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Fig. 2

A representative trajectory of HP35 adsorbing onto the graphene. ( a ) Representative snapshots at various time points. The proteins are shown in cartoons with red helix and green loop, and the graphene is shown in wheat. The aromatic residues that form the π -π stacking interactions are shown in blue, others are shown in green. ( b ) The contacting surface area of HP35 with the graphene. ( c ) The RMSD of HP35 from its native structure and the number of residues in the α -helix structure. Here, the secondary structures are determined by the DSSP program. ( d ) The distance between the graphene and the aromatic residues, including F35, W23, F10, F17, and F06. To show the adsorbing process clearer, the χ -axis had been truncated and rescaled. [41] Copyright (2011), with permission from Journal of Physical of Chemistry

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Charge

A number of studies have highlighted the importance of the GO surface charge because of its ability to affect the internalization and uptake mechanism of cells [148–150]. GO internalization was negligible in non-phagocytes, which was likely due to the strong electrostatic repulsion between the negatively charged GO and the cell surface [34]. However, others have suggested that negatively charged nanoparticles can be internalized into non-phagocytic cells by binding to available cationic sites on the cell surface and be taken up by scavenger receptors [110, 146, 150]. GO/GS particles reportedly cause morphological changes and significant lysis, leading to high haemolysis in red blood cells (RBCs). RBC membrane disruption is probably attributed to the strong electrostatic interactions between the negatively charged oxygen groups on the GO/GS surface and positively charged phosphatidylcholine lipids on the RBC outer membrane [106].

Functionalization

Studies confirmed that functionalization with PEG [52], PEGylated poly-L-lysine (PLL) [151], poly(ε -caprolactone) [152], polyvinyl alcohol [3], Pluronic [153], amine [98], carboxyl, and dextran [79] groups largely decreases the toxicity and improves the biocompatibility of graphene. In vivo results revealed that only mild chronic inflammation emerged after the subcutaneous injection of GO-Pluronic hydrogel and no noticeable short-term toxicity was tested after the intravenous injection of GO-DEX [79, 154]. PEGylated GS did not induce appreciable toxicity in mice exposed to 20 mg/kg for 3 months, as evaluated by blood biochemistry and histological examinations, and showed relatively low retention in the RES [52, 155]. Coating GO with chitosan almost eliminated the haemolytic activity in blood [39]. Moreover, the PEG coating effectively alleviated GO-induced acute tissue injuries; decreased GO aggregation and retention in the liver, lungs, and spleen; and promoted the clearance of GO [81], GO-DEX [79], and fluorinated graphene oxide (FGO) [156].

In vitro, several cell function assays showed clear evidence that the surface functionalization of pristine graphene or GO was critical for reducing the strong toxicity effects [91]. PEG-GO, PEI-GO and LA-PEG-GO damaged human lung fibroblast cells less than GO [148]. PEG-GO exhibited no cytotoxicity toward several cell cultures, such as glioblastoma cells (U87MG), breast cancer cells (MCF-7), human ovarian carcinoma cells (OVCAR-3), colon cancer cells (HCT-116), and lymphoblastoid cells (RAJI), at concentrations up to 100 μ g/mL [119, 157, 158]. GQDs-PEG exhibited very low or no toxicity against lung and cervical cancer cells even at very high concentrations (200 μ g/mL) [159]. However, as a non-biodegradable material with great potential for cellular internalization, further investigation is needed to assess the possible long-term adverse effects of functionalized graphene.

Aggregations and sedimentation

Reportedly, nanomaterials have a propensity to form aggregates rather than individual units, particularly under physiological conditions. GS surfaces allowed fewer RBCs attach comparing to GO, and GS had the lower haemolytic activity for more aqueous aggregations formation. In contrast, the fast sedimentation and aggregate formation of GS greatly inhibited the nutrient availability of human skin fibroblast cells that were grown on the bottom of wells [106]. Therefore, the aggregations and sedimentation of graphene particles exert varying effects on different cells.

Impurities

Nanomaterial purity is an important consideration because residual, contaminating metals may be responsible for the observed toxicity, rather than the nanomaterial itself, which has resulted in conflicting data on GFNs cytotoxicity [35, 160]. Traditionally prepared GO often contains high levels of Mn²⁺ and Fe²⁺, which are highly mutagenic to cells. The nonspecific release of these ions from traditionally prepared GO might lead to unusually high levels of cytotoxicity and DNA fracturing [39]. In particular, Peng et al. [161] produced high-purity GO containing only 0. 025 ppm Mn²⁺ and 0. 13 ppm Fe²⁺, and Hanene et al. [162] invented a new method to prepare high-purity, single-layer GO sheets with good aqueous dispersibility and colloidal stability. GO produced by these new methods did not induce significant cytotoxic responses (at exposure doses up to 100 μ g/mL) in vitro, and no obvious inflammatory response or granuloma formation (exposure doses up to 50 μ g/animal) were observed in vivo. Therefore, the purity of GFNs deserves attention and is a vital step towards the determination of GFNs involved in bioapplications.

Protein corona effect

Because of the high free surface charge, nanomaterials can easily form “coronas” with proteins in biological systems [163, 164]. The protein corona is suggested to affect the circulation, distribution, clearance and toxicity of nanoparticles. Several papers reported that GO forms GO-protein coronas with adsorbed plasma proteins in serum and these GO-protein coronas play an important role in deciding the fate of the GO biokinetic behaviour in vivo. Such GO-protein coronas can regulate the adhesion of GO to endothelial and immune cells through both specific and nonspecific interactions [165]. Basically, immunoglobulin G and complement proteins in the protein corona help to reorganize nanoparticles in immune cells, causing the particles to be engulfed by the RES, and IgG-coated GO was taken up by either specific or nonspecific interactions with cell membrane receptors [31, 165]. However, another study found that GO could not adhere to mucosal epithelial cells directly in the intestinal tract after the filial mice drank an aqueous GO solution because abundant proteins in the milk had adsorbed on the surface of the GO and thus inhibited their direct interaction with the mucosal epithelial cells [53]. Protein corona mitigated the cytotoxicity of GO by limiting its physical interaction with the cell membrane and reducing the cellular morphological damage in HeLa, THP-1 and A549 cells [166–168]. The cytotoxic effect was largely reduced when GO was pre-coated with FBS and incubated with cells; nearly  ∼   90 % survival was observed with 100 μ g/mL FBS-coated GO and 100 % survival with 20 μ g/mL FBS-coated GO. Similar trends were observed for GO covered by BSA [166, 169]. Consistently, additional serum could neutralize the toxicity of pristine GO in J774. A1 cells at a dose of 4 μ g/mL, which lead to a decrease in cell number of 52. 5 % compared to untreated cells [89].

After reviewing many studies, it can be concluded that the toxicity of graphene is influenced by multiple factors. Those factors combined to largely change the toxicity of GFNs in many cases. Scientific studies often need the clear identification of cause and effect, which should keep only one factor different at a time, so that the effect of that single factor can be determined. But in some papers, several factors influencing GFNs toxicity were studied at the same time, which led to confused results.