Background. Toxicity of GFNs (in vivo and in vitro)

Background

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 [1]. 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 [26]. 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 [30], oral administration [31], intravenous injection [32], intraperitoneal injection [33] and subcutaneous injection [34]. 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) [42]; collected information on the influence of GFNs in the soil and water environments [43]. 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.

Administration route

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 [49]. 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 μ m) [34]. 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.