Reduced Graphene Oxide Nanosheet for Chemo-photothermalTherapyYeong Ah Cheon, Jun Hyuk Bae, and Bong Geun Chung*

Department of Mechanical Engineering, Sogang University, Seoul 121-742, Korea

ABSTRACT: The protein-functionalized reduced graphene oxide (rGO)nanosheet is of great interest in stimuli-responsive drug delivery and controlledrelease applications. We developed doxorubicin (DOX)-loaded bovine serumalbumin (BSA)-functionalized rGO (DOX-BSA-rGO) nanosheets. To inves-tigate the reduction of BSA-functionalized GO nanosheets and drug loadingefficiency, we used X-ray photoelectron spectroscopy (XPS) and UV−visiblespectrophotometer analysis. DOX-BSA-rGO nanosheets exhibited dose-depend-ent cellular uptake without any cytotoxic effect. We also demonstrated near-infrared (NIR)-induced chemo-photothermal therapy of brain tumor cellstreated with DOX-BSA-rGO nanosheets. Therefore, this DOX-BSA-rGOnanosheet could be a powerful tool for chemo-photothermal therapyapplications.

■ INTRODUCTION

Chemotherapy has widely been used to treat tumor cells.However, conventional chemotherapy still has various limi-tations, such as hair loss, nausea, vomiting, and heart damage.1,2

To overcome these limitations, anticancer drug-loaded func-tional nanoparticle-mediated tumor therapy has previouslybeen developed.3,4 In particular, photothermal therapy couldkill the tumor cells in a localized specific region and minimizethe damage of neighbor tissues around tumor cells.5 Near-infrared (NIR) laser irradiation deeply penetrates into thetissues without any cell damage due to its long wavelength(650−900 nm).6 Carbon- or gold-based nanomaterials, whichcould highly absorb NIR wavelength, have previously been usedfor photothermal therapy applications.7−9 Despite theirpotential, the major limitation is the localized therapy, whichcan result in incomplete ablation and tumor recurrence,because the heating is only generated within the specific regionthat was exposed to NIR laser irradiation.Graphene, which is the thickness of a carbon atom layer, has

recently emerged as a promising material for photothermaltherapy applications. Due to its excellent physical and chemicalproperties (e.g., high electrical conductivity, strength),graphene has widely been used for biosensor, drug delivery,and molecular imaging applications.10,11 Compared to conven-tional nanomaterials containing sphere and core−shellstructures,12−14 two-dimensional (2D) graphene nanosheetshave great potential for high drug loading efficiency andconjugation of proteins, drugs, and fluorescent probes.15−19

Nevertheless, the bare graphene or graphene oxide (GO) isrequired for surface modification process to improve itsbiocompatibility.20−22 Recently, protein-based reduced GO(rGO) has been developed as a drug delivery carrier,photothermal agent, and bioimaging probe.23,24 Pegylatedgraphene has previously been employed for in vivo photo-

thermal therapy. Although pegylated graphene showed highefficiency of tumor passive targeting and therapy, it required alarger amount (20 mg/kg) of samples and optimal grapheneconcentrations for improving the cell viability.8,9

Bovine serum albumin (BSA), known as a reduction agentunder alkali conditions, is one of the amphiphilic adhesivepolymers that can bind hydrophilic and hydrophobicmaterials.25,26 BSA is used as a reduction binder that can loaddrugs. External stimuli (e.g., pH) have also been reported toregulate the controlled release of drugs.27−29 Recently, carbon-based materials showing high absorption at NIR wavelengthhave been used for photothermal therapy and controlled drugrelease.30−32 In particular, graphene-based photothermaltherapy has been used to reduce tissue damage around tumorcells.5 It has been known that chemo-photothermal therapyshows higher efficiency of therapy compared to singletreatment of chemotherapy or photothermal therapy.33 There-fore, an anticancer drug-loaded biocompatible graphenenanomaterial needs to be developed for chemo-photothermaltherapy applications. In this paper, we developed DOX-loadedBSA-functionalized rGO (DOX-BSA-rGO) nanosheets forchemo-photothermal therapy of brain tumor cells.

■ MATERIALS AND METHODSMaterials. Sulfuric acid (H2SO4, 95%), hydrochloric acid (HCl,

35%), and sodium hydroxide (NaOH, 97%) were purchased fromDaejung Chemicals & Metals, Korea. Potassium permanganate(KMnO4, 99.3%), hydrogen peroxide (H2O2, 34.5%) were obtainedfrom Junsei Chemical, Tokyo, and Samchun Chemical, Korea,respectively. Graphite, BSA, DOX hydrochloride, and fluoresceinisothiocyanate (FITC)-dextran were purchased from Sigma-Aldrich,

Received: January 27, 2016Published: March 1, 2016

Article

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© 2016 American Chemical Society 2731 DOI: 10.1021/acs.langmuir.6b00315Langmuir 2016, 32, 2731−2736

USA. Cell Counting Kit-8 (CCK-8) was obtained from DojindoLaboratories, Japan.Synthesis of BSA-rGO Nanosheet. GO was made by Hummer’s

method.34 Briefly, 1 g of graphite flakes was stirred in a 500 mL flaskwith 50 mL of sulfuric acid in an ice bath, and 5 g of potassiumpermanganate was subsequently added. After stirring for 1 h, themixture was warmed to 35−45 °C and was stirred for an additional 2h. Fifty milliliters of distilled water was slowly dropped into themixture solution, and the solution was then diluted by addingadditional 150 mL of distilled water under vigorous stirring. Toterminate the reaction, 10 mL of hydrogen peroxide was added andwas subsequently stirred for 15 min. The solution was washed severaltimes with 5% HCl and distilled water to remove the residual acid.Nanosized GO was obtained by further oxidation and freeze-drying.Prior to reduction of GO, graphite oxide was exfoliated to obtain GOfrom sonication (20 kHz, 500 W) in distilled water for 2 h. Onemilligram per milliliter GO was diluted in distilled water and 50 mg/mL BSA solution was subsequently added in a 70 °C water bath. Thesolution was adjusted to pH 12 rapidly with 1 M sodium hydroxide.After stabilization, the mixture was centrifuged at 15 000 rpm toeliminate the nonreactive BSA and NaOH. The precipitates wereredispersed in distilled water and were rinsed three times.Characterization of DOX-BSA-rGO Nanosheet. The morphol-

ogies of GO, BSA-rGO, and DOX-BSA-rGO nanosheets wereobserved by transmission electron microscopy (TEM, JEOL,JEM1010, Japan) with an accelerating voltage of 80 kV. XPS analysiswas performed on K-alpha (Thermo VG, UK) and T64000 (Horiba,Ar laser at 514 nm). The characteristic peak and surface charge wasanalyzed by a UV−visible spectrophotometer and Zetasizer. To studythe photothermal effect, the temperature of samples during NIRirradiation was measured at every 60 s using a digital thermometer.DOX Loading and Release from DOX-BSA-rGO Nanosheets.

The DOX-BSA-rGO nanosheet was prepared by mixing with 0.1 mg/mL of BSA-rGO. The solution was washed with phosphate bufferedsaline (PBS) to remove unbound DOX on BSA-rGO nanosheets andits precipitates were subsequently collected. A DOX release experi-ment was performed in a shaking incubator at 37 °C. For the NIR-mediated DOX release experiment, the DOX-BSA-rGO nanosheet wasdispersed in PBS (pH 5) and was subsequently irradiated by NIR (808nm, 5.5 W/cm2) light for 5 min. Supernatant was measured at 490 nm,which is the characteristic absorption peak of DOX by UV−visiblespectrophotometer. The concentration of DOX released from DOX-BSA-rGO nanosheets was quantified using a calibration curve. Wemade the calibration curve with DOX concentrations and 1 mg/mLDOX loaded into 0.1 mg/mL BSA-rGO nanosheets for 12 h. Theinitial concentrations of DOX were measured by a UV−visiblespectrophotometer to find drug loading efficiency.Cell Culture. U87MG brain tumor cells (1.6 × 104 cells/well) were

cultured in a 96-well plate with Minimum Essential Medium (MEM,Gibco, USA) containing 10% fetal bovine serum (FBS). The cells weresplit as they were 85% confluent in a well-plate. The culture mediumwas exchanged every 2 days.Cellular Uptake. A One milligram per milliliter DOX-BSA-rGO

nanosheet and 2 mg/mL FITC-dextran (10 000 Da) were dissolved indistilled water. To label FITC-dextran in the DOX-BSA-rGOnanosheet, DOX-BSA-rGO and FITC-dextran solution were stirredovernight. After overnight, the mixture was centrifuged at 14 000 rpmand was washed with PBS three times to remove unbound FITC-dextran. For cellular uptake, U87MG cells (1.6 × 103 cells/mL) wereseeded on a coverslip and were cultured for 24 h. After culturing for 24h, cells were treated with FITC-dextran-labeled DOX-BSA-rGOnanosheets at various concentrations (30−100 μg/mL) for 3 h.Cells were fixed with 4% paraformaldehyde for 15 min at roomtemperature and were stained by using Hank’s Balanced Salt Solution(HBSS) containing Alexa fluor 594-conjugated wheat germ agglutinin(Invitrogen, CA, USA) for 30 min. The cells were then permeabilizedwith 1% triton X-100 in PBS for 20 min at room temperature and weresubsequently counterstained with DAPI (Invitrogen, CA, USA) for 5min. The cellular uptake was measured by a confocal laser-scanningmicroscope (LSM 710, Carl Zeiss, Germany)

Cellular toxicity of BSA-rGO−DOX nanosheet. U87MG cellswere cultured in a 96-well plate for 24 h. After incubation for 24 h,DOX-BSA-rGO nanosheets were added to a 96-well plate. The cellswere then incubated for 6, 12, and 24 h. Ten microliter CCK-8solutions were added to the cells for 3 h, and cell viability was analyzedusing a microplate reader at 450 nm wavelength.

Cell Viability Assay. The viability of the U87MG cells culturedwith 30 μg/mL BSA-rGO and DOX-BSA-rGO nanosheets wasevaluated using a live/dead assay kit (Invitrogen, CA, USA). Toinvestigate NIR irradiation-mediated viability, the cells were incubatedfor 4 h and were subsequently irradiated by a NIR light for 3 min at apower of 5.5 W/cm2. Cells were incubated for 40 min at roomtemperature with live/dead assay solution containing 2 μM calcein AM(green) and 4 μM ethidium homodimer-1 (red). Fluorescent imageswere obtained from a confocal laser-scanning microscope. Quantitativeanalysis of cell viability was determined by CCK-8 assay.

Statistical Analysis. We performed at least three differentexperiments, and the data indicated the mean ± standard deviation.**p < 0.01 was considered to be statistically significant.

■ RESULTS AND DISCUSSIONSynthesis of DOX-BSA-rGO Nanosheets. We synthe-

sized DOX-BSA-rGO nanosheets (Figure 1). Briefly, the

graphite oxide was exfoliated to generate GO. BSA proteinswere conjugated with GO at 60 °C and pH 12 to make BSA-rGO nanosheets. Finally, DOX was bound to BSA proteins onrGO nanosheets. We used a BSA protein to make the rGOnanosheet, because BSA is a natural reduction agent. Theamphiphilic BSA protein enabled the decrease of GO-mediatedcytotoxicity and allowed for high loading efficiency ofanticancer drugs. We synthesized GO, BSA-rGO, and DOX-BSA-rGO nanosheets (Figure 2A−C). We used XPS analysis toconfirm the reduction and BSA functionality of GO (Figure2D−F). It revealed that the C−O peak of the BSA-rGOnanosheet was largely reduced and the C−N peak was newlygenerated. It also showed that the CO peak intensity of theBSA-rGO nanosheets was increased, because BSA proteinscontained amide carbon (HN-CO), suggesting that BSAproteins conjugated into GO nanosheets to form BSA-rGOnanosheets. XPS analysis of DOX-BSA-rGO nanosheets

Figure 1. Schematic illustration of the synthesis of DOX-BSA-rGOnanosheets and NIR-mediated chemo-photothermal therapy. Thegraphite oxide was exfoliated to make graphene oxide, and BSA wasused for reduction of graphene oxide at pH 12 and 60 °C. Doxorubicinwas finally conjugated with BSA-rGO nanosheets.

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represented that the total ratio was increased due to the C−Cand C−N peaks of DOX. To determine whether GOnanosheets were reduced and functionalized, the zeta potentialand UV−visible spectrophotometer analysis were employed(Figure 3). We observed the effect of pH on surface charge ofBSA protein, GO, and BSA-rGO nanosheet (Figure 3A).Interestingly, the surface charge of the BSA protein wassignificantly regulated by pH, indicating that the zeta potentialof BSA-rGO nanosheets was 28.9 mV at pH 2 and −40.3 mV atpH 7. This amphiphilic property of BSA proteins coulddetermine BSA functionality. The surface charges of BSAprotein and BSA-rGO nanosheets were similar, because BSA

proteins conjugated with rGO nanosheets. By contrast, thesurface charge of GO nanosheets was negative regardless of pH,because oxygen functional groups existed at the edge and basalplane. For the UV−visible spectrophotometer, we observed thepeak of GO nanosheets at 230 nm (marked in a dotted-line ofFigure 3B) and 300 nm due to π → π* transition from thearomatic CC bond and n → π* transition from the CObond (Figure 3B), as previously described.23 We also observedthat the sp2 bond-mediated peak was shifted at 266 nm(marked as a dotted-line in Figure 3B) in the BSA-rGOnanosheet, and the peak at 300 nm was diminished due todeoxygenation. The color of solution was changed from

Figure 2. TEM and XPS analysis. (A−C) TEM images of GO, BSA-rGO, and DOX-BSA-rGO nanosheets. (D−F) C 1s XPS spectra of GO, BSA-rGO, and DOX-BSA-rGO nanosheets.

Figure 3. Zeta potential and UV−visible spectrophotometer analysis. (A) Zeta potential analysis of GO, BSA, and BSA-rGO. (B) UV−visiblespectrophotometer analysis of GO, BSA, BSA-rGO, and DOX-BSA-rGO nanosheets. 230, 266, and 500 nm wavelengths are indicated by blackdotted-lines.

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yellowish brown to black after the BSA-mediated reductionprocess. Furthermore, we observed the peak of DOX-BSA-rGOnanosheets at 490−510 nm due to DOX absorption.Photothermal Effect. We observed that UV absorption of

BSA-rGO or DOX-BSA-rGO nanosheets was increased at 800nm (Figure 3B), because the π-network structure was restoredto increase UV absorption. It suggested that BSA-rGO orDOX-BSA-rGO nanosheets could show photothermal effectduring NIR radiation at 808 nm as previously described.35 Toinvestigate whether BSA-rGO nanosheets increased thetemperature caused by energy transfer from π-networkrestoration-mediated light absorption to heat, we applied NIRlaser (5.5 W/cm2, 808 nm) to BSA-rGO nanosheets for 300 sand analyzed the NIR-mediated temperature every 60 s. Weobserved that the temperature was proportional to concen-trations of BSA-rGO nanosheets (Figure 4A). We alsoinvestigated the effect of NIR irradiation time on temperatureof BSA-rGO nanosheets (Figure 4B). It represented that thetemperature of BSA-rGO and DOX-BSA-rGO nanosheets wasincreased with irradiation time, suggesting that a BSA-rGO orDOX-BSA-rGO nanosheet would be a great candidate as aphotothermal agent. By contrast, the temperature change ofGO nanosheets was negligible. Although DOX-rGO nano-sheets were slightly aggregated with increasing temperature,they were stable.NIR-Mediated DOX Release and Cell Viability. We

loaded DOX into amphiphilic BSA-rGO nanosheets. Theloading efficiency of DOX was approximately 77%. Weanalyzed DOX release behavior from DOX-BSA-rGO nano-sheets in a temporal manner (Figure 5). It showed that DOX atpH 5 (23% release at 6 h) was largely released from DOX-BSA-rGO nanosheets as compared to pH 7 (7% release at 6 h),because DOX was protonated to become hydrophilic at anacidic environment.36 The interaction between DOX and BSAprotein enabled the control of DOX release from DOX-BSA-rGO nanosheets as previously described.37 We also investigatedthe effect of NIR irradiation on DOX release, showing thatDOX was largely released from DOX-BSA-rGO nanosheetswhen NIR irradiation was applied (marked by black arrows ofFigure 5). The heat caused from NIR irradiation broke thebinding between DOX and BSA protein, resulting in a largerelease of DOX (50% at 6 h). This result demonstrated thatNIR irradiation approximately twice enhanced DOX release

from DOX-BSA-rGO nanosheets. We also investigated thecellular uptake using FITC-loaded DOX-BSA-rGO nanosheets(Figure 6A−C). The confocal microscopy images representedthat the cellular uptake was significantly affected byconcentrations of FITC-loaded DOX-BSA-rGO nanosheets,indicating that U87MG cells treated with 100 μg/mL FITC-loaded DOX-BSA-rGO nanosheets showed higher uptakeefficiency. Furthermore, we observed the effect of DOX-BSA-rGO nanosheets on chemotherapy (Figure 6D). CCK-8 cellviability assay demonstrated that the viability of U87MG cellstreated with DOX-BSA-rGO nanosheets (5−100 μg/mLconcentration) was decreased with incubation time regardlessof DOX concentrations. This was probably due to DOXreleased from DOX-BSA-rGO nanosheets within cell mem-brane.

Chemo-photothermal Therapy of DOX-BSA-rGONanosheet. We investigated the effect of DOX-BSA-rGOnanosheets on NIR-mediated chemo-photothermal therapy(Figure 7). U87MG cells were treated with 30 μg/mL BSA-

Figure 4. Analysis of temperature with respect to NIR irradiation time. (A) Analysis of temperature after exposure of NIR irradiation (808 nm, 5.5W/cm2) as a function of BSA-rGO concentration. (B) Temperature analysis of PBS, GO, BSA-rGO, and 30 μg/mL DOX-BSA-rGO.

Figure 5. DOX release analysis at pH 5−7 with NIR irradiation (5.5W/cm2, 5 min). Black arrows indicate that NIR irradiation is applied.DOX was largely released from BSA-rGO nanosheets at pH 5. NIRirradiation significantly increased DOX release from BSA-rGOnanosheets.

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rGO nanosheets and were subsequently exposed to NIRirradiation (5.5 W/cm2, 808 nm) every 3 min after 4 hincubation. The confocal microscopy images showed that NIRirradiation did not have any damage to cells (Figure 7A,D). Bycontrast, most cells treated with both BSA-rGO nanosheet andNIR irradiation were dead compared to neighboring cellstreated without NIR irradiation, showing that the BSA-rGOnanosheet was a nontoxic material (Figure 7B,E). Interestingly,we observed that U87MG cells treated with both 30 μg/mLDOX-BSA-rGO nanosheets and NIR irradiation were largelydead (Figure 7C,F). This was probably due to chemo-photothermal therapy, showing that NIR irradiation stimulateda large release of DOX from DOX-BSA-rGO nanosheets. CCK-8 cell viability analysis demonstrated that the viability of thecells treated with both DOX-BSA-rGO nanosheets and NIRirradiation was extremely low compared to the viability of thecells without any treatment (Figure 7G). This was probably dueto NIR irradiation that could allow for DOX release toneighboring cells. We also demonstrated that the viability(21.8%) of brain tumors treated with BSA-rGO nanosheets wasdecreased by NIR irradiation. By contrast, the brain tumorstreated with both DOX-BSA-rGO nanosheets and NIRirradiation showed the lowest cell viability (1.76%) due tochemo-photothermal therapy effect.

■ CONCLUSIONSWe synthesized functional DOX-BSA-rGO nanosheets. A NIR-mediated drug release study showed that DOX was largelyreleased from DOX-BSA-rGO nanosheets when NIR irradi-ation was applied. The heat caused from NIR irradiation brokethe binding between DOX and BSA protein, resulting in largerelease of DOX. This result demonstrated that NIR irradiation

approximately twice enhanced DOX release from DOX-BSA-rGO nanosheets. We also demonstrated that the viability ofbrain tumor cells treated with both DOX-BSA-rGO nanosheetand NIR irradiation was extremely low due to chemo-photothermal therapy effect. Therefore, this DOX-loadedBSA-rGO nanosheet could be a powerful tool for chemo-photothermal therapy applications.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: bchung@sogang.ac.kr.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis research was supported by the Research Program throughthe National Research Foundation (NRF) funded by theMinistry of Science, ICT & Future Planning (MSIP) of Korea(Grant Number 2015R1A2A1A15054236). This work wassupported by the BioNano Health-Guard Research Centerfunded by the MSIP of Korea as the Global Frontier Project(Grant number H-GUARD_2014M3A6B2060503), Republicof Korea. This research was also supported by a grant from theMarine Biotechnology Program (Grant Number 20150581,

Figure 6. Cellular uptake and viability analysis. Confocal fluorescentimages of U87MG cells with medium only (A) and after 4 hincubation with (B) 30 μg/mL and (C) 100 μg/mL of DOX-BSA-rGO nanosheets. FITC-dextran-labeled DOX-BSA-rGO nanosheetsand cell membranes are stained by green and red color, respectively.Cell nuclei are counterstained by DAPI. Scale bars are 50 μm. (D) Theviability analysis of U87MG cells with various concentrations (5−100μg/mL) of DOX-BSA-rGO nanosheets for 36 h. Figure 7. Chemo-photothermal therapy. Fluorescent images of cell

viability of U87MG cells with or without NIR irradiation (5.5 W/cm2,3 min) with 30 μg/mL BSA-rGO and DOX-BSA-rGO nanosheetsafter incubation for 6 h. Non-NIR irradiation groups: (A) normalculture medium, (B) BSA-rGO, and (C) DOX-BSA-rGO nanosheets.NIR irradiation groups: (D) normal medium, (E) BSA-rGO, and (F)DOX-BSA-rGO nanosheets. The live and dead cells are indicated withcalcein AM (green) and ethidium homodimer (red). Scale bars are 100μm. (G) Quantitative analysis of cell viability using CCK-8 assay.

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Development of technology for biohydrogen production usinghyperthermophilic archaea), funded by the Ministry of Oceansand Fisheries, Korea.

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