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Blood-air barrier. Blood-brain barrier. Blood-testis barrier. Blood-placenta barrier
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Blood-air 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 [54]. The agglomerated or dispersed particles deposit on the inner alveolar surface within the alveoli and then be engulfed by alveolar macrophages (AMs) [55]. 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 [61]. 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.
Blood-brain barrier
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 [62]. 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 [63]. In addition, graphene quantum dots (GQDs), with a small size of less than 100 nm, can cross through the blood-brain barrier [64]. 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.
Blood-testis barrier
The blood-testis and blood-epididymis barriers are well known for being some of the tightest blood-tissue barriers in the mammalian body [65]. 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 [66].
Blood-placenta barrier
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 [67]. 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 [42]. 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.
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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 [61]. 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 [49]. 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 [31].
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 [49]; however, Li et al. observed that GO accumulated in mouse liver [76]. 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 [78]. 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 [34].
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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 [57], and 46. 2 % of the intratracheally instilled FLG was excreted through the faeces 28 d after exposure [61]. In the liver, nanoparticles can be eliminated thorough the hepato-biliary pathway following the biliary duct into the duodenum [80]. 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 [81]. 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 [84]. As stated previously, nanoparticles with diameters < 100 nm can enter cells, and those with diameters < 40 nm can enter the nucleus [85]. For example, GQDs possibly penetrate cell membranes directly, rather than through energy-dependent pathways [86, 87]. Larger protein-coated graphene oxide nanoparticles (PCGO) (~1 μ m) enter cells mainly through phagocytosis, and smaller PCGO nanoparticles (~500 nm) enter cells primarily through clathrin-mediated endocytosis [88]. 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 [89]. 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 [92].
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 [34]. 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 [83]. The possible interactions and accumulation sites of GFNs are summarized in Fig. 1.
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