Biomaterials 35 (2014) 5805e5813

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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials

Multifunctional PEG-GO/CuS nanocomposites for near-infrared chemo-photothermal therapy Jing Bai, Yuwei Liu, Xiue Jiang* State Key Lab of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, Jilin, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 4 March 2014 Accepted 1 April 2014 Available online 22 April 2014

The synergistic therapy, the combination of photothermal therapy and chemotherapy, has become a potential treatment in the battles with cancer. Here, we developed a synergistic therapy tool that based on CuS nanoparticles-decorated graphene oxide functionalized with polyethylene glycol (PEG-GO/CuS) for cervical cancer treatment. The as-synthesized PEG-GO/CuS nanocomposites with excellent biocompatibility was revealed to have high storage capacity for anticancer drug of doxorubicin (Dox) and high photothermal conversion efficiency, and were effectively employed for the ablation of tumor. In addition, the therapeutic efficacy of Dox-loaded PEG-GO/CuS (PEG-GO/CuS/Dox) nanocomposites was evaluated in vitro and in vivo for cervical cancer therapy. In vitro cell cytotoxicity tests of PEG-GO/CuS/Dox demonstrate about 1.3 and 2.7-fold toxicity than PEG-GO/CuS and free Dox under 5 min irradiation with NIR laser at 1.0 W/cm2, owing to both PEG-GO/CuS-mediated photothermal ablation and cytotoxicity of light-triggered Dox release. In mouse models, mouse cervical tumor growth was found to be significantly inhibited by the chemo-photothermal effect of PEG-GO/CuS/Dox nanocomposites, resulting in effective tumor reduction. Overall, compared with chemotherapy or photothermal therapy alone, the combined treatment demonstrates better therapeutic efficacy of cancer in vitro and in vivo. These findings highlight the promise of the highly versatile multifunctional nanoparticles in biomedical application. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Graphene oxide Copper sulfide nanoparticles Drug delivery Cytotoxicity Photothermal therapy Synergistic effect

1. Introduction Cancer is considered to be one of major cause of death worldwide [1]. Over the past decades, though the doctors and patients of the whole world have been scrambling for ways to fight against cancer, success in combating cancer was limited. To date, chemotherapy is still the major therapeutic approach after surgical resection [2]. Unfortunately, the approach suffers from the harming of normal cells and tissues, insufficient dosage to diseased regions, severe adverse reactions, as well as an increased incidence of the second cancers [3,4]. In recent years, the development of nanotechnology and nanomaterials has a profound influence on nanomedicine. It is well known that nanomaterials with the match size of biological molecules can be conveniently transferred in the human environment, which open a way for detection and treatment of cancer [5,6]. In particular, multifunctional nanomaterials combined two or more functions, such as drug delivery, imaging and

* Corresponding author. Tel.: þ86 431 85262426; fax: þ86 413 85685653. E-mail address: [email protected] (X. Jiang). http://dx.doi.org/10.1016/j.biomaterials.2014.04.008 0142-9612/Ó 2014 Elsevier Ltd. All rights reserved.

hyperthermia, have the potential to improve the therapeutic efficacy of tumors. One way of enhancing therapeutic efficacy has been achieved through combining chemotherapy with hyperthermia [7]. In this approach, the nanomaterials can enhance the tumor accumulation of antitumor drugs by the enhanced permeability and retention (EPR) effect [8]. On the other hand, photothermal therapy (PTT), as a minimally invasive treatment, employs hyperthermia to kill cancer cells and improves the sensitivity of chemotherapy [9,10]. To achieve this, there has been a recent interest in developing synergistic systems capable of co-delivery of chemotherapeutic agents together with photothermal agents to the selected tumor region. To date, chemo-photothermal therapeutics have been engineered based on several noble metal supported on the silicon substrate. For example, silica/Au nanorods [11], silica/Au nanoshells [12] and silica/Pd nanosheets [13] have been fabricated and showed remarkable cancer-cell-killing efficiency. Then another kind of nanovalve was carbon-based nanomaterials [14], such as carbon nanotubes and graphene, which relied on the absorption of drug molecules via pep stacking and the superior photothermal sensitivity and exhibited a clear synergistic effect on cancer cells killing. These

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systems could strongly absorb NIR light and convert into heat to kill cancer cells. Meanwhile, the cytotoxicity of therapeutic agents is enhanced at elevated temperatures. Although synergistic effects of these systems have been proved by studies, they have to be confronted with many limitations to their wide applications. For example, silicon-based materials suffer from low drug loading capacity and fast release rates when carried molecular drugs within the porous core. Simultaneously, in order to obtain a high loading capacity for drugs, hollow mesoporous nanoparticles were usually used as carriers and obtained a hollow structure by etching mean. This is usually a complex process with certain risks, due to the use strong acid or base as an etching agent [12,13]. Some another systems show the sustained drug-release property due to their mesoporous and hollow structure, but no obvious drug cumulative release triggered by external NIR laser light was observed [12]. On the other hand, due to the low photostability of gold nanostructure, their NIR absorbance peak would diminish after laser irradiation, thus photothermal conversion capability has been greatly weakened. In addition, most of these photothermal agents are very expensive, and usually require complicated and severe synthesis processes. Also, carbon nanomaterials have shown effective synergistic effect to cancer, but they showed low photothermal conversion efficiency. Very recently, semiconductor copper sulfide (CuS) nanoparticles have been demonstrated as an attractive photothermal coupling agent owing to its low cost, low cytotoxicity and strong NIR absorption from energy band transitions [15]. However, due to the low surface area of CuS, they are not favorable for drug delivery. Thus, there is few report on drug delivery using CuS NPs for combined PTT and chemotherapy [16]. Therefore, the development of multifunctional nanomaterials with high photo-thermal conversion efficiency, high drug loading capacity, low cost and easy fabrication is highly desired. Graphene (GN) and its oxide (GO) have recently captured tremendous attention in many different fields including biomedicine [17]. With the unique two-dimensional nanosheet structure and the very high specific surface area [18], graphene sheet could serve as an ideal burgeoning support for the drug loading [19,20]. In the recent studies, GN and GO have been widely investigated for applications in drug delivery. Liu and co-workers used a facile method to prepare gelatin functionalized graphene nanosheets (gelatineGNS) [21]. GelatineGNS showed high stability in water solutions, as well as biocompatibility. Furthermore, the anticancer drug was loaded onto the gelatineGNS at a high loading capacity, and the Dox/gelatineGNS composite exhibited a high toxicity to kill MCF-7 cells. Also, Sun et al. loaded Dox on the surface of nanographene oxide by the simple p-stacking, and it desorbed at reduced pH due to its high hydrophilicity and high solubility at pH 5.5 in an intracellular endosome [22]. These works illustrate the potential for using graphene nanocarriers as an efficient drugdelivery agent for aromatic drugs. In this study, we plan to synthesize the multifunctional drug delivery system with low cytotoxicity, high photothermal conversion efficiency and stability, high drug loading capacity, pH- and heat-responsive drug release based on the combination of GO and CuS, and investigate their synergistic ablation for tumor cells in vitro and in vivo through the combination of chemotherapy and photothermal therapy. 2. Materials and methods 2.1. Chemicals and reagents Graphite powder was purchased from Aladdin Reagent Co. Ltd. Doxorubicin hydrochloride (Dox) was purchased from Beijing Huafeng United Technology Co. Methoxypolyethylene glycol amine (mPEG-NH2, Mw 10k) was purchased from Beijing JenKem Technology Co. Ltd. N-(3-dimethylamino propyl-N0 -ethylcar-bodiimide)

hydrochloride (EDC$HCl), dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)2,5-dipheny-ltetrazolium bromide (MTT) were purchased from Sigma. Dulbecco’s modified eagle medium (DMEM), penicillin, fetal bovine serum (FBS), and streptomycin were purchased from Beijing Dingguo Biotechnology Co. Other reagents were purchased from China National Medicine Corporation and used as received. Phosphate buffered saline (PBS) used in cell culture, was purchased from Invitrogen (10010). And PBS used in other experiments, was prepared by mixing stock solutions of NaH2PO4 and Na2HPO4. All solutions were freshly prepared using ultrapure water from a Millipore Milli-Q system. 2.2. Characterization The phases of the samples were identified by powder X-ray diffraction (XRD) analysis using an X-ray D/max-2200vpc (Bruker Co., German) instrument operated at 40 kV and 20 mA using Cu Ka radiation (l ¼ 0.15406 nm). To observe the detailed morphology of nanocomposites, transmission electron microscopy (TEM) was performed on a H-600 electron microscope (Hitachi, Japan) operated at 75 keV. UVevis absorption spectra were measured on UV-1700 PharmaSpec (Shimadzu, Japan) spectrometer with a 1 cm cuvette. Fluorescence measurements were performed on a LS-55 Luminescence spectrometer (PerkineElmer). The cells were imaged using a confocal laser scanning fluorescence microscope (CLSM, Leica TCS SP2, Leica Microsystems, Mannheim, Germany). Fourier transform infrared (FTIR) spectra were recorded on a Nicolet 520 FTIR spectrometer (Nicolet) equipped with a germanium attenuated total reflection (ATR) accessory, a DTGS KBr detector, and a KBr beam splitter (Bruker Co., German). 2.3. Synthesis of PEGylated graphene oxide (PEG-GO) Graphene oxide (GO) was prepared from purified natural graphite powder according to a modified Hummer’s method in a fume hood [23]. For pegylation, 1.2 g of NaOH was added to 10 mL of GO aqueous suspension (1.0 mg/mL) and the suspension was bath sonicated for about 3 h to convert OH groups to COOH via conjugation of acetic acid moieties resulting in GO-COOH [22]. The resulting solution was neutralized and purified by repeated rinsing and centrifugation. Then, 3 mL of EDC aqueous solution (2.0 mg/mL) was added to the 5 mL of GO-COOH suspension (1.0 mg/mL), and the mixture was sonicated for another 30 min. After that, 15.0 mg of 8-arm-polyethylene glycol-amine was added to the above suspension, and allowed to react overnight. The reaction is terminated by adding mercaptoethanol (4 mL). The solution was centrifuged and washed with water to remove the unreacted PEG. The final product is PEG-GO nanosheet. 2.4. Synthesis of PEG-GO/CuS nanocomposites The CuS was prepared according to established method in previous report [15]. The detailed procedure for the synthesis of PEG-GO/CuS nanocomposites was carried out as follows. In a three-neck flask, 10 mL of CuCl2$5H2O aqueous solution (1 mM) was added to 1 mL of GO-PEG aqueous suspension (1.0 mg/mL) under stirring, and kept the magnetic stirring for 30 min. Then, 100 mL of Na2S aqueous solution (0.1 M) was added into the reaction solution under stirring. After 10 min, the reaction mixture was heated to 90  C and stirred for 15 min until a dark-green solution was obtained. After the reaction, the resulting mixture was centrifuged and washed with water to remove the unreacted ions. The PEG-GO/CuS nanocomposites were obtained and stored at 4  C. 2.5. Drug loading and release study of PEG-GO/CuS nanocomposites Dox loading onto PEG-GO/CuS was achieved by simple mixing of PEG-GO/CuS with Dox solution in PBS at pH 8.0. After stirring for 24 h under dark condition, excess Dox molecules were removed by centrifugation and washed thoroughly with PBS until the supernatant became free of reddish color (corresponding to free Dox). The resulting PEG-GO/CuS/Dox complexes were redispersed in PBS for subsequent tests of Dox release and cytotoxicity in vitro. To evaluate the Dox-loading efficiency, the content of Dox in the supernatant was determined by UVevis measurements at 490 nm [24]. The Dox-loading efficiency (DLE) was calculated by equation (1): DLE% ¼

ODox  RDox  100% ODox

(1)

where ODox and RDox are the contents of the original and residual Dox solution, respectively. To measure the drug release, the PEG-GO/CuS/Dox samples were immersed in 2 mL of pH ¼ 9.0, 7.4 and 5.5 PBS at 37  C with gentle shaking. At the selected time intervals, PBS was taken out by centrifugation to test the concentration of the released Dox and replaced with 2 mL of fresh buffer solution. 2.6. Photothermal effect measurements The test solution was introduced in a quartz cuvette and exposed to the 980 nm laser at a power density of 1 W/cm2 for 10 min (Changchun New Industries Optoelec-tronics Technology, China). The temperature of the solution was monitored using a digital thermometer with a thermocouple probe (Pyrometer Instrument Company, USA).

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2.7. Cell culture and viability measurements HeLa cells (cervical cancer cell line) were cultured in a DMEM medium containing 10% fetal bovine serum, 100 units mL1 penicillin and 100 mg mL1 streptomycin at 37  C in a humidified 5% CO2 atmosphere. For cell toxicity assay, HeLa cells were seeded in a 96-well plate at a density of 1  104 cells/well and cultured in 5% CO2 at 37  C for 24 h prior to assay. After remove the DMEM medium, the cells were exposed to free Dox (10 mg/mL), PEG-GO/CuS (500 mg/mL), and PEG-GO/CuS/ Dox (500 mg/mL, the concentration of Dox was 10 mg/mL), respectively, or PEGGO/CuS at various nanomaterial concentrations (50e500 mg/mL) dispersed in the DMEM medium for 24 h. For comparison, the cells were irradiated by NIR laser at an output power of 1.0 W/cm2 with 10 min. Then, the incubation solution was removed and washed three times with PBS. 100 mL of MTT solution (0.5 mg/mL) was added to each well and incubated for 4 h. Finally, the medium was replaced with 100 mL of DMSO, and the absorbance was monitored at 570 nm using a Versamax microplate reader (BioTek Instruments Inc, USA). Results were quantified by manually subtracting the blank value and normalized against the control values. 2.8. Confocal fluorescence imaging analysis Prior to confocal microscopy study, HeLa cells were plated at a density of 5.0  105 cells per dish on glass bottom cell culture dishes (20 mm) at 37  C in a humidified atmosphere containing 5% CO2 for 24 h. The cells were washed three times with PBS, followed by incubation with 800 mL of PEG-GO/CuS (500 mg/mL) or PEG-GO/CuS/Dox (500 mg/mL, the concentration of Dox was 10 mg/mL) solution in DMEM culture medium at 37  C and 5% CO2 for 24 h. After incubation was completed, the culture medium with nanoparticles was removed and the cells were resupplied with 800 mL of fresh DMEM culture medium. For thermal therapy, corresponding culture dishes were irradiated with an 980 nm NIR laser (1 W/cm2) for 5 min. After laser irradiation, the cells were then washed three times with PBS and stained with 2 mg/mL of calcein AM in PBS for 30 min. The cells were imaged using a confocal laser scanning fluorescence microscope with 20 objective. The dye was excited at 488 nm and observed through a 500e550 nm emission band-pass. 2.9. In vivo chemo-photothermal destruction of tumors Animal care and handing procedures were conducted in conformity with national guidelines and with approval of the regional ethics committee for animal experiments. The HeLa cells (5  106) cells were harvested, suspended in PBS (10 mL), and injected subcutaneously into the right shoulder of male nude mice at the injection amount of 0.1 mL/animal. The mice were used for treatment when the tumor volume reached w50 mm3. Then twelve mice were randomly divided into 4 groups (n ¼ 3 per group), and saline solution (Group I, 0.9%), free Dox (Group II, 10.0 mg/mL), PEG-GO/CuS (Group III, 500 mg/mL) or PEG-GO/CuS/Dox (Group IV, 500 mg/mL, and the concentration of Dox was 10.0 mg/mL) were locally injected into the tumors. After 2 h of injection, the 980 nm continuous-wave NIR laser was used to irradiate tumors in group III and IV at 1.0 W/cm2 for 10 min. At 21st day, all the animals were euthanized, and the tumors were dissected. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assays were performed according to previous reports [25]. The dimension of each tumor was measured every three days using a caliper, and the tumor volumes were calculated based on the following formula: . V ¼ L  W2 2

(2)

In this equation, V, L and W are the volume, length and width of tumor, respectively. The relative tumor volume was calculated as V/V0, where V0 is the tumor volume at initiation of the treatment. 2.10. Histological analysis of tumor tissues The mice were sacrificed on the 21st day. The tumors from every group were harvested. The fresh tumor tissues were fixed in 4% neutral-buffered formalin, and then processed routinely into paraffin and used for TUNEL assay. Presence of apoptotic or necrotic cells was detected with the terminal deoxynucleotidyl transferase-mediated nick-end-labeling assay (TUNEL: ApopTagÒ Peroxidase In Situ Apoptosis Detection Kit, Chemicon-Millipore Company, Billerica, MA) according to manufacturer’s recommended procedure. Cells that stained positive for TUNEL were counted at 400 magnification in at least 5 different fields.

3. Results and discussion 3.1. Characterization of PEG-GO/CuS nanocomposites As illustrated in Scheme 1, an PEG-conjugated GO/CuS-based drug delivery system was constructed for chemo-photothermal therapy of cervical cancer. In brief, GO as carrier was synthesized by a modified Hummer’s method [23]. For pegylation, we convert hydroxyl groups to carboxylic acid (COOH) moieties and obtain the

Scheme 1. Sample preparation flowchart of PEG-GO/CuS nanocomposites.

intermediate product, named GO-COOH [22]. PEG was then conjugated onto GO-COOH nanosheets via the amide formation. An excess of copper (II) ion was then added to the PEG-GO suspension. Subsequently, the copper (II)-coated PEG-GO nanosheets were treated with sodium sulfide dissolved in a water, resulting in the formation of PEG-GO/CuS nanocomposites. To verify the successful growth of CuS nanoparticles on the surface of GO sheets directly, TEM was used to characterize the morphologies of the as-prepared composites (Fig. 1A). TEM images show that a large amount of CuS nanoparticles are well-dispersed on the surface of graphene oxide nanosheets with an average diameter of approximately 13 nm (Fig. S1), which should be attributed to the oxygen-containing functional groups (hydroxyl, epoxide and carboxylic groups) uniformly existing on the two accessible sides of GO [26]. Due to the oxygen-containing functional groups, the addition of copper precursor during the hydrothermal treatment process caused many copper ions to be adsorbed on the surface of GO nanosheets. Furthermore, the as-prepared PEG-GO/CuS nanocomposites dispersed well in PBS solution or DMEM cell culture medium and remained stable for one week without any detectable agglomeration (Fig. S2). The well stability of the PEG-GO/CuS nanocomposites solution is attributed to conjugation of eight-armed PEG-amine stars to the carboxylic acid groups on the surface of GO via carbodiimide catalyzed amide formation [19]. Such a favorable colloidal stability of PEG-GO/CuS nanocomposite solution is highly desirable for in vivo applications. In addition, successful PEGylation was evidenced by FTIR spectroscopy measurement (Fig. S3). Fig. S3 shows FTIR spectra of GO (a), GO-COOH (b), and PEG-GO sheets (c). The peak at w1730 and w1610 cm1 can be attributed to the vibration of C]O bond and the aromatic C]C stretching in the GO [22]. The bands at 1623 and 1427 cm1 are attributed to the hydrogen-bond vibration of eCOOH and symmetric (vs) stretching vibrations of the eCOO groups [27], which is consistent with obtaining intermediate product, named GO-COOH. The peaks at w2900, 1652 and 1100 cm1 in the PEG-GO samples are the characteristic peaks of eCH2, eNHeCOe and CeO bonds in PEG, respectively, which are the signatures of grafting of PEG molecules onto GO [22]. Therefore, the FTIR results confirmed that PEG has been successfully grafted onto the surface of GO nanosheets. Also, the optical property of PEG-GO/CuS nanocomposites solution was studied by using UVevis spectroscopy. Fig. 1B shows UVevis spectra of GO (a), GO-COOH (b), PEG-GO (c) and PEG-GO/CuS nanocomposites (d). The absorption of GO shows a peak at 230 nm [22], and the absorption of GO-COOH and PEG-GO also show similar absorption peaks, indicating that both synthesis of GO-COOH and PEGylation steps did not broken the structure of GO

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Fig. 1. Characterization of CuS-loaded PEG-GO nanocomposites. (A) TEM image of PEG-GO-CuS nanocomposites; (B) UVevis absorption spectra of GO (a), GO-COOH (b), PEG-GO (c) and PEG-GO/CuS nanocomposites (d) dispersed in PBS.

nanosheets. For the PEG-GO/CuS nanocomposites, the broad absorption band in the near-IR region was observed with the maximum absorption at 980 nm, which has been attributed to the characteristic of CuS nanoparticles [28]. Furthermore, the observation also demonstrated that CuS nanoparticles have been anchored on the surface of GO nanosheets. Importantly, the strong optical absorbance of PEG-GO/CuS nanocomposites in the near-IR region confirms its potential use as a PTT agent. 3.2. In vitro Dox delivery Based on the large surface of GO nanosheets, the PEG-GO/CuS nanocomposites are expected to be an ideal anticancer drug carrier. Dox, as an anti-tumor drug model, was loaded onto PEG-GO/ CuS nanocomposites by soaking PEG-GO/CuS nanocomposites with Dox in PBS (pH 8.0) overnight. The unbound drug was removed by centrifugation and repeated rinsing. Dox loading on the surface of PEG-GO/CuS was evidenced by eye due to the reddish color of the PEG-GO/CuS/Dox suspension (Fig. S4). Furthermore, a peak of Dox appears at around 504 nm in the UVevis spectrum of the PEG-GO/CuS/Dox suspension (Fig. 2A). The slight shift of the maximum peak in the UVevis spectra for the loaded Dox from 504 nm to 490 nm for free Dox may originate from the interaction of the loaded Dox drugs with the PEG-GO/CuS nanocomposites. All these results demonstrate that Dox molecules have been successfully loaded onto PEG-GO/CuS nanocomposites. Also, the loading efficiency of Dox on multi-functionalized GO was calculated by measuring the concentration of unbound drug according to the characteristic absorption of free Dox in the UVevis spectra (Fig. S5). According to equation (1), the loading efficiency of

Dox could reach 90.0%, and the drug loading content was 900 mg Dox mg1 PEG-GO/CuS nanocomposites. The result shows that the GO is indeed a promising candidate for drug carrier materials. The binding most likely comes from p-p stacking and hydrophobic interaction between multi-functionalized GO and Dox [29]. The aromatic characteristic of Dox enabled p-stacking onto the surface of graphitic sheet of PEG-GO/CuS, which was further confirmed by fluorescence quenching of Dox when loaded onto the surface of PEG-GO/CuS nanocomposites (Fig. 2B), similar to Dox loading on other carbon materials [30]. 3.3. Photothermal conversion of PEG-GO/CuS nanocomposites Having CuS nanoparticles grafted on the surface of GO, PEG-GO/ CuS nanocomposites exhibited a significant photothermal effect induced by NIR irradiation. Different concentrations of PEG-GO/CuS nanocomposites dispersed in PBS solution were irradiated using a 980 nm laser at a power of 1.0 W/cm2 for 10 min, and PBS solution and GO suspensions were used as controls (Fig. 3A). It was found that the temperature of PEG-GO/CuS solution (0.2 mg/mL) could be increased by 21.4  C within 600 s irradiation. In contrast, the temperature of buffer and 1.0 mg/mL of GO solutions was only increased by 6  C and 14  C, respectively. Moreover, with the increase of the concentration of the PEG-GO/CuS suspension (i.e., from 0.5 to 1.0 and 2.0 mg/mL), the magnitude of temperature elevation can be increased by 22.6, 24.9 and 28.7  C, respectively, in the same irradiation time of 600 s (Fig. 3A). It has been demonstrated that the cancer cells can be killed after maintenance at 42  C for 15e60 min [31]. These data suggest that PEG-GO/CuS nanocomposites can efficiently convert NIR light into heat and kill the cancer cells.

Fig. 2. Loading of Dox on PEG-GO/CuS nanocomposites. (A) UVevis absorption spectra of PEG-GO/CuS (a), free Dox (b) and PEG-GO/CuS/Dox nanocomposites (c) dispersed in PBS; (B) Fluorescence spectra of free Dox (a) and PEG-GO/CuS/Dox nanocomposites (b) dispersed in PBS.

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Fig. 3. Evaluation of photothermal conversion and stability of PEG-GO/CuS nanocomposites. (A) Temperature elevation of nanocomposites dispersed in 1 mL of PBS solution over a period of 10 min under NIR light irradiation (980 nm, 1 W/cm2) at various concentrations. PBS solution was used as a control; (B) Temperature elevation of PEG-GO/CuS nanocomposites dispersed in 1 mL of PBS solution (1 mg/mL) over five laser on/off cycles under NIR laser irradiation.

Fig. 4. The pH and heat-dependent release behavior of Dox. (A) Dox-release profiles of Dox-loaded PEG-GO/CuS nanocomposites measured at pH 5.5 (a), 7.4 (b) and 9.0 (c) in PBS buffer at 37  C. (B) NIR-triggered release of Dox from PEG-GO/CuS/Dox nanocomposites with temperature change. The samples were irradiated with an NIR laser from 0 to 10 min and 100e110 min and the laser was turned off from 10 to 100 min.

Also, another important photothermal property is the photothermal stability of PEG-GO/CuS nanocomposites. To study their photothermal stability, we recorded the time-dependent temperature of the PEG-GO/CuS dispersion upon their radiation with NIR laser for 10 min (Laser on), followed by naturally cooling of temperature to room temperature upon the switch off the NIR laser

(Laser off). This cycle was repeated five times in order to investigate the photostability of PEG-GO/CuS nanocomposites (Fig. 3B). Significantly, the photothermal effect of the PEG-GO/CuS nanocomposites did not show any decrease during the temperature elevation, which is superior to the well-known photothermal agent, Au nanorods. Usually, the Au nanorods can be melted after

Fig. 5. Cytotoxicity and therapeutic effect evaluation of the nanocomposites by MTT assays. (A) In vitro cytotoxicity of HeLa cells exposed to different concentrations of PEG-GO/CuS nanocomposites (200 mL) in DMEM for 24 h at 37  C incubation; (B) In vitro cytotoxicity of HeLa cells incubated with 200 mL of free Dox (10 mg/mL), PEG-GO/CuS nanocomposites (500 mg/mL) and PEG-GO/CuS/Dox nanocomposites (500 mg/mL, the concentration of Dox was 10 mg/mL) with (red) and without (dark gray) NIR irradiation at 1 W/cm2. Errors bars represent one standard deviation. Error bars were based on standard deviations of five parallel samples. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 6. CLSM images of HeLa cells incubated with: DMEM medium (A), PEG-GO/CuS nanocomposites (B), PEG-GO/CuS nanocomposites (C) and PEG-GO/CuS/Dox nanocomposites (D) in DMEM for 24 h at 37  C without (A, B) and with (C, D) 5 min NIR irradiation at 1 W/cm2. Red arrow indicates the laser spot; Viable cells were stained green with Calcein AM, Scale bar ¼ 100 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

irradiation cycle [32]. The super NIR photostability suggests that the PEG-GO/CuS nanocomposites are a kind of promising photothermal agent. 3.4. In vitro Dox release property As an efficient drug delivery system, the PEG-GO/CuS nanocomposites should also possess a sustained-release property. To evaluate this property, we measured the drug release behaviors at different pH values. Fig. 4A shows the release profiles of the Dox from PEG-GO/CuS/Dox nanocomposites at pH 5.5, 7.4 and 9.0 at 37  C. It can be seen that the drug release rate increased with the decrease of solution pH, and only about 17% and 10% of the total bound Dox was released from the PEG-GO/CuS/Dox nanocomposites in 45 h under neutral (pH 7.4) and basic conditions (pH 9.0), respectively. With an increase of acidity to pH 5.5, Dox was quickly released in the early stage, and about 45% of Dox was released in the 45 h, indicating the sustained drug-release behavior of the

Dox-loaded PEG-GO/CuS nanocomposites. This is because more of the eNH2 groups on Dox are protonated with the decrease of pH, which increases the hydrophilicity of Dox and leads to the release of more of the incorporated Dox. Such a release behavior of Dox from PEG-GO/CuS/Dox nanocomposites is important because the microenvironments of tumor tissue are acidic, which is beneficial for cancer therapy. Additionally, accelerated Dox release by the photothermal effect of the nanocomposites under NIR laser irradiation was also observed experimentally (Fig. 4B). As shown in Fig. 4B, a burst release of Dox molecules occurred at the first 5 min upon the NIR laser irradiation. When the NIR laser was switched off, the release rate was significantly reduced. About 10% of Dox was released from the PEG-GO/ CuS/Dox nanocomposites under 5 min NIR radiation. In comparison, the PEG-GO/CuS/Dox nanocomposites had a cumulative release of only 0.6% in the same period without NIR laser irradiation. The results suggest that the NIR light could enhance the release of Dox from the nanocomposites. Meanwhile, the temperature change was

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recorded following the laser irradiation on and off. We found that the drug-release rate is roughly coincident with the change of temperature. That is, high temperature quickly accelerated the release of Dox from the PEG-GO/CuS/Dox nanocomposites. To further verify the photothermal effect, the release of drug from the PEG-GO/CuS/Dox nanocomposites was conducted in a water bath at 45  C (Fig. S6). The release rate of Dox was accelerated by heat, which is similar to the case of laser irradiation. The results further demonstrate that photothermal heating could enhance the release of drug, thereby improving the treatment of cancer. 3.5. In vitro cytotoxicity study As discussed above, PEG-GO/CuS nanocomposite is a synergistic platform for drug carrier and photothermal therapy. To further evaluate the potential for biomedical application, we investigated the cytotoxicity of PEG-GO/CuS nanocomposites to tumor cells at different concentrations by conducting MTT assays (Fig. 5A), since only nontoxic carriers are suitable for biological applications. The PEG-GO/CuS nanocomposites were diluted with DMEM medium and incubated with HeLa cells in 5% CO2 at 37  C for 24 h. No significant cytotoxicity was determined when the incubation concentration of the PEG-GO/CuS nanocomposites was less than 100 mg/mL. Even at the highest tested dose of PEG-GO/CuS nanocomposites (500 mg/mL), the cell viability still remained above 90%. The results indicate that our as-synthesized nanomaterials have low cytotoxicity and good biocompatibility. Direct irradiation of the cells with laser in the absence of nanocomposites did not induce significant effect on cell viability (Fig. 5B). However, the laser irradiation led significant decrease of the cell viability to 40% after the cells were incubated with 500 mg/ mL of PEG-GO/CuS nanocomposites (Fig. 5B), suggesting that the heat generated from the NIR irradiation could kill large amount of cells. After 24 h incubations with free Dox, the cell viability was decreased to about 70%, which is similar to their irradiated groups. In the case of Dox-loaded PEG-GO/CuS nanocomposites, the cytotoxicity to HeLa cells was much less than free Dox under the same drug concentration (10 mg/mL), which should be attributed to less release of Dox from PEG-GO/CuS/Dox nanocomposites and low cytotoxicity of nanocomposites. However, after NIR laser irradiation, PEG-GO/CuS/Dox showed a high cytotoxicity with 75% cell death at an equivalent Dox concentration of 10 mg/mL (Fig. 5B), which was higher than separate free Dox and PEG-GO/CuS nanocomposites. This may be attributed to two facts. One reason is that the PEG-GO/CuS nanocomposites could act as an effective NIR photoabsorber to transform NIR into heat, and kill the cancer cells. On the other hand, the release of Dox is accelerated due to the elevated temperature, which enhances killing the cancer cells [33]. All the results suggest that the combination of chemotherapy and phototherapy can significantly improve the therapeutic efficacy, exhibiting a synergistic effect. To further evaluate the therapeutic effect of PEG-GO/CuS/Dox nanocomposites, fluorescent staining for the assessment of live cells was measured with confocal laser scanning microscope (CLSM). No significant difference in cell viability and density was observed between negative control and PEG-GO/CuS nanocomposites-treated cells without NIR laser irradiation (Fig. 6A and B). Conversely, as shown in Fig. 6C, HeLa cells treated with 500 mg/ mL of PEG-GO/CuS nanocomposites plus a 5 min NIR irradiation were killed within the laser spot, suggesting that PEG-GO/CuS nanocomposites can convert the NIR into the heat and destruct HeLa cells. Notably, after NIR laser irradiation (5 min, 980 nm) to the HeLa cells incubated with 500 mg/mL of PEG-GO/CuS/Dox nanocomposites, the PEG-GO/CuS/Dox nanocomposites showed a higher cell-killing efficacy for HeLa cells than the separate photothermal

Fig. 7. In vivo evaluation of chem-photothermal therapeutic effect. (A) Photographs and (B) Excised tumors from mice euthanized after the 21st day of treatment with: saline solution as control (a, 0.9%), free Dox (b, 10.0 mg/mL), PEG-GO/CuS nanocomposites (c, 500 mg/mL) and PEG-GO/CuS/Dox nanocomposites (d, 500 mg/mL, and the concentration of Dox was 10.0 mg/mL) at the injection amount of 0.1 mL/animal without (a, b) and with (c, d) 5 min NIR irradiation at 1 W/cm2; (C) Comparative therapeutic-efficacy study in vivo animal model.

therapy (without Dox) (Fig. 6D). Beyond the spot of the laser irradiation, we also found a decrease in cell viability and density with an enhanced cell-killing efficacy, which is attributed to the Dox released from PEG-GO/CuS/Dox nanocomposites upon the NIR irradiation. All the results suggest that the PEG-GO/CuS/Dox nanocomposites show a synergistic effect combining the chemotherapy and photothermal therapy. The mechanism of the synergistic effect is considered to increasing heat of PEG-GO/CuS/Dox exposed to NIR and an enhanced cytotoxicity of Dox in elevated temperature. 3.6. In vivo chemo-photothermal therapy To investigate chemo-photothermal synergistic therapy of Doxloaded PEG-GO/CuS nanocomposites, we conducted comparative efficacy studies in normal mice with tumor (Fig. 7). Twelve nude mice bearing HeLa cells at the right shoulder were randomly divided into four groups (n ¼ 3) and given intratumoral injections as following agent: 0.1 mL, 0.9% of saline solution as negative control (group I), 0.1 mL of free Dox at the concentration of 10 mg/mL (group II), 0.1 mL, 500 mg/mL of PEG-GO/CuS nanocomposites with laser treated (group III) and 0.1 mL, 500 mg/mL of PEG-GO/CuS/Dox nanocomposites with laser treated (group IV, the concentration of

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Fig. 8. TUNEL assay images of tumor tissue after the 21st day treatment from the different groups:(A) control group, (B) free Dox without irradiation, (C) PEG-GO/CuS nanocomposites with 980 nm laser irradiation at 1 W/cm2 and (D) Dox-loaded PEG-GO/CuS nanocomposites irradiation with 980 nm laser irradiation at 1 W/cm2.

Dox was 10 mg/mL). No mice died during the course of therapy. On the 21st day, mice were sacrificed, and tumors were excised. Photographs of the test mice and the tumors in each group after the treatment on the 21st day were shown in Fig. 7A and B. The sizes of tumors were measured every three days after treatment and plotted as a function of time in Fig. 7C. In the control group, tumors showed rapid growth. And, the mice treated with free Dox only exhibited no significant tumor elimination, indicating that the dosage of administered Dox is insufficient to reduce the tumor volume. While for the treatment with PEG-GO/CuS nanocomposites with laser irradiation, the tumor growth was inhibited in the first week, but the tumor began to grow again after that, suggesting the recurrence of the tumors due to the incomplete ablation of tumor cells. In contrast, after the mouse tumors were directly injected with PEGGO/CuS/Dox nanocomposites, followed by irradiation with the laser, the tumor growth was significantly suppressed, and no tumor regrowth was observed in this treatment group over a course of 21 days. Although the PEG-GO/CuS nanocomposites could only partially destruct the tumor under NIR irradiation, Dox released from the PEG-GO/CuS/Dox nanocomposites after NIR irradiation will further destroy the tumor, so as to achieve the effect of inhibiting tumor. These results clearly demonstrate the evidence of synergistic effect of combining photothermal therapy and chemotherapy in tumor growth inhibition by PEG-GO/CuS/Dox nanocomposites. The high toxicity leading to weight loss is always a great concern in vivo. In this work, neither death nor significant body weight drop was observed in all groups during the treatments, implying that the toxic side effects of treatments are not severe (Fig. S7). To further verify the synergistic therapy, we measured the histological images of tumor tissues (Fig. 8). Generally, the necrotic or apoptotic cells were stained brown, while the robust and viable

cells were stained blue. Consistent with the above observation, the TUNEL assay showed that the number of TUNEL-positive cells in the free Dox, PEG-GO/CuS nanocomposites with laser irradiation and PEG-GO/CuS/Dox nanocomposites with laser irradiation groups was dramatically increased, which is the evidence of tumor cell death, especially in the PEG-GO/CuS/Dox group. These results indicate increased toxicity of PEG-GO/CuS/Dox nanocomposites with laser irradiation. 4. Conclusions In summary, we have synthesized multifunctional PEG-GO/CuS/ Dox nanocomposites, which can combine photothermal therapy with chemotherapy in a system. The PEG-GO/CuS/Dox nanocomposites showed a high loading efficiency and exhibited pHresponsive Dox release, which was enhanced by laser irradiation. It was demonstrated in this study that the synergistic therapy is highly efficient for cancer therapy, and in vitro experimental results showed significant suppression of cell viability. Using mice cancer model, the tumor growth was effectively inhibited by the NIRinduced hyperthermia of the PEG-GO/CuS nanocomposites and enhanced drug release in PEG-GO/CuS/Dox nanocomposites. These results may raise possibilities of a potential platform using PEG-GO/ CuS/Dox nanocomposites for biomedical application. Acknowledgments This work was financially supported by the National Science Foundation for Excellent Young Scholar of China (21322510), Youth Foundation of China (21105097), Youth Foundation of Jilin Province (20140520082JH), and Natural Science Foundation of Jilin Province (201215092).

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