While the antibacterial properties of graphene oxide (GO) have been demonstrated across a spectrum of bacteria, the critical role of functional groups is unclear. To address this important issue, we utilized reduction and hydration methods to establish a GO library with different oxidation, hydroxyl, and carbon radical (•C) levels that can be used to study the impact on antibacterial activity. Using antibiotic-resistant bacteria as a test platform, we found that the •C density is most proximately associated with bacterial killing. Accordingly, hydrated GO (hGO), with the highest •C density, had the strongest antibacterial effects through membrane binding and induction of lipid peroxidation. To explore its potential applications, we demonstrated that coating of catheter and glass surfaces with hGO is capable of killing drug-resistant bacteria. In summary, •C is the principle surface moiety that can be utilized for clinical applications of GO-based antibacterial coatings.

The electronic properties of single-walled carbon nanotubes (SWCNTs) are potentially useful for electronics, optics, and sensing applications. Depending on the chirality and diameter, individual SWCNTs can be classified as semiconducting (S-SWCNT) or metallic (M-SWCNT). From a biological perspective, the hazard profiling of purified metallic versus semiconducting SWCNTs has been pursued only in bacteria, with the conclusion that aggregated M-SWCNTs are more damaging to bacterial membranes than S-SWCNTs. However, no comparative studies have been performed in a mammalian system, where most toxicity studies have been undertaken using relatively crude SWCNTs that include a M:S mix at 1:2 ratio. In order to compare the toxicological impact of SWCNTs sorted to enrich them for each of the chirality on pulmonary cells and the intact lung, we used density gradient ultracentrifugation and extensive rinsing to prepare S- and M-SWCNTs that are >98% purified. In vitro screening showed that both tube variants trigger similar amounts of interleukin 1β (IL-1β) and transforming growth factor (TGF-β1) production in THP-1 and BEAS-2B cells, without cytotoxicity. Oropharyngeal aspiration confirmed that both SWCNT variants induce comparable fibrotic effects in the lung and abundance of IL-1β and TGF-β1 release in the bronchoalveolar lavage fluid. There was also no change in the morphology, membrane integrity, and viability of E. coli, in contradistinction to the previously published effects of aggregated tubes on the bacterial membrane. Collectively, these data indicate that the electronic properties and chirality do not independently impact SWCNT toxicological impact in the lung, which is of significance to the safety assessment and incremental use of purified tubes by industry.

While two-dimensional graphene oxide (GO) is used increasingly in biomedical applications, there is uncertainty on how specific physicochemical properties relate to biocompatibility in mammalian systems. Although properties such as lateral size and the colloidal properties of the nanosheets are important, the specific material properties that we address here is the oxidation state and reactive surface groups on the planar surface. In this study, we used a GO library, comprising pristine, reduced (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. Strikingly, we observed that hGO, which exhibits the highest carbon radical density, was responsible for the generation of cell death in THP-1 and BEAS-2B cells as a consequence of lipid peroxidation of the surface membrane, membrane lysis, and cell death. In contrast, pristine GO had lesser effects, while rGO showed extensive cellular uptake with minimal effects on viability. In order to see how these in vitro effects relate to adverse outcomes in the lung, mice were exposed to GOs by oropharyngeal aspiration. Animal sacrifice after 40 h demonstrated that hGO was more prone than other materials to generate acute lung inflammation, accompanied by the highest lipid peroxidation in alveolar macrophages, cytokine production (LIX, MCP-1), and LDH release in bronchoalveolar lavage fluid. Pristine GO showed less toxicity, whereas rGO had minimal effects. We demonstrate that the surface oxidation state and carbon radical content play major roles in the induction of toxicity by GO in mammalian cells and the lung.

2D molybdenum disulfide (MoS2 ) has distinct optical and electronic properties compared to aggregated MoS2 , enabling wide use of these materials for electronic and biomedical applications. However, the hazard potential of MoS2 has not been studied extensively. Here, a comprehensive analysis of the pulmonary hazard potential of three aqueous suspended forms of MoS2 -aggregated MoS2 (Agg-MoS2 ), MoS2 exfoliated by lithiation (Lit-MoS2 ), and MoS2 dispersed by Pluronic F87 (PF87-MoS2 )-is presented. No cytotoxicity is detected in THP-1 and BEAS-2B cell lines. However, Agg-MoS2 induces strong proinflammatory and profibrogenic responses in vitro. In contrast, Lit- and PF87-MoS2 have little or no effect. In an acute toxicity study in mice, Agg-MoS2 induces acute lung inflammation, while Lit-MoS2 and PF87-MoS2 have little or no effect. In a subchronic study, there is no evidence of pulmonary fibrosis in response to all forms of MoS2 . These data suggest that exfoliation attenuates the toxicity of Agg-MoS2 , which is an important consideration toward the safety evaluation and use of nanoscale MoS2 materials for industrial and biological applications.

Engineered carbonaceous nanomaterials (ECNs), including single-wall carbon nanotubes (SWCNTs), multiwall carbon nanotubes (MWCNTs), graphene, and graphene oxide (GO), are potentially hazardous to the lung. With incremental experience in the use of predictive toxicological approaches, seeking to relate ECN physicochemical properties to adverse outcome pathways (AOPs), it is logical to explore the existence of a common AOP that allows comparative analysis of broad ECN categories. We established an ECN library comprising three different types of SWCNTs, graphene, and graphene oxide (two sizes) for comparative analysis according to a cell-based AOP that also plays a role in the pathogenesis of pulmonary fibrosis. SWCNTs synthesized by Hipco, arc discharge and Co-Mo catalyst (CoMoCAT) methods were obtained in their as-prepared (AP) state, following which they were further purified (PD) or coated with Pluronic F108 (PF108) or bovine serum albumin (BSA) to improve dispersal and colloidal stability. GO was prepared as two sizes, GO-small (S) and GO-large (L), while the graphene samples were coated with BSA and PF108 to enable dispersion in aqueous solution. In vitro screening showed that AP- and PD-SWCNTs, irrespective of the method of synthesis, as well as graphene (BSA) and GO (S and L) could trigger interleukin-1β (IL-1β) and transforming growth factor-β1 (TGF-β1) production in myeloid (THP-1) and epithelial (BEAS-2B) cell lines, respectively. Oropharyngeal aspiration in mice confirmed that AP-Hipco tubes, graphene (BSA-dispersed), GO-S and GO-L could induce IL-1β and TGF-β1 production in the lung in parallel with lung fibrosis. Notably, GO-L was the most pro-fibrogenic material based on rapid kinetics of pulmonary injury. In contrast, PF108-dispersed SWCNTs and -graphene failed to exert fibrogenic effects. Collectively, these data indicate that the dispersal state and surface reactivity of ECNs play key roles in triggering a pro-fibrogenic AOP, which could prove helpful for hazard ranking and a proposed tiered testing approach for large ECN categories.