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Combined photothermal and photodynamic therapy delivered by PEGylated MoS2 nanosheets† Teng Liu, Chao Wang, Wei Cui, Hua Gong, Chao Liang, Xiaoze Shi, Zhiwei Li, Baoquan Sun and Zhuang Liu* Single- or few-layered transitional metal dichalcogenides, as a new genus of two-dimensional nanomaterials, have attracted tremendous attention in recent years, owing to their various intriguing properties. In this study, chemically exfoliated MoS2 nanosheets are modified with lipoic acid-terminated polyethylene glycol (LA-PEG), obtaining PEGylated MoS2 (MoS2-PEG) with high stability in physiological solutions and no obvious toxicity. Taking advantage of its ultra-high surface area, the obtained MoS2PEG is able to load a photodynamic agent, chlorin e6 (Ce6), by physical adsorption. In vitro experiments reveal that Ce6 after being loaded on MoS2-PEG shows remarkably increased cellular uptake and thus

Received 5th July 2014 Accepted 21st July 2014

significantly enhanced photodynamic therapeutic efficiency. Utilizing the strong, near-infrared (NIR) absorbance of the MoS2 nanosheets, we further demonstrate photothermally enhanced photodynamic therapy using Ce6-loaded MoS2-PEG for synergistic cancer killing, in both in vitro cellular and in vivo animal experiments. Our study presents a new type of multifunctional nanocarrier for the delivery of

DOI: 10.1039/c4nr03753g

photodynamic therapy, which, if combined with photothermal therapy, appears to be an effective

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therapeutic approach for cancer treatment.

Introduction Commonly used modern clinical cancer therapies, including surgery resection, radiation therapy and chemotherapy, have many disadvantages, such as severe side effects, limited efficacy, and the tendency to induce drug resistance. Phototherapy, as a non-invasive therapeutic approach triggered by light, has recently attracted widespread interest. Photothermal therapy (PTT) and photodynamic therapy (PDT) are two major categories of phototherapy. Whereas PTT uses photo-absorbing agents to transfer light energy into heat and eradicates tumors by hyperthermia,1 PDT functions with the help of photosensitizing (PS) molecules, which under light irradiation are able to produce toxic reactive oxygen species (ROS), including singlet oxygen, to kill nearby cancer cells.2–5 However, PTT generally requires laser irradiation with high-power densities to produce a sufficient amount of heat.6 In contrast, PDT requires the effective uptake of PS agents by cancer cells and shows limited efficacy towards hypoxic tumors.7–9 Recently, a number of reports have found that mild photothermal heating (e.g. to 43  C) could enhance the cellular uptake of either chemotherapeutic drugs or photodynamic agents.10,11 The combination of photothermal and photodynamic therapy has thus been Institute of Functional Nano & So Materials (FUNSOM) & Collaborative Innovation Center of Suzhou Nano Science and Technology, Soochow University, Suzhou, Jiangsu 215123, China. E-mail: [email protected] † Electronic supplementary 10.1039/c4nr03753g

information

(ESI)

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demonstrated with various types of nanocarriers, including graphene,12–14 gold nanomaterials,15–18 conjugated polymers,19 and a few other types,20,21 aimed at killing cancer in a synergistic manner. Two-dimensional (2D) transitional metal dichalcogenide (TMDC) nanosheets, as graphene alternatives with various unique optical and electronic properties,22–26 have shown great potential in a wide range of elds, including electronic devices, transistors, energy storage devices and catalysis.27–31 Recently, a number of groups, including ours, have found that atomically thin TMDC nanosheets are also promising for applications in biomedicine. Dravid et al. discovered that defective sites resulting from the loss of sulfur atoms during the exfoliation process were available for modication by thiolated molecules.32 Utilizing the large surface area due to the 2D structure, Zhang et al. fabricated MoS2-based DNA sensors.33 Due to high absorbance in the near-infrared (NIR) region where tissues and organs are transparent, we and a few other groups used MoS2,34 WS2 (ref. 35) or Bi2Se3 (ref. 36) nanosheets as photothermal agents for cancer treatment. Our recent work utilized MoS2 nanosheets to deliver anticancer drug molecules for chemo and photothermal combination therapy, achieving remarkable synergistic effects both in vitro and in vivo.37 However, the use of TMDC nanosheets for the delivery of photodynamic therapy has not yet been demonstrated to the best of our knowledge. In this work, water-soluble MoS2 nanosheets prepared by lithium intercalation are functionalized by lipoic acid-terminated polyethylene glycol (LA-PEG). The obtained PEGylated

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MoS2 nanosheets (MoS2-PEG) are stable in physiological solutions and could be loaded with a PS agent, chlorin e6 (Ce6), for the delivery of photodynamic therapy. Compared with free Ce6, Ce6-loaded MoS2-PEG (MoS2-PEG/Ce6) exhibits enhanced cellular uptake, which could be further promoted under mild NIR photothermal heating to achieve even more effective cancer cell killing. Upon intravenous injection, MoS2-PEG/Ce6 shows highly effective accumulation in the tumor as revealed by photoacoustic imaging. Combined photothermal and photodynamic therapy is then carried out, achieving an obviously enhanced tumor growth inhibition effect compared with monotherapy.

Results and discussion Single-layered MoS2 nanosheets were prepared in a large batch using n-butyl lithium to insert and exfoliate the bulk MoS2 (ref. 38)

Fig. 1 Synthesis and characterization of MoS2-PEG. (a) A scheme showing fabrication process of MoS2-PEG. (b and c) AFM images of MoS2 nanosheets before (b) and after (c) PEGylation; below are their corresponding line profiles. (d) Photographs of MoS2 and MoS2-PEG in water or PBS. (e) UV-vis-NIR absorbance spectra of MoS2 and MoS2PEG. (f and g) IR thermal images (f) and photothermal heating curves (g) of MoS2-PEG solutions irradiated by an 808 nm laser (0.7 W cm2) for 5 min.

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(Fig. 1a). Aer removing incompletely delaminated MoS2 by centrifugation and organic residues by dialysis, these nanosheets were characterized by atomic force microscopy (AFM) (Fig. 1b), which revealed the average thickness of the as-prepared MoS2 nanosheets to be 1 nm, indicating that the majority of those nanosheets were single layered. Chemically exfoliated MoS2 nanosheets upon preparation, although soluble in water, would rapidly aggregate in the presence of salts. Therefore, a biocompatible hydrophilic polymer, PEG, was used to modify those MoS2 nanosheets, whose defects sites were anchored by sulfur atoms in LA-PEG. An AFM image of these obtained MoS2-PEG nanosheets showed an obvious size reduction (Fig. 1c), which was also conrmed by DLS data (ESI Fig. S1†). Moreover, PEGylation could greatly enhance the physiological stability of MoS2 nanosheets (Fig. 1d) while still retaining high absorbance in the NIR region (Fig. 1e). The concentration of MoS2-PEG nanosheets was quantied by its mass extinction coefficient at 800 nm (28.4 L g1 cm1),34 which was much higher than that of graphene oxide (GO) (3.6 L g1 cm1) and comparable to that of reduced GO (rGO) (24.6 L g1 cm1). As a result of such strong NIR absorbance, it was found that MoS2-PEG nanosheets appeared to be a rather effective photothermal agent under NIR laser irradiation (808 nm), which could lead to the rapid heating of MoS2-PEG solutions in a concentration-dependent manner (Fig. 1f and g). Single-layered MoS2 nanosheets, similar to graphene,39–41 have a very high surface area-to-mass ratio due to their twodimensional structure and could be loaded with aromatic chemotherapy drugs, such as doxorubicin with high loading capacity, as presented in our previous study.37 As a widely used photosensitizer for photodynamic therapy, chlorin e6 (Ce6) is able to generate cytotoxic singlet oxygen under light exposure to kill cancer cells. To load Ce6 on PEGylated MoS2 nanosheets, 0.5 mL Ce6 (5 mg mL1 in DMSO) was dropwise added into 5 mL MoS2-PEG (0.2 mg mL1) in a phosphate buffer (PB, 20 mM)

Fig. 2 Ce6 loading on MoS2-PEG. (a) A scheme showing MoS2-PEG/ Ce6 nanosheets for Ce6 loading and combined photothermal and photodynamic therapy. (b) UV-vis-NIR absorbance spectra of MoS2PEG and MoS2-PEG/Ce6. (c) SOSG test of singlet oxygen generated by free Ce6 and MoS2-PEG/Ce6 at the concentration of 1 mM Ce6 under 660 nm light exposure (5 mW cm2) for 20 min.

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at pH ¼ 6.0 (Fig. 2a). From UV-vis spectra of MoS2-PEG before and aer Ce6 loading, the Ce6 loading ratio (Ce6 : MoS2 weight ratio) was determined to be 30% (Fig. 2b), which was much higher than that on PEGylated GO (15%).12 The production of singlet oxygen (SO) was tested using the singlet oxygen sensor green (SOSG) probe. It was revealed that although the uorescence of Ce6 was signicantly quenched aer its loading onto MoS2-PEG nanosheets (ESI Fig. S2†), the SO production ability of MoS2-PEG/Ce6 under 660 nm light irradiation was still rather effective and only slightly lower than that of free Ce6 (Fig. 2c). To study the use of PEGylated MoS2 for the delivery of the photodynamic agent, we cultured 4T1 cells with free Ce6 or MoS2-PEG/Ce6 at the same Ce6 concentration (1 mM) for different periods of time. Confocal uorescence microscopy images were then recorded (Fig. 3a), revealing time-dependent uptake of both formulations of Ce6 by 4T1 cells. Notably, although the uorescence of Ce6 was partially quenched by MoS2 nanosheets in the MoS2-PEG/Ce6 formulation, cells incubated with MoS2-PEG/Ce6 still showed much stronger intracellular Ce6 uorescence compared to those incubated with free Ce6, suggesting remarkably enhanced intracellular delivery of Ce6 using MoS2-PEG as the nanocarrier. To further determine the photodynamic therapeutic efficacy, 4T1 cells were cultured with free Ce6 or MoS2-PEG/Ce6 for 12 h and then exposed to the 660 nm light to trigger photodynamic

Intracellular delivery of Ce6 for photodynamic therapy. (a) Confocal fluorescence images of 4T1 cells after incubation with free Ce6 or MoS2-PEG/Ce6 ([Ce6] ¼ 1.25 mM, [MoS2] ¼ 4 mg mL1) for different periods of time. Red and blue colors represent Ce6 fluorescence- and DAPI-stained cell nuclei, respectively. (b and c) Relative viabilities of 4T1 cells after incubation with free Ce6 or MoS2-PEG/Ce6 at various concentrations for 12 h and then treated without (b) or with (c) 660 nm light irradiation at 5 mW cm2 for 30 min. The cells were then incubated for additional 24 h before the MTT assay. Error bars were based on four parallel samples. Fig. 3

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therapy. Without light irradiation, both free Ce6 and MoS2-PEG/ Ce6 did not show any appreciable dark toxicity at the tested concentrations. On the other hand, if the cultured cells were exposed to the 660 nm light at the power density of 5 mW cm2 for 30 min, signicant concentration-dependent photodynamic cancer cell killing was then observed, with MoS2-PEG/Ce6 appearing to be much more potent than free Ce6 at the same Ce6 concentrations (Fig. 3b and c). Such dramatically enhanced photodynamic therapeutic efficacy offered by MoS2-PEG/Ce6 is attributed to the greatly promoted cellular uptake of photosensitizing molecules that produce singlet oxygen inside cells under light exposure for effective cancer cell killing. It has been found that mild hyperthermia is able to increase cell membrane permeability and promote cellular uptake of various agents.10 To test how and whether the photothermal effect attributed by MoS2 nanosheets could be utilized to further enhance the delivery of photodynamic therapy, we cultured free Ce6 or MoS2-PEG/Ce6 ([Ce6] ¼ 2 mM) with cells for 20 min with or without 808 nm laser irradiation at the power density of 0.5 W cm2, which would introduce moderate photothermal heating and increase the temperature of MoS2-PEG/Ce6 containing the cell culture medium to 43  C, a temperature not high enough to directly induce cell death.42 Flow cytometry was employed to quantitatively determine the cell uptake of Ce6 under various conditions. Whereas free Ce6-incubated cells either in dark or with irradiation showed rather weak uorescence, the uorescence of MoS2-PEG/Ce6-treated cells appeared to be much stronger and could be further enhanced by the photothermal heating with an 808 nm laser (Fig. 4a). To eliminate the quench effect of Ce6 uorescence in the MoS2-PEG/ Ce6 formulation, cancer cells were lysed aer various treatments and extracted by NaOH to deprotonate the three carboxyl acid group of Ce6, which was thus detached from MoS2-PEG and would show recovered uorescence (data not shown). Consistent to the ow cytometry and confocal data, MoS2-PEG was able to considerably shuttle more Ce6 molecules into cells compared to Ce6, and mild photothermal heating could signicantly increase the cellular uptake of MoS2-PEG/Ce6 by nearly two-fold (Fig. 4b). Next, we wondered if the combination of PTT and PDT would offer any synergistic effect in the killing of cancer cells. In our in vitro experiments, 4T1 cells were cultured with pure MoS2-PEG, Ce6 or their compounds MoS2-PEG/Ce6 ([Ce6] ¼ 2 mM) for 20 min with or without 808 nm laser irradiation (0.5 W cm2) for PTT, immediately followed by another 20 min of incubation or 660 nm laser irradiation (5 mW cm2) for PDT. Aer additional incubation for 24 h, the relative cell viabilities were then measured by the MTT assay. For cells treated with MoS2-PEG, their viabilities were maintained at high levels even aer 808 nm laser irradiation, suggesting that the mild hyperthermia under this condition was not sufficient to kill cancer cells. For cells incubated with Ce6, 661 nm laser irradiation caused 20% of cell death via ROS production, whereas 808 nm irradiation showed no inuence to the PDT efficacy as expected. In marked contrast, MoS2-PEG/ Ce6-incubated cells, if treated by both types of lasers with wavelengths at 808 nm and 660 nm to trigger photothermal and

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Fig. 4 Photothermally enhanced photodynamic therapy of 4T1 cells. (a) Flow cytometry measurement of Ce6 fluorescence intensities in 4T1 cells after 20 min incubation with free Ce6 or MoS2-PEG/Ce6 ([Ce6] ¼ 2 mM, [MoS2] ¼ 6.4 mg mL1) with or without 808 nm laser irradiation (0.5 W cm2). (b) Fluorescence intensities of cell lysates after various treatments as previously described. Compared with free Ce6, MoS2-PEG/Ce6 showed considerably higher cellular uptake that could be further enhanced by the photothermal heating. (c) Relative viabilities of 4T1 cells after incubation with MoS2-PEG, free Ce6 or MoS2-PEG/Ce6 ([Ce6] ¼ 2 mM, [MoS2] ¼ 6.4 mg mL1) for various treatments. Controls were cells treated only with materials and without light exposure. PTT and PDT were conducted by 808 nm and 660 nm light exposure, at power densities of 0.5 W cm2 and 5 mW cm2, respectively, for 20 min. Error bars were based on four parallel samples.

photodynamic effects, respectively, were effectively killed (Fig. 4c). Such remarkable synergistic therapeutic effects offered by the combination therapy could be partially attributed to the photothermally enhanced intracellular delivery of PS agents. Next, we would like to demonstrate combined photothermal and photodynamic therapy using MoS2-PEG/Ce6 in an animal tumor model. Before cancer therapy experiments, we need to conrm the tumor accumulation ability of our nanoagent. Photoacoustic tomography (PAT) functions by the detection and reconstruction of ultrasound signals resulted from thermal expansion of tissues when under irradiation with a pulsed laser.43 Compared with conventional optical imaging, PAT offers greatly improved in vivo spatial resolution and high contrast in relatively deep tissues.44 PEGylated MoS2 nanosheets with high absorbance in the NIR region are perfect contrast agents for photoacoustic tomography (PAT) imaging. Herein, mice bearing 4T1 tumors were intravenously (i.v.) injected with MoS2-PEG/ Ce6 ([MoS2-PEG] ¼ 6.85 mg kg1) and imaged under a PAT imaging system. Encouragingly, whereas only major blood vasculatures were observed before injection, prominent photoacoustic signals were seen in the tumor 24 h aer the injection of MoS2-PEG/Ce6, suggesting a rather efficient tumor uptake of those PEGylated nanosheets, likely as a result of the enhanced permeability and retention effect of cancerous tumors (Fig. 5a). IR thermal imaging was then carried out to test whether PEGylated MoS2 accumulated in the tumor would be sufficient

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Fig. 5 MoS2-PEG/Ce6 used for in vivo combination therapy. (a) Photoacoustic images of tumors in mice before and 24 h after i.v. injection of MoS2-PEG/Ce6 ([Ce6] ¼ 2.0 mg kg1, [MoS2-PEG] ¼ 6.85 mg kg1). (b) IR thermal images of tumors in mice i.v. injected with MoS2-PEG/Ce6 ([Ce6] ¼ 2.0 mg kg1, [MoS2-PEG] ¼ 6.85 mg kg1) under irradiation. The power density of 808 nm laser irradiation was 0.4 W cm2, and the duration time was 20 min. (c) Surface temperature changes of tumors monitored by the IR thermal camera during laser irradiation as indicated in (b). (d) Relative tumor volume curves of different groups of mice after the various treatments indicated. (e) Average body weight of mice after various treatments indicated. Five mice were used in each group. Error bars are based on standard errors of the mean (SEM).

to induce effective photothermal heating. 4T1-tumor-bearing mice were exposed to the 808 nm laser at a low power density of 0.5 W cm2 24 h aer i.v. injection with MoS2-PEG/Ce6. As shown in Fig. 5b, whereas the NIR irradiation of tumors on saline-injected mice showed signicant heating effect, the tumor temperature in MoS2-PEG/Ce6-injected mice rapidly increased to 44.8  C (Fig. 5c). At last, the in vivo cancer treatment efficacy of combination therapy induced by MoS2-PEG/Ce6 was evaluated. Mice bearing 4T1 tumors (volume  70 mm3) were randomly divided into six groups (n ¼ 5 per group): (1) untreated control, (2) i.v.-injected with free Ce6 and irradiated with 660 nm light (PDT), (3) i.v.injected with MoS2-PEG/Ce6, (4) i.v. injected with MoS2-PEG/ Ce6 and irradiated with 660 nm light (PDT), (5) i.v.-injected with MoS2-PEG/Ce6 and irradiated with 808 nm laser (PTT), (6) i.v.injected with MoS2-PEG/Ce6 and treated by both PDT and PTT. The doses of Ce6 and MoS2-PEG were xed at 2.0 mg kg1 and 6.85 mg kg1, respectively, in the aforementioned groups. PTT and PDT were separately introduced by 808 nm (0.45 W cm2) and 660 nm (5 mW cm2) light irradiations, respectively, for 20 min. The tumor growth in various groups was monitored aer various treatments (Fig. 5d). As expected, i.v. injection of MoS2PEG/Ce6 without light exposure showed no appreciable effect on the tumor growth. Photodynamic treatment induced by either Ce6 or MoS2-PEG/Ce6, as well as mild photothermal heating induced by MoS2-PEG/Ce6, although able to partially

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control the tumor growth in the rst few days aer treatment, appeared to not be as effective in the longer term. In contrast, tumors in mice aer MoS2-PEG/Ce6-injection and combined PTT and PDT showed signicantly delayed tumor growth, demonstrating the obvious advantage and synergistic effect of combination therapy in comparison to monotherapies. Moreover, the average body weight in each group showed no significant variation aer treatment, indicating that the combination therapy introduced by MoS2-PEG/Ce6 exerted no signicant side effects to the treated mice (Fig. 5e). Hematoxylin and eosin (H & E)-stained images (ESI Fig. S3†) of major organs of mice also indicated no obvious toxicity to normal organs of mice aer i.v. injection of MoS2-PEG/Ce6 and the combination therapy, consistent to the results in our previous study, in which PEGylated MoS2 nanocarriers exhibited no appreciable toxicity to animals as examined by blood biochemistry and complete blood panel examinations.37

Conclusions In summary, we have developed PEGylated MoS2 nanosheets as a drug carrier for the delivery of the photosensitizing agent Ce6. It is found that Ce6 molecules could be effectively shuttled into cells by MoS2-PEG for improved photodynamic cancer-cell killing. Utilizing the strong NIR absorbance of MoS2 nanosheets, photothermally enhanced photodynamic therapy is demonstrated in vitro, owing to the further increased intracellular delivery of Ce6 under mild photothermal heating. Moreover, combined PTT and PDT are further demonstrated in vivo upon systemic administration of MoS2-PEG/Ce6 and subsequent light irradiations, achieving outstanding synergistic effect in delaying tumor growth in our animal experiments. Our work shows that TMDCs with appropriate surface coatings, such as PEGyalted MoS2 as presented in this work, could be another type of 2D nanomaterials with great promise in biomedical applications.

Materials and methods 1. Materials All chemicals, unless specied otherwise, were purchased from Sigma-Aldrich and used as received. PEG polymers were purchased from PegBio, Suzhou, China. All cell culture related reagents were purchased from Hyclone. 2. Synthesis of single-layer MoS2-PEG nanosheets MoS2 nanosheets were synthesized following the modied Morrison method.45 In brief, 5 mL of 1.6 M n-butyllithium solution in hexane was added to dissolve 0.5 g MoS2 crystal, followed by stirring for 2 days inside a nitrogen glove box. Aer intercalation by lithium, MoS2 solution was centrifugated at 8000 rpm for 10 min and washed repeatedly with hexane to remove excess lithium and other organic residues. Intercalated MoS2 sample was then taken out from the glove box and immediately ultrasonicated in water for 1 h, obtaining exfoliated MoS2, which was then centrifuged in 3000 rpm to remove

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unexfoliated MoS2 and excess LiOH in the precipitates. The supernatant was dialyzed against deionized water using membranes with molecular weight cut-off (MWCO) of 14 kDa for 2 days to remove lithium compounds and other residue ions, obtaining MoS2 nanosheets dispersed in water for future use. Lipoic acid-modied PEG (LA-PEG) was synthesized following a reported protocol.46 For the PEG modication, 10 mg of LA-PEG was added into 1 mg of MoS2 nanosheets, dispersed in 2 mL of water. Aer sonication for 20 min and stirring overnight, excess PEG polymers were removed by centrifugal ltration with 100 kDa MWCO lters (Millipore) and several times of water washing. The obtained MoS2-PEG nanosheets were highly water soluble and stored at 4  C before future use. 3. Ce6 loading and releasing Ce6 loading onto MoS2-PEG nanosheets was conducted following the same protocol used in our previous work.12 Herein, we selected an appropriate ratio for efficient Ce6 loading. 0.5 mL Ce6 (5 mg mL1 in DMSO) was dropwise added into 5 mL PEGylated MoS2 nanosheets (0.2 mg mL1) in a phosphate buffer (PB, 20 mM) at pH ¼ 6.0. Aer stirring at room temperature for 24 h, excess Ce6 was washed away with water by ltering the solution through a 100 kDa Millipore lter several times. The formed MoS2-PEG/Ce6 were redispersed in 1 mL deionized water and stored at 4  C. Ce6 loading ratios were calculated by recording the UV-vis-NIR spectra of MoS2-PEG before and aer drug loading. Aer normalization with pure MoS2-PEG at 808 nm, the Ce6 absorption peak at 404 nm was used to determine the Ce6 concentration in our MoS2-PEG/Ce6 sample. To determine drug release kinetics, 1 mL MoS2-PEG/Ce6 was dialyzed against 19 mL PBS at pH ¼ 5.0 or 7.4. At xed time points, 2 mL of solution outside the dialysis bag was collected and measured by the absorbance spectrometer to determine the concentrations of released Ce6 and then poured back to ensure the constant volume. 4. Detection of singlet oxygen The generation of singlet oxygen was determined by the singlet oxygen sensor green (SOSG) dye following the standard procedure. In brief, SOSG (2.5 mM) was added to 1 mL free Ce6, MoS2PEG/Ce6 or MoS2-PEG solutions. The solution was then irradiated by a lamp passing a 660 nm band pass lter (power density ¼ 5 mW cm2) for 30 min. The presence of singlet oxygen would lead to the increase of SOSG uorescence (lex ¼ 494 nm, lem ¼ 534 nm), which was thus utilized to determine the production of SO by various samples under light exposure. 5. Cellular uptake of Ce6 4T1 murine breast cancer cell line was originally obtained from the American Type Culture Collection (ATCC) and cultured in the recommended medium under 37  C within 5% CO2 atmosphere. 4T1 cells adhered to glass slides in 24-well plates were incubated with MoS2-PEG/Ce6 or free Ce6 at the same concentration of Ce6 ([Ce6] ¼ 1.25 mM) for 30 min, 1 h, 2 h and 6 h. All Nanoscale, 2014, 6, 11219–11225 | 11223

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cells were washed twice with PBS and then xed by 0.2 mL glutaraldehyde with cell nuclei stained by 40 ,6-diamidino-2phenylindole (DAPI). Confocal uorescence imaging of cells was then performed using a Laica SP5 laser scanning confocal microscope. The uorescence of Ce6 (emission range 650– 750 nm) was excited by using a 633 nm laser.

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6. Photodynamic therapy of 4T1 cells For photodynamic therapy experiments, 4T1 cells were incubated with free Ce6 or MoS2-PEG/Ce6 at various concentrations of Ce6 for 12 h. Aerward, the cells were exposed to 660 nm light with a power density of 5 mW cm2 for 30 min. Aer being further cultured for another 24 h, the relative cell viabilities were then measured by MTT assay. Cells without light exposure were used as the control. 7. Hyperthermia-enhanced cellular uptake of Ce6 4T1 cells pre-seeded in 24-well plates were added with MoS2PEG/Ce6 or free Ce6 at the nal concentration of 2 mM Ce6, immediately followed by 808 nm laser irradiation at an intensity of 0.5 W cm2 for 20 min. Those cells cultured with materials but without irradiation were used as controls. All cells were washed twice with PBS and analyzed by a ow cytometry (FACS Calibur, Bectone Dickinson). The collected data were analyzed using the FlowJo soware. We followed a previously reported method to quantitatively measure the cellular uptake of Ce6.12 4T1 cells were cultured in 35 mm culture dishes containing MoS2-PEG/Ce6 or free Ce6 at the equivalent Ce6 concentration of 2 mM for 20 min with or without laser irradiation (808 nm, 0.5 W cm2). Cells were then washed with PBS twice, harvested using a cell scraper and then dissolved in 1 mL lysis solution (2% sodium dodecyl sulfate) for 2 h to obtain homogeneous solutions. 1 mL 0.2 M NaOH solution was then added into each culture dish for overnight incubation to extract Ce6. Such base treatments would deprotonate the three carboxyl acid group of Ce6 and thus detach it from MoS2-PEG. The uorescence spectra of the obtained solutions were measured under 404 nm excitation. For combination therapy, MoS2-PEG, Ce6, or MoS2-PEG/Ce6 ([MoS2-PEG] ¼ 6.4 mg mL1, [Ce6] ¼ 2 mM) were added into a 4T1 cell-culture solution, immediately followed by irradiation with 808 nm laser at 0.5 W cm2 for 20 min or kept under darkness as the control. Photodynamic treatment was then conducted with 660 nm light irradiation at the power density of 5 mW cm2 for 20 min. Aer additional incubation for 24 h, the relative cell viabilities were then measured by the MTT assay. 8. Combination therapy in vivo All animal experiments were carried out under the protocols approved by the Soochow University Laboratory Animal Center. To develop the tumor model, 4T1 cells (1  106) suspended in 40 mL of PBS were subcutaneously injected into the back of each Balb/c mouse. Aer the tumor volume reached 70 mm3, mice were randomly divided into six groups (n ¼ 5 per group) for various treatments as described in Fig. 5. The doses of Ce6 and MoS2-

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PEG were kept at 2.0 mg kg1 and 6.85 mg kg1, respectively. Light treatments were conducted 8 h aer the injection of various agents. We used 660 nm light irradiation for 20 min at 5 mW cm2 and 808 nm light irradiation for 20 min at 0.45 W cm2 for photodynamic and photothermal treatments, respectively. The tumor sizes were measured aer treatment by a digital caliper every 2 days for 3 weeks. The tumor volume was calculated according to the following formula: width2  length/ 2. Relative tumor volumes were calculated as V/V0 (V0 was the tumor volume when the treatment was initiated).

Acknowledgements This work was partially supported by the National Basic Research Programs of China (973 Program) (2012CB932600, 2011CB911002), the National Natural Science Foundation of China (51222203, 51132006), and a project funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.

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Nanoscale, 2014, 6, 11219–11225 | 11225

Combined photothermal and photodynamic therapy delivered by PEGylated MoS2 nanosheets.

Single- or few-layered transitional metal dichalcogenides, as a new genus of two-dimensional nanomaterials, have attracted tremendous attention in rec...
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