https://www.sciencedirect.com/scienc...302853#bib0050

Keywords
Graphene nanoporesCytotoxicityIn vivo testIn vitro testIntraperitoneal administrationBiocompatibility
1. Introduction
Graphene has become a ‘superstar’ in nanomedicine with applications to improve diagnostics, therapeutics, and genetic risk factors, owing to its multifaceted properties such as small size, large surface area-to-volume ratio, quantum size effects, and unique physicochemical properties [1], [2], [3]. One important advantage of graphene-based materials is their ability to effectively cross biological barriers such as the blood brain barrier, highlighting their potential as a drug delivery vehicle for anticancer therapeutic agents. In particular, the combined enhanced permeability and retention effect would facilitate their accumulation in tumours, releasing therapeutic levels of drugs into the target cells with reduced side effects [4]. Typically, graphene quantum dots have many properties far superior to conventional quantum dots such as photoluminescence, low toxicity and interplay between size and optical features which have been utilized as diagnostic imagining tools as well as photodynamic/photothermal therapy [5]. Similar use of three-dimensional graphene foam for stem cell therapy of stroke and its bioconjugates in regenerative medicine has been described in recent literature [6]. Recently, graphene nanopores (GNPs) have also been used in applications such as DNA sequencing [7], [8], [9] and water treatment [10], [11] and GNPs have provided unique porous frameworks [12]. Porous graphene biointerfaces have also recently been reported as an effective antimicrobial agents with highly efficiently bactericidal activities against both Gram positive and Gram negative bacteria [13], [14]. Matharu et al. [15] have reported the effects of graphene nanoplatelet-loaded polymer fibres on microbial growth of two Gram negative bacteria Escherichia coli and Pseudomonas aeruginosa. They determined the minimum inhibitory concentration of graphene-fibre which in turn can produce the highest antibacterial effects while remaining non-toxic to the normal cells. This study revealed that 8 wt% of graphene-fibres showed good antibacterial effects owing to the direct contact between bacterial cells and the sharp edges of the graphene nanostructures. This direct contact is also responsible for severe membrane deformation and the efflux of cytoplasmic material [16]. One drawback of the use of GNP's is that very few synthesis techniques are available. However, techniques such as electron beam irradiation, ion bombardment, doping, templating, chemical etching, chemical vapour deposition and other chemical methods have now been utilized for their preparation [9], [17], [18], [19], [20]. The drawbacks of these methods are the low production yield and the problems associated with their separation/purification. To address this omission, we have recently reported a study demonstrating a novel and facile approach to GNPs synthesis via thermal treatment of reduced graphene oxide without using any catalyst or template-based approach [21].

GNP, a thin, flexible material with excellent electrical addressability and robust mechanical properties is promising for label-free protein detection, DNA sequencing and high throughput wastewater based-micropollutant decontamination [7], [8], [9], [10], [11]. The high specific surface area and nanoporous framework allows direct sensing and sequencing of atomic-scale biomolecules. In recent years, cellular internalization and trans-barrier transport of micro/mesoporous graphene nanosheets have been the subject of major developments in nanobiotechnology. It is evident that nanoscale materials with a diameter less than 100 nm can enter cells, while nanoparticles smaller than 40 nm in diameter can reach the cellular nuclei. Particles with diameters below 35 nm are able to reach the brain by passing through the blood–brain barrier [22], [23], [24], while larger nanoparticles are excluded which in turn reduces the delivery of theranostic nanoparticles [25], [26], [27]. A better understating of the physiochemical properties of graphene, the interaction between graphene and cells, and possible toxicity mechanisms is of critical importance to outline potential biomedical applications of these materials. The proposed mechanism of GNPs toxicity is depicted in Fig. 1. The widespread use of graphene-based materials and their potential toxic effects are likely to exacerbate several health concerns [26], [27]. Most laboratory experiments investigating the potential applications of GNPs in life sciences have not considered the toxicity associated with GNPs in their testing regimes. Recently, however, a few studies have examined the in vitro and in vivo toxic implications of three dimensional graphene foam to investigate the bioavailability and subsequent toxicity potential [28], [29]. The pre-clinical risks, adverse effects of GNPs exposure, and approaches to minimize their health hazards still remain undefined. However, inhalation of graphene structures is believed to be a risk factor for cardiorespiratory disease. For example, inhaled graphene nanoplatelets can be transported deep within the distal regions of the lungs and trigger chronic inflammation in the respiratory tract [30]. It is generally thought that the placenta, lung, gastrointestinal tract and skin act as major barriers for many nanostructures entry into living organisms [31]. Indeed, a recent study on mice demonstrated that intratracheally delivered few-layered graphene was mainly retained in the lung, with 47% remaining after 4 weeks and this resulted dose-dependent acute lung injury and pulmonary oedema [32]. An in vitro study of the effects of graphene and graphene oxide on human skin HaCaT keratinocytes demonstrated that oxidized graphene was the most cytotoxic, inducing mitochondrial and plasma-membrane damage, and suggesting low cytotoxic effects at the skin level [33]. Reduced graphene oxide is more toxic than graphene oxide as evidenced by many studies reported recently [34], [35]. This is primarily due to its sharp edges and structural morphology. In contrast to the typically soluble nanoparticles examined in conventional toxicology investigations, graphene nanostructures have different shapes and surface areas, and which in turn can significantly influence their diffusion, dispersion, aggregation and agglomeration in plasma. Importantly, these “tunable” characteristics of graphene account for the varying toxic outcomes on the tissues. In vivo, following toxicity testing of graphene, post-mortem histological examinations of liver alterations have revealed hypertrophy of hepatocytes, necrosis and inflammatory cell infiltration in liver and kidney tissues [36]. The level of organ function and oxidative stress has been reported to affect the fate, transport and toxicity of graphene in organs but there is currently a lack of consistency in this regard [36]. Liver enzyme functions can be used to reveal the biodistribution, metabolism, and excretion patterns of graphene. Similarly, investigation of oxidative stress indicators is a commonly acknowledged mechanism adopted to investigate cellular injuries in mammals. Antioxidants act as a defence system to reinstate the cellular redox balance when oxidative stress is generated as a result of excess production of reactive oxygen species. Disruption of this critical balance in the presence of excessive reactive oxygen species triggers the activation and promotion of a pro-inflammatory cascade, which in turn may cause mitochondrial release of proapoptotic factors potentially leading to cell death. Hepatocytes are key targets for reactive oxygen species damage, and therefore liver function and biomarkers of oxidative stresses should be investigated with great care.


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Fig. 1. Schematic illustration of the potential mechanisms of action of graphene nanopores (GNPs). When graphene nanopores reach the exterior membrane of a cell, they interact with the plasma membrane or extra-cellular matrix and enter the cell, mainly through diffusion, endocytosis and/or binding to receptors. The potential toxic effects of graphene mainly depend on its physicochemical characteristics, the nature of its interaction with cells and its accumulation in specific organs. Upon interaction with light, graphene can generate reactive oxygen species, which in turn can cause oxidative stress, loss in cell functionality, pro-inflammatory responses and mitochondrial damage. Uptake of graphene into the nucleus may cause DNA-strand breaks and induction of gene expression via the activation of transcription factors, cell death and genotoxicity.

Lung cancer cell lines are commonly used to assess the cytotoxicity of an experimental material for biomedical applications. Cancer cell lines such as lung and breast were selected by National Cancer Institute as models to screen drugs/compounds as a prelude to testing in xenografts or animal models [37]. Use of human cell lines in toxicity assays allows us to selectively test mechanisms of cell toxicity in a controlled environment, which can be very difficult, time consuming and laborious to carry out in rat models. Also, animal models are genetically very different than human. Bioavailability experiment with animal models cannot explain the cellular and molecular mechanisms of interactions between cell membrane or cellular organelles and the tested compounds, which can only be achieved using well-defined and well-characterised human cell lines such as lung, breast cancer cells. Isolating primary human cell for each organ for in vitro study is not cost-effective and it also may produce huge variability due to significant heterogeneity among humans on genomic, proteomic and phenotypic levels. Human cancer cell lines hold genetic information that is well-characterised and translatable to human studies. Also, using human cancer cell lines to test for in vitro toxicity give us an indication of the range of doses that can be applied in animal models. In vivo, the efficacy of the treatments remains non-selective, non-specific and is carried out in an uncontrolled environment (e.g. genetically heterogeneous mice). Thus, we utilized human cell lines in vitro to carry out our preliminary toxicity experiments in a controlled, 2D environment which was then repeated in in vivo models to better understand the effect of the compound in a 3D, uncontrolled environment. We studied two types of lung cancer cells (e.g. adenocarcinoma and squamous cell carcinoma- both of epithelial origin) for two reasons: (a) cancer cells evolve to evade the immune system and display resistance to a number of therapeutic responses such as chemotherapy and radiotherapy, and hence possess a therapeutic challenge. Due to the occupational exposure of such graphene-based nanomaterials, it is highly likely that these nanomaterials can be inhaled in humans which may lead to respiratory pathology. Thus, testing toxic effects of this nanoparticle in lung cancer cell lines was advantageous over using somatic cell lines; and (b) unlimited proliferation is a predominant feature of all cancer cell lines compared to the limited proliferative capacity of somatic cells. During passaging, somatic cells undergo anoikis when they detach from the extracellular matrix coating, resulting in a decreased number of cells in the next passage. Consequently, the resultant cell death due to the testing compound may not be a true representation of the toxicity of a compound. Also, as the passage number increases, somatic cells may become less responsive to an exogenous therapeutic assault. These phenomena are rarely observed or are absent in cancer cells.

Clearly, in vitro and in vivo investigations into the toxicity of graphene nanostructures is becoming increasingly important. In response to this, the present study investigates the toxic effects of GNPs on lung cancer cells (SKMES-1 and A549) in vitro and in rats in vivo, specifically, biochemical, serum enzyme analyses, complete blood count as well as histological analysis have been used in this study.

2. Results
2.1. In vitro toxic effects of GNPs on lung cancer cells
Representative FACS images and analysis of one experiment of cell viability have been shown in Fig. 2. Fig. 3A demonstrates that after 24-h exposure to GNPs, the cell viability of A549 cells exhibited a significant dose-dependent reduction from 50 to 500 μg/ml. For example, after 24 h reduction in the percentage of living cell were 52.8%, 42.5% and 33.2% at concentrations 50, 250 and 500 μg/ml respectively, compared to control (0 μg/ml, ∼80%). A similar observation was made in SKMES-1 cells where GNPs concentrations 50 and above induced significant reduction of living cells. However, the reduction was not dose dependent (Fig. 3A). For example, at 50, 250 and 500 μg/ml of GNPs, percentage count for living cells were 50.8%, 46.5% and 47.4% respectively, compared to control (0 μg/ml, 70%). The number of cells undergoing early apoptosis significantly increased in a dose dependent manner following treatment with 5 μg to 500 μg/ml GNPs in both A549 and SKMES-1 cells (Fig. 3B). A dose-dependent increase in late apoptotic (Fig. 3C) and necrotic cells (Fig. 3D) was also observed in A549 cells, although no significant increase in necrosis was observed in the SKMES-1 cell line.


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Fig. 2. Representative fluorescence-activated cell sorting (FACS) analysis of cell viability, early apoptotic, late apoptotic, and necrotic cells of selected concentrations of graphene nanopores (GNPs) in two different lung cancer cell lines (A549 and SKMES-1). Data are presented as percentage of the cell population. Cell viability of A549 (upper panel) and SKMES-1 (lower panel) is shown at selected concentrations. Experiments were performed and interpreted as follows: annexin V−ve/PI−ve cells (lower left quadrant), annexin V+ve/PI−ve cells (lower right quadrant), annexin V+ve/PI+ve (upper right quadrant) and annexin V−ve/PI+ve (upper left quadrant) were considered as living, early apoptotic, late apoptotic, and necrotic cells.


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Fig. 3. Bar graph quantifying the percentage of dead, living, early-stage apoptotic, and late-stage apoptotic cells in response to different concentrations of porous graphene (GNPS). Flow cytometry analysis of A549 and SKMES-1 lung carcinoma cells stained with annexin V (apoptosis) and propidium iodide (PI; late apoptosis and necrosis) following 24 h of treatment with varying concentrations of GNPs (0–500 μg/ml). (A) graphic representation of percentage of living cells (B) early apoptosis (C) late apoptosis, (D) necrosis in response to GNPs. Data are represented as mean ± SD of three independent experiments. *p < 0.05 vs control. n.s. denotes not significant.

2.2. Effects of GNPs on body and relative organ weights
In vivo toxicity of GNPs was assessed in rats following 27-day repeated dose intraperitoneal injections. GNPs treatment did not affect the body weight of the treated rats during the 27-days exposure period for treatment with 5 mg/kg body weight either once or multiple doses (Fig. 4). No significant decrease in body weight was observed in rats administered GNPs up to 5 mg/kg. Rats in the high repeated dose group (15 mg/kg body weight) showed lower body weights after 27 days (Fig. 4) compared to the control group, but this did not reach significance. Organo-somatic indices demonstrated that organ weight did not change by the treatment of GNPs, compared to the control, supporting their low in vivo toxicity (Supplementary information Fig. 2).


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Fig. 4. Daily body weight (g) of control groups and treated groups of rats exposed to GNPs by intraperitoneal injection for 27 days. First dose was administered at day 0 for both single and multiple dose regimen, and the body weight was measured daily.

2.3. Effects of GNPs on complete blood count in the rat
To examine the in vivo cytotoxicity of GNPs, we performed a complete blood count (CBC), liver and kidney function enzymes, biomarkers of oxidative stress and histological study of vital organs of control and treated rats. Treated animals received with either 5 or 15 mg GNPs/kg body weight as either a single dose or repeated doses (8 doses spread over a 27 day period). Toxic effects of GNPs on CBC were not observed (Fig. 5A–O) although there was a slight (6%) reduction in platelet numbers in the 15 mg/kg group (Fig. 5K). The proportion of lymphocytes remained stable (Fig. 5B) and total white cell count was unaffected (Fig. 5N).


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Fig. 5. (A–N) Complete blood count in rats after 27 days of GNPs administration. Rats (n = 8 per group) were intraperitoneally injected with single doses of 5 mg/kg body weight (group 1), 15 mg/kg body weight (group 2) or multiple doses of 5 mg/kg body weight (group 3) and 15 mg/kg body weight (group 4). Values are expressed as mean ± standard deviation, for: (A) red blood cell count (RBC); (B) lymphocytosis (LYM %); (C) mid-range absolute count (MID); (D) total % of granulocytes GRA; (E) haemoglobin (HBGL); (F) mean corpuscular haemoglobin (MCH); (G) mean corpuscular haemoglobin concentration (MCHC); (H) mean corpuscular volume (MCV); (I) hematocrit (HCT); (J) red cell distribution width (RDW); (K) platelet count (PLT); (L) mean platelet component (MPC); (M) large platelet concentration ratio (LPCR); and (N) white blood cell count (WBC). Data are represented as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01 vs control. n.s. denotes not significant.

2.4. Liver and kidney function analysis
Alterations were observed in liver and kidney functions following GNPs treatment (Fig. 6) i.e., the results showed that the activities of ALT, AST, ALP enzymes were significantly increased in all groups, suggesting liver damage. Creatinine levels, indicative of kidney damage, were only significantly increased in rats treated with 15 mg/kg of GNPs repeated doses.


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Fig. 6. Liver and kidney enzyme functions results in rats 27 days post GNPs administration. Rats (n = 8 per group) were intraperitoneally injected with single doses of 5 mg/kg body weight (group 1), 15 mg/kg body weight (group 2) or multiple doses of 5 mg/kg body weight (group 3) and 15 mg/kg body weight (group 4). Values are expressed as mean ± standard deviation, for: (A) alanine transaminase (ALT), (B) aspartate transaminase (AST), (C) alkaline phosphatase (ALP) and (D) creatinine. Data are represented as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01 vs control. n.s. denotes not significant.

2.5. Histopathological changes
A comprehensive post mortem histological study was then performed to assess any tissue interactions with GNPs. Sections of heart, kidney, liver, small intestine, lung, brain and testis were examined for histopathological changes at 14 and 27 days of GNPs administration (at single or multiple doses of 5 and 15 mg/kg of body weight of rats). The histology photographs of the liver, kidney, heart and small intestine tissues after GNPs exposure of 27 days are shown in Fig. 7, Fig. 8. GNPs at all dosing regimens induced pathological changes after 27 days. Specifically, vacuolation, dilation of central vein and haemorrhage, vacuolation and dilation of central vein, damage of vacuolation, haemorrhage and degeneration of central vein, dilation of epithelial lining and hydropic degeneration oedema were observed in liver tissue. Kidney tissue of the treated groups showed acute vacuolization, dilation of epithelial lining, vacuolation and nucleus degeneration, nucleus damage, necrosis and epithelial degeneration. Heart tissue showed chemodectoma, toxic myocarditis, reddish brown atrophy; yellowish brown pigments suggesting lipofuscin granules as remnants of cell organelles and cytoplasmic material. The brain showed effects of secondary carcinoma, olegodendrocytoma small thin walled blood vessel and crytococcosis. Testicular tissue of treated groups showed spermatogenesis and vacuolation, dilation of germinal layer, degeneration of secondary spermatocytes, damage to the germinal layer and vacuolation. The lung showed damage of vacuolation, degeneration of central vein, inflammation, haemorrhage, d-shaped cells structure, hemosidophroages and lesion. These effects are presumably due to accumulation and low clearance of GNPs in the rat. After the multiple-dose exposure to GNPs, there are some histopathological changes that accumulate around the central veins of the liver. This may be ascribed to the overload of GNP particles in the liver. The histopathological changes of these organs at 14 days in the rats are shown in Supplementary information Figs. 3 and 4. No abnormal clinical signs or death was seen in the all the treated and control groups and all the rats were in good condition at the time of sacrifice.





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Graphene has become a ‘superstar’ in nanomedicine with applications to improve diagnostics, therapeutics, and genetic risk factors, owing to its multifaceted properties such as small size, large surface area-to-volume ratio, quantum size effects, and unique physicochemical properties [1], [2], [3]. One important advantage of graphene-based materials is their ability to effectively cross biological barriers such as the blood brain barrier, highlighting their potential as a drug delivery vehicle for anticancer therapeutic agents. In particular, the combined enhanced permeability and retention effect would facilitate their accumulation in tumours, releasing therapeutic levels of drugs into the target cells with reduced side effects [4]. Typically, graphene quantum dots have many properties far superior to conventional quantum dots such as photoluminescence, low toxicity and interplay between size and optical features which have been utilized as diagnostic imagining tools as well as photodynamic/photothermal therapy [5]. Similar use of three-dimensional graphene foam for stem cell therapy of stroke and its bioconjugates in regenerative medicine has been described in recent literature [6]. Recently, graphene nanopores (GNPs) have also been used in applications such as DNA sequencing [7], [8], [9] and water treatment [10], [11] and GNPs have provided unique porous frameworks [12]. Porous graphene biointerfaces have also recently been reported as an effective antimicrobial agents with highly efficiently bactericidal activities against both Gram positive and Gram negative bacteria [13], [14]. Matharu et al. [15] have reported the effects of graphene nanoplatelet-loaded polymer fibres on microbial growth of two Gram negative bacteria Escherichia coli and Pseudomonas aeruginosa. They determined the minimum inhibitory concentration of graphene-fibre which in turn can produce the highest antibacterial effects while remaining non-toxic to the normal cells. ""


One important advantage of graphene-based materials is their ability to effectively cross biological barriers such as the blood brain barrier, highlighting their potential as a drug delivery vehicle for anticancer therapeutic agents. In particular, the combined enhanced permeability and retention effect would facilitate their accumulation in tumours, releasing therapeutic levels of drugs into the target cells with reduced side effects [4]


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4. Conclusion
The present study aimed to assess the in vitro and in vivo interactions of a relatively new derivative of graphene, graphene nanopores (GNPs) in mammalian systems, for the first time and to elucidate the possible mechanism of GNPs toxicity. In vitro results showed that GNPs induced early apoptosis in both SKMES-1 and A549 lung cancer cells. However, late apoptosis was only induced at concentrations higher than 250 μg/ml, suggesting that, although GNPs at lower concentrations induced translocation of phosphatidylserine to the cell surface membrane (i.e. early apoptotic event), GNPs do not significantly disintegrate the cell membrane. Subsequently, in vivo studies indicated damage in the main organs of rats (liver, kidney, lungs, heart, brain and testis) but the possible fast clearance of GNPs through kidney. We also showed that GNPs induced oxidative stress in the liver. Blood markers remained within normal ranges following treatment. Our results show that changes in liver and kidney functions induced by the treatments were minimal. GNPs caused sub-acute toxicity at our tested doses (5 and 15 mg/kg) to the treated rats in a period of 27 days as evidenced by blood biochemistry, liver and kidney enzyme functions, oxidative stress biomarkers and histological examinations. For the first time, the in vitro and in vivo toxic effects of a porous graphene nanostructure were investigated. We found time and dose dependent toxicity of GNPs in lung cancer cell lines and rats. These findings will help elucidate how GNPs induce toxicity that may facilitate the modified and biocompatible development of porous graphene-based systems for industrial applications. The potential toxic effects posed by GNPs reveal that the toxicity of other porous derivatives of graphene such as three-dimensional graphene foam, graphene hydrogels, graphene aerogels, porous graphene nanosheets, and other composites must be evaluated to a wide range of cells and animal models to minimize their adverse effects and risks to the off-target living organisms and tissues. The assessment of biosafety and biocompatibility of graphene will certainly have an impact on commercialisation of graphene, and in opening up new gateways for their use in clinical settings. Therefore, long-term, high dose, and careful selection of administration route using different animal models are crucial before seeking any clinical application of this ‘wonder material’. """"

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"" One important advantage of graphene-based materials is their ability to effectively cross biological barriers such as the blood brain barrier,"""

And that maybe an advantage but can also be a great danger as well to the human brain...

As well a danger to the kidneys....--Tyr