Toxicity Of Graphene Oxide Research Articles

Graphene Oxide Nanoplatelets Potentiate Anticancer Effect of Cisplatin in Human Lung Cancer Cells.

Graphene oxide (GO) has been widely explored by many in drug delivery strategies and toxicity assays. The toxicity of graphene oxide depends on the size of the sheets. Smaller sheets show lower toxicity, a quality which is essential for utilization in biomedical applications. However, despite vast research on GO, anticancer properties and drug carrier capabilities of graphene oxide nanoplatelets have yet to be fully explored. Herein, we have uniquely prepared graphene oxide nanoplatelets (GONPs) from well-defined stacked graphite nanofibers (SGNF) with a base of 50 × 50 nm2 for toxicity and drug potentiation studies when coadministered with the chemotherapeutic drug cisplatin (CP) in human lung cancer cells, A549 cells. Results obtained from our studies have found that not only were GONPs able to act as drug carriers, but they can also significantly potentiate anticancer effect of CP in A549 cells.

"Ultramixing": A Simple and Effective Method To Obtain Controlled and Stable Dispersions of Graphene Oxide in Cell Culture Media.

The last decade has seen an increase in the application of graphene oxide (GO) in the biomedical field. GO has been successfully exploited for its ability to deliver many kinds of drugs into target cells. However, GO toxicity assessment is still controversial. Several studies have demonstrated that GO protein coating is crucial to alleviate the material's toxicity. Besides, coronation leads to the formation of big agglomerates, reducing the cellular uptake of the material and thus its therapeutic efficiency. In this work, we propose a simple and efficient method based on rapid (ultra-turrax, UT) mixing to control protein corona formation. Using the UT protocol, we were able to reduce GO agglomeration in the presence of proteins and obtain stable GO dispersions in cell culture media. By labelling GO with luminescent nanoparticles (quantum dots), we studied the GO internalization kinetic and efficiency. Comparing the "classic" and UT protocols, we found that the latter allows faster and more efficient internalization both in macrophages and HeLa cells without affecting cell viability. We believe that the use of UT protocol will be interesting and suitable for the preparation of next-generation GO-based drug-delivery platforms.

Kaolin alleviates the toxicity of graphene oxide for mammalian cells.

The development of novel nanoscale vehicles for drug delivery promotes the growth of interest in investigations of interaction between nanomaterials. In this paper, we report the in vitro studies of eukaryotic cell physiological response to incubation with graphene oxide and planar kaolin nanoclay. Graphene family materials, including graphene oxide (GO), hold promise for numerous applications due to their unique electronic properties. However, graphene oxide reveals toxicity to some cell lines through an unidentified mechanism. Thus, methods and agents reducing the toxicity of graphene oxide can widen its practical application. We used a colorimetric test, flow cytometry and cell index assay methods to evaluate the effects of separate and combined application of graphene oxide and kaolin on mammalian cells. We have shown that the joint application of graphene oxide and kaolin reduced the negative effects of graphene by almost 20%, most likely because of coagulation of the nanoparticles with each other, which was detected by atomic force microscopy.

Comparative toxicity evaluation of graphene oxide (GO) and zinc oxide (ZnO) nanoparticles on Drosophila melanogaster.

Engineered nanomaterials consisting of multiple nanoparticles (NPs) are finding their use in fields as wide and diverse as medicine, environment, cosmetics, energy and electronics. However, health and environmental impacts of these NPs need to be discerned individually to understand their true toxicity. Due to the promising application of upcoming material like GO-ZnO nanocomposite, the toxicity of ZnO and GO NPs was evaluated and compared individually in our study. This study compares the toxicity of Graphene Oxide (GO) NPs and Zinc Oxide (ZnO) NPs synthesized by Green method and Chemical method on Drosophila melanogaster. The GO, Chemical ZnO and Green ZnO NPs were synthesized and characterized using SEM, HR-TEM, FT-IR, UV-vis, EDX, XRD and DLS studies. NPs were comparatively analyzed for their cytotoxic and neurotoxic behaviors using different assays like MTT assay, mortality rate, larval crawling and climbing assay, total protein content analysis for evaluating the toxic potential of each of these NPs at different concentrations of use. Green ZnO were found to be least cytotoxic while Chemical ZnO caused the most cell damage. GO were found to have intermediary cytotoxicity. However, a different trend was observed with neurotoxicity wherein Green ZnO reportedly affected the neuromuscular coordination the most, while GO was found to have the least affect. This study provided insights into the different toxic effects caused by GO and ZnO NPs on Drosophila as well as comparative toxic effects of Chemical vs Green ZnO NPs.

Disinfection byproduct formation and toxicity of graphene oxide in water treatment system

Graphene oxide (GO) is a structural analog of graphene and contains numerous O-containing functional groups. As rapidly increasing production and usage of GO, it is inevitable to flow into the water and wastewater treatment system and finally oxidized by disinfectants to form DBPs. Meanwhile, as GO is a nano sized carbon material, it may also break the human digestion system when it was absorbed by human body. This study explored the DBP formation when only GO was present. Effects of Br− were also considered during the DBP formation. Both trihalomethanes (THMs) and haloacetic acids (HAAs) were formed during the chlorination and chloramination procedure, but the total concentration of THMs was at least three times higher than that of HAAs. Irradiation can significantly enhance the DBP formation via the formation of radicals. The wrinkled appearance and decomposition of aromatic ring may both be effective on the DBP formation via chlorination or bromination. The findings of this study advance knowledge on the DBP formation of GO in water treatment systems and provide insight on the toxic effects of the transformation products of GO.

Toxicity of Graphene Oxide in Nematodes with a Deficit in the Epidermal Barrier Caused by RNA Interference Knockdown of unc-52

The epidermal barrier is important for the defense of environmental organisms against the damage from engineered nanomaterials (ENMs). We employed Caenorhabditis elegans to examine the possible influence of a deficit in the epidermal barrier caused by RNA interference (RNAi) knockdown of unc-52 encoding a perlecan protein on the toxicity of graphene oxide (GO). Epidermal RNAi knockdown of unc-52 caused a functional deficit in the epidermal barrier and susceptibility to GO toxicity. Epidermal knockdown of unc-52 decreased the level of expression of fbl-1 encoding a membrane protein fibulin and sax-7 encoding a cell adhesion receptor, and epidermal knockdown of fbl-1 or sax-7 also resulted in a functional deficit in the epidermal barrier and susceptibility to GO toxicity. Additionally, epidermal knockdown of unc-52 inhibited expression of cnc-2 and prx-11 encoding two antimicrobial proteins, and epidermal knockdown of cnc-2 or prx-11 could strengthen the GO toxicity in fbl-1(RNAi) or sax-7(RNAi) nematodes. ...

Toxicity of GO to Freshwater Algae in the Presence of Al2O3 Particles with Different Morphologies: Importance of Heteroaggregation.

The roles of Al2O3 particles with different morphologies in altering graphene oxide (GO) toxicity to Chlorella pyrenoidosa were investigated. Algal growth inhibition by GO with coexisting Al2O3 particles was much lower than the sum of inhibitions from the individual materials for all the three Al2O3, showing the toxicity mitigation by Al2O3. The lowest GO toxicity was observed at the concentrations of 300, 150, and 100 mg/L for Al2O3 nanoparticles (NPs, 8-10 nm), bulk particles (BPs, 100-300 nm), and fibers (diameter: 10 nm; length: 400 nm), respectively. GO-Al2O3 heteroaggregation was responsible for the observed toxicity reduction. GO-induced algal membrane damage was suppressed by the three types of Al2O3 due to GO-Al2O3 heteroaggregation, and the reduction in intracellular reactive oxygen species generation and physical contact were confirmed as two main mechanisms. Moreover, the exposure sequence of GO and Al2O3 could highly influence the toxicity, and the simultaneous exposure of individual GO and Al2O3 showed the lowest toxicity due to minimum direct contact with algal cells. Humic acid further decreased GO-Al2O3 toxicity due to enhanced steric hindrance through surface coating of GO-Al2O3 heteroaggregates. This work provides new insights into the role of natural mineral particles in altering the environmental risk of GO.

Cytotoxicity Evaluation of Ammonia-Modified Graphene Oxide Particles in Lung Cancer Cells and Embryonic Stem Cells

Potential toxicity of graphene oxide (GO) is a subject of increasing research interest in the recent years. Here, we have evaluated the cytotoxicity of ammonia-modified GO (GO-NH2) and pristine GO particles in human lung cancer cells, A549 and embryonic stem cells, Lep3 exposed to different particles concentrations (0.1, 1, 10, 20, and 50 μg/ml) for different times (24 and 48h). Compared with GO, GO-NH2 particles possessed smaller size, positive surface charge and higher thickness. An increased propensity to aggregation in cell cultures was also found for GO-NH2 particles. Cytotoxicity evaluation revealed that GO-NH2 particles are more toxic than pristine GO. Applied at concentrations of 10, 20 and 50 μg/ml for 24h they affect significantly cell morphology of viable embryonic stem cells whereas human lung cancer A549 cells seem to be relatively more resistant to short-time exposure. After 48h exposure however cell proliferation of A549 cells was strongly suppressed in a dose-dependent manner while the proliferation ability of embryonic stem cells was not affected. These results suggested that both GO particles exert different degree of cytotoxicity which is time, dose and cell dependent. In general, ammonia-modified GO particles are more toxic than the pristine GO which should be taken into account for future biomedical applications.

Self-Damaging Aerobic Reduction of Graphene Oxide by Escherichia coli: Role of GO-Mediated Extracellular Superoxide Formation.

Microbial reduction of graphene oxide (GO) under aerobic conditions is poorly understood despite its critical role in changing GO toxicity and environmental fate. Here we show that 20 mg/L GO interacts with the membrane-bound cytochrome c of E. coli in saline, shuttling electrons from the respiratory chain to extracellular molecular oxygen. This results in the formation of superoxide anions (O2•-), which in turn reduce GO in 30 min. The critical role of superoxide was demonstrated by impeding GO reduction upon addition of superoxide dismutase, or by carrying out experiments under strictly anaerobic conditions that preclude O2•- formation. Coating GO with bovine serum albumin also stopped GO reduction, which indicates the need for direct contact between GO and the cell membrane. Cell death was observed as a consequence of GO bioreduction. Apparently, electron shuttling by GO (via membrane contact) interrupts the respiratory chain and induces oxidative stress, as indicated by a 20% decrease in electron transport activity and an increase in intracellular reactive oxygen species. This novel antimicrobial mechanism could be relevant to assess GO stability and biocompatibility, and informs potential applications for microbial control.

Immunological impact of graphene oxide sheets in the abdominal cavity is governed by surface reactivity

Graphene oxide (GO) is an oxidised form of graphene that has attracted commercial interest in multiple applications, including inks, printed electronics and spray coatings, which all raise health concerns due to potential creation of inhalable aerosols. Although a number of studies have discussed the toxicity of GO sheets, the in vivo impact of their lateral dimensions is still not clear. Here, we compared the effects of large GO sheets (l-GO, 1–20 µm) with those of small GO sheets (s-GO, < 1 µm) in terms of mesothelial damage and peritoneal inflammation, after intraperitoneal (i.p.) injection in mice. To benchmark the outcomes, long and rigid multi-walled carbon nanotubes (MWCNTs) that were shown to be associated with asbestos-like pathogenicity on the mesothelium were also tested. Our aim was to assess whether lateral dimensions can be a predictor of inflammogenicity for GO sheets in a similar fashion as length is for MWCNTs. While long MWCNTs dispersed in 0.5% BSA induced a granulomatous response on the diaphragmatic mesothelium and immune cell recruitment to the peritoneal cavity, GO sheets dispersed under similar conditions did not cause any response, regardless of their lateral dimensions. We further interrogated whether tuning the surface reactivity of GO by testing different dispersions (5% dextrose instead of 0.5% BSA) may change the biological outcome. Although the change of dispersion did not alter the impact of GO on the mesothelium (i.e. no granuloma), we observed that, when dispersed in protein-free 5% dextrose solution, s-GO elicited a greater recruitment of monocytic cells to the peritoneal cavity than l-GO, or when dispersed in protein-containing solution. Such recruitment coincided with the greater ability of s-GO to interact in vivo with peritoneal macrophages and was associated with a greater surface reactivity in comparison to l-GO. In conclusion, large dimension was not a determining factor of the immunological impact of GO sheets after i.p. administration. For an equal dose, GO sheets with lateral dimensions similar to the length of long MWCNTs were less pathogenic than the MWCNTs. On the other hand, surface reactivity and the ability of some smaller GO sheets to interact more readily with immune cells seem to be key parameters that can be tuned to improve the safety profile of GO. In particular, the choice of dispersion modality, which affected these two parameters, was found to be of crucial importance in the assessment of GO impact in this model. Overall, these findings are essential for a better understanding of the parameters governing GO toxicity and inflammation, and the rational design of safe GO-based formulations for various applications, including biomedicine.

The effects of humic acid on the toxicity of graphene oxide to Scenedesmus obliquus and Daphnia magna

The wide production and application of graphene oxide (GO) has inevitably caused its release to the aquatic ecosystem. However, the influence of natural organic matter (NOM) on the toxicity of GO to aquatic organisms needs further investigation. In this study, we conducted several toxicity tests (i.e., acute toxicity and oxidative damage) with Scenedesmus obliquus (S. obliquus) and Daphnia magna (D. magna), as well as a chronic toxicity test with D. magna, to investigate the toxicity of GO with or without the presence of humic acid (HA). Our results showed that GO induced significant toxicity to S. obliquus and D. magna, and the median lethal concentrations (72 h-LC50 and 48 h-LC50) for acute toxicity were 20.6 and 84.2 mg L−1, respectively, while the 21 d-LC50 for chronic toxicity to D. magna was 3.3 mg L−1. Additionally, HA mitigated the acute toxicity of GO to S. obliquus and D. magna by 28.6% and 32.3%, respectively, and mitigated the chronic toxicity of GO to D. magna. In the presence of HA, the decreased toxicity of GO was attributed to the alleviation of oxidative damage by HA to both S. obliquus and D. magna, the mitigation of surface envelopment to S. obliquus and the body accumulation in D. magna. Our study provides useful and basic biotoxicity data of GO with a consideration of its interaction with NOM which could aid in preventing an overestimation of the risks of GO to the natural aquatic environment.

Functional disruption in epidermal barrier enhances toxicity and accumulation of graphene oxide

In Caenorhabditis elegans, mutation of mlt-7 causes the deficits in epidermal barrier. Using the nematodes with epidermal-specific RNA interference (RNAi) knockdown of mlt-7 as a genetic tool, we found that epidermal-specific RNAi knockdown of mlt-7 resulted in a susceptibility to graphene oxide (GO) toxicity, and enhanced GO accumulation in the body. Epidermal-development related proteins of BLI-1 and IFB-1 acted as downstream targets of MLT-7, and mediated the function of MLT-7 in maintaining the epidermal barrier. Antimicrobial proteins of NLP-30 and CNC-2 also acted as downstream targets of MLT-7 in the regulation of GO toxicity. Epidermal-specific RNAi knockdown of nlp-30 or cnc-2 enhanced GO toxicity and accumulation in bli-1(RNAi) or ifb-1(RNAi) nematodes. Our data highlights the importance of maintaining normal epidermal barrier for nematodes against the GO toxicity.

Developmental basis for intestinal barrier against the toxicity of graphene oxide

BackgroundIntestinal barrier is crucial for animals against translocation of engineered nanomaterials (ENMs) into secondary targeted organs. However, the molecular mechanisms for the role of intestinal barrier against ENMs toxicity are still largely unclear. The intestine of Caenorhabditis elegans is a powerful in vivo experimental system for the study on intestinal function. In this study, we investigated the molecular basis for intestinal barrier against toxicity and translocation of graphene oxide (GO) using C. elegans as a model animal.ResultsBased on the genetic screen of genes required for the control of intestinal development at different aspects using intestine-specific RNA interference (RNAi) technique, we identified four genes (erm-1, pkc-3, hmp-2 and act-5) required for the function of intestinal barrier against GO toxicity. Under normal conditions, mutation of any of these genes altered the intestinal permeability. With the focus on PKC-3, an atypical protein kinase C, we identified an intestinal signaling cascade of PKC-3-SEC-8-WTS-1, which implies that PKC-3 might regulate intestinal permeability and GO toxicity by affecting the function of SEC-8-mediated exocyst complex and the role of WTS-1 in maintaining integrity of apical intestinal membrane. ISP-1 and SOD-3, two proteins required for the control of oxidative stress, were also identified as downstream targets for PKC-3, and functioned in parallel with WTS-1 in the regulation of GO toxicity.ConclusionsUsing C. elegans as an in vivo assay system, we found that several developmental genes required for the control of intestinal development regulated both the intestinal permeability and the GO toxicity. With the focus on PKC-3, we raised two intestinal signaling cascades, PKC-3-SEC-8-WTS-1 and PKC-3-ISP-1/SOD-3. Our results will strengthen our understanding the molecular basis for developmental machinery of intestinal barrier against GO toxicity and translocation in animals.

Characterization of the effects of trace concentrations of graphene oxide on zebrafish larvae through proteomic and standard methods

The effects of graphene oxide (GO) carbon nanomaterials on ecosystems have been well characterized, but the toxicity of GO at predicted environmental concentrations to living organisms at the protein level remain largely unknown. In the present work, the adverse effects and mechanisms of GO at predicted environmental concentrations were evaluated by integrating proteomics and standard analyses for the first time. The abundances of 243 proteins, including proteins involved in endocytosis (e.g., cltcb, arf6, capzb and dnm1a), oxidative stress (e.g., gpx4b, sod2, and prdx1), cytoskeleton assembly (e.g., krt8, krt94, lmna and vim), mitochondrial function (e.g., ndufa10, ndufa8, cox5aa, and cox6b1), Ca2+ handling (e.g., atp1b2a, atp1b1a, atp6v0a1b and ncx4a) and cardiac function (e.g., tpm4a, tpm2, tnni2a.1 and tnnt3b), were found to be notably altered in response to exposure 100 μg/L GO. The results revealed that GO caused malformation and mortality, likely through the downregulation of proteins related to actin filaments and formation of the cytoskeleton, and induced oxidative stress and mitochondrial disorders by altering the levels of antioxidant enzymes and proteins associated with the mitochondrial membrane respiratory chain. Exposure to GO also increased the heart rate of zebrafish larvae and induced pericardial edema, likely by changing the expression of proteins related to Ca2+ balance and cardiac function. This study provides new proteomic-level insights into GO toxicity against aquatic organisms, which will greatly benefit our understanding of the bio-safety of GO and its toxicity at predicted environmental concentrations.

Graphene Oxide as Scaffolds for Stem Cells: An Overview.

Graphene and graphene oxide topography have an effect on the fate of stem cells such as adhesion, differentiation, and proliferation. This overview clearly shows that a new design and manipulation of associated graphene oxide stem cell culture platforms are of paramount importance as a focus in stem cell to tissue engineering applications. This overview also proposes that a film of graphene oxide is an efficient platform to modulate structure and function of multipotent mesenchymal stem/stromal cells (MSCs) and in special human adipose tissue derived mesenchymal stromal/stem cells (AT-MSCs). The implication of graphene oxide on osteogenesis, neurogenesis, oligodendrogenesis, adipogenic and epithelial differentiation is also discussed. Graphene oxide toxicity on stem cells and the importance of GO application on ATMSCs differentiation and proliferation are final topics that are being discussed.

Kaolin Alleviates Graphene Oxide Toxicity

With increased interest in the practical use of graphene-based materials, concerns about the remediation of the environmental nanotoxicity of graphene and graphene-related materials have grown. In this study, we report that kaolin nanoclay significantly alleviates the toxicity of graphene oxide in aqueous environments. We employed the Paramecium caudatum protozoan to demonstrate the effects of equal concentrations of kaolin on the remediation of graphene oxide toxicity on survival and growth rates, chemotaxis, galvanotaxis, DNA complexation, and food vacuole formation. Importantly, the toxicity of graphene oxide coagulated with kaolin is reduced without the aggregated particles being removed from the environment.

Comparative toxicity of pristine graphene oxide and its carboxyl, imidazole or polyethylene glycol functionalized products to Daphnia magna: A two generation study

To investigate the chronic toxicity of graphene oxide (GO) and its functionalized products (GO-carboxyl, GO-imidazole and GO-polyethylene glycol), a two-generation study was conducted using the aquatic model species Daphnia magna. Each generation of daphnids were exposed for 21 days to 1.0 mg L−1 graphene material, with body length, neonate number, time of first brood and the intrinsic rate of natural increase (r) assessed as endpoints. Chronic exposure to GO, GO-carboxyl, and GO-imidazole had no adverse effect on body length or offspring number in the daphnid F0 generation, however, this exposure paradigm led to significant growth or reproduction inhibition in the following generation. Meanwhile, GO was found to show the strongest inhibitory effect, sequentially followed by GO-carboxyl and GO-imidazole. With exposure to GO-polyethylene glycol, no significant effects on growth or reproduction were observed for both F0 and F1 generation daphnids. These results reveal that carboxyl, imidazole and polyethylene glycol functional attachments alleviate the bio-toxicity of GO, especially polyethylene glycol. The increased C/O atomic ratio present in GO-carboxyl, GO-imidazole and GO-polyethylene glycol due to functionalization may mainly explain the reduced toxicity.

Short-term inhalation study of graphene oxide nanoplates

Graphene oxides possess unique physicochemical properties with important potential applications in electronics, pharmaceuticals, and medicine. However, the toxicity following inhalation exposure to graphene oxide has not yet been clarified. Therefore, this study conducted a short-term graphene oxide inhalation toxicity analysis using a nose-only inhalation exposure system and male Sprague–Dawley rats. A total of four groups (15 rats per group) were exposed: (1) control (fresh air), (2) low concentration (0.76 ± 0.16 mg/m3), (3) moderate concentration (2.60 ± 0.19 mg/m3), and (4) high concentration (9.78 ± 0.29 mg/m3). The rats were exposed to graphene oxide for 6 h/day for 5 days, followed by recovery for 1, 3, and 21 days. No significant body or organ weight changes were noted after the short-term exposure or during the recovery period. Similarly, no significant systemic effects of toxicological importance were noted in the hematological assays, bronchoalveolar lavage fluid (BAL) inflammatory markers, BAL fluid cytokines, or blood biochemical assays following the graphene oxide exposure or during the post-exposure observation period. Moreover, no significant differences were observed in the BAL cell differentials, such as lymphocytes, macrophages, or polymorphonuclear cells. Graphene oxide-ingested alveolar macrophages as a spontaneous clearance reaction were observed in the lungs of all the concentration groups from post 1 day to post 21 days. Histopathological examination of the liver and kidneys did not reveal any significant test-article-relevant histopathological lesions. Importantly, similar to previously reported graphene inhalation data, this short-term nose-only inhalation study found only minimal or unnoticeable graphene oxide toxicity in the lungs and other organs.

Systematic toxicity investigation of graphene oxide: evaluation of assay selection, cell type, exposure period and flake size.

Understanding the toxicity of nanomaterials is essential for the safe and sustainable development of new applications. This is particularly true for a nanomaterial as widely used as graphene oxide (GO), which is utilized as films for electronics, membranes for filtration, drug carriers and more. Despite this, the current literature presents conflicting results on the overall toxicity of GO. Here, the cytotoxicity of three sizes of commercially available GO was investigated on six cell lines, as values of NOAEL/LOAEL. The effectiveness of four viability assays was also evaluated. The overall toxicity of GO greatly varied between cell lines; the suspension cells showed a greater response to the GO treatment compared to the adherent cell lines. Time dependent cytotoxicity was also cell line dependent, with only one cell line demonstrating obvious dependence. The six cell lines were also tested to evaluate their response to varying GO flake sizes: the suspension/phagocytic cells showed little variation in viability, while a difference was observed for the adherent/non-phagocytic cell lines. By systematically studying the effect of dose, GO size and treatment time for the six cell lines by using commercially available GO samples, we eliminate many of the variables which may result in the conflicting reports on the cytotoxicity of GO in the literature.

Graphene oxide interactions with co-existing heavy metal cations: adsorption, colloidal properties and joint toxicity

Schematic diagram of the combined toxicity of graphene oxide and heavy metal cations towards bacteria.