribution [small GO (GO-S) 100–500 nm, and large GO (GO-L) 1–10 μm], by using a wide range of assays including whole-genome microarray analysis and single-cell mass cytometry. 2 ), these materials resulted more specific, affecting the production of only a few cytokines in selected cell subpopulations. Taken together, these studies confirmed that the functionalization of GO significantly affected the number of transcripts altered by graphene. High-throughput technologies have revolutionized the analysis of immune cells and their complex interactions. Consequently, a comprehensive analysis of how the immune system interacts with nanomaterials is only possible by the adoption of system biology approaches and high-throughput tools that permit multiplex analysis of cell type, cell activation state, and soluble mediators of stimulation/inhibition of immune cells. In a recent study conducted in the framework of the JTC 2015 FLAGERA call (G-Immunomics project), Orecchioniinvestigated the effects on human immune cells of two types of thoroughly characterized GO sheets, differing in their lateral size distribution [small GO (GO-S) 100–500 nm, and large GO (GO-L) 1–10 μm], by using a wide range of assays including whole-genome microarray analysis and single-cell mass cytometry. (134) Exposure of peripheral blood mononuclear cells from healthy donors to small GO sheets was found to have a more significant impact when compared to large GO sheets, as reflected in the upregulation of critical genes implicated in immune responses and the release of the pro-inflammatory cytokines, IL-1β and TNF-α. These findings were confirmed by genomics approaches using Jurkat T cells as representative for the adaptive immune system and THP-1 cells, a monocytic cell line representative of the innate immune system. The microarray studies identified the activation of some relevant immune pathways correlated with T cell chemotaxis/T cell migration, regulation of T cell chemotaxis, and leukocyte chemotaxis pathways. (134) This work suggested that small GO could elicit an innate and also an adaptive response boosting a strong recruitment of immune cells, potentially providing a first step toward unconventional strategies for nanobased immunotherapeutics. Moreover, by using single-cell mass cytometry, multidimensional cytometry experiments in which simultaneous investigations of 15 immune cell populations with interrogation of 30 markers at the single-cell level was possible. (134) GO caused a broad, non-cell-specific activation triggering the production of all cytokines analyzed in a wide variety of cell populations. Following surface functionalization of the two GO with amino groups (GO-NH), these materials resulted more specific, affecting the production of only a few cytokines in selected cell subpopulations. Taken together, these studies confirmed that the functionalization of GO significantly affected the number of transcripts altered by graphene. (134) Moreover, functionalization of GO with amino groups increased the biocompatibility. The study lays the foundation for an innovative approach for multidimensional, high-throughput analysis of the effects of GBMs on immune cells ( Figure 4 ).

Figure 4 Figure 4. Dissecting the immunological impact of graphene using single-cell mass cytometry. SPADE (spanning tree progression analysis of density-normalized events) clustering algorithm analysis of significantly secreted cytokines. The tree plots show the different immune cell subpopulations, and the size of each cluster in the tree indicates the relative frequency of cells that fall within the dimensional confines of the node boundaries. Node color is scaled to the median intensity of marker expression of the cells within each node, expressed as a percentage of the maximum value in the data set: (a) IL-6; (b) TNF-α, and (c) MIP-1β for GO (left) and GO-NH 2 (right). Reprinted with permission from ref (134). Copyright 2017 Nature Publishing Group.