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. 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.

包括氧化石墨烯（graphene oxide, GO）在内的石墨烯基材料（graphene-based materials, GBMs）的安全性评估，对于其在社会诸多领域的安全应用至关重要。尤为关键的是，特定材料属性与生物学效应之间的关联仍有待进一步阐明。本研究通过小鼠单次鼻腔滴注给药，对比了氧化石墨烯片层横向尺寸对其急性与慢性肺部应答的影响。尽管微米级氧化石墨烯（1~30 μm）在肺部的转运能力弱于纳米级氧化石墨烯，但其诱导的肺部炎症反应更为强烈。全基因组RNA测序结果亦显示，氧化石墨烯的生物学效应存在显著的尺寸依赖性，这与组织病理学检测结果相符。尽管大尺寸氧化石墨烯（而非最小尺寸的10~300 nm氧化石墨烯）可诱导形成可持续长达90天的肉芽肿，但未观察到肺部纤维化现象。拉曼光谱成像结果证实，氧化石墨烯的原位生物转化模式与尺寸无关，这可部分解释上述实验现象。本研究结果表明，横向尺寸是影响氧化石墨烯肺部应答的核心因素，提示在喷涂、涂装等易产生气溶胶分散体的生产车间或应用场景中，应避免人员吸入微米级氧化石墨烯。本研究结果可为基于证据的风险评估体系构建提供支撑，同时为面向石墨烯基材料增强型产品与应用的安全设计（safer-by-design）方案落地提供参考，以保障从业人员与终端用户的健康安全。本研究旨在探究小鼠单次暴露于氧化石墨烯后，其横向尺寸是否会对氧化石墨烯的短期与长期肺部应答产生显著影响。基于已有文献，本研究提出假设：单次鼻腔滴注（intranasal, i.n.），剂量为50 μg/只小鼠后，微米级氧化石墨烯片层诱导的肺部损伤效应将比纳米级氧化石墨烯更为显著。所有实验开展前，均将6~8周龄的雌性C57BL/6小鼠（Envigo, UK）适应性饲养7天。每组实验动物数量为4~5只。所有氧化石墨烯材料均于给药前约30分钟，分散于超纯水配制的5%（m/v，质量体积比）葡萄糖水溶液（5% dextrose）中，浓度为1 mg/mL。多壁碳纳米管（multi-walled carbon nanotubes, MWCNTs）则以相同浓度分散于添加了0.5%（m/v）牛血清白蛋白（bovine serum albumin, BSA）的0.9%（m/v）氯化钠水溶液中，随后在80 W功率下进行水浴超声10分钟（VWR, UK）。实验动物通过吸入2.5%异氟烷（以2 L/min流速流经100%氧气）进行麻醉。实验组小鼠经双侧鼻腔滴注50 μL相应的纳米材料分散液（即50 μg/只小鼠）。对照组小鼠则给予等量的5%葡萄糖溶液。给药过程中，实验动物取仰卧位并倾斜约60°，以确保完全滴注给药剂量。滴注后密切观察小鼠直至完全苏醒，该过程通常在5分钟内完成。分别于暴露后第1、7或28天，通过腹腔注射2 mL戊巴比妥钠实施安乐死。在各预设时间点，小心分离肺部与大气道，并摘取各肺叶的小块组织用于RNA提取。组织样本置于RNAlater试剂（Invitrogen, Thermo Fisher Scientific, UK）中，并立即置于冰上。使用TissueLyser LT匀浆器（QIAGEN, UK）对RNAlater溶液中的样本进行匀浆处理。将匀浆后的样本加载至含硅胶膜的离心柱中，按照操作手册使用PureLink RNA Mini试剂盒（Invitrogen, Thermo Fisher Scientific, UK）提取总RNA。使用Biophotometer Plus分光光度计（Eppendorf AG, Germany），通过检测230 nm、260 nm与280 nm处的光密度值，计算总RNA的浓度与纯度。使用Agilent 2200生物分析仪评估RNA总量与RNA完整性指数（RNA integrity number, RIN）。RIN值低于8的RNA样本将被弃用。提取的RNA样本保存于-80℃冰箱，直至使用HiSeq2500测序仪进行RNA测序。