Journal of Photochemistry and Photobiology B: Biology 138 (2014) 191–201

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Redox-responsive biodegradable PEGylated nanographene oxide for efficiently chemo-photothermal therapy: A comparative study with non-biodegradable PEGylated nanographene oxide Honglian Xiong, Zhouyi Guo, Wen Zhang, Huiqing Zhong, Songhao Liu, Yanhong Ji ⇑ MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China

a r t i c l e

i n f o

Article history: Received 17 February 2014 Received in revised form 28 May 2014 Accepted 31 May 2014 Available online 13 June 2014 Keywords: Graphene oxide Sheddable poly(ethylene glycol) Glutathione (GSH) Disulfide bonds Chemo-photothermal therapy

a b s t r a c t Nanographene oxide (NGO) with a non-sheddable poly(ethylene glycol) (PEG) coating has been used for chemo-photothermal therapy. However, the drug release of PEGylated NGO (NGO-PEG) with an amine bond is adversely affected by the diffusion barrier effect of PEG shells. Here, we developed a simple new method for the preparation of biodegradable PEGylated NGO conjugates (NGO-SS-PEG) with cleavable disulfide bonds for rapid drug release and more efficiently chemo-photothermal therapy. The glutathione (GSH)-induced and photothermal-mediated intracellular release of doxorubicin (DOX) from NGO-SS-PEG was studied in A549 cells using confocal laser scanning microscopy and flow cytometry analysis. In vivo cytotoxicity experiments were performed on chemo-photothermal therapy. Furthermore, we presented a comparative study of intracellular drug release and biological efficacy between NGO-SS-PEG/DOX and NGO-PEG/DOX. The results demonstrated that the rapid drug release from the NGO-SS-PEG conjugates with sheddable PEG was triggered upon the stimulus of high GSH levels inside A549 cells. Interesting, the DOX release mediated by the photothermal effect from the NGO-SS-PEG conjugates was found to be more obvious than that for NGO-PEG. Additionally, NGO-SS-PEG showed a higher efficacy than NGO-PEG for anti-tumor therapy compared with NGO-PEG. Thus, NGO-SS-PEG can improve therapeutic efficacy and is an attractive drug nanocarrier. Ó 2014 Published by Elsevier B.V.

1. Introduction Chemo-photothermal therapy, the combination of chemotherapy and photothermal therapy, has been proven to be an effective strategy to improve the efficacy of cancer therapy [1–3]. A wide variety of nanoparticles, including graphene oxide (GO) [4,5], cabon nanotubes [6] and gold nanorods [7], have been employed to deliver both heat and drugs simultaneously to the selected tumor sites, due to their strong absorbance in the near-infrared (NIR) region and targeted delivery. However, nanoparticles with a sheddable coating for more efficiently chemo-photothermal therapy are unexplored. Nanographene oxide (NGO), a two-dimensional material featuring a variety of reactive oxygen functional groups such as epoxy, hydroxyl groups on the basal plane and carboxylic acid groups at the sheet edges [8–10], has attracted tremendous attention in drug delivery [11–13] and NIR photothermal therapy [10,14]. Dai et al. have fabricated poly(ethylene glycol) (PEG) functionalized NGO ⇑ Corresponding author. Tel.: +86 20 85211436 8605. E-mail address: [email protected] (Y. Ji). http://dx.doi.org/10.1016/j.jphotobiol.2014.05.023 1011-1344/Ó 2014 Published by Elsevier B.V.

(NGO-PEG) for cancer drug delivery [15], and Liu et al. have reported the influence of the surface chemistry and the size of PEGylated NGO on photothermal therapy [10]. To further improve the efficacy of cancer therapy and reduce side effects, the co-delivery of a drug and heat to tumor sites using NGO has been developed. Recently, we exhibited the synergistic effect of PEGylated NGO for combined chemotherapy and photothermal therapy [5]. Immediately following this work, we developed targeted chemo-photothermal therapy based on folic-acidfunctionalized NGO [16]. The above work also demonstrated that it is critical for NGO functionalized with hydrophilic coatings to improve its stability and solubility in physiology environments for efficiently biomedical application, because NGO is prone to aggregate in the physiological environments due to the screening of electrostatic charges and nonspecific binding with protein [15,17]. Despite the significant advantages of PEGylated NGO over NGO, the PEG coating adversely inhibits the rapid drug release because of its diffusion barrier [18,19], which has hampered its biological application. To accelerate drug release from conjugates, sheddable shell functionalized micelles [20,21] and nanoparticles [22] have

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been developed by the formation of redox-responsive cleavable disulfide linkages. As is well known, there is a large glutathione (GSH) concentration gradient between the cell exterior (2 lM) and interior (2–10 mM) [22]. In particular, the GSH level in certain tumors is at least four times higher than that of normal cells [21,23]. When nanoparticles with a disulfide-linked shell enter cancer cells, intracellular GSH can cleave disulfide bonds by a redox reaction [24], resulting in rapid drug release from the conjugates, which could maintain a stable concentration for a long time. For effective therapy, rapid intracellular drug release is highly preferred upon immediate arrival in tumor site of the delivery system [25]. Thus, this sharp contrast in the GSH concentration may serve as an ideal stimulus for triggering intracellular drug release [10,26]. So far, functional NGO conjugates with sheddable shells have only been reported by Wen et al. [27] and Cho and Choi [24]. The nanocarriers delivered only drug to the tumor sites without any heat. However, studies have not yet been performed on graphene oxide nanosheets with redox-responsive sheddable shells for chemo-photothermal therapy and on the influence of the photothermal effect on the detachment of PEG shells. Here, we utilized a simple new method for the preparation of PEG modified NGO by cleavable disulfide linkages (NGO-SS-PEG). The process involved exfoliation by sonication, the carboxylation of NGO and the conjugation of PEG-SH to NGO-SH by disulfide bonds. Additionally, we investigated the influence of the photothermal effect on the detachment of PEG shells and the ability of NGO-SS-PEG to improve the efficacy of chemo-photothermal therapy. Doxorubicin (DOX) release from NGO-SS-PEG was significantly faster than that from NGO-PEG upon the stimulus of high GSH levels inside A549 cells. According to a previous report, intracellular GSH levels in human lung adenocarcinoma cell line (A549) used in our studies is seven times higher than that in a normal human lung fibroblast line (CCL-210) [28]. Additionally, a more rapid release of DOX was observed because the photothermal effect mediated an increase in the cleavage of disulfide bonds. The obtained NGO-SS-PEG/DOX could deliver both heat and drugs to the tumor sites and showed greater efficacy against tumor cells compared to that of NGO-PEG. It is anticipated that NGO with detachable PEG coatings has the potential for the efficient chemo-photothermal ablation of tumor cells. 2. Materials and methods 2.1. Materials Graphite flakes were purchased from Shanghai Yifan Graphite Co., Ltd. Methoxypoly(ethylene glycol)-thiol (mPEG-SH, Mw = 5000) was purchased from Laysan Bio Inc (America). Gibco Invitrogen Corp. supplied 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Chloroacetic acid (ClCH2COOH), Methoxypoly(ethylene glycol)-amine (mPEG-NH2, Mw = 5000), sodium hydrogensulfide (NaSH) and doxorubicin hydrochloride (DOX) were obtained from Sigma–Aldrich. All aqueous solutions were prepared with Milli-Q water. The dialysis bags (MWCO = 100,000) were purchased from Spectrum Laboratories Inc. 2.2. Synthesis of NGO–COOH Graphene oxide (NGO) was prepared from native graphite flakes according to the modified Hummer’s method [29,30]. NGO was obtained by the sonication of GO (400 W, 3 h). For NGO carboxylation, NaOH (1.2 g) and ClCH2COOH (1.0 g) were added to an NGO aqueous suspension (10 mL, 2 mg /mL), followed by bath sonication (150 W, 3 h) to convert OH groups into COOH groups. The NGO–COOH solutions were neutralized with diluted hydrochloric

acid and purified by repetitive rinsing and filtrations, producing a well-dispersed NGO–COOH aqueous solution. 2.3. Synthesis of NGO-SS-PEG and NGO-PEG The preparation of NGO-SS-PEG was carried out by the formation of disulfide bonds between mPEG-SH and NGO-SH using air oxidation [31,32]. Briefly, mPEG-SH (50 mg) in an aqueous solution was first added to the NGO–COOH solutions (20 mL, 0.5 mg/mL), followed by 10 min of sonication. NaSH (200 mg) in an aqueous solution was then added gradually under vigorous stirring, and the resulting mixture was then sonicated at 40 °C for 1 h. Subsequently, the mixture was stirred for 10 h at 55 °C to produce thiol groups on the NGO–COOH surfaces by reaction with NaSH according to previous reports [33,34] and to conjugate mPEG-SH to NGO-SH by a disulfide linkage using air as a oxidizing agent. Then, another 50 mg of PEG-SH was added under vigorous stirring for an additional 5 h of reaction at 55 °C. This solution was stirred for 24 h at room temperature for a more complete reaction. The final product (NGO-SS-PEG) was obtained by repeated washing and dialysis against deionized water for 48 h to remove unreacted chemicals and impurities and then was kept at 4 °C in the dark. For NGO-PEG preparation, EDC (10 mg) and NHS (10 mg) were added to the NGO–COOH suspension (10 mL, 1 mg/mL) at pH 5.6, and the mixture was sonicated for 5 min. mPEG-NH2 (100 mg) was then added to the above suspension and stirred for 24 h at room temperature. The final product (NGO-PEG) was obtained by washing with deionized water several times and dialysis for 48 h. 2.4. Characterization The morphology and the size of NGO-PEG and NGO-SS-PEG were observed using an atomic force microscope (AFM) (Multimode Nanoscope V, Veeco Instruments, USA). The hydrodynamic diameters and polydispersity index (PDI) were obtained by dynamic light scattering (DLS) using a Malvern Nano-zs90 instrument. Raman spectra of the samples were measured by a confocal Raman spectroscope (Renishaw InVia, Derbyshire, UK) with a 785 nm laser source. The absorption spectra were characterized by an ultraviolet–visible (UV–Vis) spectrometer (UV-1700, Shimadzu, Japan). Fourier transform infrared (FTIR) spectra were recorded on a Nicolet 6700 FTIR spectrometer. 2.5. Stability and GSH-induced destabilization of NGO-PEG and NGOSS-PEG in physiological solutions To evaluate their stability, NGO–COOH, NGO-PEG and NGO-SSPEG were separately dispersed in phosphate-buffered saline (PBS) or Dulbecco’s modified Eagle’s medium (DMEM). After 24 h, photos were recorded by a Canon camera. The stability of NGO-PEG and NGO-SS-PEG in PBS (pH 7.4) was obtained by DLS measurement. To investigate the redox-responsive PEG detachment, the stability of NGO-SS-PEG or NGO-PEG in PBS containing 10 mM GSH (pH 7.4) was also observed. Briefly, GSH (the final concentration: 10 mM) was add to 0.2 mg/mL of NGO-SS-PEG/DOX or NGO-PEG/ DOX in PBS (pH 7.4). The solution was then placed in a shaking bed at 37 °C with a rotation speed of 100 rpm for 4 h. Photos were recorded. 2.6. Laser irradiation and temperature measurements For temperature measurements, NGO-PEG and NGO-SS-PEG were both diluted to10 mg/L with PBS containing 5% fetal calf serum (FCS; Sigma–Aldrich). The suspensions (500 lL) in 15 mL conical-bottom centrifuge glass tubes were exposed to an 808 nm continuous-wave NIR laser (2 W/cm2, 1–5 min). The

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temperature was measured by a thermocouple immersed into the suspensions. 2.7. DOX loading and GSH-induced DOX release in vitro from NGO-SSPEG or NGO-PEG DOX loading was performed by simply mixing 0.4 mg/mL DOX with 0.2 mg/mL NGO-SS-PEG or NGO-PEG overnight at pH 8.0 at room temperature in the dark. Unbound DOX was removed by repeated washing and ultrafiltration through Amicon 100 kDa centrifugal filters (Millipore). The resultant NGO-SS-PEG/DOX or NGO-PEG/DOX composites were re-suspended and stored at 4 °C in the dark. The amount of DOX loaded onto NGO-SS-PEG or NGO-PEG was measured by the absorbance peak at 490 nm after subtracting the absorbance of NGO-PEG or NGO-PEG at that wavelength with a molar extinction coefficient of 1.05  104 mol/(L cm). The experiment was repeated three times. The DOX release was investigated by UV–Vis spectroscopy. NGOSS-PEG/DOX and NGO-PEG/DOX solutions were separately placed into dialysis bags, which were immersed into four mediums: 50 mL PBS containing 0 or 10 mM GSH at pH 7.4 (physiological pH value) and PBS with 0 mM and 10 mM GSH at pH 5.5 (tumor pH value), respectively. The GSH concentrations simulated intracellular (0 mM) and extracellular (10 mM) GSH concentrations, respectively. Subsequently, the release reservoir was placed in a shaking bed at 37 °C at a speed of 100 rpm. The drug release was assumed to start as soon as the dialysis bags were placed into the reservoir. At regular time intervals, samples (1 mL) were taken out for measurement and replaced with an equal volume of fresh medium. The amount of released DOX was quantified using UV–Vis spectroscopy. To measure the DOX release mediated by the photothermal effect, NGO-SS-PEG/DOX and NGO-SS-PEG (50 mg/L, in terms of the NGO concentration) were separately irradiated by a NIR laser (2 W/cm2 for 3 min) before being placing in PBS containing 10 mM GSH at pH 5.5. The release reservoir was kept under constant stirring, and at various time points, samples was taken out for characterization. The concentration of released DOX was quantified using UV–Vis spectroscopy.

35 mm glass-bottom dishes for 24 h of incubation. The cells were then treated with 20 mg/L of NGO-PEG or NGO-SS-PEG for 4 h. Subsequently, the culture medium was removed; the cells were rinsed twice again with PBS and imaged by CLSM. For the flow cytometry analysis (fluorescence-activated cell sorting, FACS), A549 cells were grown in 6-well plates (1.0  105 cells per well) and incubated for 24 h. Then, the culture medium was replaced with a fresh one containing 20 mg/ L NGO-SS-PEG or NGO-PEG. After 4 h of incubation, the cells were washed twice with cold PBS, fixed with 2% paraformaldehyde, and then transferred to FACS tubes for measurement using a flow cytometer. 2.10. Redox-dependent intracellular drug release Redox-dependent intracellular drug release was studied by CLSM (488 nm excitation) and FACS (488 nm excitation). For CLSM, A549 cells were plated in 35 mm glass-bottom culture dishes for 24 h of incubation. The cells were then treated with NGO-PEG/ DOX or NGO-SS-PEG/DOX for 4 h (the DOX concentration: 10 mg/ L). Subsequently, the culture medium was removed; the cells were rinsed twice again with PBS and imaged by CLSM. For measuring intracellular DOX release mediated by the photothermal effect, A549 cells were plated in 35 mm glass-bottom culture dishes for 24 h of incubation. The cells were then treated with NGO-PEG/DOX or NGO-SS-PEG/DOX for 3 h (the DOX concentration: 10 mg/L). Then, the culture medium was replaced with 0.5 mL fresh medium, and the cells were either irradiated or not irradiated by an 808 nm continuous-wave NIR laser with the power density of 2 W/cm2 for 5 min, followed by an additional 2 h of incubation. Subsequently, the culture medium was removed; the cells were rinsed twice again with PBS and imaged by CLSM. For FACS, A549 cells were grown in 6-well plates (1.0  105 cells per well) and were incubated for 24 h. Then, the culture medium was replaced with a fresh medium containing NGO-SS-PEG/DOX or NGO-PEG/DOX (the DOX concentration: 10 mg/L). After 4 h of incubation at 37 °C, the cells were washed twice with cold PBS, fixed with 2% paraformaldehyde, and then transferred to FACS tubes for measurement using a flow cytometer.

2.8. Cell culture and cell viability measurement

3. Results and discussion

A549 cells were cultured in DMEM supplemented with 10% fetal bovine serum, penicillin (100 units/mL), streptomycin (100 mg/mL) in 5% CO2 and 95% air at 37 °C in a humidified incubator. Cell viability was studied by a MTT assay. A549 cells were plated in the 96-well plates (1  104 per well) with five replicate wells for each sample and incubated for 24 h prior to treatment. Each experiment was repeated five times. Free DOX, NGO-SS-PEG, and NGO-SS-PEG/DOX were added to the cells with various test concentrations (DOX concentrations: 2, 10 and 20 mg/L) in the culture medium. Cells cultured in the medium without drugs were taken as the control. After 12 h of incubation, the cells were either exposed or not exposed to 808 nm NIR irradiation with a power density of 2 W/cm2 for 3 min and incubated for a further 12 h. Cell viability was measured using a MTT assay. To determine the cell toxicity of the NGO nanoparticles, A549 cells were exposed to NGO-PEG or NGO-SS-PEG with various concentrations (10, 20, 30, 40 and 50 mg/L). Cell viability was observed after 24 h of incubation.

3.1. Synthesis and Characterization of NGO-PEG and NGO-SS-PEG

2.9. Cellular uptake and internalization The cellular uptake was studied using a confocal laser scanning microscope (488 nm excitation) (Leica TCS SP5 II, Germany) and a flow cytometer (488 nm excitation, BD FACSCantoII). For confocal laser scanning microscopy (CLSM), A549 cells were plated in

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Fig. 1 shows the synthetic route for the preparation of NGO-SSPEG conjugates. The process involved exfoliation by sonication, the carboxylation of NGO and the conjugation of PEG-SH to NGO-SH by disulfide bonds. The synthesis of NGO-PEG was as described in our previous paper [5]. AFM was used to characterize the morphology of NGO-PEG and NGO-SS-PEG. As shown in Fig. 2a, the lateral size of the NGO-PEG and NGO-SS-PEG was less than 200 nm. The height of NGO-PEG or NGO-SS-PEG was approximately 2.5 nm, mainly due to the attachment of the PEG coating onto both planes of the NGO sheets. A thickness of 1–2 nm for NGO is the characteristic of a single or two layered sheet, according to previous work [35–37]. The results displayed that both NGO-SS-PEG and NGO-PEG consisted of a single- or two-layered structure and the aqueous solutions were well dispersed. Fig. 2c reveals the size distribution curves of NGO-PEG and NGO-SS-PEG obtained from DLS measurements. The corresponding PDI are 0.144 and 0.172, respectively. Fig. 2b shows the FTIR spectra of NGO, NGO-PEG, NGO-SS-PEG, and GSH-reduced NGO-SS-PEG. The FTIR spectrum of NGO exhibited a strong OAH vibration at (3405 cm 1). The spectrum of NGO also indicated the existence of CAH (2900 cm 1), C@C (1622 cm 1), and C@O (1729 cm 1) [38]. As shown in the spectrum of NGOPEG, the appearance of the strong CAH vibrations (2900 cm 1)

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Fig. 1. Schematic illustration for the preparation of NGO-SS-PEG conjugates. 1

and CAOAC bonds (1110 cm ) on the PEG chains and a characteristic COANH vibration (1650 cm 1) suggested the successful conjugation of mPEG-NH2 to NGO by the formation of amide carbonyl bonds (COANH) [30]. The spectrum of NGO-SS-PEG demonstrated that the strong CAH vibrations and the CAOAC bonds on PEG chains emerged, whereas the COANH vibration was not present in the NGO-SS-PEG group. These results revealed that PEG-SH was successfully conjugated to NGO through the formation of disulfide linkages. However, it was not possible to observe the disulfide linkage in the FTIR spectra due to a weak infrared response. To further confirm the successful PEG-SH conjugation onto NGO, NGO-SS-PEG was dispersed in an aqueous solution containing 10 mM GSH, which was placed in a shaking bed at 37 °C at a speed of 100 rpm for 4 h to produce GSH-reduced NGO-SS-PEG. From the spectrum of GSH-reduced NGO-SS-PEG, the peaks from PEG disappeared and a broad peak at 3447 cm 1 was observed, indicating PEG detachment and NGO aggregation induced by the reductive cleavage of disulfide bonds. As seen in the UV–Vis spectrum (Fig. 2d), the absorption peak of NGO was 232 nm [29]. The characteristic absorption peak of NGOSS-PEG and NGO-PEG was observed at approximately 232 nm. However, NGO-SS-PEG and NGO-PEG both have a higher optical absorption in Vis–NIR region than NGO at the same concentration [29]. The inset showed that NGO-PEG was light brown; NGO-SSPEG showed a darker brownish colour than NGO-PEG (Fig. 2d). Raman spectroscopy was used to investigate the structural properties of the nanomaterials (Fig. 2e). The Raman spectrum of NGO displayed a broad graphite lattice (G band) at 1603 cm 1and a disorder-induced band (D band) at 1354 cm 1 [39]. However, in the Raman spectrum of NGO-SS-PEG, the intensity ratio of the D band and the G band (ID/IG) increased from 0.76 to 0.84. Furthermore, an increased ID/IG value was observed in the spectrum of NGO-PEG.

3.2. Stability and GSH-induced destabilization of NGO-SS-PEG and NGO-PEG in physiological solutions To evaluate the stability of the nanoparticles, NGO–COOH, NGO-PEG and NGO-SS-PEG were separately dispersed in PBS or

DMEM, respectively. After 24 h, no changes were observed in the NGO-PEG and the NGO-SS-PEG solutions, indicating high stability and solubility in physiological solutions, whereas obvious aggregation was observed in the NGO solutions (Fig. 3a). To investigate the redox-responsive detachment of the PEG shells, GSH (the final concentration: 10 mM) was added to 0.2 mg/mL of NGO-SS-PEG/DOX and NGO-PEG/DOX in PBS, respectively. As seen in Fig. 3b, the GSH-induced precipitation of NGO-SS-PEG was significant, whereas no aggregation was found in NGO-PEG solutions, mainly due to GSH-induced cleavage of the disulfide bonds, as a result the PEG coating detached from the planes of the NGO sheets. Fig. 3c revealed the size change of NGO-PEG and NGO-SS-PEG over the course of 15 days, respectively. NGO-PEG and NGO-SS-mPEG are both stable in PBS.

3.3. Photothermal sensitivity of NGO-SS-PEG and NGO-PEG The photothermal responses of 20 mg/L of NGO-PEG and NGOSS-PEG solutions were compared using a 2 W/cm2 NIR laser at 808 nm. As shown in Fig. 4, both suspensions demonstrated time-dependent temperature increases in response to the NIR irradiation and their extraordinary photothermal energy conversion efficiency (Fig. 4). The temperatures rapidly reached approximately 70 °C within 5 min of irradiation. However, no significant difference in the photothermal sensitivity was observed between NGO-PEG and NGO-SS-PEG.

3.4. DOX loading and GSH-induced DOX release from NGO-SS-PEG or NGO-PEG DOX was loaded onto NGO-SS-PEG or NGO-PEG by simply mixing 0.4 mg/mL DOX with 0.2 mg/mL NGO-SS-PEG or NGO-PEG at pH 8.0 overnight. The unbound DOX was removed. The loading amount of NGO-SS-PEG and NGO-PEG are both calculated to be 110 and 117% wt% estimated from the weight ratio between drug and carrier, respectively. However, for comparison, DOX was loaded onto NGO-SS-PEG or NGO-PEG by mixing 0.2 mg/mL DOX

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Fig. 2. Characterization of NGO-PEG and NGO-SS-PEG: (a) AFM images of NGO-SS-PEG and NGO-PEG. (b) FTIR spectra of NGO, NGO-PEG, NGO-SS-PEG, and GSH-reduced NGO-SS-PEG. (c) Size distribution curves obtained from DLS measurements. PDI = 0.144 for NGO-PEG; PDI = 0.172 for NGO-SS-PEG. (d) UV–Vis spectra of NGO, NGO-SS-PEG, and NGO-PEG aqueous solutions at a concentration of 10 mg/L. The inset showed the photographs of NGO, NGO-PEG, and NGO-SS-PEG aqueous solutions. (e) Raman spectra of NGO, NGO-PEG, and NGO-SS-PEG aqueous solutions.

with 0.22 mg/mL NGO-SS-PEG or NGO-PEG to obtain the loading mount of 100%. According to previous studies, DOX loading onto NGO can be attributed to p–p stacking and hydrophobic interactions [18]. DOX loading onto NGO-PEG or NGO-SS-PEG was evidenced by the deep red appearance. The formation of NGO-PEG/DOX and NGO-SS-PEG/DOX suspensions was further confirmed by UV–Vis measurement. As shown in the UV–Vis spectra, the UV–Vis characteristic peak of DOX was at approximately 490 nm (Fig. 5a). However, the DOX absorption peak of NGO-SS-PEG/DOX red shifted from 485 to 495 nm (Fig. 5a), which is attributed to the groundstate electron donor–acceptor interaction between NGO and DOX [11,40,41], further indicating the successful DOX loading. Additionally, a red shift of the DOX characteristic peak was observed in the spectrum of NGO-PEG/DOX. The release profiles of DOX from NGO-SS-PEG or NGO-PEG were investigated by UV–Vis spectroscopy in four media: PBS with 0 mM or 10 mM GSH at pH 7.4 and PBS with 0 mM or 10 mM GSH at pH 5.5. As shown in the release profiles (Fig. 5b and c),

DOX release from NGO-PEG or NGO-SS-PEG at pH 5.5 was much faster than that at pH 7.4 in the presence and absence of GSH, indicating a pH-dependent drug release. The pH-responsive drug release was attributed to the protonation effect of DOX and increased water solubility at acidic conditions [42]. More interestingly, approximately 40% of the DOX from NGO-SS-PEG or NGOPEG were released in the first 48 h in PBS without GSH at pH 5.5 (the simulated tumor intracellular pH value and the extracellular GSH concentrations) (Fig. 5b and c), respectively, indicating that there was no obvious difference in the DOX-release rates between two types of nanoparticles in the absence of GSH. However, upon 48 h exposure to PBS with 10 mM GSH at pH 5.5, the DOX release ratio of NGO-SS-PEG increased to approximately 58% (Fig. 5c), whereas that of NGO-PEG remained unchanged (Fig. 5b), indicating a significantly accelerated process in the presence of GSH for NGOSS-PEG, likely due to the GSH-induced cleavage of disulfide bonds and the subsequent shedding of the PEG shell from both planes of the NGO sheets. In the control group, only approximately 10% of the DOX was released from NGO-SS-PEG or NGO-PEG in PBS with

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Fig. 3. (a) Photos of NGO–COOH, NGO-PEG and NGO-SS-PEG separately dispersed in PBS or DMEM. (b) Photos of NGO-PEG and NGO-SS-PEG in PBS with and without GSH, respectively. GSH-reduced NGO-SS-PEG was obviously aggregated. (c) The size change of NGO-PEG and NGO-SS-mPEG determined by DLS over the course of 15 days, respectively. NGO-PEG and NGO-SS-mPEG are both stable in PBS.

before being placed into the dialysis bag. As shown in Fig. 5d, more than 65% of the DOX was rapidly released from NGO-SS-PEG/DOX after12 h of laser irradiation, and the profile then gradually plateaued in the following 60 h, whereas approximately 48% of the DOX was rapidly released form NGO-PEG/DOX at 24 h, and then the profile plateaued. These results indicated that the laser irradiation can accelerate the drug release from a graphene oxide nanocarrier. However, the photothermal effect for NGO-SS-PEG/DOX was more significant, likely due to the accelerated detachment of the disulfide-linked PEG diffusion barriers for the following reasons: (i) hyperthermia elevated the GSH content in cancer cells, and redox agent, GSH, cleave the disulfide bonds, thereby increasing the release of DOX from the internalized carriers. (ii) Heat stimulated glutathione-mediated degradation of disulfides.

3.5. In vitro cytotoxicity study

Fig. 4. Temperature change curves of water, NGO-PEG, and NGO-SS-PEG in PBS with 5% FCS exposed to the 808 nm laser at a power density of 2 W/cm2.

0 mM GSH or 10 mM GSH at pH 7.4, corresponding to normal and the tumor extracellular microenvironment [22]. These results indicated that the release ratio of DOX was very slow in the presence and absence of GSH in the extracellular microenvironment and was slight difference between samples treated with or without redox trigger; nevertheless, it is not as pronounced as pH triggering drug release. The possible reasons are weak hydrophilic and water soluble of DOX at high pH or nanoparticle aggregation induced by GSH addition are not favourable for drug diffusion. However, the aggregation is expected to be greatly reduced due to non-free diffusion and low concentration of NGO in the in vitro and in vivo applications. Furthermore, the DOX release mediated by the photothermal effect under acidic conditions (pH 5.5) was also studied (Fig. 5d). NGO-SS-PEG/DOX and NGO-PEG/DOX (50 mg/L, in terms of the NGO concentrations) in PBS containing 10 mM GSH at pH 5.5 were both irradiated with a NIR laser (808 nm, 2 W/cm2 for 3 min)

The in vitro cytotoxicity of A549 cells was determined by a MTT assay. A549 cells were incubated with various concentrations of NGO-PEG or NGO-SS-PEG (10, 20, 30, 40, and 50 mg/L) for 24 h. As depicted in Fig. 6a, the viability of A549 cells still remained above 97% when the concentrations of NGO-PEG and NGO-SSPEG were separately increased to 50 mg/L, indicating the low cytotoxicity of the NGO nanocarriers. To evaluate the efficiency of NGO-PEG/DOX and NGO-SS-PEG/ DOX for the chemo-photothermal ablation of tumor cells, A549 cells were incubated with 2, 10, and 20 mg/L of free DOX, NGOPEG/DOX, or NGO-SS-PEG/DOX for 24 h prior to NIR irradiation (in terms of the DOX concentrations) (Fig. 6b). The dose-dependent cytotoxicity of all groups was observed. At an equivalent DOX concentration of 2 mg/L (Fig. 6b), the inhibition rate of free DOX was 27.8%, whereas those of NGO-PEG/DOX and NGO-SS-PEG were 20% and 25.6%, respectively. The results indicated that the cytotoxicity of free DOX was higher, because the release of DOX from the NGO conjugates might experience a sustained release process due to a time-consuming DOX release. At the DOX concentrations of 10 and 20 mg/L (Fig. 6b), the inhibition rates were increased to approximately 75% and 89%, respectively, for the cells treated with NGO-SS-PEG/DOX, the inhibition rates were approximately 66% and 79.4%, respectively, for the cells

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Fig. 5. (a) UV–Vis spectra of DOX, NGO-PEG/DOX, and NGO-SS-PEG/DOX aqueous solutions. The inset showed a photograph of (1) DOX, (2) NGO-PEG/DOX, and (3) NGO-SSPEG/DOX). (b) The drug release profiles for NGO-PEG/DOX solutions at pH 7.4 and 5.5 containing 0 and 10 mM GSH (c) GSH-mediated drug release for NGO-SS-PEG/DOX solutions at pH 7.4 and 5.5 containing 0 and 10 mM GSH. (d) The GSH-mediated release profile of NGO-SS-PEG/DOX and NGO-PEG/DOX in PBS at pH 5.5 containing10 mM GSH under laser irradiation (2 W/cm2, 3 min).

treated with NGO-PEG/DOX, and the inhibition rates of the corresponding free DOX were 52% and 70%, respectively. These results suggested the ability of drug delivery to tumor cells and an enhanced cell-killing effect for both NGO-SS-PEG and NGO-PEG. Similar results were observed in our previous study [5]. Obviously, however, these effects of NGO-SS-PEG were higher than for NGOPEG/DOX, due to the GSH-induced reductive cleavage of the disulfide-linked PEG diffusion barriers. After irradiation by NIR light (2 W/cm2, 3 min), at DOX concentrations of 10 mg/L, the inhibition rate of the NGO-SS-PEG/DOX+NIR group was increased to 90%, which was significantly higher than that of the NGO-PEG/DOX+NIR group (79%), as shown in Fig. 6c. It was possible that the NGO-SS-PEG/DOX+NIR group and the NGO-PEG/DOX+NIR group can deliver both heat and drug to tumor cells and significantly improved the efficacy of cancer treatment. More importantly, a more rapid DOX release from NGO-SSPEG/DOX under NIR irradiation was observed in comparison with NGO-PEG/DOX, which was likely due to a photothermal-induced increase in the disulfide-bond cleavage, thereby increasing the PEG coating detachment from the planes of the NGO sheets. However, as shown in Fig. 6c, at a DOX concentration of 20 mg/L with laser irradiation, the difference in the cytotoxicity between NGO-PEG/DOX and NGO-SS-PEG/DOX was not obvious, probably due to the massive uptake caused by the high DOX loading concentrations and nanoparticle concentrations. DOX is known to be an effective anticancer drug, but for cancer therapy, rapid drug release after the arrival of nanocarriers to the tumor sites may enhance the therapeutic efficacy as well as reduce the probability of drug resistance in cells. Therefore, the conjugation of PEG onto NGO

by cleavable disulfide linkages is a very attractive approach to trigger rapid drug release from nanomaterials and improve their therapeutic efficacy. 3.6. Cellular uptake and internalization To assess the cellular uptake and intracellular distribution of PEGylated NGO by various covalent bonds using CLSM, A549 cells were incubated with NGO-PEG and NGO-SS-PEG at the same concentration of 20 mg/L for 4 h. Simultaneously with the fluorescence images, differential interference contrast (DIC) images were taken also. A549 cells without nanoparticle treatment are used as the control. As shown in CLSM images (Fig. 7a), weak fluorescence signals were observed in A549 cell cytoplasm, but the fluorescence intensities of NGO-PEG and NGO-SS-PEG exhibited no significant difference. These experimental results clearly indicated the cellular uptake for NGO-PEG and NGO-SS-PEG in A549 cells. It has been known that PEG functionalization can prolong the circulation time of nanomaterials, avoid their rapid uptake by macrophages of the mononuclear phagocyte system, and enhance their accumulation in the tumor sites by the enhanced permeability and retention effect [43–45]. To further confirm the intracellular uptake of nanocarriers, FACS was used. Fig. 7b shows that the flow cytometry histograms of fluorescence from A549 cells incubated with NGO-PEG and NGO-SS-PEG at a concentration of 20 mg/L for 4 h. The cells without nanoparticle treatment were used as the control. FACS image demonstrated the increased fluorescence intensities inside A549

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Fig. 6. (a) Cell viability of A549 cells with various concentrations of NGO-SS-PEG and NGO-PEG solutions, indicating the low cytotoxicity of both NGO-PEG and NGO-SS-PEG. (b) Relative cell viability of A549 cells for 24 h after treatment with free DOX, NGO-PEG/DOX or NGO-SS-PEG/DOX. (c) Relative cell viability of A549 cells for 24 h after treatment with NGO-PEG, NGO-PEG/DOX, NGO-SS-PEG and NGO-SS-PEG/DOX with 2 W/cm2 of 808 nm NIR irradiation for 3 min at various concentrations. All data are presented as the mean ± SD. Each experiment was repeated three times. **P < 0.05 versus the corresponding NGO-PEG/DOX groups. Differences were considered statistically significant by Student’s t-test. *P < 0.05 versus the corresponding NGO-PEG/DOX+NIR groups.

Fig. 7. (a) CLSM images of A549 cells incubated without nanoparticle treatment (control), with NGO-PEG or with NGO-SS-PEG (from left to right, the NGO concentration: 20 mg/L) for 4 h under488 nm excitation. The upper panel is confocal fluorescence images. The upper panel is confocal fluorescence images. The lower panel is the merged images of DIC and fluorescence. The experiments are repeated three times. (b) The flow cytometry histograms of A549 cells incubated with NGO-PEG or NGO-SS-PEG (the NGO concentration: 20 mg/L) for 4 h at a 488 nm excitation wavelength. A549 cells without treatment were taken as the control. The experiments are repeated three times.

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cells treated with NGO-PEG or NGO-SS-PEG cells, indicating the cellular uptake. However, the fluorescence intensity did not display a significant difference between the cells incubated with NGO-PEG and with NGO-SS-PEG, which was consistent with the cell uptake behavior observed by CLSM. Here, we mainly investigated the cellular uptake of functionalized NGO. NGO were usually internalized via the endocytosis process, while free DOX was transported into cells via passive diffusion [46]. Previous report shows that graphene has an optimal radius of about 25–100 nm for endocytosis, which are equivalent to sheet diameters of 50–200 nm [47]. Many more small nanosheets than large ones are endocytosed [48]. However, below the minimum size, carbon nanotubes cannot be internalized through endocytosis. In our study, the size of NGO-SS-PEG is no less than that of NGO-PEG (Fig. 2c). The lateral dimension of Functionalize NGO ranged from 50 to 500 nm. The size distribution of NGO complex shows the feasibility of the comparative study. 3.7. Redox-dependent biological efficacy of DOX-loaded NGO-SS-PEG For the evaluation of therapeutic efficacy, the DOX release was investigated using a fluorescence spectrometer (LS55, Perkin Elmer, USA). As seen in the fluorescence spectra (Fig. 8a), a DOX aqueous solution had a broad fluorescence band from 500 to 700 nm under 488 nm excitation. Significant fluorescence quenching was observed for both NGO-SS-PEG/DOX and NGO-PEG/DOX,

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which is attributed to the photoinduced electron-transfer effect along the NGO/DOX interface [11]. The intracellular DOX release of NGO-PEG/DOX and NGO-SSPEG/DOX was measured using CLSM and FACS. For the CLSM study, A549 cells were plated in glass-bottom culture dishes and treated with NGO-PEG/DOX or NGO-SS-PEG/DOX (the DOX concentration: 10 mg/L) for 4 h. Simultaneously with the fluorescence images, DIC images were taken also. As shown in the CLSM images (Fig. 8b), stronger DOX fluorescence signals were observed in A549 cells incubated with NGO-SS-PEG/DOX than for NGO-PEG/DOX, indicating enhanced DOX release. It was noteworthy that massive DOX fluorescence was observed in the nucleus of A549 cells treated with NGO-SS-PEG/DOX. Strong DOX fluorescence was also observed in perinuclear and the cytosol region when delivered by NGO due to sustained release of DOX. It is necessary for DOX to be released from NGO complexes and then enter into the nuclei, because DOX-loaded nanoparticles were too large to enter the nucleus directly [49,50]. Moreover, as shown in the flow cytometry histograms (Figs. 7b and 8c), the fluorescence intensity of NGO-SSPEG and NGO-PEG were both far less than that of DOX, thereby being considered negligible. This result likely resulted from the fact that DOX was rapidly released from NGO-SS-PEG due to the GSHresponsive disulfide linkage cleavage between PEG and NGO sheets and was then transported into the nucleus. The DOX release from NGO-SS-PEG and accumulation is critical in the nucleus for anticancer activity because it requires interaction with DNA [51,52].

Fig. 8. (a) Fluorescence spectra of DOX, NGO-SS-PEG and NGO-PEG in water at a 488 nm excitation wavelength. Significant fluorescence quenching was observed. (b) CLSM images of A549 cells incubated with NGO-PEG/DOX or NGO-SS-PEG/DOX (the DOX concentration: 10 mg/L) for 4 h under an excitation of 488 nm and CLSM images of A549 cells incubated with NGO-PEG/DOX or NGO-SS-PEG/DOX for 3 h, irradiated 3 min, and further incubated 2 h. The upper panel is confocal fluorescence images. The lower panel is the merged images of DIC and fluorescence. The experiments are repeated three times. (c) The flow cytometry histograms of A549 cells incubated with NGO-PEG or NGOSS-PEG for 4 h (the DOX concentration: 10 mg/L) under 488 nm excitation. A549 cells, which did not undergo treatment, was used as the control. The experiments are repeated three times. (d) The corresponding mean DOX fluorescence intensity of A549 cells.

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DOX release mediated by the photothermal effect was also determined by CLSM. A549 cells were treated with NGO-PEG/ DOX or NGO-SS-PEG/DOX (the DOX concentration: 10 mg/L) for 3 h prior to NIR irradiation followed by a further 2 h of incubation. As depicted in the CLSM images of NGO-SS-PEG/DOX+NIR group, significantly stronger DOX fluorescence was observed in A549 cells, and cell death occurred. On the other hand, obvious DOX fluorescence was also found in both the cytoplasm and the nucleus of NGO-PEG/DOX treated A549 cells (Fig. 8b), likely due to cell damage induced by the photothermal effect. These results further demonstrated the excellent efficacy of the chemo-photothermal therapy against tumor cells and the enhanced DOX release from NGO-SS-PEG/D by photothermal-effect mediated PEG shell detachment. FACS was used to further confirm the intracellular DOX release in A549 cells. A549 cells were treated with NGO-PEG/DOX or NGOSS-PEG/DOX (in terms of the DOX concentration: 10 mg/L) for 4 h. Consistent with the results of CLSM, FACS revealed a significant DOX fluorescence intensity difference between NGO-PEG/DOXand NGO-SS-PEG/DOX-treated A549 cells (Fig. 8c), thus indicating enhanced DOX release from NGO-SS-PEG/DOX due to the disulfide bond cleavage. The DOX fluorescence intensity from A549 cells incubated with NGO-SS-PEG/DOX was approximately 1.25 times that of cells incubated with NGO-PEG/DOX (Fig. 8d). 4. Conclusion In this study, we have developed a NGO-SS-PEG conjugate composed of a sheddable PEG shell and NGO by a disulfide linkage, which can carry heat and drugs specifically to tumor cells, and we have investigated their chemo-photothermal therapeutic efficacy. Intracellular DOX-release experiments demonstrated a rapid of drug release from NGO-SS-PEG conjugates with sheddable PEG upon the stimulus of high GSH levels inside A549 cells. This occurred regardless of whether NGO-SS-PEG was under laser irradiation, although the drug release from irradiated samples was more obvious. Furthermore, NGO-SS-PEG/DOX under NIR irradiation exhibited the higher cytotoxicity to A549 cells compared to NGO-PEG/DOX. Altogether, this work demonstrates the use of NGO-SS-PEG as a nanocarrier for the delivery of drugs and heat to tumor cells, which maybe an effective method to facilitate chemo-photothermal cancer therapy and reduce the side effects of drugs. Acknowledgments This work was supported by the National Natural Science Foundation of China (Nos. 61275187, 61378089, 61335011, and 31300691), and the Specialized Research Fund for the Doctoral Program of Higher Education of China (Nos. 20114407110001 and 200805740003). References [1] H. Liu, D. Chen, L. Li, T. Liu, L. Tan, X. Wu, et al., Multifunctional gold nanoshells on silica nanorattles: a platform for the combination of photothermal therapy and chemotherapy with low systemic toxicity, Angew. Chem. 123 (4) (2011) 921–925. [2] S.M. Lee, H. Park, K.H. Yoo, Synergistic cancer therapeutic effects of locally delivered drug and heat using multifunctional nanoparticles, Adv. Mater. 22 (36) (2010) 4049–4053. [3] H. Park, J. Yang, J. Lee, S. Haam, I.H. Choi, K.H. Yoo, Multifunctional nanoparticles for combined doxorubicin and photothermal treatments, ACS Nano 3 (10) (2009) 2919–2926. [4] X. Ma, H. Tao, K. Yang, L. Feng, L. Cheng, X. Shi, et al., A functionalized graphene oxide-iron oxide nanocomposite for magnetically targeted drug delivery, photothermal therapy, and magnetic resonance imaging, Nano Res. 5 (3) (2012) 199–212.

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