Size‐Dependent Pulmonary Impact of Thin Graphene Oxide Sheets in Mice: Toward Safe‐by‐Design

Abstract Safety assessment of graphene‐based materials (GBMs) including graphene oxide (GO) is essential for their safe use across many sectors of society. In particular, the link between specific material properties and biological effects needs to be further elucidated. Here, the effects of lateral dimensions of GO sheets in acute and chronic pulmonary responses after single intranasal instillation in mice are compared. Micrometer‐sized GO induces stronger pulmonary inflammation than nanometer‐sized GO, despite reduced translocation to the lungs. Genome‐wide RNA sequencing also reveals distinct size‐dependent effects of GO, in agreement with the histopathological results. Although large GO, but not the smallest GO, triggers the formation of granulomas that persists for up to 90 days, no pulmonary fibrosis is observed. These latter results can be partly explained by Raman imaging, which evidences the progressive biotransformation of GO into less graphitic structures. The findings demonstrate that lateral dimensions play a fundamental role in the pulmonary response to GO, and suggest that airborne exposure to micrometer‐sized GO should be avoided in the production plant or applications, where aerosolized dispersions are likely to occur. These results are important toward the implementation of a safer‐by‐design approach for GBM products and applications, for the benefit of workers and end‐users.


SUPPLEMENTARY TABLE LEGENDS
. GO uptake by different organs after intranasal instillation. Amount of instilled dose was normalised by dry tissue weight (mg/g). Values correspond to mean ± SD. Table S4. Antibody-drug conjugates used to stain lung cells for flow cytometry. Stability in physiological media such as 50% serum or PBS was determined in vitro, after incubating dispersions for up to 7 days at a temperature of 37 o C. All GO-DOTA probes exhibited high radiolabelling stability in physiological media.
6 Figure S2. Biodistribution of GO-DOTA[ 115 In] after intranasal instillation. Mice were instilled with DOTA-functionalised GO sheets labelled with naturally occurring 115 In, and organs were collected 1 and 7 days after exposure. Quantification of 115 In by ICP-MS in relevant extra-pulmonary organs revealed size-dependent clearance profiles, with l-GO being noticeably found in nasal cavity and GI tract, whereas s-GO translocated to the kidney and 7 us-GO translocated to the brain. No significant accumulation in the reticuloendothelial system was detected at the considered time points. Moreover, amount of GO sheets in each lung lobe was quantified to assert whether their distribution in the lungs was homogenous.
No significant differences were detected between left and right lungs, although patterns of accumulation appeared to be random. Individual data points corresponding to each animal are plotted alongside mean values ± SD (n = 4). Data are presented as % instilled dose (ID) per gram of dry tissue. Data were analysed using a 2-way ANOVA test with post hoc Sidak's multiple comparisons test. Significant differences between treatments are plotted with (*), whereas differences over time are plotted with (#). In both cases, statistical significance is reported as: (*), p < 0.05; (**), p < 0.01; (***), p < 0.001. Reference Raman spectrum of GO. (B) Raman spectra were acquired on sections of lungs from mice exposed to GO. Raman mapping analysis compares each acquired individual spectrum for each pixel of the map with this specific reference spectrum. Correlation was performed within a fixed region between 1145 and 1810 cm -1 (highlighted in grey), in order to exclude the interference of background signal from the tissue and emphasise the presence of the D and G bands. Raman mapping of lung sections from mice exposed to 5% dextrose did not evidence any sign of Raman-positive areas corresponding to the presence of GO.
Scale bars = 200 µm. Figure S5. Raman mapping of lung sections from mice exposed to l-GO. Lungs were harvested from mice exposed to l-GO at days 1, 7 and 28 post exposure. (A) Raman spectroscopy revealed large areas of strong correlation indicating the presence of GO, as shown by the correlation maps overlaying the region of interest (ROI) in bright-field images.

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The decreased correlation at days 7 and 28 suggests the occurrence of clearance and/or material biotransformation. Scale bars = 200 µm. (B) Evolution of the structure of l-GO in the lungs was assessed by calculating the ratio between the intensities of the D and G bands (I D /I G ). Position of the G band was also evaluated to determine the emergence of the D' band.
Individual data points correspond to 10 acquired spectra obtained from Raman maps, alongside mean values ± SD. Statistical analysis was performed using a Kruskal-Wallis test with post hoc Dunn's multiple comparisons test: (*), p < 0.05.
13 Figure S6. Raman mapping of lung sections from mice exposed to s-GO. Lungs were harvested from mice exposed to l-GO at days 1, 7 and 28 post exposure. (A) Raman spectroscopy revealed large areas of strong correlation indicating the presence of GO, as shown by the correlation maps overlaying the region of interest (ROI) in bright-field images.
14 The decreased correlation at days 7 and 28 suggests the occurrence of clearance and/or material biotransformation. Scale bars = 200 µm. (B) Evolution of the structure of s-GO in the lungs was assessed by calculating the ratio between the intensities of the D and G bands (I D /I G ). Position of the G band was also evaluated to determine the emergence of the D' band.
Individual data points correspond to 10 acquired spectra obtained from Raman maps, alongside mean values ± SD. Statistical analysis was performed using a Kruskal-Wallis test with post hoc Dunn's multiple comparisons test. No statistical significance was obtained.
15 Figure S7. Raman mapping of lung sections from mice exposed to us-GO. Lungs were harvested from mice exposed to l-GO at days 1, 7 and 28 post exposure.  despite an increased abundance of PMN in mice exposed to l-GO, 7 days after instillation.  The top diseases and biofunctions pathways identified by using the IPA software in lung tissue samples of mice exposed for 1, 7, and 28 days to a single dose of us-GO. DEGs having 28 ≥ 0.5 log fold change and ≥ 0.05 FDR were included in the analysis. Network analysis of DEGs identified in the lungs of mice at 28 days post-exposure was generated by IPA to predict which activators affected gene expression.

Relative abundance of lymphocytes (Lφ) and monocytic cells (Mφ
29 Figure S15. Significantly affected gene expression pathways upon exposure to s-GO. The top diseases and biofunctions pathways identified by using the IPA software in lung tissue samples of mice exposed for 1, 7, and 28 days to a single dose of s-GO. DEGs having ≥ 0.5 log fold change and ≥ 0.05 FDR were included in the analysis. Network analysis of DEGs identified in the lungs of mice at 28 days post-exposure was generated by IPA to predict which activators affected gene expression. where represents the deposited fraction of GO in the lower respiratory tract, which is the total relative instilled dose detected in lungs and trachea (see Table S1, Supporting Information). Human deposition efficiency of inhaled particles was estimated to be about 30% 24 . Interspecies extrapolation considered the normalisation of lung burden by their respective airway surface area, which is 102.2 m 2 for humans and 0.05 m 2 for mice 71 .