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Facile synthesis of biocompatible MoSe2 nanoparticles for efficient targeted photothermal therapy of human lung cancer

Two-dimensional molybdenum selenide (MoSe2) nanosheets have been explored for their use as photothermal agents in tumor therapy although several biocompatibility and stability issues have hindered their use. Herein, a facile strategy to prepare PLGA–MoSe2 mixture coated with a folate (FA)modified PEG-lipid shell led to the synthesis of multifunctional nanoparticles (FA–PL–MoSe2). The PEGlipid shells endowed the insoluble MoSe2 nanosheets with excellent colloidal stability. FA–PL–MoSe2 nanoparticles did not only exhibit good photothermal properties, but also excellent photothermal stability. The FA conjugation promoted a high nanoparticle specificity to target human lung cancer SPCA-1 cells as well as tumor tissue. In vitro cellular experiments demonstrated that FA–PL–MoSe2 had minor cytotoxicity, great biocompatibility, and could efficiently kill tumor cells under near-infrared laser (NIR, 808 nm) irradiation. Following about 1 month of treatment, FA–PL–MoSe2 showed good hemo-/ histocompatibility combined with NIR irradiation and an improved tumor inhibition effect compared to PL–MoSe2, with no relapse and no significant systemic in vivo toxicity.


Introduction
Lung cancer incidence is increasing worldwide, resulting in millions of deaths annually. 1,2 Currently, common treatments of lung cancer include surgical resection, 3 radiofrequency ablation, 4 and chemotherapy. 5 However, these therapeutic strategies have detrimental side effects such as surgical wounds, compromised cardiopulmonary function, medical comorbidities, 6 pneumothorax, 7,8 and multidrug resistance, 9,10 among others. Thus, these shortcomings require the development of alternative minimally invasive treatments. In recent years, photothermal therapy combined with NIR laser irradiation has been reported as a minimally invasive cancer treatment, attracting great research interest. [11][12][13][14] Highly efficient photothermal therapy needs excellent photothermal agents able to absorb the NIR laser and converting it into hyperthermia to destroy tumor cells. 15,16 To date, two-dimensional (2D) layered transition metal dichalcogenides have drawn great attention as photothermal agents for photothermal therapy due to their novel electronic and optical properties. 17,18 They are generally prepared with the formula MX 2 (M, transition metal atoms; X, chalcogen atoms), and include MoS 2 , WS 2 , TiS 2 , among others. [19][20][21][22] The exfoliated transition-metal dichalcogenides (MoS 2 , WS 2 , and WSe 2 ) have been reported to have lower cytotoxicity than those of classic 2D materials such as graphene and its analogues. 23 Among transition metal dichalcogenide materials, molybdenum selenide (MoSe 2 ) nanosheets possess an obvious NIR absorption peak, indicating their great potential as NIR photothermal transducers. [24][25][26] However, their poor dispersibility in water and lack of active specicity hinder its bioapplication in tumor photothermal therapy. Further processing techniques, such as ultrasonication to produce MoSe 2 nanodots and poly (vinylpyrrolidone) modication, have been reported to lead to the formation of biocompatible nanosheets. [24][25][26] Therefore, the advent of more facile processing strategies and assembly techniques for MoSe 2 nanosheets formation with excellent biocompatibility and multifunctionality is urgently required.
Herein, a facile self-assembly method based on PLGA and PEG-lipid was used to prepare biocompatible MoSe 2 nanoparticles (FA-PL-MoSe 2 , Fig. 1) consisting of a PLGA/MoSe 2 core and a PEG-lipid shell. The PEG-lipid coating endows these nanosheets with excellent hydrophilicity. Since tumor targeting is a promising strategy to better exploit the potential of photothermal agents in photothermal therapy, in order to enhance the tumor therapeutic effect, folate (FA), a water-soluble vitamin essential for human health, was conjugated onto the shells to target the FA receptors overexpressed in lung cancer cells. [27][28][29] The toxicity and biocompatibility of FA-PL-MoSe 2 were systematically evaluated in vitro and in vivo and lung cancer cells and tumor tissues were treated with FA-PL-MoSe 2 under 808 nm laser irradiation in order to conrm the photothermal therapy effect.

Preparation of FA-PL-MoSe 2
MoSe 2 nanosheets were prepared according to the previously reported methods. 24,25 In detail, 50 mg MoSe 2 powder was added into 50 mL water, and then the aqueous dispersion was sonicated in an ice-bath by using tip ultrasonic processor (Scientz-IID, 950 W, 25 kHz) with 70% amplitude for 10 h (the sonication was pulsed for 2 s on and 3 s off). The MoSe 2 suspension was centrifuged at 4500 rpm for 20 min to separate the large aggregates. Aerwards, the supernatant was then mixed with 10 mg mL À1 PLGA and sonicated in an ice-bath with 50% amplitude for 3 h to obtain the emulsion. And then, the emul-sied mixture was added into 12 mL of deionized water containing soybean phosphatidylcholine and DSPE-PEG 2000 -FA at a mass ratio of 2 : 1 under gentle stirring. The FA-PL-MoSe 2 was washed for three times using an Amicon Ultra-4 centrifugal lter (Millipore, Billerica, MA) with a molecular weight cutoff of 100 kDa. As a control, the nanoparticles without FA target molecules were prepared using the same method in which the DSPE-PEG 2000 -FA was replaced by DSPE-PEG 2000 -COOH. The puried FA-PL-MoSe 2 was stored at 4 C before use.

Characterizations
Transmission electron microscope (TEM) images were obtained on a JEOL JEM2011 at 200 kV. Atomic force microscopy (AFM) images were recorded by a Multimode V8 in tapping mode. Dynamic light scattering (DLS) and zeta potential characterization were performed on a Malvern Zetasizer Nano ZS (ZS90). UV-vis absorption spectra ranging from 900 to 400 nm were recorded using a Lambda 750 spectrophotometer (Perki-nElmer). The content of Mo in cells and tissue was measured by inductively coupled plasma-atomic emission spectrometry (ICP-AES; Hitachi, P4010). Photothermal effect measurements were performed under irradiation by 808 nm diode laser (Changchun New Industries Optoelectronics Technology Co. Ltd., MDL-III-808R). During NIR irradiation, variation of the temperature was recorded every 30 s using a thermocouple thermometer (Fluke).

Cell culture and cellular uptake
Human lung cancer SPC-A-1 cells were provided by the American Type Culture Collection (ATCC) and cultured in RPMI-1640 media supplemented with 10% FBS and 1% penicillin-streptomycin at 37 C with 5% CO 2 .
For cellular uptake, SPC-A-1 cells (2 Â 10 5 cells per well) were seeded in a 6-well cell culture plate and incubated for 24 h. Aerwards, the media were replaced with fresh RPMI-1640 media containing FA-PL-MoSe 2 and then cultured for an extra 3 h. Aer discarding the media, cells were gently rinsed with PBS for three times, homogenized, and treated with 1 mL aqua regia solution for 4 h. Mo content internalized by cells was detected by an ICP-AES. In addition, the cells treated with FA-PL-MoSe 2 were collected for Bio-TEM to observe the location of the nanoparticles inner and outer cells. The detailed Bio-TEM was performed according to previous report. 30 To further observe the cellular uptake, ICG was used to label the FA-PL-MoSe 2 . SPC-A-1 cells adhered to glass slides in 6-well plates and were incubated with free ICG, PL-MoSe 2 , FA-PL-MoSe 2 and FA-PL-MoSe 2 + FA blocking at the same concentration of ICG (0.05 mg mL À1 ) for 3 h, respectively. The cells were then washed with PBS thrice and xed by 0.2 mL of glutaraldehyde, followed by staining with DAPI for 10 min. The uorescence images of cells were captured using the laser scanning microscope.

In vitro biocompatibility
In vitro hemolysis assay was conducted according to previous work. 31 In detail, 0.2 mL separated red blood cells (RBCs) were mixed with 0.8 mL FA-PL-MoSe 2 (in PBS) at predetermined concentrations (50 mg mL À1 , 100 mg mL À1 , 150 mg mL À1 , 200 mg mL À1 and 250 mg mL À1 ). RBCs incubated with deionized water or PBS were used as positive or negative control, respectively. Aer standing incubated at 37 C for 1 h, the above set of suspensions were centrifuged (10 000 rpm, 1 min) and the absorbance of the supernatants at 541 nm was monitored by a UV-vis spectrometer. The hemolytic percentage (HP) was calculated using the following equation.
where A t , A pc and A nc are the absorbance of the supernatant at 541 nm of the test sample, positive and negative controls, respectively. In addition, the cytotoxicity of FA-PL-MoSe 2 was conducted by using a standard CCK-8 assay (Bestbio, China). SPC-A-1 cells (1 Â 10 5 cells per mL, 0.5 mL) were seeded in 96-well plate and cultured for 24 h. Aer discarding the old media, fresh media containing 0.01, 0.05, 0.1, 0.3 and 0.5 mg mL À1 of FA-PL-MoSe 2 were incubated with SPC-A-1 cells for 24 h. PBS was used to mildly wash the cells three times. A 100 mL CCK-8 working solution (10% CCK-8 + 90% RPMI-1640) was then added to each well, followed by incubation at 37 C for 1 h. The absorbance value at 450 nm was detected using a microplate reader (Innite 200 Pro, Tecan, Austria). Photos of the cells aer 24 h treatment with 0.05 and 0.5 mg mL À1 of FA-PL-MoSe 2 were collected by an optical microscope.

In vitro photothermal therapy
The in vitro photothermal performance of FA-PL-MoSe 2 was tested. In detail, 100 mL FA-PL-MoSe 2 dispersions with different Mo concentrations were placed in each well of 96-well plate and irradiated under 808 nm laser irradiation for 5 min. The realtime temperature and thermal images of the test samples at different time points (once every 30 seconds) were recorded using the thermocouple thermometer and infrared thermal camera (Fluke TI10, USA), respectively.
For in vitro photothermal therapy, SPC-A-1 cells were seeded in 96-well plates (2 Â 10 4 cells per well). Aer 24 h incubation, the cells were treated with fresh media containing PL-MoSe 2 and FA-PL-MoSe 2 at different Mo concentrations (PBS was used for the control group). Aer another 12 h, the media were discarded and the free PL-MoSe 2 and FA-PL-MoSe 2 outside the cells were removed via PBS washing. Aer addition of fresh media, the cells were irradiated by an 808 nm laser (1 W cm À2 , 5 min). The treated cells were incubated for another 24 h, and cell viability was then measured by a standard CCK-8 assay.

Blood analysis
Five healthy Balb/c nude mice were intravenously injected with 150 mL FA-PL-MoSe 2 at a Mo dose of 15 mg kg À1 . Mice treated with saline were used as blank control group. On the day 1, day 7, and day 24 post injection, the mice were sacriced to collect blood. The whole blood was divided into two parts. The one was centrifuged and the blood supernatant was used to analyze the serum biochemistry parameters including aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The other was used for complete blood counts evaluations including white blood cell (WBC), red blood cells (RBC), hemoglobin (HGB), mean platelet volume (MPV), mean corpuscular hemoglobin (MCH), hematocrit (HCT), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV) and platelet (PLT).

Animal model and in vivo biodistribution
Balb/c nude mice (3)(4)(5) week old) were used in vivo experiments and were purchased from Charles River Laboratories (Beijing, China). 1 Â 10 6 SPC-A-1 cells in 150 mL PBS were subcutaneous injected into the back of Balb/c nude mouse to establish animal tumor model. Aer tumor volume reached 80 mm, 3 the mice were used for further in vivo experiments. All welfare and experimental procedures in this study were performed in accordance with the policies of National Ministry of Health and approved by the Ethics Committee of Xinxiang Medical University.
Systemic biodistribution of FA-PL-MoSe 2 was performed in tumor-bearing mice. The tissues including the tumor, heart, liver, spleen, lung, and kidney were weighed and digested by aqua regia solution overnight at 1 h, 1 day, 7 days, and 24 days post intravenous injection of FA-PL-MoSe 2 (15 mg kg À1 ). The Mo content in different tissues was then analyzed by an ICP-AES.

In vivo photothermal therapy
Prior to in vivo photothermal therapy, tumor-bearing mice treated with saline (blank control), PL-MoSe 2 and FA-PL-MoSe 2 . Aer 24 h intravenous injection, tumor region was irradiated under 808 nm laser irradiation (1 W cm À2 ) for 5 min, and simultaneously the real-time images were captured by an infrared thermal camera. Moreover, tumor-bearing mice (n ¼ 5 per group) were treated with saline, FA-PL-MoSe 2 , PL-MoSe 2 + NIR and FA-PL-MoSe 2 + NIR (with 5 mg kg À1 of MoSe 2 ). Aer 24 h intravenous injection, tumor region was irradiated under 808 nm laser irradiation (1 W cm À2 ) for 5 min. During the irradiation, the temperature and thermal images of the tumor region at different time points (once every 30 seconds) were recorded using the thermocouple thermometer and infrared thermal camera, respectively. During the treatment, the relative tumor volume (V/V 0 , where V 0 represents tumor volume when the treatment was initiated, i.e., day 0), body weight of each mouse were monitored every four days.

Histology examination
Healthy Balb/c nude mice were intravenously injected with 150 mL of saline and FA-PL-MoSe 2 (15 mg kg À1 ) for 1 day or 24 days. And then, mice were sacriced and the major organs including the heart, liver, spleen, lung, and kidney were collected. The obtained organs were xed with 4% paraformaldehyde overnight. Aerwards, these organs were dehydrated in 25% sucrose, sectioned into 5 mm slices and stained with hematoxylin and eosin (H&E). The stained sections were imaged under an inverted phase contrast microscope.

Results and discussion
Preparation and characterization of FA-PL-MoSe 2 In brief, unstable MoSe 2 nanosheets were prepared from commercial bulk MoSe 2 under ultrasonication, and then stabilized by a PLGA core and PEG-lipid shell, whilst simultaneously being modied by the target molecule FA (Fig. 1a).
The morphology of the as-prepared MoSe 2 nanosheets was observed by TEM to be that of ake-like particles; highresolution TEM images showed a lattice spacing of 0.21 nm (Fig. 2a and b); DLS analysis showed an average diameter of approximately 20 nm (Fig. 2c). Following PEG-lipid shell formation, the FA-PL-MoSe 2 nanoparticles were almost spherical according to AFM analysis and approximately 200 nm in average diameter as evaluated by DLS analysis (Fig. 2d, e and S1 †). The zeta potential of FA-PL-MoSe 2 was determined to be À71 to À18.5 mV (Fig. 2f and S2 †).
Prior to preparing the insoluble MoSe 2 nanosheets for biomedical applications, a PEG-lipid shell was assembled around the MoSe 2 nanosheets via a self-assembly method to enhance its biocompatibility and physiologic stability. MoSe 2 nanosheets and FA-PL-MoSe 2 were mixed with water, FBS, cell media, PBS, or saline. The average size of the FA-PL-MoSe 2 nanoparticles in these media featured almost no changes over 1 week (Fig. 3a). However, for bare MoSe 2 nanosheets, the nanoparticles inevitably aggregated and precipitated in these media due to the electron screen effect, 32 which results in a signicant increase of hydrodynamic size (Fig. 3b). These results provide further evidence for the high physiologic stability of FA-PL-MoSe 2 , most likely due to the PEG-lipid shell around the MoSe 2 nanosheets. [33][34][35]   mg mL À1 ; as the control, the temperature of pure water increased by approximately 1 C under the same conditions. Fig. 4c depicts the photostability of FA-PL-MoSe 2 , indicating that it retained its initial excellent photothermal effects without any weakening of temperature elevation aer ve cycles of irradiation. Furthermore, FA-PL-MoSe 2 presented with similar absorbance spectra before and aer ve cycles of NIR irradiation (Fig. 4d). These results demonstrate that FA-PL-MoSe 2 has remarkable photostability and an excellent photothermal conversion effect.    similar to that of the negative PBS-treated group, suggesting an excellent hemocompatibility of FA-PL-MoSe 2 . 38,39 Moreover, the cytotoxicity of the nanoparticles on SPC-A-1 cells was evaluated by the CCK-8 assay. As shown in Fig. 6b, FA-PL-MoSe 2 below 0.5 mg mL À1 exhibited signicantly low toxicity to cells, with a higher than 90% cell viability. The cell morphology of SPC-A-1 cells aer 24 h of treatment with FA-PL-MoSe 2 (0.05 and 0.5 mg mL À1 ) exhibited no appreciable change, and was similar to the control group (Fig. 6c-e). These results indicate that the FA-PL-MoSe 2 has a great biocompatibility in vitro.

In vitro photothermal therapy
Adherent cells were rstly incubated with PBS, PL-MoSe 2 , or FA-PL-MoSe 2 solution (100 mg mL À1 of MoSe 2 ) for 3 h, respectively. The cells were then treated by 808 nm irradiation (1.0 W cm À2 ) for 5 min. As shown in Fig. 7a, cells treated with FA-PL-MoSe 2 showed the highest temperature increase aer 5 min irradiation compared to PL-MoSe 2 -and PBS-treated cells, with the highest temperature increase being of 22 C as detected by a thermometer (Fig. 7b). The cells in these three groups were cultured for a further 21 h, and their viabilities were detected by the CCK-8 assay. As shown in Fig. 7c, the FA-PL-MoSe 2 -treated SPC-A-1 cells showed a most signicant concentrationdependent cell death due to the FA targeting facilitating the internalization of FA-PL-MoSe 2 and thus the generation of more heat to kill cells.

In vivo biocompatibility
As shown in Fig. 8a, there were no distinct weight changes over 24 days treatment in the control Balb/c mice nor in those treated with FA-PL-MoSe 2 , PL-MoSe 2 + NIR, and FA-PL-MoSe 2 + NIR. As illustrated in Fig. 8b- d, complete blood counts (including  WBC, RBC, HGB, MPV, MCH, HCT, MCHC, MCV, and PLT), and blood biochemistry (including AST and ALT) in FA-PL-MoSe 2treated mice over 24 days showed no signicant difference compared to the control (saline treated group). Fig. 8e showed no apparent pathological tissue damage or abnormality in major organs (heart, liver, spleen, lung, and kidney) in FA-PL-MoSe 2 -treated mice according to H&E staining results. These results demonstrated that FA-PL-MoSe 2 showed low toxicity and excellent in vivo biocompatibility.

In vivo photothermal therapy
Tumor-bearing mice were intravenously injected with saline, PL-MoSe 2 , and FA-PL-MoSe 2 and, 24 h later, the tumor regions were irradiated by 808 nm laser (1 W cm À2 ) for 5 min. As shown in Fig. 9a and b, NIR irradiation induced the highest temperature increase (by about 20 C) in FA-PL-MoSe 2 -treated tumor regions compared with saline or PL-MoSe 2 -treated groups.
The biodistribution of Mo element in various tissues including the heart, liver, spleen, lung, kidney, and tumor at 1 h and 24 h, 7 days and 24 days following an intravenous injection of FA-PL-MoSe 2 is shown in Fig. 9c. For the rst 24 h postinjection, the majority of the Mo was accumulated in the tumor tissue except for liver and spleen. Aer 24 h, the Mo level in all tissues decreased with time, while in the tumor, it remained relatively high over seven days, indicating that the nanoparticles could be eliminated from these major organs thus avoiding potential toxic and side effects. These results were likely due to the fact that the PEG-lipid coating can improve the blood circulation duration and effectively decrease the macrophage clearance of nanoparticles by the reticuloendothelial system as well as the FA tumor targeting effect enhancing tumor accumulation. 40,41 Fig. 9d shows the in vivo photothermal therapy effect of FA-PL-MoSe 2 . Due to the excellent targeting and photothermal effects, tumor-bearing mice treated with FA-PL-MoSe 2 combined with 808 nm laser showed signicant tumor ablation and growth suppression, and no tumor relapse was observed at 1 month post treatment.

Conclusions
In summary, an excellent photothermal therapy agent based on PEG-lipid coated MoSe 2 nanosheets was successfully synthesized and used for efficient tumor targeted photothermal therapy. The PLGA core and PEG-lipid shell provided the unstable MoSe 2 nanosheets with outstanding physiological stability and photostability, as well as in vitro and in vivo biocompatibility. The inner MoSe 2 core can easily absorb and convert NIR laser into hyperthermia, whereas the outer FA coating can facilitate the internalization of nanoparticles into the cells via receptor-mediated endocytosis. In addition, in vitro and in vivo studies showed that, compared with PL-MoSe 2 , FA-PL-MoSe 2 coupled with NIR irradiation signicantly induced a temperature increase being of about 20 C and thus had an excellent targeting anticancer efficacy without any toxicity to healthy tissues. Therefore, considering all of the above desirable characteristics, the FA-PL-MoSe 2 nanoparticles will be a promising agent for cancer photothermal therapy.