Radar-like MoS₂ nanoparticles as a highly efficient 808 nm laser-induced photothermal agent for cancer therapy

Abstract

MoS₂, a typical transition-metal dichalcogenide, has attracted increasing attention in the field of biomedicine due to its preeminent properties. In this paper, MoS₂ nanoparticles with radar-like shapes were demonstrated as 808 nm laser-induced photothermal agents with outstanding photothermal performance. According to our calculations, the radar-like morphology endowed the MoS₂ nanoparticles with higher photothermal conversion efficiency (53.3%) compared to other morphologies including nanoflowers, microspheres and irregular nanoparticles. Cytotoxicity assay indicated that the radar-like nanoparticles were biocompatible. Results of in vitro photothermal therapy and apoptosis assay indicated that tumor cells treated with MoS₂ radar-like nanoparticles can be seriously damaged. Photothermal therapy on tumor-bearing mice was carried out to further verify the anti-cancer properties of the MoS₂ radar-like nanoparticles. All the aforementioned results suggested that the MoS₂ radar-like nanoparticles exhibit enormous potential for cancer photothermal therapy.

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1. Introduction

Therapy technology based on thermal energy has attracted extensive attention in recent years in the medical field, particularly oncotherapy.¹ Photothermal therapy (PTT) is a non-invasive approach using light-absorbing material to convert luminous energy into heat.^2,3 Near-infrared (NIR)-induced PTT, which uses an NIR laser (emission wavelength in the range of 700–1100 nm) for treatment, has generated research interests because of the low absorbance of NIR irradiation in biological tissues.^4–6 Lasers with wavelengths of 808 nm can provide a moderate depth of penetration and power; hence, using 808 nm lasers to generate heat energy during therapy is appropriate.^7,8

Photothermal agents (PTAs), which are required for efficient PTT, play a significant role in the conversion process.⁹ Some organic chromophores (indocyanine green, polypyrrole)^7,10,11 and carbon-based materials (graphene oxide, reduced graphene oxide and carbon nanotubes)^12–14 were developed as PTAs owing to their intrinsic high absorbances in the NIR region. Noble metal materials including Pd nanosheets and Au nanoparticles (nanorods, nanocages, nanoshells, hollow nanospheres, nanorattles)^3,15–22 have also received considerable interest in this field. Their tunable optical properties based on surface plasmon resonance (SPR) effects make them promising PTAs. In addition, certain semiconductors have been reported as potential PTAs.^23–25 Particularly, flower-like CuS superstructures composed of nanoparticles generate stronger light absorbance than their building blocks, indicating that a designed morphology may result in a higher photothermal conversion efficiency (PCE).²⁶

Transition-metal dichalcogenides (TMDs), a branch of two-dimension materials that have emerged in the past decades, are receiving extensive attention by researchers.²⁷ Molybdenum disulfide (MoS₂), a typical TMD, has been widely applied in transistors, hydrogen evolution, lithium-ion batteries, photodetectors, and gas sensors.^28–33 The remarkable photothermal performance of MoS₂ was first reported for chemically exfoliated MoS₂ nanosheets.³⁴ This research opened up a new path for the application of MoS₂ in PTT. Due to its favorable biocompatibility^35–37 and strong absorbance in the NIR window, various studies on MoS₂ have been recently carried out in the field of biomedicine.^38–42

Herein, MoS₂ radar-like nanoparticles (RNPs) were synthesized using a facile and surfactant-free hydrothermal method and demonstrated as an outstanding 808 nm laser-induced photothermal agent. The photostability and cytotoxicity of the MoS₂ RNPs were then investigated. To demonstrate the anti-cancer performance of the MoS₂ RNPs, they were used for the in vitro and in vivo PTT of tumor cells, and apoptosis assay was carried out.

2. Experimental section

2.1 Reagents and materials

All the chemicals were analytical grade and used without further purification. Sodium molybdate dehydrate (Na₂MoO₄·2H₂O) was purchased from Sinopharm Chemical Reagent Co. Ltd. Heptamolybdate tetrahydrate ((NH₄)₆Mo₇O₂₄·4H₂O), thioacetamide (CH₃CSNH₂) and thiourea (CS(NH₂)₂) were purchased from Aladdin Reagent Co. Ltd. Deionized water (18.25 MΩ cm) was used during the experiments.

2.2 Synthesis of MoS₂ RNPs

Typically, 0.4 mmol of sodium molybdate dihydrate (Na₂MoO₄·2H₂O) and 1 mmol of thioacetamide (CH₃CSNH₂) were dissolved in 30 ml deionized water under magnetic stirring, and the transparent mixture solution was then transferred into a Teflon-lined stainless-steel autoclave. After sealing, the autoclave was heated to 200 °C for 36 h. The resulting products was washed several times with deionized water and dried overnight at 60 °C.

2.3 Characterization

The X-ray diffraction (XRD) patterns of the above products were obtained using a Bruker D8 advanced X-ray diffractometer with graphite monochromatized Cu K_α radiation (λ = 1.5418 Å). Scanning electron microscopy (SEM) was performed using a JEOL JSM-6700F field-emission scanning electron microscope (operating voltage = 5 kV). Transmission electron microscopy (TEM) images were obtained on a JEOL JEM-2100 transmission electron microscope (operating voltage = 200 kV). Element mapping patterns were obtained using an X-ray micro-analyzer attached to the JEM-2100 transmission electron microscope. Absorbance spectra were collected using a Purkinje TU-1901 spectrometer.

2.4 Laser heating of MoS₂ in water

Photothermal heating effects were measured using an infrared laser system (808 nm, BWT, China) operating in continuous-wave mode (power density = 0.5 W cm⁻²). After equilibrating to ambient temperature, a 10 mm-diameter circular well containing 0.3 ml of aqueous dispersion was placed perpendicularly under the laser beam, and an infrared thermal imaging camera (S6-a, IRS, China) was then used to record the temperature.

2.5 Cytotoxicity assays

The in vitro cytotoxicity of the MoS₂ RNPs was measured by cell counting kit-8 (CCK-8) assay (Bestbio, China), similar to in a previous study.⁴³ 4T1 cells growing in log phase were seeded onto a 96-well plate at a density of 5000 per well. The previous media were replaced with MoS₂ RNPs solutions (100, 50, 25 or 10 ppm) and then incubated for 24, 36 or 48 h at 37 °C under 5% CO₂. After incubation, the solutions were removed, and each well was washed three times with phosphate-buffered saline (PBS) solution. Ten microliters of CCK-8 reagents was then added in each well, and the plate was kept at 37 °C for an additional 1 h. A microplate reader (EMax Plus, Molecular Devices, USA) was used to measure the optical density (OD) value at a wavelength of 450 nm.

2.6 Photothermal therapy in vitro

4T1 cells were seeded onto a 24-well plate at a density of 10 000 per well. The previous media were replaced with PBS and MoS₂ RNPs solutions (100 ppm) and incubated for 1 h. After incubation, each well was separately placed under the irradiation of an 808 nm laser (power density = 0.5 W cm⁻²) for 300 s. The cells were washed three times with PBS solution before being stained by 0.4% trypan blue solution. An inverted biological microscope (IX-51, Olympus, Japan) was used to obtain the images of the labeled cells.

2.7 Apoptosis assay

An FITC Annexin V Apoptosis Detection Kit I (BD, USA) was used for flow cytometry. 4T1 cells were seeded onto a six-well plate at a density of 30 000 per well. The previous media were replaced with PBS and MoS₂ RNP solutions (100 ppm) and separately irradiated using an 808 nm laser (power density = 0.5 W cm⁻²) for 300 s. The cells were collected and washed twice with cold PBS solution. After resuspension in 100 μl binding buffer, 5 μl Annexin V-FITC and 5 μl propidium iodide (PI) were added for staining. After 15 min, the cells were washed and again resuspended in 400 μl binding buffer. A flow cytometer (FACSCalibur, BD, USA) was used to obtain the apoptosis data.

2.8 Photothermal therapy in vivo

All animal experiments in this study were performed according to protocols approved by the Animal Center of Qilu Hospital of Shandong University and were in accordance with the policies of the National Ministry of Health. Mice were injected subcutaneously with 5000 4T1 cells in the left hind leg and fed for 21 d before the experiment. The mice were randomly divided into two groups and then injected intratumorally with 0.1 ml 100 ppm MoS₂ RNPs PBS dispersion (experimental group) or 0.1 ml PBS solution (control group). After 2 h, half of the mice in each group were irradiated with an 808 nm laser (power density = 0.5 W cm⁻²) for 600 s with the laser spot focusing on the tumor. All groups were then fed under the same conditions until execution. The tumors removed from the mice were embedded in paraffin, sectioned into slices, and then stained with hematoxylin/eosin. A biological microscope (BX-53, Olympus, Japan) was used to obtain histological images.

3. Results and discussion

3.1 Characteristics

As shown in the XRD patterns (Fig. S1a†), all the main peaks can be indexed to hexagonal phase MoS₂ (JCPDS 73-1508). Element mapping was used to further verify the composition of the MoS₂ RNPs (Fig. S2†). SEM and TEM measurements were performed to reveal the morphologies of the products. A radar-like shape (Fig. 1a and b) with a diameters of approximately 200 nm and an edge thickness around 10 nm was observed. The SEM images of other morphologies can be found in the ESI (Fig. S1(b–d)†). Fig. 1c shows the absorbance spectra of MoS₂ RNPs aqueous dispersions at different concentrations under room temperature. As shown in the inset of Fig. 1c, the transparent solutions indicated the good water solubility of the MoS₂ RNPs.

Fig. 1 (a) SEM image of the MoS₂ RNPs. (b) TEM image of an MoS₂ RNP; the inset images reveal that the edge thickness is around 10 nm. (c) Absorbance profile of MoS₂ RNPs at different concentrations. The inset is the photograph of each solution.

3.2 In vitro photothermal performance experiment

In order to investigate the photothermal heating of the MoS₂ RNPs, solutions of a series of concentrations were prepared and irradiated by a continuous-wave laser (wavelength = 808 nm, power density = 0.5 W cm⁻²), and the temperature change of each solution was measured as a function of time. In order to ensure the accuracy of measurement, an infrared thermal camera was used instead of thermocouple to avoid interaction between the laser and the couple. Fig. 2a and b show the remarkable photothermal performance of the MoS₂ RNPs. As observed, the aqueous solution with a concentration of 100 ppm shows the highest increase in temperature (up to 23 °C) after irradiation for 300 s under the 808 nm laser, which is nearly four times as great as that reported in a previous study (6 °C in 300 s).³⁸ We also measured the photothermal performance of MoS₂ INPs, nanoflowers and microspheres under same conditions, as shown in Fig. 3a. It has been suggested in previous studies that the morphology and structure have noticeable influences on the optical properties of nanoparticles.^26,44,45 Here, the superior photothermal performance could be attributed to the radar-like morphology, which acted as a reflector for the incoming laser (as shown in Fig. 2c) and hence improved the average frequency of laser exposure of a single particle.

Fig. 2 (a) Temperature changes of aqueous solutions of MoS₂ RNPs with different concentrations under 808 nm laser irradiation as a function of time. The inset is a plot of the temperature change over 300 s versus RNP concentration. (b) Infrared thermal images of MoS₂ RNP aqueous solutions (100 ppm) irradiated for the noted period of time. (c) Schematic pattern of the laser reflection on an MoS₂ nanosheet (left) and RNP (right); n stands for the average frequency of laser exposure. (d) The temperature change of MoS₂ RNP aqueous solution (100 ppm) under 808 nm laser irradiation for 10 ON/OFF power cycles. (e) Absorbance profile of MoS₂ RNP aqueous solution (100 ppm) before and after 10 laser power ON/OFF cycles. No significant change was observed according to the absorbance curve before and after the cycles.

Fig. 3 (a) Temperature changes of aqueous solutions (100 ppm) of MoS₂ RNPs, MoS₂ nanoflowers and MoS₂ microspheres under 808 nm laser irradiation as a function of time. (b) The temperature change of the MoS₂ RNP aqueous solution (100 ppm) upon irradiation with the 808 nm laser for 600 s and then shutting down the laser power. (c) Calculation of time constant τ_s by applying the linear time data of cooling versus negative natural logarithm of dimensionless driving force temperature θ. (d) Plot of PCE for MoS₂ RNPs, MoS₂ nanoflakes (literature⁴²), MoS₂ nanosheets (literature⁴⁰), MoS₂ INPs, MoS₂ nanoflowers and MoS₂ microspheres.

The photostability of the MoS₂ RNPs was then studied. An aqueous solution (concentration = 100 ppm) was irradiated with an 808 nm laser (power density = 0.5 W cm⁻²) for 10 cycles of power ON/OFF. For each cycle, the solution was irradiated for 300 s and then allowed to cool naturally for 300 s. The temperature change of the entire process was recorded as shown in Fig. 2d. No significant change was observed in absorbance after 10 ON/OFF cycles (Fig. 2e).

3.3 Calculation of photothermal conversion efficiency

Since PCE is a critical factor for the heating performance and PTT application, calculations were carried out to further verify the superior properties of MoS₂ RNPs. To achieve the measurement, we use a method similar to that reported in the literature.⁴⁶ Aqueous dispersions were continuously irradiated with an 808 nm laser for 600 s; thereafter, the laser was shut off, and the dispersion was cooled naturally. The temperature change of the entire process was recorded three times under same conditions, as shown in Fig. 3c and S3(a and c).†

According to Roper's report,⁴⁷ the PCE can be calculated using eqn (1):

where η_T is the photothermal conversion efficiency; T_max and T_amb are the maximum system temperature and ambient temperature, respectively; Q₀ stands for the heat loss caused by the absorbance of the container, which was measured independently to be 25.1 mW using pure water instead of the dispersions; I is the laser power (500 mW), A_λ is the absorbance of the MoS₂ RNPs (0.628, wavelength = 808 nm); h is the heat-transfer coefficient, and S is the radiative heat transfer surface area. The product hS can be calculated using eqn (2):where m_D and C_D stand for the mass and heat capacity of the solvent, respectively. τ_s is the time constant of the sample system and can be determined by applying cooling time data versus the negative natural logarithm of θ (as shown in Fig. 3c and Fig. S3b, d and f†), where θ is a dimensionless driving force temperature and can be calculated by eqn (3) as

Hence, the average PCE of the MoS₂ RNPs was calculated to be 53.3%, which is higher than those of single-layer MoS₂ (24.4%)⁴⁰ and MoS₂ nanoflakes (27.6%)⁴² reported in previous studies. For comparison, the PCEs of other morphologies were subsequently determined under the same experimental conditions (Fig. S4†) to be 23.1% (MoS₂ INPs), 15.2% (MoS₂ microspheres) and 18.2% (MoS₂ nanoflowers), indicating the superior photothermal conversion performance of the MoS₂ RNPs (Fig. 3d).

3.4 Cytotoxicity assay and PTT performance in vitro

CCK-8 assay was used to reveal the biocompatibility of the MoS₂ RNPs. No significant cytotoxicity was observed, even in the presence of the highest concentration (100 ppm) of MoS₂ RNPs; the corresponding cell viability was above 80% after 48 h of incubation (Fig. 4a).

Fig. 4 (a) Cell viability assay of the 4T1 cells treated with different concentrations of MoS₂ RNPs for different periods of time. (b) Images of 4T1 cells before (1 and 2) and after (3 and 4) irradiation with an 808 nm laser (power density = 0.5 W cm⁻²) for 300 s; groups 2 and 4 were treated with a 100 ppm MoS₂ RNPs solution, while groups 1 and 3 were treated with PBS. Cells dyed by trypan blue were identified as dead cells. (c and d) Flow cytometric analysis for apoptosis of 4T1 cells treated with PBS (c) and MoS₂ RNPs (d) after irradiation with an 808 nm laser (power density = 0.5 W cm⁻²) for 300 s; dots located in Q2 and Q3 correspond to early apoptosis and late apoptosis cells, respectively.

Since the MoS₂ RNPs do inconspicuous damage to the tumor cells without laser irradiation, we then studied the efficacy of PTT in vitro. As seen in Fig. 4b, the dead cells were dyed by trypan blue, while the viable cells were not. After irradiating with the 808 nm laser (power density = 0.5 W cm⁻²) for 300 s, the amount of dead cells among the cells treated with MoS₂ RNPs increased.

3.5 Apoptosis assay

It should be noted that cell apoptosis plays a significant role during cancer cell growth.^48,49 A study on laser-induced apoptosis was carried out in order to investigate the contribution of MoS₂ RNPs during the apoptosis process. Cells were irradiated by an 808 nm laser (power density = 0.5 W cm⁻²) for 300 s before the measurement. Flow cytometry was used to determine the cell apoptosis amount, as shown in Fig. 4c and d; each dot is a signal collected from a cell. Cells in the lower left quadrant (Q4) are considered viable, while cells in lower right (Q3), upper right (Q2) and upper left (Q1) quadrants correspond to early apoptosis, late apoptosis and dead, respectively. Here, a significant increase in apoptosis rate was observed in the cells treated with MoS₂ RNPs; the sum of Q2 and Q3 increased from 14.4% (control) to 54.3% (experimental), which demonstrated that MoS₂ RNPs can cause serious tumor cell apoptosis under irradiation with an 808 nm laser.

3.6 PTT performance in vivo

To evaluate the therapy efficiency for cancer cells in vivo, 4T1 tumor-bearing mice were injected intratumorally with PBS or MoS₂ RNP solutions and irradiated with an 808 nm laser for 600 s. Due to the possible radiation injury caused by the high-power-density laser, the power density used herein (0.5 W cm⁻²) was much lower than that used in previous studies.⁷ Infrared thermal images before and after irradiation showed that tumor tissues were efficiently heated in the experimental group. The following histological examination indicated severe necrosis in tumors treated with MoS₂ RNPs; we observed large-scale karyorrhexis 1 d after treatment (Fig. 5b(4)). The changes in tumor volume indicated that tumor growth was inhibited after treatment with MoS₂ RNPs and laser irradiation (Fig. S7†).

Fig. 5 (a) Infrared thermal images of 4T1 tumor-bearing mice before (1 and 2) and after (3 and 4) irradiation with an 808 nm laser (power density = 0.5 W cm⁻²) for 600 s. Tumor tissues (on left hind leg) treated with MoS₂ RNPs were efficiently heated. (b) Histological images of tumor tissues obtained from the control group (1 and 3) and the experimental group (2 and 4) 1 d after treatment. Panels 3 and 4 show enlarged versions of 1 and 2. Karyorrhexis of tumor cells can be observed in panel 4.

4. Conclusions

In summary, radar-like MoS₂ nanoparticles were developed through a facile hydrothermal method as a highly efficient 808 nm laser-induced photothermal agent with superior conversion efficiency (53.3%). When an aqueous solution of the as-synthesized MoS₂ RNPs (100 ppm) was irradiated with an 808 nm laser for 300 s, the increase of the solution temperature can reach 23 °C. The outstanding PCE of the MoS₂ RNPs can be attributed to the radar-like structures, which acted as reflectors. In addition, the MoS₂ RNPs displayed excellent photostability and biocompatibility. PTT of tumor cells in vitro and apoptosis assay indicated that the tumor cells were efficiently destroyed using MoS₂ RNPs as an 808 nm laser agent. Furthermore, in vivo tumor cell PTT performance was investigated by irradiating tumor-bearing mice using an 808 nm laser at a low power density (0.5 W cm⁻²). The therapeutic outcomes indicated that tumor tissue cells in vivo can also be killed. All the aforementioned results suggest that MoS₂ RNPs are promising 808 nm agents for future PTT applications.

Acknowledgements

We are grateful for the financial support from the National Basic Research Program of China (973 Program 2013CB934301) and the National Natural Science Foundation of China (NSFC21377068, 21575077).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra03226e

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