Letter Cite This: Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/journal/estlcu
Kaolin Alleviates Graphene Oxide Toxicity Marina Kryuchkova and Rawil Fakhrullin* Institute of Fundamental Medicine and Biology, Kazan Federal University, Kreml uramı 18, Kazan, Republic of Tatarstan, Russian Federation 420008 S Supporting Information *
ABSTRACT: 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.
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INTRODUCTION Graphene and graphene oxide-based materials1 are intensively gaining popularity among researchers as novel and promising materials for electronics,2 energy,3 catalysis,4 nanoarchetictonics,5 and environmental6 and biomedical7 applications. The wider application areas and increased level of production, however, suggest an increased risk of environmental exposure to graphene oxide, including aqueous habitats.8 Graphene oxide is a toxic material;9 numerous studies have demonstrated the adverse effects of graphene oxide on human keratinocytes,10 blood cells,11 microorganisms,12,13 and mammals.14 In particular, aqueous organisms, both marine15 and freshwater,16 can be severely affected by graphene oxide exposure. Therefore, it is challenging to envisage a safe and effective approach to alleviating graphene oxide toxicity in aqueous ecosystems. Importantly, the remediation of graphene oxide toxicity should be ideally performed in an 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 to reducing the toxicity of graphene oxide without removal of adsorbed graphene oxide from the environment have been suggested so far. Here we 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 Paramecium caudatum protozoans, increasing survival and growth rates, normalizing chemotaxis and galvanotaxis, improving food digestion, and reducing the number of © XXXX American Chemical Society
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 Chemicals. Kaolin nanoclay and a 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. P. caudatum Culture. P. caudatum protozoan cells were cultivated in aqueous growth medium supplemented with NaCl (1.0 g L−1), KCl (0.1 g L−1), NaHCO3 (0.2 g L−1), MgSO4 (0.1 g L−1), and CaCl2 (0.1 g L−1) at 22−24 °C. Protozoans were fed Saccharomyces cerevisiae yeast added to the growth medium. P. caudatum cells were harvested by centrifugation (3000 rpm for 10 min) in logarithmic growth phase and washed with medium. 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 protists. Investigation of the Alleviation of in Vivo Toxicity. The toxicity of kaolin and graphene oxide was evaluated by adding their pure or equally mixed suspensions to P. caudatum growth medium at concentrations of 10, 25, 50, 100, 500, and Received: Revised: Accepted: Published: A
March 12, 2018 April 3, 2018 April 5, 2018 April 5, 2018 DOI: 10.1021/acs.estlett.8b00135 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX
Letter
Environmental Science & Technology Letters 1000 μg mL−1. The cells were exposed to kaolin and graphene oxide for 24−96 h. The protozoans had access to yeast food during the exposure. All experiments were performed in triplicate, and data are presented as means ± the standard deviation. Chemotaxis Assay. For the chemotaxis assay, two adjacent growth medium 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, and 500 μg mL−1), and the second was the pure medium with yeast cells (food source). Ten cells were placed into the nanoparticle-doped medium drop, and then the drops were connected with the liquid medium bridge, allowing the free propulsion of protists between the drops. Chemotaxis was evaluated microscopically by counting the number of the cells in each drop over 24 h. The behavioral reaction was considered positive if the cells remained inside the nanoparticlesupplemented drop, whereas for negative chemotaxis, the cells migrated actively into the pure medium. Survival Rate. The cells (n = 10−15) were collected manually from the stock culture (24 h 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 at the final growth volume. The protozoans were incubated at 22−24 °C for 24−96 h. Viable and nonviable cells were counted using a stereomicroscope; those cells that were immobile and did not preserve the typical shape were considered dead. Control experiments were performed using only the pure growth medium. The survival rate (N, %) was calculated as follows:
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 an electric field (1.5 V, for 10 min). After migration of the cells, cellular fractions were collected using an eight-channel pipet, to separate the cells into eight fractions. Then the cells were transferred into individual wells and counted using a stereomicroscope. Macronucleus Morphology. The cells were incubated for 24−96 h in growth medium supplemented with 10, 50, 25, 50, and 100 μg mL−1 kaolin, graphene oxide, or a mixture of kaolin and graphene oxide. The morphology of the macronuclei was investigated using a fluorescence microscope after staining with DAPI dye (0.1 mg mL−1). Thirty individual cells were investigated for each concentration that was studied. The bright field and fluorescence images were then overlaid, and the overall shape and the numerical dimensions of the macronucleus (length, width, and stretching index) were estimated from the images. Characterization Techniques. Aqueous hydrodynamic diameters and ζ potentials of nanoparticles were measured in water at 25 °C 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 (AFM) 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 of 2 nm). Diluted dispersions of particles were dropped onto clean glass substrates and dried in air. The raw AFM images that were obtained were processed using NanoScope Analysis version 1.6 (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 minimize dust interference.
N = N2/N1 × 100 where N2 is the average number of protozoans at the end of the experiment 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, their mixtures, and growth medium (including yeast food) were added to wells. The effects of nanoparticles on fertility were evaluated as the reduction of the binary fission rate compared to that of control cells. Investigation of Phagocytosis. The effects of kaolin, graphene oxide, and their mixtures (at 10, 50, 25, 50, and 100 μg mL−1) on the intensity of phagocytosis in P. caudatum cells were investigated by counting the number of food vacuoles in the cells after the uptake of nanoparticles. Protozoans (50 cells) were collected manually and placed in culture wells supplemented with nanomaterials. The cells were incubated for 24−96 h, and then 10−15 protozoans were isolated and stained using 1% aqueous Congo Red dye that targets predominantly food vacuoles. Then the cells were fixed using 2.5% glutaraldehyde [in 0.1 M phosphate buffer (pH 7.4)] and washed with water. The number of vacuoles was counted using microphotographs. Galvanotaxis Assay. For the galvanotaxis (electric fieldstimulated motility) assay, the cells were incubated for 24 h 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 of medium supplemented with nanomaterials and incubated for 24 h. Then the cells were transferred
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RESULTS AND DISCUSSION Here we report the effective remediation of graphene oxide toxicity by kaolin clay particles. In our previous study,18 we investigated the toxicity of clay nanoparticles employing graphene oxide nanoplatelets as a control material with a known high toxicity toward freshwater protists. After the experiments that included exposure to pure nanoparticles, we decided to evaluate the synergistic effects of mixed suspensions containing nanoclay and graphene oxide in equal concentrations. 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 with respect to graphene oxide-induced toxicity. We evaluated the remediation of graphene oxide toxicity by kaolin particles using P. caudatum as an in vivo model. P. caudatum, motile unicellular microscopic organisms (Figure S2), feed on yeast while taking up other particles suspended in the growth medium. If pure kaolin or graphene oxide particles are added to the medium, the protozoans will ingest them during grazing. The first insight into the antidotelike effect of kaolin nanoclay was obtained during a simple B
DOI: 10.1021/acs.estlett.8b00135 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX
Letter
Environmental Science & Technology Letters
Figure 1. Kaolin alleviates the toxic effects of graphene oxide on (a) the survival rate and (b) the growth rate in exposed P. caudatum cells.
behavioral chemotaxis test, during which the protists were allowed to choose freely the medium 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 medium droplets, avoiding pristine graphene oxide droplets (Figure S3). Next, we 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 h, and then the dead, deformed, and nonmotile cells were counted. We found that pure graphene oxide was toxic at concentrations of 500 and 1000 μg mL−1 (Figure 1a) during a 24 h incubation. Apart from evidently dead cells, considerable retardation of motility was observed. However, the toxicity of graphene oxide was alleviated by the equal concentrations of kaolin present in the medium. At 1000 μg mL−1 graphene oxide, the mortality of P. caudatum was 54.6%, while an equal concentration of kaolin added to graphene oxide reduced this number to only 7%. Even more pronounced toxicity remediation was observed during the reproduction test (Figure 1b). We observed the growth 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% of the control, and at higher concentrations, reproduction was completely suppressed. Kaolin effectively remediated the reproductive toxicity; at 500 and 1000 μg mL−1, the growth rate was ∼30%, while at lower concentrations, the growth rate was the same as in control samples. Similar results were obtained for longer incubation periods (data not shown). The effects of graphene oxide on growth rate suggest that the toxicity mechanisms might be related to DNA complexation and damage.19 Indeed, graphene oxide affects the macronucleus morphology and size of P. caudatum (Figure 2a). During a 24 h treatment with 100 μg mL−1 graphene oxide, the average nucleus length was 34% greater than that of and the total nucleus area 21% larger than that of the nontreated cells. At the same time, we observed a 10% reduction in macronucleus width, outlining the rod-shaped nucleus deformity characterized with a small length:width ratio.20 Similar effects were observed for other graphene oxide concentrations. However, kaolin added to graphene oxide at an equal concentration completely
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 100 μg mL−1 graphene oxide and (b) normal nucleus morphology in a cell treated with a graphene oxide/kaolin mixture (100 μg mL−1).
eliminated the toxic effects of graphene oxide on DNA (Figure 2b). Next, we evaluated uptake and food vacuole formation in P. caudatum exposed to graphene oxide and kaolin. We used S. cerevisiae yeast as a food source to investigate the toxic effects of graphene oxide and their alleviation by kaolin (Figure 3a−d). Normally, P. caudatum rely on the 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 a number smaller (by 44%) than the number for control cells. Addition of higher concentrations of graphene oxide caused further decreases in the rate of food vacuole formation. However, when kaolin was added to the food mixture doped with graphene oxide, this led to a 20% increase in the number of food vacuoles compared with that in cells treated with graphene oxide. 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 C
DOI: 10.1021/acs.estlett.8b00135 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX
Letter
Environmental Science & Technology Letters
Figure 3. Dark field microscopy images of P. caudatum: (a) nontreated, (b) treated with kaolin (100 μg mL−1) alone (note developed food vacuoles), (c) treated with graphene oxide (100 μg mL−1) alone (no food vacuoles observed), and (d) treated with a 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 the distribution of kaolin and graphene oxide in a P. caudatum cell.
Figure 4. Adsorption of graphene oxide on kaolin nanoparticles. (a) Photograph demonstrating the change in color of kaolin/graphene oxide mixtures after centrifugation. (b) Adsorption (removal) of graphene oxide by kaolin measured using the optical absorbance at 277 nm. Atomic force microscopy images of kaolin−graphene oxide complexation: (c) topography image and (d) corresponding adhesion force map.
alleviated the inhibitory effect (22% inhibition). Kaolin alone did not significantly inhibit galvanotaxis in P. caudatum protozoans within the studied concentration range. Our findings confirm that kaolin reduces significantly the membrane-related toxic effects of graphene oxide. From our results, it appears that kaolin strongly interacts with graphene oxide, leading to attenuation of its chemical properties. The mechanisms of graphene-related material nanotoxicity are not yet fully understood; however, the influence of size and morphology of graphene oxide particles was suggested.9 Kaolin has been previously reported to remediate silver nanoparticle toxicity in aqueous environments.24 Both materials used in our study had similar size distributions (1.9−2.2 μm hydrodynamic diameters) and exhibited negative ζ 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 goethite.25 In our study, however, we found that kaolin strongly aggregates with graphene oxide (Figure 4). Within the
allows for imaging of the distribution of nanomaterials in live organisms,22 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 3e,f). We found that graphene oxide maps overlapped with kaolin maps, diffusely distributed both in food vacuoles, near the macronucleus, and in the cytoplasm. It is noteworthy that nanoparticles were also detected close to the cellular membrane, which explains well our results (Figure S4) on the effects of graphene oxide and kaolin on the electrically forced motility of P. caudatum (galvanotaxis). 23 The galvanotaxis study was performed after incubation of protozoans for 24 h with graphene oxide, kaolin, or a mixture of both. Then the cells were placed into the electrode-equipped vessel where the voltage (1.5 V) was applied for 10 min. Normally, the voltage-stimulated cells travel to the 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 mL−1), this somewhat D
DOI: 10.1021/acs.estlett.8b00135 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX
Environmental Science & Technology Letters
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ACKNOWLEDGMENTS The work was 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 (6.7743.2017/6.7 ).
range of concentrations studied, 90−100% of graphene oxide was coagulated by kaolin and could be removed from the medium via centrifugation or even via gravity sedimentation. AFM imaging and adhesion force mapping (in peak force tapping mode) were used to confirm the coagulation of graphene oxide with kaolin particles (Figure 4d). 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
