Table 2 Toxicity of GFNs in cell models

Table 2 Toxicity of GFNs in cell models

Full size table

Origins of GFNs toxicity

Reportedly, the characteristics of graphene, including its concentration, lateral dimension, surface structure, functional groups, purity and protein corona, strongly influence its toxicity in biological systems [2, 7, 104, 126–129].

Concentration

Numerous results have shown that graphene materials cause dose-dependent toxicity in animals and cells, such as liver and kidney injury, lung granuloma formation, decreased cell viability and cell apoptosis [130–134]. In vivo studies, GO did not exhibit obvious toxicity in mice exposed to a low dose (0. 1 mg) and middle dose (0. 25 mg) but induced chronic toxicity at a high dose (0. 4 mg). The high content of GO mainly deposited in the lungs, liver, spleen, and kidneys and was difficult to be cleaned by the kidneys via a single tail vein injection [135]. Intriguingly, increasing the dose resulted in a dramatic decrease in the hepatic uptake but an increase in the pulmonary uptake of s-GO by intravenous injection [31], because the high dose of GO potentially surpassed the uptake saturation or depleted the mass of plasma opsonins, which consequently suppressed the hepatic uptake. Moreover, an in vitro study reported that 20 μ g/mL GO nanosheets exhibited no cytotoxicity in A549 within 2 h of incubation, but a higher concentration (85 μ g/mL) decreased the cell viability to 50 % within 24 h [136, 137]. Lü et al. also demonstrated that GO had no obvious cytotoxicity at low concentrations for 96 h in a human neuroblastoma SH-SY5Y cell line, but the viability of cells sharply decreased to 20 % after treatment with 100 mg/mL GO for 96 h of incubation [123]. The results in HeLa cells, NIH-3 T3 cells, and breast cancer cells (SKBR3, MCF7) treated with graphene nanoribbons also showed a dose- (10–400 mg/ml) and time-dependent (12–48 h) decrease in cell viability [138]. Increasing concentrations of GO entered the lysosomes, mitochondria, endoplasm, and cell nucleus [119]. Several data indicated that rGO caused apoptosis-mediated cell death at a lower dose and early time point but that necrosis was prevalent with the increase in time/dose [110, 135].

Lateral dimension

Nanoparticles with sizes < 100 nm can enter the cell, < 40 nm can enter nucleus, and smaller than < 35 nm can cross the blood brain barrier [85]. One study showed that GO (588, 556, 148 nm) did not enter A549 cells and had no obvious cytotoxicity [112]. When the diameter of graphene is between 100  ~  500 nm, the smallest size may cause the most severe toxicity, and when the diameter is below 40 nm, the smallest sizes may be the safest. For instance, rGO with a diameter of 11  ±  4 nm could enter into the nucleus of the hMSCs and cause chromosomal aberrations and DNA fragmentation at very low concentrations of 0. 1 and 1. 0 mg/mL in 1 h. However, rGO sheets with diameters of 3. 8  ±  0. 4 nm exhibited no notable genotoxicity in hMSCs even at a high dose of 100 mg/mL after 24 h [118].

In an in vivo study, s-GO (100–500 nm) preferentially accumulated in the liver, whereas l-GO (1–5 μ m) was mainly located in the lungs because l-GO formed larger GO-protein complexes that were filtered out by the pulmonary capillary vessels after intravenously injection [31]. Given the relative lateral sizes (205. 8 nm, 146. 8 nm and 33. 78 nm) of the three GO nanosheets at the same concentration, smaller GO experiences much greater uptake than larger GO in Hela cells [139]. The high uptake of s-GO changed in the microenvironment of cells and consequently induced the greatest viability loss and most serious oxidative stress among three sizes of GO samples [119]. As a result, one study delineated that GO size-dependently induced the M1 polarization of macrophages and pro-inflammatory responses in vitro and in vivo. Larger GO showed stronger adsorption onto the plasma membrane with less phagocytosis, eliciting robust interactions with TLRs and activating NF-κ B pathways, compared to smaller GO sheets, which were more likely taken up by cells [94]. To further uncover the detailed mechanism underlying these effects, more studies are needed to illustrate the vital mechanism of the lateral size of graphene materials.

Surface structure

GFNs possess widely varying surface chemistries. For example, the pristine graphene surface is hydrophobic, GO surface is partially hydrophobic with carboxylate groups [140–142], and rGO has intermediate hydrophilicity [143]. GFNs were observed to disrupt the function and structure of cell membranes and proteins probably by exceptionally strong molecular interactions with cells [2, 91]. For instance, rGO bonded to cell membranes, stimulated receptors and activated mitochondrial pathways, inducing apoptosis [110, 111, 144]. Limited evidence showed that GO is smaller and less toxic than rGO because of the high oxygen content, smoother edges, and hydrophilic properties of the former species [104, 145, 146]. Because of the different surface oxidation states of GO and rGO, GO possessing distinct hydrophilicity might be internalized and taken up by HepG2 cells easily. On the contrary, rGO with evident hydrophobicity, could be adsorbed and aggregated at cell surfaces without (or with lower) uptake [110]. Due to strong π -π stacking interactions, graphene is highly capability of breaking many residues of the protein, particularly the aromatic ones, such as the villin headpiece (HP), F10, W23, and F35. The protein’s secondary and tertiary structures are largely lying on the graphene surface, disrupting the structure and function of the protein [41] (Fig. 2). In addition, GO can insert between the base pairs of double-stranded DNA and disturb the flow of genetic information at the molecular level, which might be one of the main causes of the mutagenic effect of GO [7, 112, 146, 147].