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Toxicology Letters 200 (2011) 201–210

Contents lists available at ScienceDirect

Toxicology Letters

journa l homepage: www.e lsev ier .com/ locate / tox le t

n vitro toxicity evaluation of graphene oxide on A549 cells

anli Changa, Sheng-Tao Yanga,b, Jia-Hui Liua,b, Erya Donga, Yanwen Wanga,oneng Caoa,∗, Yuanfang Liua,b, Haifang Wanga,∗

Institute of Nanochemistry and Nanobiology, Shanghai University, Shanghai 200444, ChinaBeijing National Laboratory for Molecular Sciences and College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China

r t i c l e i n f o

rticle history:eceived 28 July 2010eceived in revised form 11 October 2010ccepted 24 November 2010

a b s t r a c t

Graphene and its derivatives have attracted great research interest for their potential applications inelectronics, energy, materials and biomedical areas. However, little information of their toxicity and bio-compatibility is available. Herein, we performed a comprehensive study on the toxicity of graphene oxide(GO) by examining the influences of GO on the morphology, viability, mortality and membrane integrity

vailable online 2 December 2010

eywords:raphene oxideiocompatibilityoxicityize effect

of A549 cells. The results suggest that GO does not enter A549 cell and has no obvious cytotoxicity. ButGO can cause a dose-dependent oxidative stress in cell and induce a slight loss of cell viability at highconcentration. These effects are dose and size related, and should be considered in the development ofbio-applications of GO. Overall, GO is a pretty safe material at cellular level, which is confirmed by thefavorable cell growth on GO film.

© 2010 Elsevier Ireland Ltd. All rights reserved.

ell growth substrate

. Introduction

Because of their unique physicochemical properties, graphenend its derivatives have attracted tremendous research interestAllen et al., 2010; Geim, 2009; Rao et al., 2009). They hold greatromise in electronics, energy, materials and biomedical areasAllen et al., 2010; Geim, 2009; Neto et al., 2009; Rao et al., 2009).raphene oxide (GO) is one of the most important graphene deriva-

ives and has been extensively studied in recent years (Park anduoff, 2009). We reported that GO could be used to produce directlyhe graphene-based composites (Cao et al., 2010). Beyond that,O has been also used in many areas, including hydrogen storage

Wang et al., 2009b), catalysis (Scheuermann et al., 2009), trans-arent film (Dikin et al., 2007) and electrode (Eda et al., 2008).

n particular, GO is a potential candidate for biological applica-ions, such as drug delivery and bio-analysis (Liu et al., 2008; Lut al., 2010; Sun et al., 2008; Yang et al., 2009b, 2010; Zhang et al.,010a). For example, Liu et al. (2008) found that GO could deliveroxorubicin into cancer cells for the therapeutic purpose.

Many studies have shown that nanomaterials might have side-ffects on health (Aillon et al., 2009; Oberdörster et al., 2005; Xiat al., 2009). For instance, we have reported the toxicity and reten-ion of carbon nanotubes (CNTs) in vitro and in vivo (Deng et al.,

∗ Corresponding authors. Fax: +86 21 66135275.E-mail addresses: ancao@shu.edu.cn (A. Cao), hwang@shu.edu.cn (H. Wang).

378-4274/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.toxlet.2010.11.016

2007; Jia et al., 2005; Wang et al., 2004, 2008, 2009a, 2009c; Yanget al., 2008a, 2008b, 2009a). The thorough understanding of thebiological behavior of nanomaterials guarantees the sustainablenanotechnology (Aillon et al., 2009; Hussain et al., 2009; Nel et al.,2006; Oberdörster et al., 2005; Xia et al., 2009). However, for thenewly developed graphene and its derivatives, such information isgenerally lacking to date.

Herein, we performed a systematic study on the toxicity of GOat cell level. The morphology, viability, mortality and membraneintegrity of A549 cells, a human lung carcinoma epithelial cell line,were evaluated after GO exposure. The results suggest that, GO hasno obvious toxicity to A549 cells, though GO induces the cellu-lar oxidative stress even at low concentration and induce a slightdecrease of the cell viability at high concentration. The transmissionelectron microscopy (TEM) investigation suggests that GO couldhardly enter cells. The size of GO sheets has effect on the toxicity ofGO at high concentration, that is larger sheets have better biocom-patibility. The good biocompatibility of GO allows it to be used forvarious biomedical purposes in future. Preliminarily, we show theGO film is a good substrate for cell growth.

2. Materials and methods

2.1. Preparation and characterization of GO

Natural graphite powder (≤30 �m, with purity higher than 99.85 wt.%) waspurchased from Sinopharm Chemical Reagent Co., Ltd., China. The preparationof GO followed the modified Hummer method (Hummers and Offerman, 1958;Kovtyukhova et al., 1999), which is described in Supplementary Data. The obtained

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o suspension was further heated to 120 ◦C for 20 min to get GO mixture (m-GO).fter cooling to room temperature, the suspension was centrifuged at 18,000 rpm

or 50 min to obtain the s-GO (supernatant, GO with smaller size) and l-GO (residue,O with larger size) samples.

The three GO samples were characterized by TEM (JEM-200CX, JEOL, Japan),tomic force microscopy (AFM, SPM-9600, Shimadzu, Japan), Fourier transformnfrared spectroscopy (FTIR, Avatar 370, Thermo Nicolet, USA), Raman spectroscopyRenishaw Invia Plus laser Raman spectrometer, Renishaw, UK) and X-ray photo-lectron spectroscopy (XPS, AXIS Ultra instrument, Kratos, UK). The particle sizeistribution and zeta potential in water were measured by Nanosizer (Zetasizer000 HS, Malvern, UK).

GO were dispersed in ultra-pure water to prepare the stock solution (1.0 mg/mL).he stock solution was sonicated for 1 h (40 kHz, 50 W) and diluted to differentoncentrations with F-12K culture medium just prior to the cell exposure.

.2. Cell culture

A549 cell line is one popular cell line in nanotoxicology studies with a cell cycleime of 22 h (Pulskamp et al., 2007; Herzog et al., 2007). A549 cells were kindlyrovided by Dr. Y. Zhong at Shanghai University, China. A549 cells were cultured in-12K culture medium supplemented with 10% (v/v) fetal bovine serum (Lanzhouational Hyclone Bio-Engineering Co. Ltd., China) at 37 ◦C in a humidified atmo-

phere of 5% CO2/95% air.

.3. Cell morphology and ultrastructure

A549 cells were plated in the 96-well plates (5 × 103 cells per well) and incu-ated for 24 h. m-GO, s-GO and l-GO were introduced separately to cells with aredetermined concentration in culture medium. Cells cultured in the mediumithout adding GO were taken as the control. The cell morphology was recordednder an optical microscopy at 24 h postexposure.

To investigate the cellular ultrastructure of GO-treated A549 cells, thin-sectionsf cells were investigated under TEM. A549 cells were plated in the 6-well plates1 × 105 cells per well) and incubated for 24 h. GO samples were introduced to theells with a final concentration of 200 �g/mL. Cells without GO exposure were takens the control. After 24 h exposure, the cells were washed with ice-cold PBS for threeimes. After the centrifugation (4000 rpm × 10 min), cells were collected, prefixedith 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide, dehydrated in a graded

lcohol series, embedded in epoxy resin, and cut with an ultramicrotome. Thin-ections poststained with uranyl acetate and lead citrate were inspected with TEM.

.4. Cell viability

The cell viability was evaluated by CCK-8 assay (Dojindo Molecular Technolo-ies, Inc.). A549 cells were plated in the 96-well plates (5 × 103 cells per well) andncubated for 24 h. m-GO, s-GO and l-GO were introduced separately to cells withifferent test concentrations (10, 25, 50, 100 and 200 �g/mL) in culture medium.ells cultured in the medium without adding GO were taken as the control. After4, 48 and 72 h incubation, the cells were washed with D-Hanks buffer solution.wo hundred microlitres of CCK-8 solution was added to each well and incubatedor an additional 3 h at 37 ◦C. The optical density (OD) of each well at 450 nm wasecorded on a Microplate Reader (Thermo, Varioskan Flash). The cell viability (%f control) is expressed as the percentage of (ODtest − ODblank)/(ODcontrol − ODblank),here ODtest is the optical density of the cells exposed to GO sample, ODcontrol is the

ptical density of the control sample and ODblank is the optical density of the wellsithout A549 cells.

In a separate experiment, to test the effect of the adsorption of culture mediumy GO on the toxicity, the GO samples (10, 25, 50, 100 and 200 �g/mL) were incu-ated in culture medium (cell-free) at 37 ◦C under 5% CO2/95% air for 24 h. Then, theixtures were centrifuged at 4000 rpm for 5 min to remove precipitate (GO). TheO free supernatants were collected and introduced to A549 cells (5 × 103 cells perell). After 24 h incubation, the cell viability was assayed by CCK-8 assay.

.5. Cell mortality

The cell mortality was evaluated by Trypan blue assay (Beyotime Institute ofiotechnology, China). A549 cells were plated in the 6-well plates (1 × 105 cells perell) and incubated for 24 h. Then, GO was introduced to cells with different concen-

rations (10, 25, 50, 100 and 200 �g/mL) in culture medium. Cells cultured in the freeedium were taken as the control. Twenty-four hours later, the supernatant was

ollected and the cells were detached with 300 �L trypsin–EDTA solution. The mix-ure of the supernatant and detached cells was centrifugated at 1200 rpm for 5 min.hen, the residue was added with 800 �L Trypan blue solution and dispersed. Aftermin staining, cells were counted using cytometer. The dead cells were stained withlue color. Cell mortality (%) is expressed as percentage of the dead cell number/theotal cell number.

ers 200 (2011) 201–210

2.6. Membrane integrity

LDH test-kit (CytoTox 96® Non-Radioactive Cytotoxicity Assay, Promega Co.)was used to assess the cell membrane integrity. A549 cells were plated in the 96-wellplates (5 × 103 cells per well) and incubated for 24 h. GO samples were introducedseparately to the cells with different concentrations (10, 25, 50, 100 and 200 �g/mL)and incubated for another 24 h. The positive control was prepared by adding 10 �Lof lysis solution to the control cells at 45 min prior to the centrifugation. Then,the centrifugation (1200 rpm × 5 min) was performed. One hundred microlitres ofsupernatant was taken out from each well for LDH assay following the instructionof the kit. The absorbance at 490 nm was recorded on a Microplate Reader (Thermo,Varioskan Flash). The LDH leakage (% of positive control) is expressed as the percent-age of (ODtest − ODblank)/(ODpositive − ODblank), where ODtest is the optical density ofthe control cells or cells exposed to GO, ODpositive is the optical density of the positivecontrol cells and ODblank is the optical density of the wells without A549 cells.

2.7. Apoptosis assay

Apoptosis kit (FITC Annexin V Apoptosis Detection Kit I, BD Biosciences, USA)was employed to detect apoptotic and necrotic cells. The manual of the kit wasstrictly followed. Briefly, A549 cells were plated in the 6-well plates (1 × 105 cellsper well) and incubated for 24 h. The GO samples were introduced to the cells atdifferent concentrations (10, 100 and 200 �g/mL) and incubated for another 24 h.The positive control was prepared by culturing the control cells in medium con-taining 200 mM H2O2 for 30 min. A549 cells were collected, washed twice with coldD-hanks buffer solution, and re-suspended in binding buffer (1 × 106 cells/mL). After100 �L of A549 cells was transferred to a tube, 5 �L of FITC-conjugated Annexin V(Annexin V-FITC) and 5 �L of propidium iodide (PI) were added followed by incuba-tion for 15 min at room temperature in the dark. The stained A549 cells were dilutedby the binding buffer and directly analyzed by the fluorescence-activated cell sort-ing method (FACS, FACSCalibur, BD Biosciences, USA). The cells were set as positivedepending on the fluorescence intensity of Annexin V-FITC or PI. The positive ofAnnexin V-FITC indicates the out-releasing of phospholipid phosphatidylserine (PS),which happens in the early stage of apoptosis. The positive of PI indicates the dam-age of cell membrane, which occurs either in the end stage of apoptosis, in necrosisor in dead cells. Therefore, the apoptotic cells were identified as Annexin V-FITC+

and PI− . The nonviable cells were identified as Annexin V-FITC+ and PI+ and viablecells as Annexin V-FITC− and PI− .

2.8. Reactive oxygen species (ROS) assay

The oxidant-sensitive dye DCFH-DA was used for ROS detection (Reactive Oxy-gen Species Assay Kit, Beyotime Institute of Biotechnology, China). A549 cells wereplated in the 96-well plates (5 × 103 cells per well) and incubated for 24 h. GO sam-ples were introduced to the cells with different concentrations (10, 25, 50, 100and 200 �g/mL) and incubated for another 24 h. The positive controls were pre-pared by culturing the normal cells with culture medium containing 200 mM H2O2

at 1 h prior to the addition of DCFH-DA probe. Then, the culture medium for allcells was replaced by 100 �L of new culture medium containing 20 �M DCFH-DA.The cells were washed with D-Hanks buffer solution for three times 1 h later. Afteradding 100 �L of D-Hanks buffer solution to each well, the fluorescence intensitywas monitored by a Microplate Reader. The ROS level is expressed as the ratio of(Ftest − Fblank)/(Fcontrol − Fblank), where Ftest is the fluorescence intensity of the cellsexposed to GO or the positive control, Fcontrol is the fluorescence intensity of thecontrol cells and Fblank is the fluorescence intensity of the wells without A549 cells.

In order to test the ROS generation of GO in culture medium (cell free), the GOsamples (0, 10, 25, 50, 100 and 200 �g/mL) were incubated in F-12K medium sup-plemented with 10% (v/v) fetal bovine serum for 24 h. The ROS level was measuredfollowing the protocol developed by Lu et al. (2009). Briefly, 0.1 mM DCFH-DA waschemically hydrolyzed to 2′ , 7′-dichlorofluorescein (DCFH) at pH 7.0 with 0.01 MNaOH at room temperature (30 min in the dark). The chemical reaction was stoppedby adding 200 �L PBS. After adding 50 �L DCFH solution to GO in culture medium,the mixture was incubated at 37 ◦C for 1 h and centrifuged at 4000 rpm for 5 min.The fluorescence generated by the DCFH oxidation was measured on a MicroplateReader.

2.9. Cell growth on GO films

GO films were prepared by evaporating 4 mL of m-GO (1 mg/mL) in a 35 mmculture dish under 80 ◦C. A549 cells were plated in the GO coated dish (2 × 105 cellsper dish) and incubated for 24 h for morphology observation. A549 cells cultured innormal dishes were taken as the control.

2.10. Statistical analysis

Except ultrastructural investigation, six parallel tests were conducted for eachsample. All data are presented as the mean with the standard deviation (mean ± SD).Significance has been calculated using Student’s t-test. * denotes a statistical signif-icance (*≤0.05 and **≤0.01) vs the control.

Y. Chang et al. / Toxicology Letters 200 (2011) 201–210 203

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Fig. 1. Characterization of GO samples. (a–c) AFM images of l-GO

. Results

.1. Characterization of GO

Except for the size, the three GO samples, m-GO, l-GO and s-GO,re very similar. Fig. 1 shows the representative AFM images of thehree GO samples. Most of GO sheets exist as single or few layers.he thickness of the GO layer is around 0.9 nm according to AFMeasurement (Fig. S1). Both large and small sheets are presented

n m-GO (430 ± 300 nm). The size of l-GO sheets (780 ± 410 nm) isarger than that of s-GO sheets (160 ± 90 nm). In aqueous suspen-ion, the average hydrodynamic diameter (Dh) is 588 nm for m-GO,56 nm for l-GO, 148 nm for s-GO, according to Nanosizer measure-ents (Table S1). Three GO samples have very similar FTIR spectra

Fig. S2). The broad absorption at 3400 cm−1 suggests the existencef –COOH and –OH groups. The absorption at 1720 cm−1 corre-ponds to C O bonds. The oxygen contents based on XPS analysisre 33.1% for l-GO, 37.0% for s-GO and 35.8% for m-GO (Fig. S3). The

D/IG values in Raman spectra, which indicate the defect content,re very close among the three samples (Fig. 1d and Table S1).

.2. Cell morphology

The morphology is one important indicator of the status of cells.

he cell morphological changes after GO exposure were recorded toemonstrate the effect of GO on A549 cells directly (Fig. 2). There iso obvious difference between the GO-treated cells and the controlells. Most cells adhere to the substrate tightly and are in normalpindle-shape.

GO (b) and m-GO (c); (d) Raman spectra of l-GO, s-GO and m-GO.

We also checked the influence of GO on the cell attachment fol-lowing Wang et al.’s method with some modifications (Wang et al.,2010). Compared with control cells, GO-treated cells do not showany difference in their adhesion to the culture dish (Fig. S7).

3.3. Cell viability

The cell viability is assayed to estimate the toxicity of GO sam-ples quantitatively by CCK-8 assay (Fig. 3), in which the formationof formazan dye depends on the mitochondria activity. As a whole,the viability loss is dose-related. At higher GO concentrations, theviability loss is observed. Size is another factor on viability. Theinfluence of l-GO and m-GO on the viability of A549 cells is tiny.Even at the highest concentration of 200 �g/mL, more than 80% ofthe cell viability remains. However, s-GO induces more viabilityloss than l-GO and m-GO. At 200 �g/mL of GO, the cell viability is67% at 24 h postexposure. Culture period has little influence on theviability. Similar results were obtained from 24, 48 and 72 h expo-sure (Figs. 3, S4 and S5). Therefore, the followed experiments wereperformed by 24 h exposure.

Nutrient depletion induced by nanomaterial adsorption is a wellrecognized reason for the nanotoxicity. Therefore, we tested theinfluence of culture medium adsorption on the toxicity of GO. F-12Kmedium was pre-treated with GO samples separately for 24 h and

the supernatants were collected for A549 cell culture. If the cellsdid not survive in the GO-pretreated culture medium, we couldconclude that the adsorption of nutrients on GO contributes to thetoxicity. But the cells grew just as well as the control cells. The cellviability does not decrease along with GO concentrations (Fig. 4).

204 Y. Chang et al. / Toxicology Letters 200 (2011) 201–210

Fig. 2. Optical microscopy images of GO-treated A549 c

Fig. 3. The viability of A549 cells after exposed to GO for 24 h.

ells. (a) l-GO; (b) s-GO; (c) m-GO; (d) the control.

This reveals that the adsorption of nutrients on GO sheets frommedium does not affect the status of cells under our experimentcondition.

3.4. Cell mortality

While viability shows the activity of cell mitochondria, themortality indicates the death of cell. Here, the cell mortality ismonitored by Trypan blue exclusion assay, in which the dead cellsare stained into blue while the live ones remain unchanged. Themortality is expressed by the ratio of dead cells in all cells. Whilethere is the viability loss induced by GO exposure, compared tothe control, no mortality increase of A549 cells is observed afterGO treatment (Fig. 5). The mortality remains around 1.5% upon theexposure, nearly the same as that of the control (1.4%).

3.5. Membrane integrity

When the membrane is damaged, the intracellular LDHmolecules would be released into the culture medium. Therefore,LDH level out of cells reflects the cell membrane integrity. Inter-estingly, GO exposure does not induce, but restrains LDH leakage

(Fig. 6). The LDH levels of GO-treated cells are even slightly lowerthan that of the control cells (7.5%). For example, at GO concen-tration of 200 �g/mL, the LDH leakage level is around 6% for theGO-treated cells. In contrast, the size of GO samples has ignorableinfluence on the LDH leakage.

Y. Chang et al. / Toxicology Letters 200 (2011) 201–210 205

Fig. 4. The viability of A549 cells after exposed to the supernatant of GO pre-incubated medium for 24 h.

Fig. 5. The influence of GO on the mortality of A549 cells.

Fig. 6. The influence of GO on the membrane integrity of A549 cells.

3.6. Cell apoptosis

GO does not induce any apoptosis or necrosis of A549 cells(Fig. 7). The apoptosis level is not relevant to the dose or the size ofthe GO samples. At the concentration of 200 �g/mL, the apoptosisrates (1.1–2.4%) are still comparative with that of the control cells(1.5%). In contrast, the positive control cells, which treated with200 mM H2O2 for 30 min, show much serious apoptosis (32.4%) andnecrosis (54.3%).

3.7. Ultrastructure investigation

The ultra-section of A549 cells was observed under TEM for theuptake of GO and the changes of ultrastructure. All GO treated cellsshow similar structures to the control cells (Fig. 8). GO exposuredoes not have any obvious impact on the ultrastructure of A549cells. We did not find any GO sheets inside cells, either.

3.8. ROS level

The ROS generation is one commonly proposed toxicologicalmechanism of nanoparticles. The GO exposure induces oxidativestress in A549 cells even at low concentrations (Fig. 9). GO withhigher concentrations induces more ROS. Among the three GO sam-ples, s-GO causes the most serious oxidative stress. For example, at200 �g/mL, the ROS level for s-GO treated cells is 3.9 times of con-trol, while it is 2.6 for l-GO treated cells and 2.1 for m-GO treatedcells. However, for the positive control, the ROS level is 12.0 timesof control, much higher than that of GO-treated cells under the

same condition. There is no meaningful difference between the cellsexposed to l-GO and m-GO.

In the F-12K medium (cell free), GO induces the GO dose-dependent ROS generation. Higher GO dose brings on the higherlevel of ROS (Fig. 10). However, it is l-GO, not s-GO or m-GO,

206 Y. Chang et al. / Toxicology Letters 200 (2011) 201–210

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ig. 7. FACS results of the Annexin V-FITC and PI assay. (a–d) Scatter diagrams of cnd f) The summary of the apoptosis rate (e) and necrosis rate (f) of A549 cells afte

hows high ability in generating ROS. At 200 �g/mL, the fluores-ence intensity from l-GO sample is 50% higher than that from s-GOr m-GO.

.9. Cell growth on GO substrate

The cells grow very well on the GO film. The density and mor-hology of the cells cultured on GO film are comparative to thosef the cells cultured in normal culture dish (Fig. 11). The thicknessf the GO film is around several tens of micrometers. Due to the

posed to 200 �g/mL of l-GO (a), s-GO (b), m-GO (c) and the negative control (d). (esed to GO for 24 h.

dark brown color of GO film, the contrast of Fig. 11a is not as goodas that of the control.

4. Discussion

Nanomaterials have unique physicochemical properties and areapplied in various areas. However, their biological properties inorganisms will finally determine their destiny in future. Com-pared to available results of carbon based nanomaterials, such asfullerene, CNT, carbon nanofibre and carbon nanoparticle (Jia et al.,

Y. Chang et al. / Toxicology Letters 200 (2011) 201–210 207

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Fig. 9. The influence of GO on the ROS level of A549 cells.

ig. 8. TEM images of the m-GO treated A549 cells (a) and the control cells (b).

005; Lewinski et al., 2008; Lindberg et al., 2009; Liu et al., 2010;ian et al., 2006), our results indicate that GO has pretty good bio-ompatibility to A549 cells.

A systematic study was performed to evaluate the toxic-ty/biocompatibility of GO to A549 cell, a widely used model celline for the toxicity study. The results collectively indicate that GO isighly biocompatible, which is consistent with the GO drug deliverytudies (Liu et al., 2008; Lu et al., 2010; Sun et al., 2008; Yang et al.,009b; Zhang et al., 2010a). In addition to the literature reportinghe good biocompatibility of GO, there is literature reporting thatO has higher toxicity to cells and animals at high concentrations

Agarwal et al., 2010; Hu et al., 2010; Wang et al., 2010). For exam-le, Wang et al. found that GO is toxic to human fibroblast cells athe concentration of 50 �g/mL and higher. The inconsistency might

ome from the GO synthesis/film preparation, and the testing mod-ls. The good biocompatibility of GO sheets is also reflected by theell growth on GO film. Unlike Agarwal’s report (Agarwal et al.,010), we found that A549 cells grew very well on the GO film

Fig. 10. GO induced ROS generation in F-12K culture medium.

without obvious toxicity. Our study suggests that GO could be used

as the cell growth substrate.

CNT is the closest material of graphene (Geim and Novoselov,2007). The toxicity of CNTs is heavily influenced by their function-alization degree (Sayes et al., 2006). For example, carboxylation

208 Y. Chang et al. / Toxicology Lett

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ity/apoptosis of GO-treated A549 cells is the same as that of the

ig. 11. The microscopic images of cells grown on the GO film (a) and in normal cellulture dish (b).

f CNTs makes CNTs abundant in oxygen atoms, and decreasesheir toxicity. GO contains many oxygen atoms in the forms of car-oxyl groups, epoxy groups and hydroxyl groups (Dreyer et al.,010). The functionalization degree of GO is generally higherhan that of carboxylated CNTs according to the oxygen content.herefore, the good biocompatibility of GO is generally expectedo this regard. Comparing to the very recent toxicity results ofraphene (Zhang et al., 2010b), we find that GO has much loweroxicity, which is indicated by results of the viability assay andDH leakage assay. This supports the phenomenon obtained fromNTs studies, i.e. functionalization decreases the toxicity of CNTs.nother aspect might contribute to the high biocompatibility is the

wo-dimensional structure of GO. The distinct difference betweenO and CNTs is GO’s two-dimensional structure and CNTs’ one-

imension (Geim and Novoselov, 2007). Although the effect ofhape on the toxicity is still unknown in detail to date, many pre-ious results show that the shape affects the biological fate ofanomaterials (Oh et al., 2010; Simon-Deckers et al., 2009).

ers 200 (2011) 201–210

In addition, GO is not found in cells by TEM investigation. Thismight contribute to the high biocompatibility of GO, too. It is dif-ficult to investigate the monolayer GO sheet in biological samplesunder TEM. However, GO folds and aggregates when being addedinto the culture medium. The aggregates in culture medium madeGO distinguishable under TEM (Fig. S6), compared with it in purewater. Therefore, if there is any in cells, we may find it easily. Forexample, Wang et al. observed GO aggregates in human fibroblastcells (Wang et al., 2010). Based on all experimental observationsin this study, GO is hardly swallowed by A549 cells. The differencebetween our results and Wang et al.’s results might come from thedifferent sample properties and cell lines. In the drug/DNA deliverystudies, GO was found entering the cells with cargo (Liu et al., 2008;Lu et al., 2010; Sun et al., 2008). But, the size of GO they used is lessthan 100 nm, even reached 5 nm. The uptake of carbon nanomateri-als by cells is a widely observed phenomenon (Lewinski et al., 2008;Raffa et al., 2010). In particular, the negative charged fullerene andCNTs are easily swallowed by different cells (Li et al., 2008b; Wanget al., 2009a, 2009c). However, the uptake of nanomaterials is reg-ulated by their size. For example, the CNTs accumulation in cells islength-dependent (Becker et al., 2007; Jin et al., 2009; Raffa et al.,2008). CNTs with length longer than 2 �m can hardly enter cells(Raffa et al., 2008). Therefore, the size of Go might be the controlfactor of inhibiting the endocytosis of GO. The aggregation of GOin culture medium may take the consequences too (Wick et al.,2007), though the hydrodynamic diameters of three GO samplesare less than 2 �m in pure water. We propose that the shape, sizeand aggregation of GO sheets affect the uptake.

Considering that GO is not observed inside the A549 cells,GO more possibly interact with the cells on the cellular surfaceor via other pathway indirectly. The interaction on the cellularsurface may be reflected by the membrane integrity evaluation.Surprisingly, the LDH leakage levels of cells treated with high con-centration GO are lower than that of the control cells. It could hardlybe regarded that GO exposure improves the membrane integrity,but most likely the leakage tunnels are partially blocked by GOcovering. This hints that GO might partially block the substanceexchange of A549 cells. The reduced LDH leakage might be a distinctcharacter of sheet-like GO, since it is not reported in the toxicitystudy of fullerene, CNTs and other carbon nanoparticles.

As for the indirect interaction, one possibility is that GO absorbsthe nutrients in culture medium and then the depletion of nutri-ents induces the oxidative stress and toxicity to A549 cells. Suchtoxicity mechanism has been reported in the study of CNTs (Guoet al., 2008; Liu et al., 2009). Guo et al. reported that the depletionof nutrients by the absorption onto CNTs led to severe toxicity toHepG2 cells (Guo et al., 2008). The theoretical calculations have pre-dicted the absorption of amino acid and other biological moleculesonto graphene (Qin et al., 2010; Rajesh et al., 2009). We mixed GOand culture medium for 24 h, then centrifuged the mixture to pre-cipitate GO. The supernatant was used to culture cells. No toxicityto A549 cells was found (Fig. 4), compared with the cells incu-bated with the normal culture medium. Therefore, the absorptionof nutrients from the culture medium does not influence A549 cellsunder the experimental condition in this study.

Another possibility is that GO influences the cell adhesion abilityof A549 cells. However, GO shows ignorable influence on the celladhesion ability of A549 cells (Fig. S7). The unaffected adhesionability is also indicated in the GO film evaluation. Our results clearlysuggest that cells adhere to GO membrane steadily.

Although GO hardly enters A549 cells and the mortal-

control cells, GO induces statistically significant ROS generation,even at low concentration. Oxidative stress is a well recognizedtoxicological mechanism of various nanoparticles (Lewinski et al.,2008; Li et al., 2008a; Pulskamp et al., 2007; Yang et al., 2008b).

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t low dose, GO induces ROS generation, but no obvious toxicity isbserved. Similarly, oxidative stress was observed while no toxic-ty of CNTs to cells presented in Pulskamp et al.’s study (Pulskampt al., 2007). The oxidative stress may contribute to the slight via-ility decrease of GO at high concentration. The oxidative stress

nduced by GO is moderately low when comparing to fullerene andNTs (Lewinski et al., 2008). Further, ROS generate when incubat-

ng GO with the culture medium alone (cell free) and ROS level isO concentration depended. Hence, the intracellular ROS is most

ikely induced by the external ROS. To make such a mechanismlear, more efforts are required.

. Conclusions

In summary, the toxicity of GO to A549 cells was evaluated byarious cytotoxicity methods. GO hardly enters cells and showsood biocompatibility. GO has potential being the substrate for theell growth. However, GO arouses oxidative stress, and induces thelight decrease of the cell viability at high GO dose. The effect ofO on A549 cells is dose and size related. Our results are essential

or the biomedical applications and safety assessment of GO andould stimulate more toxicology evaluations of graphene and itserivatives.

onflict of interest

There are no conflicts of interest.

cknowledgements

We acknowledge financial support from the China Nat-ral Science Foundation (No. 21071094), the National Basicesearch Program of China (973 Program Nos. 2011CB933402 and009CB930200), Shanghai MEC (11ZZ82) and Shanghai Leadingcademic Disciplines (S30109).

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.toxlet.2010.11.016.

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