Due to their unique physicochemical properties, graphene-family nanomaterials (GFNs) are widely used in many fields, especially in biomedical applications. Currently, many studies have investigated the biocompatibility and toxicity of GFNs in vivo and in intro. Generally, GFNs may exert different degrees of toxicity in animals or cell models by following with different administration routes and penetrating through physiological barriers, subsequently being distributed in tissues or located in cells, eventually being excreted out of the bodies. This review collects studies on the toxic effects of GFNs in several organs and cell models. We also point out that various factors determine the toxicity of GFNs including the lateral size, surface structure, functionalization, charge, impurities, aggregations, and corona effect ect. In addition, several typical mechanisms underlying GFN toxicity have been revealed, for instance, physical destruction, oxidative stress, DNA damage, inflammatory response, apoptosis, autophagy, and necrosis. In these mechanisms, (toll-like receptors-) TLR-, transforming growth factor β- (TGF-β-) and tumor necrosis factor-alpha (TNF-α) dependent-pathways are involved in the signalling pathway network, and oxidative stress plays a crucial role in these pathways. In this review, we summarize the available information on regulating factors and the mechanisms of GFNs toxicity, and propose some challenges and suggestions for further investigations of GFNs, with the aim of completing the toxicology mechanisms, and providing suggestions to improve the biological safety of GFNs and facilitate their wide application.
Graphene, which is isolated from crystalline graphite, is a flat monolayer composed of single-atom-thick, two-dimensional sheets of a hexagonally arranged honeycomb lattice . Because of its unique structural, specific surface area and mechanical characteristics, the functions and applications of graphene have gained considerable attention since the discovery of the material in 2004 [2, 3]. Graphene and its derivatives include monolayer graphene, few-layer graphene (FLG), graphene oxide (GO), reduced graphene oxide (rGO), graphene nanosheets (GNS), and graphene nanoribbons, etc. [4–7]. GO is one of the most vital chemical graphene derivatives of the graphene-family nanomaterials (GFNs), which attracts increasing attention for its potential biomedical applications. Graphene-based materials usually have sizes ranging from several to hundreds of nanometer and are 1-10 nm thick [8, 9], which is also the definition of ‘nanoparticles’ or ‘nanomaterials’. Due to their exceptional physical and chemical properties, graphene materials have been widely used in various fields, including energy storage; nanoelectronic devices; batteries [10–12]; and biomedical applications, such as antibacterials [13, 14], biosensors [15–18], cell imaging [19, 20], drug delivery [8, 21, 22], and tissue engineering [23–25].
Along with the application and production of GFNs increasing, the risk of unintentional occupational or environmental exposure to GFNs is increasing . And recently, there are some investigation on GFNs exposure in occupational settings and published data showed that the occupational exposure of GFNs had potential toxicity to the workers and researchers [27–29]. GFNs can be delivered into bodies by intratracheal instillation , oral administration , intravenous injection , intraperitoneal injection  and subcutaneous injection . GFNs can induce acute and chronic injuries in tissues by penetrating through the blood-air barrier, blood-testis barrier, blood-brain barrier, and blood-placenta barrier etc. and accumulating in the lung, liver, and spleen etc. For example, some graphene nanomaterials aerosols can be inhaled and substantial deposition in the respiratory tract, and they can easily penetrate through the tracheobronchial airways and then transit down to the lower lung airways, resulting in the subsequent formation of granulomas, lung fibrosis and adverse health effects to exposed persons [2, 29]. Several reviews have outlined the unique properties [35, 36] and summarized the latest potential biological applications of GFNs for drug delivery, gene delivery, biosensors, tissue engineering, and neurosurgery [37–39]; assessed the biocompatibility of GFNs in cells (bacterial, mammalian and plant) [7, 40, 41] and animals (mice and zebrafish) ; collected information on the influence of GFNs in the soil and water environments . Although these reviews discussed the related safety profiles and nanotoxicology of GFNs, the specific conclusions and detailed mechanisms of toxicity were insufficient, and the mechanisms of toxicity were not summarized completely. The toxicological mechanisms of GFNs demonstrated in recent studies mainly contain inflammatory response, DNA damage, apoptosis, autophagy and necrosis etc., and those mechanisms can be collected to further explore the complex signalling pathways network regulating the toxicity of GFNs. It needs to point out that there are several factors which largely influence the toxicity of GFNs, such as the concentration, lateral dimension, surface structure and functionalization etc. Herein, this review presents a comprehensive summary of the available information on the mechanisms and regulating factors of GFNs toxicity in vitro and in vivo via different experimental methods, with the goals of providing suggestions for further studies of GFNs and completing the toxicology mechanisms to improve the biological safety of GFNs and facilitate their wide application.
Toxicity of GFNs (in vivo and in vitro)
GFNs penetrate through the physiological barriers or cellular structures by different exposure ways or administration routes and entry the body or cells, eventually resulting in toxicity in vivo and in vitro. The varying administration routes and entry paths, different tissue distribution and excretion, even the various cell uptake patterns and locations, may determine the degree of the toxicity of GFNs [44–46]. So to make them clear may be helpful to better understand the laws of the occurrence and development of GFNs toxicity.
The common administration routes in animal models include airway exposure (intranasal insufflation, intratracheal instillation, and inhalation), oral administration, intravenous injection, intraperitoneal injection and subcutaneous injection. The major exposure route for GFNs in the working environment is airway exposure, thus inhalation and intratracheal instillation are used mostly in mice to simulate human exposure to GFNs. Though the inhalation method provides the most realistic simulation to real life exposure, instillation is more effective and time-saving method, and GFNs was found that causing longer inflammation period using instillation (intratracheal instillation, intrapleural installation and pharyngeal aspiration) than inhalation [24, 30, 47, 48]. GFNs were investigated to deposit in the lungs and accumulate to a high level, which retained for more than 3 months in the lungs with slow clearing after intratracheal instillation . Intravenous injection is also widely used to assess the toxicity of graphene nanomaterials, and graphene circulates through the body of mice in 30 min, accumulating at a working concentration in the liver and bladder [32, 50–52]. However, GO derivatives had rather finite intestinal adsorption and were rapidly excreted in adult mice via oral administration [31, 53]. Nano-sized GO (350 nm) caused less mononuclear cells to infiltrate subcutaneous adipose tissue after subcutaneous injection in the neck region compared to micron-sized GO (2 μm) . GO agglomerated near the injection site after intraperitoneal injection, and numerous smaller aggregates settled in the proximity of the liver and spleen serosa [31, 33]. Experiments on skin contact with or skin permeation of GFNs were not found in the papers reviewed here, and there is insufficient evidence available to conclude that graphene can penetrate intact skin or skin lesions. The route of nasal drops, which has been widely used to test the neurotoxicity or brain injury potential of other nanomaterials, was not mentioned in the papers reviewed here.
GFNs entry paths
GFNs reach various locations through blood circulation or biological barriers after entering the body, which results in varying degrees of retention in different organs. Due to their nanosize, GFNs can reach deeper organs by passing through the normal physiological barriers, such as the blood-air barrier, blood-testis barrier, blood-brain barrier and blood-placental barrier.
The lungs are a potential entrance for graphene nanoparticles into the human body through airway. The inhaled GO nanosheets can destroy the ultrastructure and biophysical properties of pulmonary surfactant (PS) film, which is the first line of host defense, and emerge their potential toxicity . The agglomerated or dispersed particles deposit on the inner alveolar surface within the alveoli and then be engulfed by alveolar macrophages (AMs) . Clearance in the lungs is facilitated by the mucociliary escalator, AMs, or epithelial layer [56–58]. However, some small, inhaled nanoparticles infiltrate the intact lung epithelial barrier and can then transiently enter the alveolar epithelium or the interstitium [59, 60]. Intratracheally instilled graphene can redistribute to the liver and spleen by passing through the air-blood barrier . The study of blood-air barrier may draw an intensive attention, since the researchers and workers occupational exposure of GFNs usually through inhalation. To make clear how the blood-air barrier plays a role in the toxicity of GFNs may become a research hot topic.
The intricate arrangement of the blood-brain barrier, consisting of numbers of membrane receptors and highly selective carriers, only exerts subtle influence on blood circulation and the brain microenvironment compared to the peripheral vascular endothelium . The research on the mechanism of blood-brain barrier had made some progress involved in diseases and nanotoxicity. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) revealed that rGO, with an average diameter of 342 ± 23.5 nm, permeated through the paracellular pathway into the inter-endothelial cleft in a time-dependent manner by decreasing the blood-brain barrier paracellular tightness . In addition, graphene quantum dots (GQDs), with a small size of less than 100 nm, can cross through the blood-brain barrier . Studies on how graphene materials pass through the blood-brain barrier and cause neurotoxicity are very rare, and more data are needed to draw a conclusion.
The blood-testis and blood-epididymis barriers are well known for being some of the tightest blood-tissue barriers in the mammalian body . GO particles with diameters of 54.9 ± 23.1 nm had difficulty penetrating the blood-testis and blood-epididymis barriers after intra-abdominal injection, and the sperm quality of the mice was not obviously affected even at 300 mg/kg dosage .
The placental barrier is indispensable in maintaining pregnancy, as it mediates the exchange of nutrients and metabolic waste products, exerts vital metabolic functions and secretes hormones . A recent review suggested that the placenta does not provide a tight barrier against the transfer of nanoparticles to foetuses, specifically against the distribution of carbonaceous nanoparticles to and in the foetus . It was suggested that rGO and gold particles (diameter of 13 nm) are barely present or are absent in the placenta and foetus in late gestation after intravenous injection [44, 68]. However, other reports showed that transplacental transfer does occur in late gestational stages [69, 70]. Much attention had been paid to the developmental toxicity of nanomaterials, and reports showed that many nanoparticles did cross the placental barrier and strongly influenced the development of embryos [71–75]. But studies of the exposure to graphene materials through the placenta barrier are deficient, and how these particles transfer to embryos should be evaluated in detail in the future.
These four barriers were the most frequently mentioned barriers in the literature, and other barriers have not been evaluated in recent studies, such as skin barriers, which have not been mentioned in any of the hundreds of GFNs toxicity studies searched. Moreover, the mechanism by which GFNs pass through these barriers is not well understood, and more systematic investigations are urgently needed.
Distribution and excretion of GFNs in tissue
The absorption, distribution, and excretion of graphene nanoparticles may be affected by various factors including the administration routes, physicochemical properties, particle agglomeration and surface coating of GFNs.
The different administration routes influence the distribution of GFNs, for example, intratracheally instilled FLG passing through the air-blood barrier mainly accumulated and was retained in the lungs, with 47 % remaining after 4 weeks . Intravenously administered GO entered the body through blood circulation and was highly retained in the lung, liver, spleen and bone marrow, and inflammatory cell infiltration, granuloma formation and pulmonary edema were observed in the lungs of mice after intravenous injection of 10 mg kg/body weight GO . Similarly, high accumulation of PEGylated GO derivatives was observed in the reticuloendothelial (RES) system including liver and spleen after intraperitoneal injection. In contrast, GO-PEG and FLG did not show detectable gastrointestinal tract absorption or tissue uptake via oral administration .
The different properties of GFNs, such as their size, dose and functional groups, always lead to inconsistent results in the distribution profiles of graphene. For instance, Zhang et al. found that GO was mainly entrapped in mouse lungs ; however, Li et al. observed that GO accumulated in mouse liver . Notably, small GO sheets, with diameters of 10–30 nm, were mainly distributed in the liver and spleen, whereas larger GO sheets (10–800 nm) mainly accumulated in the lungs [49, 52, 77]. If the size of GO is larger than the size of the vessels, GO usually becomes stuck in the arteries and capillaries in the proximity of the injection site. The accumulation of GO in the lungs was shown to increase with an increase in the injected dose and size, but that in the liver significantly decreased . Coating biocompatible polymers onto GO also affects the biodistribution, for instance, the intravenous injection of GO-PEG and GO-dextran (GO-DEX) accumulate in the reticuloendothelial system (RES), including the liver and spleen, without short-term toxicity [31, 79]. Moreover, the charge of plasma proteins and adsorption of GO by plasma proteins also affects the biodistribution .
The excretion and clearance of GFNs vary in different organs. In the lungs, observations indicated that NGO is drawn into and cleared by AMs, which might be eliminated from the sputum through mucociliary clearance or other ways , and 46.2 % of the intratracheally instilled FLG was excreted through the faeces 28 d after exposure . In the liver, nanoparticles can be eliminated thorough the hepato-biliary pathway following the biliary duct into the duodenum . In addition, PEGylated GNS that mainly accumulates in the liver and spleen can be gradually cleared, likely by both renal and faecal excretion. As recently reviewed, GO sheets larger than 200 nm are trapped by splenic physical filtration, but small sizes (approximately 8 nm) can penetrate the renal tubules into the urine and be rapidly removed without obvious toxicity . The excretion paths of GFNs have not yet been clearly explained, but renal and faecal routes appear to be the main elimination routes for graphene.
Recently, the distribution and excretion/toxicity strategy has become an important part of nano-toxicological studies. To date, several controversial results regarding the distribution and excretion of graphene in vivo have been reported in several papers, and a systematic evaluation of the toxicokinetics of GFNs is still needed. The metabolism and excretion of nanomaterials are long-period processes, however, the recent studies of GFNs had been limited to short-term toxicological assessments, and the long-term accumulation and toxicity of GFNs on different tissues remain unknown. Therefore, long-term studies on the deposition and excretion of GFNs need to be performed using different cells and animals to ensure the materials’ biosafety before utilization in human biomedical applications.
Uptake and location of GFNs in cells
The uptake and location of GFNs have also been observed to exert different effects in different cell lines. Graphene is taken up into cells via various routes [82, 83]. Basically, the physicochemical parameters such as the size, shape, coating, charge, hydrodynamic diameter, isoelectric point, and pH gradient are important to allow GO to pass through the cell membrane . As stated previously, nanoparticles with diameters <100 nm can enter cells, and those with diameters <40 nm can enter the nucleus . For example, GQDs possibly penetrate cell membranes directly, rather than through energy-dependent pathways [86, 87]. Larger protein-coated graphene oxide nanoparticles (PCGO) (~1 μm) enter cells mainly through phagocytosis, and smaller PCGO nanoparticles (~500 nm) enter cells primarily through clathrin-mediated endocytosis . GO sheets could adhere and wrap around the cell membrane, insert in the lipid bilayer or be internalized into the cell as a consequence of interactions with cells . Similarly, PEGylated reduced graphene oxide (PrGO) and rGO were shown to adhere onto the lipid bilayer cell membrane prominently due to the interaction of hydrophobic, unmodified graphitic domains with the cell membrane [90, 91]. Consequently, it was suggested that prolonged exposure to or a high concentration of graphene induces physical or biological damage to the cell membrane, along with destabilization of actin filaments and the cytoskeleton .
Current data demonstrates that GO sheets interact with the plasma membrane and are phagocytosed by macrophages. Three major receptors on macrophages take part in the phagocytosis of GNS: the Fcg receptor (FcgR), mannose receptor (MR), and complement receptor (CR). Furthermore, FcgR is a key receptor in the mediated phagocytic pathway [90, 93, 94]. The protein corona of GO promotes the recognition by macrophage receptors, especially the IgG contained within the protein corona. Macrophages were observed to undergo prodigious morphological changes upon contact with GO . After internalization, graphene accumulated in the cell cytoplasm, perinuclear space, and nucleus, which induced cytotoxicity in murine macrophages by increasing intracellular ROS through depletion of the mitochondrial membrane potential and by triggering apoptosis through activation of the mitochondrial pathway . The possible interactions and accumulation sites of GFNs are summarized in Fig. 1.