Next-generation sequencing reveals size-dependent pulmonary impact of thin graphene oxide sheets in mice: towards safe-by-design

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 we compared the effects of lateral dimensions of GO sheets in acute and chronic pulmonary responses after single intranasal instillation in mice. Micrometre-sized GO (1–30 µm) induced stronger pulmonary inflammation than nanometre-sized GO, despite a reduced translocation to the lungs. Genome-wide RNA sequencing also revealed distinct size dependent effects of GO, in agreement with the histopathological results. Although large GO, but not the smallest GO (10–300 nm), triggered the formation of granulomas that persisted up to 90 days, no pulmonary fibrosis was observed. These results could be partly explained by the pattern of size-independent in situ biotransformation of GO, as evidenced by Raman spectroscopy imaging. Our 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 production plant or applications, such as spray coating or painting, where aerosolised dispersions are likely to occur. These results are important for the development of evidence-based risk assessment, and provide information towards the implementation of a safer-by-design approach for GBM enhanced products and applications, for the benefit of workers and end-users. Overall design: The aim of the present study was to investigate whether lateral dimensions play a significant role in the pulmonary response to GO at short- and long-term after single exposure in mice. Considering the literature, we hypothesised that micrometre-sized GO sheets would induce a more deleterious pulmonary response than nanometre-sized GO after single intranasal (i.n.) instillation (50 µg/mouse). Six to 8-week old female C57BL/6 mice (Envigo, UK) were allowed to acclimatise for 7 days prior to any experiment. Experiments were carried out using 4-5 animals per group. All GO materials were dispersed in an aqueous solution of 5% (m/v) dextrose in ultrapure water (5% dextrose), at a concentration of 1 mg/mL, about 30 min before administration. MWCNTs were dispersed at the same concentration in an aqueous solution of 0.9% (m/v) sodium chloride supplemented with 0.5% (m/v) bovine serum albumin (BSA), followed by bath sonication for 10 min at 80 W (VWR, UK). Animals were anesthesised by inhalation of 2.5% isoflurane in 100% oxygen flowing at 2 L/min. Mice in experimental groups were instilled with 50 µL of the respective nanomaterial dispersions (i.e., 50 µg/mouse), which were equally distributed in each nostril. The same volume of 5% dextrose solution was given to the control group. During administration, the animals were held in a supine position, tilted to about 60°, in order to effectively introduce the full dose. Mice were observed until full recovery, which occurred within 5 min after instillation. Mice were sacrificed at days 1, 7, or 28 days after exposure by overdose via intraperitoneal injection of 2 mL pentobarbitone. At all considered time points, the lungs were carefully dissected from the large airways and small portions of each lobe were extracted for RNA extraction. Tissue samples were placed in RNAlater (Invitrogen, Thermo Fisher Scientific, UK), and placed immediately on ice. Samples in RNAlater solution were homogenised using a TissueLyser LT (QIAGEN, UK). Homogenised samples were then loaded onto spin cartridges containing silica membranes, for extraction of total RNA performed using a PureLink RNA Mini kit (Invitrogen, Thermo Fisher Scientific, UK) according to the manufacturer's instructions. Total RNA concentration and purity were calculated by measuring the optical density at 230, 260 and 280 nm, using a Biophotometer Plus spectrophotometer (Eppendorf AG, Germany). RNA quantity and RNA integrity number (RIN) were assessed by Agilent 2200 bioanalyzer. Sample RNA with RIN values below 8 was discarded. The isolated RNA samples were stored at -80°C until RNA sequencing was carried out using HiSeq2500 sequencer.