Research Article Received: 25 February 2014,
Revised: 25 June 2014,
Accepted: 21 July 2014
Published online in Wiley Online Library: 16 September 2014
(wileyonlinelibrary.com) DOI 10.1002/jat.3066
Structural disruption increases toxicity of graphene nanoribbons Sayan Mullick Chowdhurya, Subham Dasguptab, Anne E. McElroyb and Balaji Sitharamana* ABSTRACT: The increased utilization of graphene nanoribbons (GNRs) for biomedical and material science applications necessitates the thorough evaluation of potential toxicity of these materials under both intentional and accidental exposure scenarios. We here investigated the effects of structural disruption of GNRs (induced by low-energy bath and high-energy probe sonication) to in vitro (human cell lines), and in vivo (Oryzias latipes embryo) biological systems. Our results demonstrate that low concentration (20 μg ml 1) suspensions of GNRs prepared by as little as 1 min of probe sonication can cause significant decreases in the overall metabolic state of cells in vitro, and increased embryo/larval mortality in vivo, as compared to bath sonicated or unsonicated suspensions. Structural analysis indicates that probe sonication leads to disruption in GNR structure and production of smaller carbonaceous debris, which may be the cause of the toxicity observed. These results point out the importance of assessing post-production structural modifications for any application using nanomaterials. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web-site. Keywords: carbon nanomaterials; graphene; embryo toxicity; Japanese medaka; cytotoxicity; sonication
Introduction
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*Correspondence to: Balaji Sitharaman, Department of Biomedical Engineering, Bioengineering Building, Room 115, Stony Brook University, Stony Brook, NY 11794–5281, USA. Email: [email protected] a Department of Biomedical Engineering, Stony Brook University, Stony Brook, NY, USA b School of Marine and Atmospheric Sciences, Stony Brook University, Stony Brook, NY, USA
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The exciting physio-chemical properties and numerous applications of graphene in material science (Krishnamoorthy et al., 2011; Xu et al., 2012; Yin et al., 2012; Zhang and Song, 2012) and medicine (Huang et al., 2011; Paratala et al., 2012; Sun et al., 2008; Yang et al., 2009) have prompted an increase in the research on and industrial use of graphene-based materials. These particles have not only proved valuable in materials research, but are also in the process of being developed for drug delivery, imaging and other therapeutic applications. Yet, reports indicate that graphene nanoparticles, depending on their chemical compositions and method of synthesis, show diverse effects in terms of oxidative stress, uptake and toxicity in cells and tissues (Mullick Chowdhury et al., 2013; Sasidharan et al., 2011). For a large number of biomedical and material science applications, hydrophobic graphene nanoparticles need to be solubilized or form stable suspensions in aqueous solutions. Recently, oxidized graphene nanoribbons (O-GNRs) synthesized from multiwalled carbon nanotubes (MWCNT) have been shown to be more stable in water than graphene oxide nanoplatelets (O-GNP) synthesized from graphite, using the modified Hummer’s method (Kosynkin et al., 2009). Nevertheless, similar to other carbon nanomaterials, high-energy sonication is required to obtain semi-stable dispersions of the solid graphene sheets (O-GNRs and O-GNPs) in aqueous solutions. Moreover, the shear forces associated with sonication driven exfoliation of separate graphene nanoparticles may result in structural disruption of the graphene structure in the process (Compton et al., 2012; Gu et al., 2009; Li et al., 2008). Although sonication improves the dispersibility of solid aggregates, achieving long-term stability of these graphene suspensions in physiological solutions remains a challenge (Schipper et al., 2008).
With the advancement of use of graphene-based nanomaterials in research and industry, they may ultimately end up in landfills and eventually in nearby surface and groundwater bodies (Lanphere et al., 2014; Lin et al., 2010). Graphene nanoparticles have been showed to be stable in surface water and thus could ultimately have adverse effects in the surface and subsurface organisms in natural water bodies (Lanphere et al., 2014). Comparative studies of graphene-based nanoparticles in both cell lines and zebrafish embryos indicate that the shape of the materials influences both absorption and toxicity (Chen et al., 2012; Talukdar et al., 2014). Recently a number of studies have evaluated the biodistribution and toxicity of graphene-based nanoparticles in cell lines, and small animals (Chang et al., 2011; Kanakia et al., 2014; Wang et al., 2011; Zhang et al., 2010). In comparison to O-GNPs, very few in vitro or in vivo toxicity studies have been reported with O-GNRs (Lu et al., 2014; Mullick Chowdhury et al., 2013, 2014). These studies report a size- and concentration-dependent decrease in cell viability (Seabra et al., 2014). Although, some initial cell studies suggested O-GNPs cannot enter cells and can only cause oxidative stress on the cell membrane (Chang et al., 2011; Wang et al., 2011), subsequent studies have shown that O-GNPs can enter cells through membrane penetration (Li et al., 2013). These internalized graphene
S. Mullick Chowdhury et al. nanostructures, depending on their shape have been shown to cause mitochondrial injury (Zhang et al., 2010) and cell cycle alterations (Matesanz et al., 2013). Although, a recent study reported that O-GNPs do not cause hemolysis (Chowdhury et al., 2013; Sasidharan et al., 2012), another study reported that both bath and probe sonication of O-GNP result in increased hemolysis of red blood cells (Liao et al., 2011). However, these sonicated O-GNPs were less toxic to fibroblasts as compared to unsonicated aggregated O-GNP sheets (Liao et al., 2011). Studies conducted using embryos of the zebrafish (Danio rerio) report mixed results on effects observed, likely associated with structural differences in the graphene-based nanomaterials being analyzed or the method of preparation. Zebrafish embryos exposed to 50 mg l 1 of O-GNPs showed significant hatching delays as well morphological defects in the hatched larvae (Chen et al., 2012). The same study also showed agglomerates of O-GNPs in the outer layer of the chorion after exposure to 25 mg l 1 of OGNP. In contrast, Gollavelli and Ling (2012) found no developmental defects or mortalities in zebrafish embryos microinjected with multi-functionalized graphene, but showed the nanomaterials to be widely biodistributed in the fish. Another study (Liu et al., 2013) found similar results with O-GNPs showing no effects on zebrafish embryos and larvae, as well as rapid elimination of the nanomaterials. The influence of 24–48 h (long-term) sonication in nitric acid during synthesis of the MWCNT was investigated in another study where significant early developmental delays and increased abnormalities of the brain, notochord, eyes and yolk sac of zebrafish embryos injected with these materials were observed (Cheng and Cheng, 2012). These authors also showed that the toxicity was associated with breakdown of MWCNT in the sonicated solutions. This study demonstrated the ability of sonication to increase the toxicity of nanomaterials; however, due to the mode of exposure (injection) and the length of sonication (hours), it is not representative of more natural exposure pathways or commonly used sonication methods. Despite the common application of high-energy sonication in creating dispersions of carbon-based nanomaterials, very few published studies have explicitly examined the influence of these treatments on toxicity. In this study we assessed the influence of short-term (1–20 min), low-energy (bath) and high-energy (probe) sonication on the toxicity of 20 μg ml 1 solutions of O-GNR in vitro and in vivo using human cell lines and fish embryos respectively. Alveolar basal epithelial carcinoma cells (A549), and Michigan Cancer Foundation breast cancer cells (MCF7) were exposed to O-GNR particles, and the associated toxicity was evaluated using lactate dehydrogenase (LDH) release assays for cell viability, intracellular reactive oxygen species (ROS) generation assays, and WST-1 and Presto blue assays for overall cellular metabolism. The toxicological impact of the sonicated samples on the survival of Japanese medaka (Oryzias latipes) embryos was also tested. The impacts of the different sonication treatments on the structure of O-GNR were assessed by Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR) and atomic force microscopy (AFM).
at a concentration of 100 μg ml 1 of cell culture media and 20 μg ml 1 of embryo rearing media (in mM: 17.1 NaCl, 272 CaCl2.2H2O, 402 KCl, 661 MgSO4.7H2O; pH 6.3) by bath sonicating them for 5 min (Ultrasonicator FS30H; Fisher Scientific, Pittsburgh, PA, USA) and were examined either directly, after further bath sonication (100 W) for 5 or 20 min, or after probe sonication for either 1, 5 or 10 min (2 s on–1 s off pulse, 225 W, Ultrasonicator LPX 750; Cole Parmer, Chicago, IL, USA). Some solutions were centrifuged at 20 800 g for 15 min to remove non-dispersed aggregated material before testing. Resuspended pellets were also evaluated, made up at concentrations equivalent to those they were prepared from. All solutions were used within 10 days of preparation. Raman Spectroscopy Each uncentrifuged sonicated O-GNR sample was diluted to 10 μg ml 1 using isopropanol, and 5 μl were drop cast on to a silicon wafer (Ted Pella, Redding, CA, USA) and dried overnight. Raman spectra of samples coated on the silicon wafer were obtained using an Enwave Pro Raman-L Spectrophotometer (Irvine, CA, USA) equipped with a charge-couple device detector, and a 532 nm laser, at 20% of maximum laser strength (500 mW). Moreover, Raman spectra were obtained on a solid O-GNR sample on a microscopic slide covered with aluminum foil. Averages of 10 readings on a particular sample were used to plot the spectra. Fourier Transform Infrared Spectroscopy Five mg samples of bath and probe-sonicated samples (in deionized water) were freeze-dried for 48 h and used for FTIR analyses along with unsonicated solid samples. A Thermo Fisher Nicolet 6700 FTIR spectrometer (Thermo Fisher Scientific, Waltham, MA) equipped with a SmartOrbit attenuated total reflectance accessory with a type IIa diamond element was used to obtain FTIR spectra of all the samples. Atomic Force Microscopy Sonicated O-GNR samples were centrifuged at 10 621 g for 30 min. A total of 2 μl of the supernatant was diluted using 8 μl 1 : 1 ethanol/water mixture and drop cast on to silicon wafers and dried overnight before imaging. Pellets obtained upon centrifugation were resuspended in 1 ml of 1 : 1 ethanol/water mixture and treated the same way. AFM images of all suspensions were obtained using a Nano Surf Easy Scan 2 Atomic Force Microscopy (NanoScience Instruments Inc., Phoenix, AZ, USA), operating in tapping mode, using a V-shaped cantilever. Cell Culture
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Methods
MCF7 and A549 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and incubated in a humidified atmosphere of 95% O2 and 5% CO2 at 37 °C. A549 and MCF7 cells were cultured in Dulbecco’s Modified Eagle Medium and RPMI 1600 medium, respectively. Both media were supplemented with 10% fetal bovine serum.
Preparation of Sonicated Oxidized Graphene Nanoribbon Solutions
Presto Blue Assay
O-GNR were synthesized using a protocol involving the unzipping of MWCNTs by KMnO4, as previously described (Mullick Chowdhury et al., 2013). O-GNR suspensions were made
Cellular metabolism was examined in all three cell lines after 24 h exposure to 20 μg /ml 1 of the various uncentrifuged sonicated O-GNR suspensions (bath sonicated 5 min, bath
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Structural disruption increases toxicity of graphene nanoribbons sonicated 20 min and probe sonicated for 1, 5 or 10 min) using the Presto blue assay (Invitrogen, Grand Island, NY, USA). Cells were plated at 1 × 104 cells per well in 96-well plates (BD Biosciences, San Jose, CA, USA) and incubated for 24 h. The medium was replaced in each well before the start of the assay, and 50 μl of the various sonicated O-GNR suspensions (100 μg ml 1) were added to each well for a final treatment concentration of 20 μg ml 1. The cells were incubated at 37 °C for 24 h, and subsequently the medium with O-GNRs was removed and the wells were rinsed three times with Dulbecco’s phosphate-buffered saline to remove any remnant O-GNRs before adding 100 μl of fresh media and 10 μl of Presto Blue reagent. The O-GNRs were washed out to minimize background fluorescence from the nanostructures. The plates were again incubated for 1 h at 37 °C. Fluorescence readings of the wells were made at 530 nm Ex/580 nm Em using a Cytofluor fluorescence multi-well plate reader (Series H4000 PerSeptive Biosystems, Framingham, MA, USA). Excitation of any remnant extracellular or intracellular graphene structures at wavelengths between 500 and 600 nm leads to low-intensity emission at near infra-red wavelengths (beyond 780 nm) and is unlikely to interfere with the fluorescence measurements associated with this assay (Loh et al., 2010). Fluorescence readings of the media alone were used for baseline correction, and unexposed cells were used as the control. The cell viability in terms of percentage of control cells was expressed as the percentage of (Ftest – Fblank)/(Fcontrol).
Lactic Dehydrogenase Release Assay Cell death and lysis of cells exposed to sonicated O-GNR samples were evaluated by lactate dehydrogenase assays (Sigma-Aldrich, NY, USA). MCF7 cells were plated at a density of 15 × 104 cells per well in 24-well cell culture plates, and incubated for 24 h, after which the cells were treated with 50 μl of the uncentrifuged sonicated samples and incubated for 24 or 72 h. At the end of the incubation period, media were collected from the individual wells and centrifuged at 153 g for 5 min. A total of 50 μl of the media supernatant were added to a fresh 96-well plate along with the LDH assay reagent (100 μl in each well), and incubated for 45 min. The absorbance values were recorded at 490 nm, with absorbance of the culture media being used for baseline correction, and untreated cells serving as the control. The LDH leakage (percentage of the positive control) was expressed as the percentage of Asample/Acontrol, where Asample is the optical density of sonicated O-GNR samples and Acontrol is the optical density of the control cells.
Cell Count
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O-GNR solution toxicity was assessed in vivo using embryos from Japanese medaka, maintained according to Stony Brook University IACUC approved protocols. One day postfertilization (dpf) embryos were used for all analyses. For the first experiments, individual embryos were exposed in 96-well plates (Falcon; Becton Dickinson, Franklin Lake, NJ, USA) (Fig. 7). In the last experiment three replicate groups (n = 10 embryos/group) were exposed to 10 ml of O-GNR solutions in 20 ml vials. The embryos were exposed separately to complete O-GNR solutions, supernatant of O-GNR solutions postcentrifugation and in some cases pellets resuspended in embryo rearing media (Morin et al., 2011). All exposures were conducted at 25 °C with gentle agitation (220 rpm on an orbital shaker) and a 16 : 8 light/dark cycle. All solutions were replaced every other day, and the embryos were exposed for a total of 6 days. At 7 dpf, all embryos were transferred to embryo rearing media until they hatched, and the combined embryo/larval survival was assessed at 12 dpf.
Transmission Electron Microscopy MCF7 cells were plated on ACLARW film (Electron Microscopy Sciences, Hatford, PA, USA) at a density of 106 cells per film, and exposed to O-GNR (at 20 μg ml 1) for 24 h. Medaka embryos were treated with O-GNR (at 20 μg ml 1) for 6 days or left untreated. After exposure periods of either 24 h or 6 days, cells and embryos were fixed with 2.5% electron microscopy grade glutaraldehyde (Electron Microscopy Sciences) in 0.1 M PBS. After fixation, the films containing fixed cells and embryos (sliced into half using a thin blade) were placed in 2% osmium tetroxide in 0.1 M PBS, treated with multiple (graded) ethanol washes for dehydration and embedded in Durcupan resin (Sigma-Aldrich, St. Louis, MO, USA). Blocks containing high cell densities and embryo slices containing the chorion were cut into 80 nm ultrathin sections using an Ultracut E microtome (Reichert-Jung, Cambridge, UK), and placed on Formvar-coated copper grids. The sections were then viewed with a Tecnai Bio Twin G transmission electron microscope (FEI, Hillsboro, OR, USA), at 80 kV. Digital images were acquired using an XR-60 charge-couple device digital camera system (AMT, Woburn, MA, USA). Ten images each for treated and untreated MCF7 cells and one section from four different treated and untreated embryos were analyzed. Statistics All in vitro data are presented as mean + SD. The Presto blue assay and cell counting were repeated with independent cell preparations on 3 different days (n = 3), and all other assays were repeated with independent cell preparations on 4 different days (n = 4). The mean of readings obtained from three parallel wells per exposure condition per day was considered as one independent observation (n = 1). Square root transformation was used to transform percentage data to approximate a normal distribution, and these transformed data were used for one-way ANOVA, followed by Tukey–Kramer post hoc analyses. Survival of fish embryos exposed to O-GNR solutions was evaluated by logistic regression analysis or by ANOVA followed by Tukey–Kramer post hoc analysis. All analyses were performed using Statistica software (Statsoft, Tulsa, OK, USA).
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MCF7 cells were plated at a density of 5 × 104 cells per well in 24-well cell culture plates and incubated for 24 h, followed by treatment with the uncentrifuged sonicated samples (50 μl) and incubation for 24 and 72 h. Following incubation, the media was removed and wells were washed three times with Dulbecco’s phosphate-buffered saline to remove any remnant O-GNR. The cells were then detached from the wells and counted using a hemocytometer.
In Vivo Medaka Fish Embryo Survival Assay
S. Mullick Chowdhury et al.
Results Atomic Force Microscopy Figure 1 shows representative AFM images obtained from the supernatant of O-GNR samples after different sonication treatments followed by centrifugation. Figure 1(A) shows representative O-GNRs (black arrows) observed in the supernatant of bath-sonicated samples post centrifugation. Little or no carbon debris was observed for this treatment. Figure 1(B) shows O-GNR samples probe sonicated for 1 min. Shorter, more globular, carbonaceous structures (black arrows) were observed. The approximate diameter of the globular structures was
250–400 nm. Figure 1(C) shows O-GNR samples probe sonicated for 5 min. The image shows the presence of smaller disc-like structures (black arrows) approximately 150–250 nm in diameter. Figure 1(D) shows the presence of a large number of even smaller sized particles and carbon debris (black arrows) in O-GNR solution probe sonicated for 10 min. AFM data are numerically compared in Fig. 1(E), which shows the average length of 15 O-GNRs in each sonication type obtained from three to four AFM images. Bath-sonicated samples showed an average length of 744 ± 178 nm, and the sample probes sonicated for 1, 5 and 10 min showed average lengths of 323 ± 50 nm, 201 ± 28 and 100 ± 10 nm, respectively,
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Figure 1. Representative atomic force microscopy images of the oxidized graphene nanoribbon (O-GNR) supernatants after different sonication treatments and centrifugation. Scale bars on all images represent 1 μm. (A) O-GNRs in a bath-sonicated (250 W, 20 min sonication) sample showing predominantly intact particles (black arrows). (B,C) O-GNRs sonicated for 1 and 5 min, respectively, showing the presence of round carbon structures (black arrows). (D) O-GNR sonicated for 10 min, showing the presence of small carbon debris (black arrows). (E) Bar diagram showing size distribution of O-GNR supernatants after different sonication treatments and centrifugation. The calculations were done using 15 O-GNRs from multiple atomic force microscopy images (n = 15). Data presented as means + SD. * represent a significant difference (P < 0.05) compared to bath-sonicated O-GNR. #Significant difference (P < 0.05) compared to 1 min probe-sonicated O-GNR. †Significant difference (P < 0.05) compared to 5 min probesonicated O-GNR.
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Structural disruption increases toxicity of graphene nanoribbons indicating a significant decrease in lengths with increased sonication time and intensity. A suspension made from the resuspended pellet obtained after centrifugation of bath-sonicated O-GNR samples is shown in Fig. 2(A). Large aggregated structures (>1000 nm in diameter) of O-GNR were mainly observed (black arrows). Figure 2(B) shows a suspension made from the resuspended pellet
obtained after centrifugation of O-GNR samples that were probe sonicated for 1 min. Large aggregated structures of O-GNR were observed (500–1500 nm) (black arrows), along with small aggregates (green arrows) similar in size to those observed in the supernatant of this suspension (100–250 nm in diameter) (Fig. 1B). Figure 2(C) shows the suspension of the resuspended pellets obtained after centrifugation of the
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Figure 2. Representative atomic force microscopy images of resuspended pellets of oxidized graphene nanoribbons (O-GNRs) after different sonication treatments and centrifugation. Scale bars on all images represent 1 μm. (A) Bath-sonicated O-GNR aggregates resuspended post-centrifugation. (B–D) O-GNR resuspended after 1, 5 and 10 min of probe sonication, respectively, showing both large (black arrows) and small (green arrows) aggregates. (E) Bar diagram showing size distribution of resuspended O-GNR pellets after different sonication treatments and centrifugation. The calculations were done using 15 O-GNRs from multiple atomic force microscopy images (n = 15). Data presented as means + SD. *Significant difference (P < 0.05) compared to bath-sonicated O-GNR. #Significant difference (P < 0.05) compared to 1 min probe-sonicated O-GNR.
S. Mullick Chowdhury et al. O-GNR samples that were probe sonicated for 5 min. Very few large aggregates of O-GNR were observed (300–900 nm in diameter, black arrows) along with small aggregates (100–250 nm in diameter, green arrows). The large aggregates were smaller than those observed in samples that were probe sonicated for 1 min. Figure 2(D) shows the sizes of the O-GNR aggregates from the resuspended pellets obtained after centrifugation of O-GNR samples that were probe sonicated for 10 min. Similar to the 5 min sonicated samples, very few large aggregates of O-GNR were observed (200–500 nm in diameter, black arrows) along with small aggregates (50–150 nm in diameter, green arrows). Both the large and small aggregates were much smaller than the O-GNR aggregates from the 1 and 5 min probe-sonicated samples. Figure 2(E) shows the statistical analysis of length of 15 O-GNR aggregates in each sonication type obtained from multiple AFM images. Bath-sonicated samples showed an average length of 1535 ± 314 nm, and samples probe sonicated for 1, 5 and 10 minutes showed average lengths of 997 ± 358 nm, 566 ± 291 and 194 ± 130 nm,
respectively, indicating a gradual decrease in lengths of samples probe sonicated for ≥ 1 min. Raman Spectroscopy Figure 3 shows the Raman spectroscopic analyses of solid O-GNR and O-GNR suspensions after different types of sonication. As seen in Fig. 3(A), the five different sonicated samples (both bath and probe sonicated) showed a distinct increase in a peak at 1150 cm 1, with increasing time and intensity of sonication as compared to the solid unsonicated sample. Moreover, an increase in the D/G band ratio was observed with increasing intensity and sonication time, indicating sonication-related structural disruption (Fig. 3B). Fourier Transform Infrared Spectroscopy Figure 3(C) shows the FTIR spectra of unsonicated and sonicated O-GNR samples. Characteristic troughs for graphene oxide were observed for the unsonicated solid sample at 1732 cm 1
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Figure 3. (A) Representative Raman spectra of oxidized graphene nanoribbons and (B) ratios of D band and G band intensity for samples that were unsonicated, bath sonicated for 5 or 20 min, or probe sonicated for 1, 5 or 10 min. (C) Representative Fourier transform infra-red spectra of oxidized graphene nanoribbon samples that were unsonicated, bath sonicated for 20 min or probe sonicated for 1, 5 or 10 min.
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Structural disruption increases toxicity of graphene nanoribbons corresponding to C = O stretching vibrations of COOH groups (indicated with black arrow), 1605 cm 1 corresponding to O–H deformation vibrations of COOH groups (indicated with black arrow), 1400 cm 1 corresponding to O–H deformation vibrations of tertiary C–OH groups (indicated with orange arrow) and at 1070 cm 1 corresponding to C–O stretching vibrations of epoxy groups (indicated with blue arrow). Increase in sonication intensity and time resulted in gradual decrease of all these troughs. The FTIR spectra with O-GNR samples probe sonicated for 10 min decreased the most. Presto Blue Assay The presto blue assay is based on the principle that metabolically active cells can convert the cell permeable dye resazurin to a pink fluorescent product, with the amount of dye converted being directly proportional to the metabolic state of the cells, especially mitochondrial integrity (Lien et al., 2012). Figure 4(A,B) shows the state of cellular metabolism in A549 and MCF7 cells exposed to sonicated O-GNR samples. Both cell lines showed reduced metabolism in response to O-GNR, with the MCF7 cells being more sensitive to the effects of
sonication. In these cells, treatment with O-GNR dispersions that were bath sonicated for 20 min resulted in a decrease of the cellular metabolism values to approximately 75% of the values of untreated cells (Fig. 4B). Despite the initial decrease in cellular metabolism, increases in the intensity and/or time of sonication did not result in further effects on cellular metabolism. A549 cells showed a similar decrease in cellular metabolism only after exposure to probe-sonicated O-GNRs (Fig. 4A). In these cells, the adverse effects appeared to be time-dependent, with the largest decrease in cellular metabolism (approximately 60% of the cellular metabolism of untreated cells) observed in response to the O-GNR dispersions probe that were sonicated for ≥ 5 min.
Transmission Electron Microscopy of MCF7 Cells Figure 4(C,D) shows the interaction of MCF7 cells with O-GNR. Figure 4(C) shows the presence of smaller O-GNR particles inside MCF7 cells (black arrows). Red arrows indicate the cell membrane and yellow arrow indicates the nucleus. Figure 4(D) suggests that larger O-GNR aggregates fail to enter the cells but can cause membrane damage (black arrows).
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Figure 4. Presto blue assay of (A) A549 cells and (B) MCF7 cells treated with bath- and probe-sonicated oxidized graphene nanoribbons (O-GNRs), evaluated after 24 h incubation with the cells. The assay was repeated on three independent cell preparations conducted on different days (n = 3). Data are normalized to assay values obtained from untreated cells and presented as means + SD. *Significant difference (P < 0.05) compared to the unexposed control cells. Representative transmission electron microscopy images of MCF7 cells treated with O-GNRs for 24 h showing (C) small O-GNR particles inside cells (black arrow) (D) larger O-GNR aggregates (black arrow) fail to enter cells but can cause membrane damage. Red arrows indicate the cell membrane and yellow arrow indicates the nucleus.
S. Mullick Chowdhury et al. Lactic Dehydrogenase Release Assay The LDH assay is based on the principle that dying and unhealthy cells release LDH though their ruptured membranes. Thus, enhanced cell death results in a greater release of LDH into the cell media. However, basal levels of LDH in the media are present even in normal untreated cells and increase as the cells proliferate. Figure 5(A) and (B) show the LDH release from MCF7 cells after exposure to sonicated O-GNR dispersions for 24 and 72 h, respectively. After 24 h of exposure, no significant increase in LDH release was noted in MCF7 cells. However, after 72 h, a decrease of approximately 50% in LDH release compared to the untreated control cells was observed in the media of cells treated with O-GNR dispersions probe sonicated for 10 min.
Cell Count Figure 6(A) and (B) show the cell count for MCF7 cells treated with the various sonicated O-GNR dispersions for 24 and 72 h, respectively. Mean population doubling time of MCF7 cells is
reported to be ~24 h (Sutherland et al., 1983). After 24 h, no significant change in cell count was observed regardless of treatment. However, after 72 h, a significant decrease in cell count (approximately 44%) was observed for cells treated with O-GNR dispersions that were probe sonicated for 10 min (14 × 104 cells) compared to cells treated with 5 min bath-sonicated O-GNR dispersions (25 × 104 cells), indicating that cell growth was inhibited between the 24 h and 72 h time points.
Medaka Embryo Mortality Exposure to complete sonicated samples and supernatant. OGNR dispersions that were bath sonicated for 20 min did not increase embryo mortality as compared to unexposed controls. In comparison, probe-sonicated O-GNR solutions caused a significant sonication time-dependent increase in embryo mortality. Dispersions that were probe sonicated for 1, 5 and 10 min reduced embryo viability to approximately 75%, 55% and 40% of control values, respectively (Fig. 7A). Bath sonication resulted in no apparent increase in toxicity
Figure 5. Toxicity of bath- and probe-sonicated oxidized graphene nanoribbon samples in MCF7 cells evaluated after (A) 24 and (B) 72 h of incubation using the LDH release assay. The assay was repeated on four independent cell preparations conducted on different days (n = 4). Data are normalized to assay values obtained from untreated cells and presented as means + SD. *Significant difference (P < 0.05) compared to the unexposed control cells. LDH, lactate dehydrogenase.
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Figure 6. Cell count of MCF7 cells treated with bath- and probe-sonicated oxidized graphene nanoribbons, evaluated after (A) 24 and (B) 72 h of incubation. The assay was repeated on three independent cell preparations conducted on different days (n = 3). Data are presented as means + SD. *Significant difference (P < 0.05) compared to the unexposed control cells.
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Structural disruption increases toxicity of graphene nanoribbons
Figure 7. Embryonic survival as a function of sonication time and intensity. Significantly increased mortality of embryos with increasing time of 3 3 sonication was observed (P = 5.3 × 10 for A and P = 3.3 × 10 for B). (A) The solutions were not centrifuged post-sonication; (B) the solutions were 5 centrifuged post-sonication. When the two data sets were combined, the sonication time remained significant (P = 5.5 × 10 ) while centrifugation 1 was not (P = 5.4 × 10 ), indicating that the observed toxicity was likely due to smaller materials not removed by centrifugation. Representative transmission electron microscopy section of a medaka embryo showing (C) untreated medaka embryo membrane (chorion) (yellow arrow) and (D) presence of oxidized graphene nanoribbons (O-GNRs) (black arrows) inside chorion (yellow arrow) from embryos treated with O-GNR. Red arrows indicate structural damage to the chorion.
compared to the controls (Fig. 7A), and was hence not further evaluated. Experiments conducted on probe-sonicated solutions that were centrifuged to remove undispersed O-GNR material produced nearly identical results (Fig. 7B). Sonication time significantly enhanced toxicity in both experiments (P = 5.3 × 10 3 for Fig. 7A, and P = 3.3 × 10 3 for Fig. 7B). Given the similarity in toxicity observed upon exposure to O-GNRs, regardless of whether or not they were centrifuged before testing, the analysis was repeated with the combined data set where both sonication and centrifugation status were evaluated as separate factors. With the combined data set, sonication time was highly significant (P = 5.5 × 10 5), while centrifugation status was not (P = 5.4 × 10 1), suggesting that there is no significant difference between the toxicity of centrifuged versus uncentrifuged O-GNR suspensions.
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Transmission Electron Microscopy of Medaka Embryo Figure 7(C,D) shows the effect of interaction of medaka embryo chorion with O-GNRs. Figure 7(C) shows an intact chorion (yellow arrow) not treated with O-GNRs. Figure 7(D) shows that O-GNRs (black arrows) can enter the chorion (yellow arrow) and can cause membrane damage (red arrows).
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Exposure to resuspended pellets. Based on the results obtained above (Fig. 7), the toxicities of complete sonicated samples (uncentrifuged), supernatant (after centrifugation) and resuspended pellets for probe-sonicated O-GNR dispersions (sonicated for 1, 5 and 10 min) were compared (Fig. 8) and normalized to control toxicity (which was 0 in all cases).
Similar to the results of the analyses shown in Fig. 7, probe sonication was found to increase toxicity. Additionally, as previously noted, toxicity resulting from the supernatant did not differ from that of the complete sample. However, the resuspended pellet resulted in a significant increase in embryo toxicity for the 1 and 5 min probe-sonicated samples compared to both the complete O-GNR solutions and the supernatants. Although the mean embryonic survival resulting from exposure to the resuspended pellet for the 10 min probe-sonicated samples was lower than that observed from exposure to the complete sample or the supernatant after centrifugation, the increase in toxicity was not statistically significant.
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Figure 8. Embryonic survival, presented as the percentage of control values, after exposure to uncentrifuged probe-sonicated oxidized graphene nanoribbons (O-GNRs), supernatant from centrifuged probe-sonicated O-GNR samples, and the resuspended pellet from centrifuged probe-sonicated O-GNR samples, with samples being probe sonicated for 1, 5 or 10 min. Two-way ANOVA revealed that the nature of the O-GNR solution (uncentrifuged, centrifuged or resuspended), as well as the time of sonication significantly affected embryo survival 7 6 (P = 6.15 × 10 and 1.16 × 10 , respectively). #Significant difference (P < 0.05) between the toxicity of the supernatant and the resuspended pellet obtained from centrifuged probe-sonicated samples by one-way ANOVA.
Discussion
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The in vitro and in vivo toxic effects of short-term (1–20 min), low-energy (bath) and high-energy (probe) sonication of OGNR were evaluated in this study. In all experiments, nominal O-GNR concentrations of 20 μg ml 1 were used, as this concentration has been previously demonstrated to be non-toxic to cells in vitro (Mullick Chowdhury et al., 2013), as well as to fish embryos in vivo (data not shown). The O-GNRs used in the experiments were synthesized from MWCNTs and had an average size of 1–2.5 μm (Figs 1A and 2A). AFM images indicated that both the supernatants and resuspended pellets of probe-sonicated samples generated smaller structures, whereas the sizes of these structures were inversely related to the sonication time. This observation is consistent with recent studies that have reported that high-energy sonication can lead to breakdown of graphene oxide nanosheets (Compton et al., 2012). Structural analyses by Raman spectroscopy showed the characteristic D band (1340 cm 1) produced due to defects in the O-GNR structure, and a G band (1580 cm 1) resulting from the graphitic hexagonal structures. In the O-GNR samples that were bath or probe sonicated in water, the G band appeared shifted to 1626 cm 1, indicating an increase in single sheet graphene structures (Ferrari et al., 2006). With increasing intensity and time of sonication, the relative height of the G band decreased, consequently resulting in an increased intensity ratio of the G and D bands (Fig. 3B), which is indicative of an increase in structural defects. Furthermore, an increase in the intensity of a band at 1150 cm 1 was observed, which can be attributed to the breakdown of the structure to form hydrogenated carbon structures with alternate C = C and C-C bonds associated with increased time and intensity of sonication (Subramanyam et al., 1997). The data obtained from Raman analysis are corroborated by FTIR
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spectra of the unsonicated solid samples and dried sonicated samples, which show a gradual decrease in characteristic troughs of graphene oxide with increased intensity and time of sonication (Fig. 3C). This is indicative of loss of structural integrity of the graphene structure. It is likely that these alterations in structure due to sonication intensity and time are responsible for the enhanced toxicity observed with multiple endpoints. The significant decreases observed in cellular metabolism in vitro (Fig. 4) and embryo survival in vivo (Figs 7 and 8) upon exposure to probe-sonicated O-GNRs suggest that the smaller particles disrupt the cellular machinery through interactions with cellular organelles or the cytoskeleton upon uptake, possibly in a manner similar to that of other nanoparticles (Dam et al., 2012; Gupta et al., 2004; Johnson-Lyles et al., 2010). TEM results from MCF7 cells showed uptake of only small particles and further supported this hypothesis (Fig. 4C,D). Although no cytotoxicity was observed in vitro even after 72 h exposure to the O-GNR dispersions, a ~50% decrease in basal LDH release was observed in samples probe sonicated for 10 min, suggesting a reduction in cell proliferation (Fig. 5B). This observation was further supported by cell count analyses, which showed a decrease in the number of MCF7 cells treated with 10 min probe-sonicated samples for 72 h (approximately 44% of 5 min bath-sonicated O-GNR treated cells; Fig. 6B). ROS production in the treated cells was almost three times higher than in unexposed cells, although no significant differences in ROS levels were observed among the different sonication treatments (Fig. S1). Thus, ROS production is likely not the mechanism responsible for the effects observed in this study. Moreover, data from the WST-1 assay (Fig. S2) conducted after 2 h exposures also failed to show significant effects of O-GNR treatment, suggesting that sonication has no immediate effects on overall cellular metabolism. Taken together, these results indicate that, while exposure to sonicated O-GNR can cause chronic reductions in cellular metabolism, it does not cause immediate cell death. Japanese medaka embryos were also found to be sensitive to the toxic effects of probe-sonicated O-GNR (Fig. 7A), and this is consistent with previous studies reporting significant toxicity in zebrafish embryos injected with MWCNT subjected to highenergy sonication for prolonged periods (24–48 h) (Cheng and Cheng, 2012). Our findings demonstrate enhanced toxicity after more realistic aqueous exposures to O-GNR that is probe sonicated for as little as 1 min, indicating that toxicity resulting from standard processing procedures and exposure scenarios is reasonable. Enhanced toxicity of sonicated nanomaterials has been mentioned in a previous report on aquatic species (Oberdörster et al., 2006), yet no data were provided to support this claim. The results from the present study indicate that evaluation of the effects of post-production processing on nanomaterial toxicity should be part of routine analysis. The similar toxicity profiles of the centrifuged and uncentrifuged samples supports the hypothesis that smaller, unaggregated and dispersed structures or carbon debris are responsible for the enhanced toxicity observed in sonicated OGNR solutions (Fig. 7A,B). The observed enhanced toxicity could be due to greater bioavailability of smaller particles, toxic interactions with the chorion and/or enhanced cellular toxicity. TEM images of control and O-GNR-treated medaka embryos indicates that O-GNR can enter the chorion of treated medaka embryos and can cause structural damage (Fig. 7C,D). Although exposure
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Structural disruption increases toxicity of graphene nanoribbons to bath-sonicated O-GNR suspensions did not increase embryo mortality (Fig. 7A), it did lead to precocious hatching (by approximately 2 days) (Fig. S3). This has been previously observed by Wu et al. (2010) in medaka embryos and by Lee et al. (2007) in zebrafish embryos after exposure to silver nanoparticles, although it is in contrast to a recent report of delayed hatch in zebrafish embryos by Chen et al. (2012). In a natural setting, less developed fry may have reduced abilities to swim and/or capture food, putting them at increased risk of starvation or predation. Early hatching may be attributed to the docking of small nanoparticle aggregates into the chorion pore channels, leading to disruption of the membrane transport, and disintegration of the chorion structure (Wu et al., 2010). Chen et al. (2012) also reported agglomerates of O-GNRs in the outer layer of zebrafish chorions, but hypothesized the chorion acted as a protective barrier to these large particles. It is likely that the smaller size of the probe-sonicated O-GNRs examining in our study allowed for greater penetration and, thus, effects. The finding that the resuspended pellet alone could cause significantly enhanced toxicity, compared to 1 and 5 min probe-sonicated uncentrifuged samples or their supernatants (Fig. 8), was somewhat surprising. However, AFM images of the resuspended pellets revealed that they contained both large and small aggregates of O-GNRs, which differ structurally (Fig. 2A–E), and are likely more toxic compared to aggregates and particles of uncentrifuged bath- and probesonicated samples. This is similar to observations reported by Liao et al. (2011), who demonstrated that graphene sheet aggregates were more toxic to fibroblasts compared to sonicated samples. It is possible that these different types of aggregates have different mechanisms of action, and further detailed investigations will be needed to elucidate and differentiate these mechanisms.
Conclusions
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This work was supported by the National Institutes of Health (grant no. 1DP2OD007394-01), and the Wallace H. Coulter Foundation.
Conflict of Interest The Authors did not report any conflict of interest.
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Our findings clearly indicate that with increasing sonication intensity and time, smaller carbon particles are being produced. Further, these suspensions are capable of causing reductions in overall cellular metabolism and cell proliferation in in vitro assays, which are likely associated with disruptions of the cellular machinery. Although significant, these effects did not result in short-term cell death in this study. In vivo studies with medaka embryos further showed that exposure to similar concentrations of probe-sonicated O-GNR results in precocious hatching and decreased survival, clearly demonstrating the effects of these suspensions on an intact organism. The in vivo results indicate that there may be environmental consequences of exposure to these materials, and the enhanced toxicity of resuspended probe-sonicated O-GNR aggregates suggests that other aggregated O-GNR structures may be a concern. Further studies are needed to elucidate the mechanisms associated with embryo toxicity. Based on the results of this study, structural disruption of O-GNR and similar nanostructures should be avoided, particularly for biomedical applications. These results may also apply to other carbon-based nanoparticles, and thus indicate a need to examine the potential influence of post-production processing for all nanomaterial applications.
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Supporting Information Additional supporting information may be found in the online version of this article at the publisher’s web-site.
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J. Appl. Toxicol. 2014; 34: 1235–1246
Revised: 25 June 2014,
Accepted: 21 July 2014
Published online in Wiley Online Library: 16 September 2014
(wileyonlinelibrary.com) DOI 10.1002/jat.3066
Structural disruption increases toxicity of graphene nanoribbons Sayan Mullick Chowdhurya, Subham Dasguptab, Anne E. McElroyb and Balaji Sitharamana* ABSTRACT: The increased utilization of graphene nanoribbons (GNRs) for biomedical and material science applications necessitates the thorough evaluation of potential toxicity of these materials under both intentional and accidental exposure scenarios. We here investigated the effects of structural disruption of GNRs (induced by low-energy bath and high-energy probe sonication) to in vitro (human cell lines), and in vivo (Oryzias latipes embryo) biological systems. Our results demonstrate that low concentration (20 μg ml 1) suspensions of GNRs prepared by as little as 1 min of probe sonication can cause significant decreases in the overall metabolic state of cells in vitro, and increased embryo/larval mortality in vivo, as compared to bath sonicated or unsonicated suspensions. Structural analysis indicates that probe sonication leads to disruption in GNR structure and production of smaller carbonaceous debris, which may be the cause of the toxicity observed. These results point out the importance of assessing post-production structural modifications for any application using nanomaterials. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web-site. Keywords: carbon nanomaterials; graphene; embryo toxicity; Japanese medaka; cytotoxicity; sonication
Introduction
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*Correspondence to: Balaji Sitharaman, Department of Biomedical Engineering, Bioengineering Building, Room 115, Stony Brook University, Stony Brook, NY 11794–5281, USA. Email: [email protected] a Department of Biomedical Engineering, Stony Brook University, Stony Brook, NY, USA b School of Marine and Atmospheric Sciences, Stony Brook University, Stony Brook, NY, USA
Copyright © 2014 John Wiley & Sons, Ltd.
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The exciting physio-chemical properties and numerous applications of graphene in material science (Krishnamoorthy et al., 2011; Xu et al., 2012; Yin et al., 2012; Zhang and Song, 2012) and medicine (Huang et al., 2011; Paratala et al., 2012; Sun et al., 2008; Yang et al., 2009) have prompted an increase in the research on and industrial use of graphene-based materials. These particles have not only proved valuable in materials research, but are also in the process of being developed for drug delivery, imaging and other therapeutic applications. Yet, reports indicate that graphene nanoparticles, depending on their chemical compositions and method of synthesis, show diverse effects in terms of oxidative stress, uptake and toxicity in cells and tissues (Mullick Chowdhury et al., 2013; Sasidharan et al., 2011). For a large number of biomedical and material science applications, hydrophobic graphene nanoparticles need to be solubilized or form stable suspensions in aqueous solutions. Recently, oxidized graphene nanoribbons (O-GNRs) synthesized from multiwalled carbon nanotubes (MWCNT) have been shown to be more stable in water than graphene oxide nanoplatelets (O-GNP) synthesized from graphite, using the modified Hummer’s method (Kosynkin et al., 2009). Nevertheless, similar to other carbon nanomaterials, high-energy sonication is required to obtain semi-stable dispersions of the solid graphene sheets (O-GNRs and O-GNPs) in aqueous solutions. Moreover, the shear forces associated with sonication driven exfoliation of separate graphene nanoparticles may result in structural disruption of the graphene structure in the process (Compton et al., 2012; Gu et al., 2009; Li et al., 2008). Although sonication improves the dispersibility of solid aggregates, achieving long-term stability of these graphene suspensions in physiological solutions remains a challenge (Schipper et al., 2008).
With the advancement of use of graphene-based nanomaterials in research and industry, they may ultimately end up in landfills and eventually in nearby surface and groundwater bodies (Lanphere et al., 2014; Lin et al., 2010). Graphene nanoparticles have been showed to be stable in surface water and thus could ultimately have adverse effects in the surface and subsurface organisms in natural water bodies (Lanphere et al., 2014). Comparative studies of graphene-based nanoparticles in both cell lines and zebrafish embryos indicate that the shape of the materials influences both absorption and toxicity (Chen et al., 2012; Talukdar et al., 2014). Recently a number of studies have evaluated the biodistribution and toxicity of graphene-based nanoparticles in cell lines, and small animals (Chang et al., 2011; Kanakia et al., 2014; Wang et al., 2011; Zhang et al., 2010). In comparison to O-GNPs, very few in vitro or in vivo toxicity studies have been reported with O-GNRs (Lu et al., 2014; Mullick Chowdhury et al., 2013, 2014). These studies report a size- and concentration-dependent decrease in cell viability (Seabra et al., 2014). Although, some initial cell studies suggested O-GNPs cannot enter cells and can only cause oxidative stress on the cell membrane (Chang et al., 2011; Wang et al., 2011), subsequent studies have shown that O-GNPs can enter cells through membrane penetration (Li et al., 2013). These internalized graphene
S. Mullick Chowdhury et al. nanostructures, depending on their shape have been shown to cause mitochondrial injury (Zhang et al., 2010) and cell cycle alterations (Matesanz et al., 2013). Although, a recent study reported that O-GNPs do not cause hemolysis (Chowdhury et al., 2013; Sasidharan et al., 2012), another study reported that both bath and probe sonication of O-GNP result in increased hemolysis of red blood cells (Liao et al., 2011). However, these sonicated O-GNPs were less toxic to fibroblasts as compared to unsonicated aggregated O-GNP sheets (Liao et al., 2011). Studies conducted using embryos of the zebrafish (Danio rerio) report mixed results on effects observed, likely associated with structural differences in the graphene-based nanomaterials being analyzed or the method of preparation. Zebrafish embryos exposed to 50 mg l 1 of O-GNPs showed significant hatching delays as well morphological defects in the hatched larvae (Chen et al., 2012). The same study also showed agglomerates of O-GNPs in the outer layer of the chorion after exposure to 25 mg l 1 of OGNP. In contrast, Gollavelli and Ling (2012) found no developmental defects or mortalities in zebrafish embryos microinjected with multi-functionalized graphene, but showed the nanomaterials to be widely biodistributed in the fish. Another study (Liu et al., 2013) found similar results with O-GNPs showing no effects on zebrafish embryos and larvae, as well as rapid elimination of the nanomaterials. The influence of 24–48 h (long-term) sonication in nitric acid during synthesis of the MWCNT was investigated in another study where significant early developmental delays and increased abnormalities of the brain, notochord, eyes and yolk sac of zebrafish embryos injected with these materials were observed (Cheng and Cheng, 2012). These authors also showed that the toxicity was associated with breakdown of MWCNT in the sonicated solutions. This study demonstrated the ability of sonication to increase the toxicity of nanomaterials; however, due to the mode of exposure (injection) and the length of sonication (hours), it is not representative of more natural exposure pathways or commonly used sonication methods. Despite the common application of high-energy sonication in creating dispersions of carbon-based nanomaterials, very few published studies have explicitly examined the influence of these treatments on toxicity. In this study we assessed the influence of short-term (1–20 min), low-energy (bath) and high-energy (probe) sonication on the toxicity of 20 μg ml 1 solutions of O-GNR in vitro and in vivo using human cell lines and fish embryos respectively. Alveolar basal epithelial carcinoma cells (A549), and Michigan Cancer Foundation breast cancer cells (MCF7) were exposed to O-GNR particles, and the associated toxicity was evaluated using lactate dehydrogenase (LDH) release assays for cell viability, intracellular reactive oxygen species (ROS) generation assays, and WST-1 and Presto blue assays for overall cellular metabolism. The toxicological impact of the sonicated samples on the survival of Japanese medaka (Oryzias latipes) embryos was also tested. The impacts of the different sonication treatments on the structure of O-GNR were assessed by Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR) and atomic force microscopy (AFM).
at a concentration of 100 μg ml 1 of cell culture media and 20 μg ml 1 of embryo rearing media (in mM: 17.1 NaCl, 272 CaCl2.2H2O, 402 KCl, 661 MgSO4.7H2O; pH 6.3) by bath sonicating them for 5 min (Ultrasonicator FS30H; Fisher Scientific, Pittsburgh, PA, USA) and were examined either directly, after further bath sonication (100 W) for 5 or 20 min, or after probe sonication for either 1, 5 or 10 min (2 s on–1 s off pulse, 225 W, Ultrasonicator LPX 750; Cole Parmer, Chicago, IL, USA). Some solutions were centrifuged at 20 800 g for 15 min to remove non-dispersed aggregated material before testing. Resuspended pellets were also evaluated, made up at concentrations equivalent to those they were prepared from. All solutions were used within 10 days of preparation. Raman Spectroscopy Each uncentrifuged sonicated O-GNR sample was diluted to 10 μg ml 1 using isopropanol, and 5 μl were drop cast on to a silicon wafer (Ted Pella, Redding, CA, USA) and dried overnight. Raman spectra of samples coated on the silicon wafer were obtained using an Enwave Pro Raman-L Spectrophotometer (Irvine, CA, USA) equipped with a charge-couple device detector, and a 532 nm laser, at 20% of maximum laser strength (500 mW). Moreover, Raman spectra were obtained on a solid O-GNR sample on a microscopic slide covered with aluminum foil. Averages of 10 readings on a particular sample were used to plot the spectra. Fourier Transform Infrared Spectroscopy Five mg samples of bath and probe-sonicated samples (in deionized water) were freeze-dried for 48 h and used for FTIR analyses along with unsonicated solid samples. A Thermo Fisher Nicolet 6700 FTIR spectrometer (Thermo Fisher Scientific, Waltham, MA) equipped with a SmartOrbit attenuated total reflectance accessory with a type IIa diamond element was used to obtain FTIR spectra of all the samples. Atomic Force Microscopy Sonicated O-GNR samples were centrifuged at 10 621 g for 30 min. A total of 2 μl of the supernatant was diluted using 8 μl 1 : 1 ethanol/water mixture and drop cast on to silicon wafers and dried overnight before imaging. Pellets obtained upon centrifugation were resuspended in 1 ml of 1 : 1 ethanol/water mixture and treated the same way. AFM images of all suspensions were obtained using a Nano Surf Easy Scan 2 Atomic Force Microscopy (NanoScience Instruments Inc., Phoenix, AZ, USA), operating in tapping mode, using a V-shaped cantilever. Cell Culture
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Methods
MCF7 and A549 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and incubated in a humidified atmosphere of 95% O2 and 5% CO2 at 37 °C. A549 and MCF7 cells were cultured in Dulbecco’s Modified Eagle Medium and RPMI 1600 medium, respectively. Both media were supplemented with 10% fetal bovine serum.
Preparation of Sonicated Oxidized Graphene Nanoribbon Solutions
Presto Blue Assay
O-GNR were synthesized using a protocol involving the unzipping of MWCNTs by KMnO4, as previously described (Mullick Chowdhury et al., 2013). O-GNR suspensions were made
Cellular metabolism was examined in all three cell lines after 24 h exposure to 20 μg /ml 1 of the various uncentrifuged sonicated O-GNR suspensions (bath sonicated 5 min, bath
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Structural disruption increases toxicity of graphene nanoribbons sonicated 20 min and probe sonicated for 1, 5 or 10 min) using the Presto blue assay (Invitrogen, Grand Island, NY, USA). Cells were plated at 1 × 104 cells per well in 96-well plates (BD Biosciences, San Jose, CA, USA) and incubated for 24 h. The medium was replaced in each well before the start of the assay, and 50 μl of the various sonicated O-GNR suspensions (100 μg ml 1) were added to each well for a final treatment concentration of 20 μg ml 1. The cells were incubated at 37 °C for 24 h, and subsequently the medium with O-GNRs was removed and the wells were rinsed three times with Dulbecco’s phosphate-buffered saline to remove any remnant O-GNRs before adding 100 μl of fresh media and 10 μl of Presto Blue reagent. The O-GNRs were washed out to minimize background fluorescence from the nanostructures. The plates were again incubated for 1 h at 37 °C. Fluorescence readings of the wells were made at 530 nm Ex/580 nm Em using a Cytofluor fluorescence multi-well plate reader (Series H4000 PerSeptive Biosystems, Framingham, MA, USA). Excitation of any remnant extracellular or intracellular graphene structures at wavelengths between 500 and 600 nm leads to low-intensity emission at near infra-red wavelengths (beyond 780 nm) and is unlikely to interfere with the fluorescence measurements associated with this assay (Loh et al., 2010). Fluorescence readings of the media alone were used for baseline correction, and unexposed cells were used as the control. The cell viability in terms of percentage of control cells was expressed as the percentage of (Ftest – Fblank)/(Fcontrol).
Lactic Dehydrogenase Release Assay Cell death and lysis of cells exposed to sonicated O-GNR samples were evaluated by lactate dehydrogenase assays (Sigma-Aldrich, NY, USA). MCF7 cells were plated at a density of 15 × 104 cells per well in 24-well cell culture plates, and incubated for 24 h, after which the cells were treated with 50 μl of the uncentrifuged sonicated samples and incubated for 24 or 72 h. At the end of the incubation period, media were collected from the individual wells and centrifuged at 153 g for 5 min. A total of 50 μl of the media supernatant were added to a fresh 96-well plate along with the LDH assay reagent (100 μl in each well), and incubated for 45 min. The absorbance values were recorded at 490 nm, with absorbance of the culture media being used for baseline correction, and untreated cells serving as the control. The LDH leakage (percentage of the positive control) was expressed as the percentage of Asample/Acontrol, where Asample is the optical density of sonicated O-GNR samples and Acontrol is the optical density of the control cells.
Cell Count
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O-GNR solution toxicity was assessed in vivo using embryos from Japanese medaka, maintained according to Stony Brook University IACUC approved protocols. One day postfertilization (dpf) embryos were used for all analyses. For the first experiments, individual embryos were exposed in 96-well plates (Falcon; Becton Dickinson, Franklin Lake, NJ, USA) (Fig. 7). In the last experiment three replicate groups (n = 10 embryos/group) were exposed to 10 ml of O-GNR solutions in 20 ml vials. The embryos were exposed separately to complete O-GNR solutions, supernatant of O-GNR solutions postcentrifugation and in some cases pellets resuspended in embryo rearing media (Morin et al., 2011). All exposures were conducted at 25 °C with gentle agitation (220 rpm on an orbital shaker) and a 16 : 8 light/dark cycle. All solutions were replaced every other day, and the embryos were exposed for a total of 6 days. At 7 dpf, all embryos were transferred to embryo rearing media until they hatched, and the combined embryo/larval survival was assessed at 12 dpf.
Transmission Electron Microscopy MCF7 cells were plated on ACLARW film (Electron Microscopy Sciences, Hatford, PA, USA) at a density of 106 cells per film, and exposed to O-GNR (at 20 μg ml 1) for 24 h. Medaka embryos were treated with O-GNR (at 20 μg ml 1) for 6 days or left untreated. After exposure periods of either 24 h or 6 days, cells and embryos were fixed with 2.5% electron microscopy grade glutaraldehyde (Electron Microscopy Sciences) in 0.1 M PBS. After fixation, the films containing fixed cells and embryos (sliced into half using a thin blade) were placed in 2% osmium tetroxide in 0.1 M PBS, treated with multiple (graded) ethanol washes for dehydration and embedded in Durcupan resin (Sigma-Aldrich, St. Louis, MO, USA). Blocks containing high cell densities and embryo slices containing the chorion were cut into 80 nm ultrathin sections using an Ultracut E microtome (Reichert-Jung, Cambridge, UK), and placed on Formvar-coated copper grids. The sections were then viewed with a Tecnai Bio Twin G transmission electron microscope (FEI, Hillsboro, OR, USA), at 80 kV. Digital images were acquired using an XR-60 charge-couple device digital camera system (AMT, Woburn, MA, USA). Ten images each for treated and untreated MCF7 cells and one section from four different treated and untreated embryos were analyzed. Statistics All in vitro data are presented as mean + SD. The Presto blue assay and cell counting were repeated with independent cell preparations on 3 different days (n = 3), and all other assays were repeated with independent cell preparations on 4 different days (n = 4). The mean of readings obtained from three parallel wells per exposure condition per day was considered as one independent observation (n = 1). Square root transformation was used to transform percentage data to approximate a normal distribution, and these transformed data were used for one-way ANOVA, followed by Tukey–Kramer post hoc analyses. Survival of fish embryos exposed to O-GNR solutions was evaluated by logistic regression analysis or by ANOVA followed by Tukey–Kramer post hoc analysis. All analyses were performed using Statistica software (Statsoft, Tulsa, OK, USA).
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MCF7 cells were plated at a density of 5 × 104 cells per well in 24-well cell culture plates and incubated for 24 h, followed by treatment with the uncentrifuged sonicated samples (50 μl) and incubation for 24 and 72 h. Following incubation, the media was removed and wells were washed three times with Dulbecco’s phosphate-buffered saline to remove any remnant O-GNR. The cells were then detached from the wells and counted using a hemocytometer.
In Vivo Medaka Fish Embryo Survival Assay
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Results Atomic Force Microscopy Figure 1 shows representative AFM images obtained from the supernatant of O-GNR samples after different sonication treatments followed by centrifugation. Figure 1(A) shows representative O-GNRs (black arrows) observed in the supernatant of bath-sonicated samples post centrifugation. Little or no carbon debris was observed for this treatment. Figure 1(B) shows O-GNR samples probe sonicated for 1 min. Shorter, more globular, carbonaceous structures (black arrows) were observed. The approximate diameter of the globular structures was
250–400 nm. Figure 1(C) shows O-GNR samples probe sonicated for 5 min. The image shows the presence of smaller disc-like structures (black arrows) approximately 150–250 nm in diameter. Figure 1(D) shows the presence of a large number of even smaller sized particles and carbon debris (black arrows) in O-GNR solution probe sonicated for 10 min. AFM data are numerically compared in Fig. 1(E), which shows the average length of 15 O-GNRs in each sonication type obtained from three to four AFM images. Bath-sonicated samples showed an average length of 744 ± 178 nm, and the sample probes sonicated for 1, 5 and 10 min showed average lengths of 323 ± 50 nm, 201 ± 28 and 100 ± 10 nm, respectively,
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Figure 1. Representative atomic force microscopy images of the oxidized graphene nanoribbon (O-GNR) supernatants after different sonication treatments and centrifugation. Scale bars on all images represent 1 μm. (A) O-GNRs in a bath-sonicated (250 W, 20 min sonication) sample showing predominantly intact particles (black arrows). (B,C) O-GNRs sonicated for 1 and 5 min, respectively, showing the presence of round carbon structures (black arrows). (D) O-GNR sonicated for 10 min, showing the presence of small carbon debris (black arrows). (E) Bar diagram showing size distribution of O-GNR supernatants after different sonication treatments and centrifugation. The calculations were done using 15 O-GNRs from multiple atomic force microscopy images (n = 15). Data presented as means + SD. * represent a significant difference (P < 0.05) compared to bath-sonicated O-GNR. #Significant difference (P < 0.05) compared to 1 min probe-sonicated O-GNR. †Significant difference (P < 0.05) compared to 5 min probesonicated O-GNR.
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Structural disruption increases toxicity of graphene nanoribbons indicating a significant decrease in lengths with increased sonication time and intensity. A suspension made from the resuspended pellet obtained after centrifugation of bath-sonicated O-GNR samples is shown in Fig. 2(A). Large aggregated structures (>1000 nm in diameter) of O-GNR were mainly observed (black arrows). Figure 2(B) shows a suspension made from the resuspended pellet
obtained after centrifugation of O-GNR samples that were probe sonicated for 1 min. Large aggregated structures of O-GNR were observed (500–1500 nm) (black arrows), along with small aggregates (green arrows) similar in size to those observed in the supernatant of this suspension (100–250 nm in diameter) (Fig. 1B). Figure 2(C) shows the suspension of the resuspended pellets obtained after centrifugation of the
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Figure 2. Representative atomic force microscopy images of resuspended pellets of oxidized graphene nanoribbons (O-GNRs) after different sonication treatments and centrifugation. Scale bars on all images represent 1 μm. (A) Bath-sonicated O-GNR aggregates resuspended post-centrifugation. (B–D) O-GNR resuspended after 1, 5 and 10 min of probe sonication, respectively, showing both large (black arrows) and small (green arrows) aggregates. (E) Bar diagram showing size distribution of resuspended O-GNR pellets after different sonication treatments and centrifugation. The calculations were done using 15 O-GNRs from multiple atomic force microscopy images (n = 15). Data presented as means + SD. *Significant difference (P < 0.05) compared to bath-sonicated O-GNR. #Significant difference (P < 0.05) compared to 1 min probe-sonicated O-GNR.
S. Mullick Chowdhury et al. O-GNR samples that were probe sonicated for 5 min. Very few large aggregates of O-GNR were observed (300–900 nm in diameter, black arrows) along with small aggregates (100–250 nm in diameter, green arrows). The large aggregates were smaller than those observed in samples that were probe sonicated for 1 min. Figure 2(D) shows the sizes of the O-GNR aggregates from the resuspended pellets obtained after centrifugation of O-GNR samples that were probe sonicated for 10 min. Similar to the 5 min sonicated samples, very few large aggregates of O-GNR were observed (200–500 nm in diameter, black arrows) along with small aggregates (50–150 nm in diameter, green arrows). Both the large and small aggregates were much smaller than the O-GNR aggregates from the 1 and 5 min probe-sonicated samples. Figure 2(E) shows the statistical analysis of length of 15 O-GNR aggregates in each sonication type obtained from multiple AFM images. Bath-sonicated samples showed an average length of 1535 ± 314 nm, and samples probe sonicated for 1, 5 and 10 minutes showed average lengths of 997 ± 358 nm, 566 ± 291 and 194 ± 130 nm,
respectively, indicating a gradual decrease in lengths of samples probe sonicated for ≥ 1 min. Raman Spectroscopy Figure 3 shows the Raman spectroscopic analyses of solid O-GNR and O-GNR suspensions after different types of sonication. As seen in Fig. 3(A), the five different sonicated samples (both bath and probe sonicated) showed a distinct increase in a peak at 1150 cm 1, with increasing time and intensity of sonication as compared to the solid unsonicated sample. Moreover, an increase in the D/G band ratio was observed with increasing intensity and sonication time, indicating sonication-related structural disruption (Fig. 3B). Fourier Transform Infrared Spectroscopy Figure 3(C) shows the FTIR spectra of unsonicated and sonicated O-GNR samples. Characteristic troughs for graphene oxide were observed for the unsonicated solid sample at 1732 cm 1
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Figure 3. (A) Representative Raman spectra of oxidized graphene nanoribbons and (B) ratios of D band and G band intensity for samples that were unsonicated, bath sonicated for 5 or 20 min, or probe sonicated for 1, 5 or 10 min. (C) Representative Fourier transform infra-red spectra of oxidized graphene nanoribbon samples that were unsonicated, bath sonicated for 20 min or probe sonicated for 1, 5 or 10 min.
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Structural disruption increases toxicity of graphene nanoribbons corresponding to C = O stretching vibrations of COOH groups (indicated with black arrow), 1605 cm 1 corresponding to O–H deformation vibrations of COOH groups (indicated with black arrow), 1400 cm 1 corresponding to O–H deformation vibrations of tertiary C–OH groups (indicated with orange arrow) and at 1070 cm 1 corresponding to C–O stretching vibrations of epoxy groups (indicated with blue arrow). Increase in sonication intensity and time resulted in gradual decrease of all these troughs. The FTIR spectra with O-GNR samples probe sonicated for 10 min decreased the most. Presto Blue Assay The presto blue assay is based on the principle that metabolically active cells can convert the cell permeable dye resazurin to a pink fluorescent product, with the amount of dye converted being directly proportional to the metabolic state of the cells, especially mitochondrial integrity (Lien et al., 2012). Figure 4(A,B) shows the state of cellular metabolism in A549 and MCF7 cells exposed to sonicated O-GNR samples. Both cell lines showed reduced metabolism in response to O-GNR, with the MCF7 cells being more sensitive to the effects of
sonication. In these cells, treatment with O-GNR dispersions that were bath sonicated for 20 min resulted in a decrease of the cellular metabolism values to approximately 75% of the values of untreated cells (Fig. 4B). Despite the initial decrease in cellular metabolism, increases in the intensity and/or time of sonication did not result in further effects on cellular metabolism. A549 cells showed a similar decrease in cellular metabolism only after exposure to probe-sonicated O-GNRs (Fig. 4A). In these cells, the adverse effects appeared to be time-dependent, with the largest decrease in cellular metabolism (approximately 60% of the cellular metabolism of untreated cells) observed in response to the O-GNR dispersions probe that were sonicated for ≥ 5 min.
Transmission Electron Microscopy of MCF7 Cells Figure 4(C,D) shows the interaction of MCF7 cells with O-GNR. Figure 4(C) shows the presence of smaller O-GNR particles inside MCF7 cells (black arrows). Red arrows indicate the cell membrane and yellow arrow indicates the nucleus. Figure 4(D) suggests that larger O-GNR aggregates fail to enter the cells but can cause membrane damage (black arrows).
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Figure 4. Presto blue assay of (A) A549 cells and (B) MCF7 cells treated with bath- and probe-sonicated oxidized graphene nanoribbons (O-GNRs), evaluated after 24 h incubation with the cells. The assay was repeated on three independent cell preparations conducted on different days (n = 3). Data are normalized to assay values obtained from untreated cells and presented as means + SD. *Significant difference (P < 0.05) compared to the unexposed control cells. Representative transmission electron microscopy images of MCF7 cells treated with O-GNRs for 24 h showing (C) small O-GNR particles inside cells (black arrow) (D) larger O-GNR aggregates (black arrow) fail to enter cells but can cause membrane damage. Red arrows indicate the cell membrane and yellow arrow indicates the nucleus.
S. Mullick Chowdhury et al. Lactic Dehydrogenase Release Assay The LDH assay is based on the principle that dying and unhealthy cells release LDH though their ruptured membranes. Thus, enhanced cell death results in a greater release of LDH into the cell media. However, basal levels of LDH in the media are present even in normal untreated cells and increase as the cells proliferate. Figure 5(A) and (B) show the LDH release from MCF7 cells after exposure to sonicated O-GNR dispersions for 24 and 72 h, respectively. After 24 h of exposure, no significant increase in LDH release was noted in MCF7 cells. However, after 72 h, a decrease of approximately 50% in LDH release compared to the untreated control cells was observed in the media of cells treated with O-GNR dispersions probe sonicated for 10 min.
Cell Count Figure 6(A) and (B) show the cell count for MCF7 cells treated with the various sonicated O-GNR dispersions for 24 and 72 h, respectively. Mean population doubling time of MCF7 cells is
reported to be ~24 h (Sutherland et al., 1983). After 24 h, no significant change in cell count was observed regardless of treatment. However, after 72 h, a significant decrease in cell count (approximately 44%) was observed for cells treated with O-GNR dispersions that were probe sonicated for 10 min (14 × 104 cells) compared to cells treated with 5 min bath-sonicated O-GNR dispersions (25 × 104 cells), indicating that cell growth was inhibited between the 24 h and 72 h time points.
Medaka Embryo Mortality Exposure to complete sonicated samples and supernatant. OGNR dispersions that were bath sonicated for 20 min did not increase embryo mortality as compared to unexposed controls. In comparison, probe-sonicated O-GNR solutions caused a significant sonication time-dependent increase in embryo mortality. Dispersions that were probe sonicated for 1, 5 and 10 min reduced embryo viability to approximately 75%, 55% and 40% of control values, respectively (Fig. 7A). Bath sonication resulted in no apparent increase in toxicity
Figure 5. Toxicity of bath- and probe-sonicated oxidized graphene nanoribbon samples in MCF7 cells evaluated after (A) 24 and (B) 72 h of incubation using the LDH release assay. The assay was repeated on four independent cell preparations conducted on different days (n = 4). Data are normalized to assay values obtained from untreated cells and presented as means + SD. *Significant difference (P < 0.05) compared to the unexposed control cells. LDH, lactate dehydrogenase.
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Figure 6. Cell count of MCF7 cells treated with bath- and probe-sonicated oxidized graphene nanoribbons, evaluated after (A) 24 and (B) 72 h of incubation. The assay was repeated on three independent cell preparations conducted on different days (n = 3). Data are presented as means + SD. *Significant difference (P < 0.05) compared to the unexposed control cells.
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Structural disruption increases toxicity of graphene nanoribbons
Figure 7. Embryonic survival as a function of sonication time and intensity. Significantly increased mortality of embryos with increasing time of 3 3 sonication was observed (P = 5.3 × 10 for A and P = 3.3 × 10 for B). (A) The solutions were not centrifuged post-sonication; (B) the solutions were 5 centrifuged post-sonication. When the two data sets were combined, the sonication time remained significant (P = 5.5 × 10 ) while centrifugation 1 was not (P = 5.4 × 10 ), indicating that the observed toxicity was likely due to smaller materials not removed by centrifugation. Representative transmission electron microscopy section of a medaka embryo showing (C) untreated medaka embryo membrane (chorion) (yellow arrow) and (D) presence of oxidized graphene nanoribbons (O-GNRs) (black arrows) inside chorion (yellow arrow) from embryos treated with O-GNR. Red arrows indicate structural damage to the chorion.
compared to the controls (Fig. 7A), and was hence not further evaluated. Experiments conducted on probe-sonicated solutions that were centrifuged to remove undispersed O-GNR material produced nearly identical results (Fig. 7B). Sonication time significantly enhanced toxicity in both experiments (P = 5.3 × 10 3 for Fig. 7A, and P = 3.3 × 10 3 for Fig. 7B). Given the similarity in toxicity observed upon exposure to O-GNRs, regardless of whether or not they were centrifuged before testing, the analysis was repeated with the combined data set where both sonication and centrifugation status were evaluated as separate factors. With the combined data set, sonication time was highly significant (P = 5.5 × 10 5), while centrifugation status was not (P = 5.4 × 10 1), suggesting that there is no significant difference between the toxicity of centrifuged versus uncentrifuged O-GNR suspensions.
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Transmission Electron Microscopy of Medaka Embryo Figure 7(C,D) shows the effect of interaction of medaka embryo chorion with O-GNRs. Figure 7(C) shows an intact chorion (yellow arrow) not treated with O-GNRs. Figure 7(D) shows that O-GNRs (black arrows) can enter the chorion (yellow arrow) and can cause membrane damage (red arrows).
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Exposure to resuspended pellets. Based on the results obtained above (Fig. 7), the toxicities of complete sonicated samples (uncentrifuged), supernatant (after centrifugation) and resuspended pellets for probe-sonicated O-GNR dispersions (sonicated for 1, 5 and 10 min) were compared (Fig. 8) and normalized to control toxicity (which was 0 in all cases).
Similar to the results of the analyses shown in Fig. 7, probe sonication was found to increase toxicity. Additionally, as previously noted, toxicity resulting from the supernatant did not differ from that of the complete sample. However, the resuspended pellet resulted in a significant increase in embryo toxicity for the 1 and 5 min probe-sonicated samples compared to both the complete O-GNR solutions and the supernatants. Although the mean embryonic survival resulting from exposure to the resuspended pellet for the 10 min probe-sonicated samples was lower than that observed from exposure to the complete sample or the supernatant after centrifugation, the increase in toxicity was not statistically significant.
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Figure 8. Embryonic survival, presented as the percentage of control values, after exposure to uncentrifuged probe-sonicated oxidized graphene nanoribbons (O-GNRs), supernatant from centrifuged probe-sonicated O-GNR samples, and the resuspended pellet from centrifuged probe-sonicated O-GNR samples, with samples being probe sonicated for 1, 5 or 10 min. Two-way ANOVA revealed that the nature of the O-GNR solution (uncentrifuged, centrifuged or resuspended), as well as the time of sonication significantly affected embryo survival 7 6 (P = 6.15 × 10 and 1.16 × 10 , respectively). #Significant difference (P < 0.05) between the toxicity of the supernatant and the resuspended pellet obtained from centrifuged probe-sonicated samples by one-way ANOVA.
Discussion
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The in vitro and in vivo toxic effects of short-term (1–20 min), low-energy (bath) and high-energy (probe) sonication of OGNR were evaluated in this study. In all experiments, nominal O-GNR concentrations of 20 μg ml 1 were used, as this concentration has been previously demonstrated to be non-toxic to cells in vitro (Mullick Chowdhury et al., 2013), as well as to fish embryos in vivo (data not shown). The O-GNRs used in the experiments were synthesized from MWCNTs and had an average size of 1–2.5 μm (Figs 1A and 2A). AFM images indicated that both the supernatants and resuspended pellets of probe-sonicated samples generated smaller structures, whereas the sizes of these structures were inversely related to the sonication time. This observation is consistent with recent studies that have reported that high-energy sonication can lead to breakdown of graphene oxide nanosheets (Compton et al., 2012). Structural analyses by Raman spectroscopy showed the characteristic D band (1340 cm 1) produced due to defects in the O-GNR structure, and a G band (1580 cm 1) resulting from the graphitic hexagonal structures. In the O-GNR samples that were bath or probe sonicated in water, the G band appeared shifted to 1626 cm 1, indicating an increase in single sheet graphene structures (Ferrari et al., 2006). With increasing intensity and time of sonication, the relative height of the G band decreased, consequently resulting in an increased intensity ratio of the G and D bands (Fig. 3B), which is indicative of an increase in structural defects. Furthermore, an increase in the intensity of a band at 1150 cm 1 was observed, which can be attributed to the breakdown of the structure to form hydrogenated carbon structures with alternate C = C and C-C bonds associated with increased time and intensity of sonication (Subramanyam et al., 1997). The data obtained from Raman analysis are corroborated by FTIR
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spectra of the unsonicated solid samples and dried sonicated samples, which show a gradual decrease in characteristic troughs of graphene oxide with increased intensity and time of sonication (Fig. 3C). This is indicative of loss of structural integrity of the graphene structure. It is likely that these alterations in structure due to sonication intensity and time are responsible for the enhanced toxicity observed with multiple endpoints. The significant decreases observed in cellular metabolism in vitro (Fig. 4) and embryo survival in vivo (Figs 7 and 8) upon exposure to probe-sonicated O-GNRs suggest that the smaller particles disrupt the cellular machinery through interactions with cellular organelles or the cytoskeleton upon uptake, possibly in a manner similar to that of other nanoparticles (Dam et al., 2012; Gupta et al., 2004; Johnson-Lyles et al., 2010). TEM results from MCF7 cells showed uptake of only small particles and further supported this hypothesis (Fig. 4C,D). Although no cytotoxicity was observed in vitro even after 72 h exposure to the O-GNR dispersions, a ~50% decrease in basal LDH release was observed in samples probe sonicated for 10 min, suggesting a reduction in cell proliferation (Fig. 5B). This observation was further supported by cell count analyses, which showed a decrease in the number of MCF7 cells treated with 10 min probe-sonicated samples for 72 h (approximately 44% of 5 min bath-sonicated O-GNR treated cells; Fig. 6B). ROS production in the treated cells was almost three times higher than in unexposed cells, although no significant differences in ROS levels were observed among the different sonication treatments (Fig. S1). Thus, ROS production is likely not the mechanism responsible for the effects observed in this study. Moreover, data from the WST-1 assay (Fig. S2) conducted after 2 h exposures also failed to show significant effects of O-GNR treatment, suggesting that sonication has no immediate effects on overall cellular metabolism. Taken together, these results indicate that, while exposure to sonicated O-GNR can cause chronic reductions in cellular metabolism, it does not cause immediate cell death. Japanese medaka embryos were also found to be sensitive to the toxic effects of probe-sonicated O-GNR (Fig. 7A), and this is consistent with previous studies reporting significant toxicity in zebrafish embryos injected with MWCNT subjected to highenergy sonication for prolonged periods (24–48 h) (Cheng and Cheng, 2012). Our findings demonstrate enhanced toxicity after more realistic aqueous exposures to O-GNR that is probe sonicated for as little as 1 min, indicating that toxicity resulting from standard processing procedures and exposure scenarios is reasonable. Enhanced toxicity of sonicated nanomaterials has been mentioned in a previous report on aquatic species (Oberdörster et al., 2006), yet no data were provided to support this claim. The results from the present study indicate that evaluation of the effects of post-production processing on nanomaterial toxicity should be part of routine analysis. The similar toxicity profiles of the centrifuged and uncentrifuged samples supports the hypothesis that smaller, unaggregated and dispersed structures or carbon debris are responsible for the enhanced toxicity observed in sonicated OGNR solutions (Fig. 7A,B). The observed enhanced toxicity could be due to greater bioavailability of smaller particles, toxic interactions with the chorion and/or enhanced cellular toxicity. TEM images of control and O-GNR-treated medaka embryos indicates that O-GNR can enter the chorion of treated medaka embryos and can cause structural damage (Fig. 7C,D). Although exposure
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Structural disruption increases toxicity of graphene nanoribbons to bath-sonicated O-GNR suspensions did not increase embryo mortality (Fig. 7A), it did lead to precocious hatching (by approximately 2 days) (Fig. S3). This has been previously observed by Wu et al. (2010) in medaka embryos and by Lee et al. (2007) in zebrafish embryos after exposure to silver nanoparticles, although it is in contrast to a recent report of delayed hatch in zebrafish embryos by Chen et al. (2012). In a natural setting, less developed fry may have reduced abilities to swim and/or capture food, putting them at increased risk of starvation or predation. Early hatching may be attributed to the docking of small nanoparticle aggregates into the chorion pore channels, leading to disruption of the membrane transport, and disintegration of the chorion structure (Wu et al., 2010). Chen et al. (2012) also reported agglomerates of O-GNRs in the outer layer of zebrafish chorions, but hypothesized the chorion acted as a protective barrier to these large particles. It is likely that the smaller size of the probe-sonicated O-GNRs examining in our study allowed for greater penetration and, thus, effects. The finding that the resuspended pellet alone could cause significantly enhanced toxicity, compared to 1 and 5 min probe-sonicated uncentrifuged samples or their supernatants (Fig. 8), was somewhat surprising. However, AFM images of the resuspended pellets revealed that they contained both large and small aggregates of O-GNRs, which differ structurally (Fig. 2A–E), and are likely more toxic compared to aggregates and particles of uncentrifuged bath- and probesonicated samples. This is similar to observations reported by Liao et al. (2011), who demonstrated that graphene sheet aggregates were more toxic to fibroblasts compared to sonicated samples. It is possible that these different types of aggregates have different mechanisms of action, and further detailed investigations will be needed to elucidate and differentiate these mechanisms.
Conclusions
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This work was supported by the National Institutes of Health (grant no. 1DP2OD007394-01), and the Wallace H. Coulter Foundation.
Conflict of Interest The Authors did not report any conflict of interest.
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Our findings clearly indicate that with increasing sonication intensity and time, smaller carbon particles are being produced. Further, these suspensions are capable of causing reductions in overall cellular metabolism and cell proliferation in in vitro assays, which are likely associated with disruptions of the cellular machinery. Although significant, these effects did not result in short-term cell death in this study. In vivo studies with medaka embryos further showed that exposure to similar concentrations of probe-sonicated O-GNR results in precocious hatching and decreased survival, clearly demonstrating the effects of these suspensions on an intact organism. The in vivo results indicate that there may be environmental consequences of exposure to these materials, and the enhanced toxicity of resuspended probe-sonicated O-GNR aggregates suggests that other aggregated O-GNR structures may be a concern. Further studies are needed to elucidate the mechanisms associated with embryo toxicity. Based on the results of this study, structural disruption of O-GNR and similar nanostructures should be avoided, particularly for biomedical applications. These results may also apply to other carbon-based nanoparticles, and thus indicate a need to examine the potential influence of post-production processing for all nanomaterial applications.
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