Graphene has drawn a lot of interest in the material community due to unique physicochemical properties. Owing to a high surface area to volume ratio and free oxygen groups, the oxidized derivative, graphene oxide (GO) has promising potential as a drug delivery system. Here, the lung tolerability of two distinct GO varying in lateral dimensions is investigated, to reveal the most suitable candidate platform for pulmonary drug delivery. Following repeated chronic pulmonary exposure of mice to GO sheet suspensions, the innate and adaptive immune responses are studied. An acute and transient influx of neutrophils and eosinophils in the alveolar space, together with the replacement of alveolar macrophages by interstitial ones and a significant activation toward anti-inflammatory subsets, are found for both GO materials. Micrometric GO give rise to persistent multinucleated macrophages and granulomas. However, neither adaptive immune response nor lung tissue remodeling are induced after exposure to micrometric GO. Concurrently, milder effects and faster tissue recovery, both associated to a faster clearance from the respiratory tract, are found for nanometric GO, suggesting a greater lung tolerability. Taken together, these results highlight the importance of dimensions in the design of biocompatible 2D materials for pulmonary drug delivery system.

Background A key aspect of any new material safety assessment is the evaluation of their in vivo genotoxicity. Graphene oxide (GO) has been studied for many promising applications, but there are remaining concerns about its safety profile, especially after inhalation. Herein we tested whether GO lateral dimension, comparing micrometric (LGO) and nanometric (USGO) GO sheets, has a role in the formation of DNA double strand breaks in mouse lungs. We used spatial resolution and differential cell type analysis to measure DNA damages in both epithelial and immune cells, after either single or repeated exposure. Results GO induced DNA damages were size and dose dependent, in both exposure scenario. After single exposure to a high dose, both USGO and LGO induced significant DNA damage in the lung parenchyma, but only during the acute phase response (p < 0.05 for USGO; p < 0.01 for LGO). This was followed by a fast lung recovery at day 7 and 28 for both GOs. When evaluating the chronic impact of GO after repeated exposure, only a high dose of LGO induced long-term DNA damages in lung alveolar epithelia (at 84 days, p < 0.05). Regardless of size, low dose GO did not induce any significant DNA damage after repeated exposure. A multiparametric correlation analysis of our repeated exposure data revealed that transient or persistent inflammation and oxidative stress were associated to either recovery or persistent DNA damages. For USGO, recovery from DNA damage was correlated to efficient recovery from acute inflammation (i.e., significant secretion of SAA3, p < 0.001; infiltration of neutrophils, p < 0.01). In contrast, the persistence of LGO in lungs was associated to a long-lasting presence of multinucleated macrophages (up to 84 days, p < 0.05), an underlying inflammation (IL-1α secretion up to 28 days, p < 0.05) and the presence of persistent DNA damages at 84 days. Conclusions Overall these results highlight the importance of the exposure scenario used. We showed that LGO was more genotoxic after repeated exposure than single exposure due to persistent lung inflammation. These findings are important in the context of human health risk assessment and toward establishing recommendations for a safe use of graphene based materials in the workplace.

Graphene‐based materials (GBMs) have promising applications in various sectors, including pulmonary nanomedicine. Nevertheless, the influence of GBM physicochemical characteristics on their fate and impact in lung has not been thoroughly addressed. To fill this gap, the biological response, distribution, and bio‐persistence of four different GBMs in mouse lungs up to 28 days after single oropharyngeal aspiration are investigated. None of the GBMs, varying in size (large versus small) and carbon to oxygen ratio as well as thickness (few‐layers graphene (FLG) versus thin graphene oxide (GO)), induce a strong pulmonary immune response. However, recruited neutrophils internalize nanosheets better and degrade GBMs faster than macrophages, revealing their crucial role in the elimination of small GBMs. In contrast, large GO sheets induce more damages due to a hindered degradation and long‐term persistence in macrophages. Overall, small dimensions appear to be a leading feature in the design of safe GBM pulmonary nanovectors due to an enhanced degradation in phagocytes and a faster clearance from the lungs for small GBMs. Thickness also plays an important role, since decreased material loading in alveolar phagocytes and faster elimination are found for FLGs compared to thinner GOs. These results are important for designing safer‐by‐design GBMs for biomedical application.

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BackgroundA key aspect of any new material safety assessment is the evaluation of their in vivo genotoxicity. Graphene oxide (GO) has been studied for many promising applications, but there are remaining concerns about its safety profile, especially after inhalation. Herein we tested whether GO lateral dimension, comparing micrometric (LGO) and nanometric (USGO) GO sheets, could have an impact on the formation of DNA double strand breaks in mouse lungs. We used spatial resolution and differential cell type analysis to decipher between DNA damages on epithelial and immune cells. Single as well as multiple lung exposures were performed to evaluate the importance of using more realistic scenario when evaluating the potential genotoxicity of materials.ResultsDNA damages induced by GO were size and dose dependent, in both single and repeated exposure scenario. A fast recovery was found after single exposure, irrespective of the dimensions of the GO sheets. However, the LGO induced long-term DNA damages in lung alveolar epithelia, at 84 days after repeated exposure. A multiparametric correlation analysis of repeated exposure data revealed that transient or persistent inflammation and oxidative stress were associated to either recovery or persistent DNA damages. For USGO, recovery from DNA damages was correlated to efficient recovery from acute inflammation. In contrast, the persistence of LGO in the lungs and the long-lasting presence of multinucleated macrophages were associated to persistent DNA damages. Conclusions Overall these results highlight the importance of the exposure scenario used. We showed that LGO was more genotoxic after repeated exposure than single exposure due to persistent lung inflammation. These findings are important in the context of human health risk assessment and toward establishing recommendations for a safe use of graphene based materials in the workplace.

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