Toxicity in the central nervous system

Toxicity in the central nervous system

Graphene has largely benefited neurosurgery with the application of drug/gene delivery for brain tumour treatment, intracranial and spinal biocompatible devices, biosensing and bioimaging techniques. Studies regarding the potentialities or risks of graphene in the brain have emerged. In the chicken embryo model, pristine graphene flakes decreased the ribonucleic acid level and the rate of deoxyribonucleic acid synthesis, leading to harmful effects on brain tissue development and the atypical ultrastructure was observed in the brain [101]. The recent researches of GFNs in the central nervous system are mostly involved in the application rather than the toxicity. The data of the toxic study on GFNs is underway.

Toxicity in reproduction and development system

Pristine graphene reduced the vascularization of the heart and the density of branched vessels after injection into fertilized chicken eggs followed by incubation for 19 d [101]. GO and rGO damage zebrafish embryos by influencing the embryo hatching rate and body length in a concentration-dependent manner. Although no obvious malformation or mortality was observed in exposed zebrafish embryos [102], GO adhered to and was wrapped in the chorion of the zebrafish embryos, causing remarkable hypoxia and hatching delay. GO aggregates were retained in many organelles, such as the eyes, heart, yolk sac, and tail of the embryos, and apoptosis and reactive oxygen species (ROS) generation were observed in these regions [103].

The GFNs exert different toxicological effects on male or female reproductive system. Data showed that GO exerted very low or nearly no toxic effects on male reproduction even at a high dose via intra-abdominal injection [66]. Additionally, rGO did not change the serum estrogen levels of non-pregnant female mice. The condition is different in the female mouse: mouse dams could give birth to healthy offspring after rGO injection before mating or during early gestation, and only a few abnormal foetuses were present among the rGO-injected dam litters. However, the pregnant mice had abortions at all dose, and most pregnant mice died when the high dose of rGO was injected during late gestation [44]. Notably, the development of offspring in the high dosage group was delayed during the lactation period. The high dose of GO decreased the maternal mice’s water consumption by oral exposure, which reduced milk production and thus postponed the growth of offspring [53]. Though the findings indicate that GFNs are potentially harmful to development, but data on reproductive and developmental toxicity are still deficient. Studies of the influence of GFNs on male and female reproduction and development are still required to elucidate the underlying toxicity mechanism.

Influence of haemocompatibility

GO release into the blood is ineluctable. The haemocompatibility of GO was found to be dependent on the functional coating and the exposure conditions. GO with submicron size resulted in the greatest haemolytic activity, while aggregated graphene induced the lowest haemolytic reaction. Pristine graphene and GO demonstrated haemolytic effect up to 75 μ g/mL [104]. GO-polyethylenimine (GO-PEI) exhibited notable toxicity by binding to HSA, even at 1. 6 μ g/mL [105]. Carboxylated graphene oxide (GO-COOH) showed significant cytotoxicity toward T lymphocytes at concentrations above 50 μ g/mL and had good biocompatibility below 25 μ g/mL, whereas GO-chitosan nearly inhibited haemolytic activity [106]. Until now, the corresponding risk of haemocompatibility has remained largely unknown.

In conclusion, the lung injury induced by GFNs has been studied in several studies, the results of which have demonstrated inflammatory cell infiltration, pulmonary edema and granuloma formation in the lungs. However, only a few specific studies have evaluated in other organs, such as the liver, spleen, and kidney, and the injury symptoms, damage index and level of damage to these internal organs were not fully investigated. Moreover, studies on the neurotoxicity of GFNs are quite rare; no data has revealed which nerves or brain areas experience damage, nor have the related behavioural manifestations been studied. The developmental toxicity of GFNs may induce structural abnormalities, growth retardation, behavioural and functional abnormalities, and even death. A study on the reproductive and developmental toxicity of GFNs will be extremely significant and gain extensive attention in the future. Almost all the GFNs toxicity studies were short-period experiments, and no studies have investigated long-term chronic toxic injury. However, based on studies of other nanomaterials toxicity, long-term GFNs exposure may be an important factor harming health [107–109]. Therefore, the long-term study of GFNs is necessary.

Toxicity of GFNs in cell models

The cytotoxicity of GFNs in vitro has been verified in various cells to change the cell viability and morphology, destroy the membrane integrity, and induce DNA damage [110–112]. GO or rGO decrease cell adhesion; induce cell apoptosis; and enter lysosomes, mitochondria, cell nuclei, and endoplasm [113]. GQDs entered cells and induced DNA damage by the increased expression of p53, Rad 51, and OGG1 proteins in NIH-3 T3 cells [87]. However, GQDs did not pose significant toxicity to human breast cancer cell lines (at a dose of 50 μ g/mL) or human neural stem cells (at a dose of 250 μ g/mL) [114, 115]. GO derivatives dramatically decreased the expression of differential genes that are responsible for the structure and function of the cell membrane, such as regulation of the actin cytoskeleton, focal adhesion and endocytosis [89]. In rat pheochromocytoma cells (PC12 cells), graphene and rGO caused cytotoxic effects and mitochondrial injury, such as the release of lactate dehydrogenase (LDH), an increase in the activation of caspase-3, and the generation of ROS [82, 116].

Graphene can increase cell viability [117] or cause cell death [118] depending on the cell line, type of graphene material and the doseage. GO cytotoxicity was observed in human fibroblasts and lung epithelial cells at concentrations above 20 μ g/mL after 24 h, but minimal toxicity was found in A549 cells at concentrations higher than 50 μ g/mL [119]. The biological responses induced by GO such as ROS, malondialdehyde (MDA), and LDH increased, whereas superoxide dismutase (SOD) decreased dose-dependently in HeLa cells [120]. However, GO-molecular beacon (GO-MB) showed low cytotoxicity even at 20 μ g/mL in HeLa cells [121]. GO decreased the viability of A549 cells, while the same concentration and time of exposure increased the cell viability of CaCo2 colorectal carcinoma cells [122]. Another study reported that GO dramatically enhanced the differentiation of SH-SY5Y, accompanied by increasing neurite length and the expression of neuronal marker MAP2 at low concentrations but that GO suppressed the viability of SH-SY5Y cells at high doses (≥ 80 mg/mL) [123]. Functionalized coatings on GO, such as GO-PEG [124] and GO-chitosan [125], can profoundly attenuate the particles’ cytotoxicity by inhibiting the interactions between cells.

The toxicity of GFNs in vitro is summarized in Table 2. Data on the cytotoxicity of graphene nanomaterials are contrasting, and varying characteristics influence the results. The mechanisms and influencing factors of toxicity need to be elucidated in detail.