Kaolin Alleviates Graphene Oxide Toxicity - Environmental Science

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Kaolin alleviates graphene oxide toxicity Marina Kryuchkova, and Rawil F. Fakhrullin Environ. Sci. Technol. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.estlett.8b00135 • Publication Date (Web): 05 Apr 2018 Downloaded from http://pubs.acs.org on April 6, 2018

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Environmental Science & Technology Letters

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Kaolin Alleviates Graphene Oxide Toxicity

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Marina Kryuchkova, Rawil Fakhrullin*

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Institute of Fundamental Medicine and Biology, Kazan Federal University, Kreml uramı 18, Kazan, Republic of Tatarstan, Russian Federation, 420008

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*Corresponding author, e-mail: [email protected], Telephone: (843) 5905506

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Abstract

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

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Introduction

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Graphene and graphene oxide-based materials1 are intensively gaining popularity among researchers as novel and promising materials for electronics2, energy3, catalysis4, nanoarchetictonics5, environmental6 and biomedical7 applications. The wider application areas and increased production, however, implies the increased risk of graphene oxide exposure to the environment, including aqueous habitats8. Graphene oxide is a toxic material9, numerous studies have demonstrated the adverse effects of graphene oxide on human keratinocytes10, blood cells11, microorganisms12,13 and mammals14. In particular, aqueous organisms, both marine15 and freshwater16 can be severely affected during graphene oxide exposure. Therefore, it is challenging to envisage a safe and effective approach to alleviate graphene oxide toxicity in aqueous ecosystems. Importantly, the remediation of graphene oxide toxicity should be ideally performed in the antidote-like way, without the physical removal of the adsorbed nanomaterial, making it very attractive for practical environmental applications. To the best of our knowledge no approaches have been suggested so far to reduce the toxicity of graphene oxide without removal of adsorbed graphene oxide from the environment.

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Here were report the effective alleviation of graphene oxide toxicity by kaolin nanoclay. We found that kaolin coagulates with graphene oxide in water, producing relatively large complexes. This reduces the adverse effects of graphene oxide on P. caudatums protozoans, increasing survival and growth rate, normalising chemotaxis and galvanotaxis, improving food digestion and reducing macronucleus deformities. Overall, our results indicate that kaolin may find applications in environmental remediation of graphene-related materials.

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Materials and Methods

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Chemicals Kaolin nanoclay and graphene oxide aqueous solution were purchased from Sigma-Aldrich. Graphene oxide was sonicated for 5 min prior to use. All other chemicals were purchased from Sigma-Aldrich unless noted otherwise. Paramecium caudatum culture P. caudatum protozoan cells were cultivated in aqueous growth media supplemented by NaCl (1.0 g L-1); KCl (0.1 g L-1); NaHCO3 (0.2 g L-1); MgSO4 (0.1 g L-1); CaCl2 (0.1 g L-1) at 22-24 °С. Protozoans were fed by Saccharomyces cerevisiae yeast added into growth media. P. caudatum cells were harvested by centrifugation (3000 rpm, 10 min) at the logarithmic growth phase and washed with media. For certain experiments and imaging, the ciliates were picked up manually, using a micropipette. A Carl Zeiss Stemi 2000C stereomicroscope was used for the routine observation of the animals. In vivo toxicity alleviation investigation The toxicity of kaolin and graphene oxide was evaluated by adding their pure or equally mixed suspensions into P. caudatum growth media at 10; 25; 50; 100; 500 and 1000 µg mL-1. The cells were exposed to kaolin and graphene oxide for 24 – 96 hours. The protozoans had access to yeast food during the exposure. All experiments were performed in triplicates, data presented as mean±standard deviation. Chemotaxis assay For chemotaxis assay, two adjacent growth media drops (0.01 mL) were placed onto a microscopy glass slide and positioned under the stereomicroscope. The first drop contained the appropriate concentration of kaolin, graphene oxide, or their mixture (at 10, 25, 50, 100, 500 µg mL-1), the second was the pure media with yeast cells (food source). 10 cells were placed into the nanoparticle-doped media drop, then the drops were connected with the liquid media bridge, allowing the free propulsion of animals between the drops. The chemotaxis was evaluated microscopically by counting the number of the cells in each drop during 24 hours. The behavioural reaction was considered positive if the cells remained inside the nanoparticles-supplemented drop, whereas for negative chemotaxis the cells migrated actively into the pure media. Survival rate The cells (n=10-15) were collected manually from the stock culture (24 hour growth) and were inoculated into the wells of cell culture plates. Then 20 µL of kaolin, graphene oxide and their mixtures were added to reach the appropriate concentration on the final growth volume. The protozoans were incubated at 22-24°С during 24-96 hours. Viable and non-viable cells were counted using a stereomicroscope, those cells which were immobile and did not preserve the typical shape were considered as dead. Control experiments were performed using the pure growth media only. The survival rate (N, %) was calculated as follows: N = N2/N1×100 where N2 is the average number of protozoans at the end and N1 is the average number of protozoans at the start of the experiment. Vitality of protozoans The reproduction activity (vitality) was investigated by placing a single protozoan cell into cell culture plate wells (24-well plates were used) and monitoring the progeny growth rate. The appropriate volumes of kaolin, graphene oxide and their mixtures and growth media (including yeast food) were added into wells. The 2 ACS Paragon Plus Environment

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effects of nanoparticles on fertility were evaluated as the reduction of binary fission rate compared to control cells.

Phagocytosis investigation The effects of kaolin, graphene oxide and their mixtures (at 10, 50, 25, 50, 100 µg mL-1) on the intensity of phagocytosis in P. caudatum cells was investigated by counting the number of food vacuoles in cells after nanoparticles uptake. Protozoans (50 cells) were collected manually and placed into culture wells supplemented nanomaterials. The cells were incubated for 24-96 hours, then 10-15 protozoans were isolated and stained using 1% aqueous Congo Red dye which targets predominantly food vacuoles. Then the cells were fixated using 2.5% glutaraldehyde (in 0.1 M phosphate buffer, pH 7.4) and washed with water. The vacuole number was counted using microphotographs. Galvanotaxis assay For galvanotaxis (electric field stimulated motility) assay the cells were incubated for 24 with kaolin, graphene oxide and their mixtures (at 10, 50, 25, 50, 100 and 1000 µg mL-1)17. Protozoans (100 cells) were suspended in 2 mL media supplemented with nanomaterials, and incubated for 24 hours. Then the cells were transferred into a V-shaped vessel (70 mm) equipped with copper cathode and anode wires. A Keithley 2450 Source Meter SMU Instrument was used to generate electric field (1.5 V, for 10 mins). After migration of the cells, cellular fractions were collected using 8-channel pipette, to separate the cells into 8 fractions. Then the cells were transferred into individual wells and counted using a stereomicroscope. Macronucleus morphology The cells were incubated for 24-96 hours in growth media supplemented with 10, 50, 25, 50, 100 µg mL-1 kaolin, graphene oxide or the mixture of kaolin and graphene oxide. The macronuclei morphology were investigated using a fluorescence microscope after staining with DAPI dye (0.1 mg mL-1). 30 individual cells were investigated for each concentration studied. The bright field and fluorescence images were then overplayed, the overall shape and the numerical dimensions of the macronucleus (length, width, stretching index) were estimated from the images. Characterisation techniques Aqueous hydrodynamic diameters and zeta-potential of nanoparticles were measured in water at 25 °С using a Malvern Zetasizer Nano ZS instrument and standard U-shaped plastic cells. Bright field and fluorescence microscopy images were collected using a Carl Zeiss Axio Imager microscope equipped with an AxioCam HRC CCD camera. Optical microscopy images were processed using ZEN software (Carl Zeiss). Atomic Force Microscopy images of kaolin and graphene oxide were obtained using a Dimension FastScan instrument (Bruker) operated by Bruker Nanoscope software in ScanAsyst™ mode in air using silicon-nitride ScanAsyst Air probes (nominal tip radius 2 nm). Diluted dispersions of particles were dropped onto clean glass substrates and dried in air. The raw AFM images obtained were processed using NanoScope Analysis v.1.6. software (Bruker). Dark field microscopy images were obtained using an Olympus BX51 upright microscope equipped with a CytoViva® enhanced dark-field condenser and a DAGE CCD camera. Hyperspectral images (hypercubes) were collected using a PixelFly PCO CCD camera coupled to a VIS-near IR spectrometer. Dust-free Nexterion® glass slides and coverslips (Schott) were used for dark field microscopy imaging to minimise dust interference. 3 ACS Paragon Plus Environment


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Results and Discussion

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Here we report the effective remediation of graphene oxide toxicity by kaolin clay particles. In our previous study18 we investigated the toxicity of clay nanoparticles employing graphene oxide nanoplatelets as a control material with known high toxicity towards freshwater protists. After the experiments with pure nanoparticles exposure, we decided to evaluate the synergistic effects of mixed suspensions containing nanoclay and graphene oxide in equal concentrations. Atomic force microscopy (AFM) images of the nanoparticles used in this study are shown in Figure S1. Surprisingly we found that unlike other nanoclay used, kaolin platelets exhibited very pronounced remediation effects towards graphene oxide induced toxicity. We evaluated the remediation of graphene oxide toxicity by kaolin particles using Paramecium caudatum as an in vivo model. P. caudatum, motile unicellular microscopic organisms (Figure S2), feed on yeast taking up other particles suspended in the growth media. If pure kaolin or graphene oxide particles are added to the media, the protozoans will ingest them during grazing. The first insight on the antidote-like effect of kaolin nanoclay was obtained during a simple behavioural chemotaxis test, when the animals were allowed to choose freely the media droplets containing either graphene oxide or graphene oxide supplemented with an equal kaolin concentration. Microscopic observations have confirmed that most of the P. caudatum protists were confined to the graphene oxide and kaolin containing media droplets, avoiding pristine graphene oxide (Figure. S3).

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Next, we have applied a set of toxicity evaluation tests to evaluate the toxicity remediation effects of kaolin on graphene oxide. We have found graphene oxide to be highly toxic to P. caudatum within a range of concentrations, which were used in this study (10 – 1000 µg mL-1). Equal concentrations of kaolin were added to graphene oxide suspensions, sonicated, co-incubated for 6 h, and then added to protists (10-15 cells per vessel). The cells were cultivated for 24-96 hours, then the dead deformed and non-motile cells were counted. We found that pure graphene oxide was toxic at 500 and 1000 µg mL-1 (Figure 1 a) during 24 h incubation. Apart from evidently dead cells, the considerable motility retardation was observed. However, the toxicity of graphene oxide was alleviated by the equal concentrations of kaolin present in the media. At 1000 µg mL-1 of graphene oxide the mortality of P. caudatum was 54.6%, while the equal concentration of kaolin added to graphene oxide reduced this number for only 7%. Even more pronounced toxicity remediation was observed during the reproduction test (Figure 1 b). We observed the growth rate of cells exposed to pure and kaolin-doped graphene oxide suspensions for 24 h. Graphene oxide severely affects reproduction of P. caudatum even at relatively low concentrations, at 10 µg mL-1 the growth rate was 67% from control, at higher concentrations reproduction was completely suppressed. Kaolin effectively remediated the reproductive toxicity, at 500 and 1000 µg mL-1 growth rate was within ~30%, while at lower concentration the growth rate was the same as in control

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samples. Similar results were obtained for longer incubation periods (data not shown).

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Figure 1. Kaolin alleviates the toxic effects of graphene oxide on survival rate (a) and growth rate (b) in exposed P. caudatum cells.

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The effects of graphene oxide on growth rate suggest that the toxicity mechanisms might be related to DNA complexation and damage19. Indeed, graphene oxide affects the macronucleus morphology and size in P. caudatum (Figure 2 a). During 24 h of graphene oxide treatment at 100 µg mL-1, nucleus average length was 34% longer and total nucleus area 21% larger than that in the non-treated cells. At the same time, we have observed the 10% reduction of macronucleus width, outlining the rodshaped nucleus deformity characterised with low length to width ratio20. Similar effects were observed for other concentrations of graphene oxide. However, kaolin added to graphene oxide at the equal concentrations completely removed the toxic effects of graphene oxide on DNA (Figure 2 b).



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Figure 2. Optical and merged fluorescence microscopy images of DAPI-stained macronuclei in P. caudatum: a) - P. caudatum macronucleus exhibiting the rod shape deformity caused by graphene oxide at 100 µg mL-1; b) – normal nucleus morphology in cell treated by graphene oxide and kaolin mixture (100 µg mL-1)

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Next, we evaluated the uptake and food vacuole formation in P. caudatum exposed to graphene oxide and kaolin. We used Saccharomyces cerevisiae yeast as food source to investigate the toxic effects of graphene oxide and their alleviation by kaolin (Figure 3 a-d). Normally, P. caudatum rely on formation of food vacuoles for digestion of microbial cells. Kaolin per se does not inhibit the formation of food vacuoles, whereas cells treated with graphene oxide (100 µg mL-1) for 24 h showed reduced number (by 44%) than control cells. Addition of higher concentrations of graphene oxide caused further reduction of food vacuole formation. However, if kaolin was added to the food mixture doped with graphene oxide, this lead to 20% increase of food vacuoles number if compared with graphene oxide treated cells.


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Figure 3. Dark-field microscopy images of P. caudatum: a) – non-treated; b) treated by kaolin (100 µg mL-1) alone (note developed food vacuoles); c) – treated by graphene oxide (100 µg mL-1) alone (no food vacuoles observed); d) – treated by mixture of kaolin and graphene oxide (100 µg mL-1) (food vacuoles observed); e) – reflected light spectrum of pure graphene oxide; f) – reflected light spectrum of pure kaolin; g) – hyperspectral map of kaolin and graphene oxide distribution in P. caudatum cell.

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We also employed dark field microscopy coupled with hyperspectral imaging21 to investigate the distribution of graphene oxide and kaolin in P. caudatum cells. This approach allows for imaging of nanomaterials distribution in live organisms22, unlike electron microscopy imaging. Spectral libraries of kaolin and graphene oxide were collected and then applied to map the distribution of both materials in live P. caudatum cells (Figure 3 e-f). We found that graphene oxide maps were overlapped with kaolin maps, diffusely distributed both in food vacuoles, near the macronucleus and in cytoplasm. Noteworthy, nanoparticles were also detected close to cellular membrane, which explains well our results (Figure S4) on the effects of graphene oxide and kaolin on electrically forced motility of P. caudatum (galvanotaxis).23 Galvanotaxis study was performed after 24 h incubation of protozoans with graphene oxide, kaolin, or mixture of both. Then the cells were placed into the electrodeequipped vessel where the voltage (1.5 V) was applied for 10 min. Normally, the voltage-stimulated cells travel to anode, however toxic materials may inhibit galvanotaxis by blocking potassium channels in protozoans’ cilia membranes. We found that graphene oxide alone at the highest concentration studied (1 mg mL-1) severely inhibited (56%) cellular galvanotaxis, however if kaolin was added (1 mg mL1 ), this somewhat alleviated the inhibitory effect (22% inhibition). Kaolin alone did not significantly inhibit galvanotaxis in P. caudatum protozoans within the concentrations studied. Our findings confirm that kaolin reduces significantly the membrane-related toxic effects of graphene oxide.

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From our results it appears that kaolin strongly interacts with graphene oxide, leading to attenuation of its chemical properties. The mechanisms of graphene-related materials nanotoxicity are not fully understood yet, however the influence of size and morphology of graphene oxide particles was suggested9. Kaolin has been previously reported to remediate silver nanoparticles toxicity in aqueous environments.24 Both 7 ACS Paragon Plus Environment


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materials used in our study had similar size distribution (1.9 – 2.2 µm hydrodynamic diameter) and exhibited negative zeta potentials (- 47 mV for graphene oxide and – 22 mV for kaolin). This suggests that there will be no effective electrostatic interaction between similarly-charged particles, which was demonstrated earlier for heteroaggregation of graphene oxide with montmorillonite, kaolinite, and goethite25. In our study, however, we found that kaolin strongly aggregates with graphene oxide (Figure 4). Within the range of concentrations studied 90-100% of graphene oxide was coagulated by kaolin and could be removed from the media via centrifugation or even via gravity sedimentation. AFM imaging and adhesion force mapping (in Peak Force Tapping mode) was applied to confirm the coagulation of graphene oxide with kaolin particles (Figure 4 d). AFM topography images and adhesion force maps show thin graphene oxide sheets deposited onto relatively large nanoclay particles. Previously, it has been reported that kaolin platelets exhibit partially positively charged edges at pH below 726,27, thus facilitating the aggregation of graphene oxide. Apart from optical absorbance and AFM study, the increase of hydrodynamic diameter value (ca. 4 µm) with remaining overall negative zeta-potential (- 38 mV) of the resulting coagulated particles confirms the edge aggregation of graphene oxide with kaolin.

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Figure 4. Graphene oxide adsorption on kaolin nanoparticles: a) a photograph demonstrating the colour change in kaolin/graphene oxide mixtures after centrifugation; b) – absorption (removal) of graphene oxide by kaolin as measure using optical absorbance at 277 nm; atomic force microscopy images of kaolingraphene oxide complexation: c) – topography image, d) – corresponding adhesion force map.

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As our data confirm, kaolin alleviates the toxicity of adsorbed graphene oxide without its removal from the media where the protozoans are cultivated, indicating that kaolin acts as an antidote to alleviate graphene oxide toxicity. We have evaluated several other nanoclays (halloysite and montmorillonite) in a set of preliminary studies, though some minor toxicity alleviations were also detected, the best performance was demonstrated by kaolin. Apparently, the corresponding geometry of kaolin and graphene oxide sheets along with larger area of positively-charged edges facilitate the adsorption of graphene oxide onto kaolin if compared with other nanoclays. Currently, the exact toxicity mechanisms of graphene-related materials, including graphene oxide, are not elucidated yet.9 In case of chemically pure graphene oxide 8 ACS Paragon Plus Environment

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not modified with surfactants or polymers, several toxicity pathways have been proposed, including oxidative stress28, DNA damage19, membrane damage29 and cell wall damage due to sharp edges of the particles30. Our results indicate that most of these toxicity factors are effectively alleviated by complexation with kaolin, which apparently arrests the reactively-active sites on graphene oxide nanoparticles, thus reducing its chemical activity. Further studies are necessary to find out if this remediation effect of kaolin is universal and can be applied for other model systems (i.e. human cells and higher multicellular organisms), the exact mechanisms of toxicity alleviation are to be discovered. Our current findings suggest that kaolin nanoclay, cheap and environmentally-friendly material, may find applications in remediation of toxicity of graphene-related materials in freshwater environments.

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Acknowledgement

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The work is performed according to the Russian Government Program of Competitive Growth of Kazan Federal University. This work was funded by the subsidy allocated to Kazan Federal University for the state assignment in the sphere of scientific activities (6.7743.2017/BY).

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Supporting information: additional supporting figures, as noted in the text.

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