Combined Chemo-photothermal Antitumor Therapy Using

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Combined chemo-photothermal anti-tumor therapy using molybdenum disulfide modified with hyperbranched polyglycidyl Kewei Wang, Qianqian Chen, Wei Xue, Sha Li, and Zonghua Liu ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00499 • Publication Date (Web): 07 Sep 2017 Downloaded from http://pubs.acs.org on September 12, 2017

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ACS Biomaterials Science & Engineering

Combined

chemo-photothermal

anti-tumor

therapy

using

molybdenum

disulfide modified with hyperbranched polyglycidyl

Kewei Wang, Qianqian Chen, Wei Xue, Sha Li *, Zonghua Liu * Key Laboratory of Biomaterials of Guangdong Higher Education Institutes, Department of Biomedical Engineering, Jinan University, No. 601 West Huangpu Road, Guangzhou, 510632, China

* Corresponding authors: Zonghua Liu ([email protected])

Sha Li ([email protected])

ACS Paragon Plus Environment


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ABSTRACT In the treatment of cancers, molybdenum disulfide (MoS2) has shown great potential as a photo absorbing agent in photothermal therapy and meanwhile as an anti-tumor drug delivery system in chemotherapy. However, the poor dispersibility and stability of MoS2 in aqueous solutions limit its applications in cancer therapy. To overcome the shortcomings, MoS2 was modified mainly by surface adsorption of linear polymers, such as chitosan and polyethylene glycol. As reported, the linear polymers could be more rapidly cleared from blood circulation than their branched counterparts. Herein, we developed hyperbranched polyglycidyl (HPG)-modified MoS2 (MoS2-HPG) by absorbing HPG on the MoS2 surface. The MoS2-HPG as a novel photo absorbing agent was also used as a nano-scaled carrier to load anti-tumor drug doxorubicin hydrochloride (DOX) (MoS2-HPG-DOX) for combined chemo-photothermal therapy. The physicochemical and photothermal properties of MoS2-HPG were measured, and the results indicate that MoS2-HPG had good dispersion and stability in aqueous solutions, and also high photothermal conversion efficiency. MoS2-HPG displayed good biocompatibility in hemocompatibility and cytotoxicity evaluations in vitro. Furthermore, the combined chemo-photothermal therapy by using MoS2-HPG-DOX demonstrated better anti-cancer effect than the individual chemotherapy or photothermal therapy alone. From the results, MoS2-HPG with combined chemo-photothermal therapy could be developed as a promising therapeutic formulation for clinical cancer treatment.

KEYWORDS: molybdenum disulfide; photothermal therapy; chemotherapy; drug delivery


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1. INTRODUCTION To date, cancer remains a major threat to human health and life. To treat various cancers, three conventional methods (surgery, chemotherapy, and radiotherapy) have been widely used in clinic, which could alleviate symptoms to different extents but usually could not completely cure cancers. To fight cancers, some novel anti-cancer methods have been developed,1-4 such as photothermal therapy and biotherapy. More importantly, combined therapy of the above-mentioned methods has obtained more and more attention, which unites the multiple merits of the different methods and hence displays better therapeutic efficacy against cancers.5-8 In recent years, photothermal therapy as a novel cancer treatment method has gained eye-catching attention, due to its non-traumatic administration, less side effects, convenient implementation and so on.9-10 In photothermal therapy, photo absorbing agents are used to transform near-infrared (NIR) light energy to heat to destroy cancerous cells by hyperthermia, which as a result achieves anti-tumor therapeutic effect.2, 11 Typically, NIR in the wavelength range of 700-1100 nm is used, because it can effectively penetrate through tissues but rarely absorbed.12-15 In practice, the key of photothermal therapy is to find suitable photo absorbing agents with both outstanding photothermal conversion efficiency as well as satisfactory biosafety. Up to now, a lot of photo absorbing agents have been developed, such as gold nanoparticles,16 graphene17 and indocyanine green.18 Molybdenum disulfide (MoS2), one of the two-dimensional transitional metal di-chalcogenide nanosheets, has outstanding performance in the fields of photothermal therapy, drug delivery, and bioimaging,9, 19-20 etc. As a photo absorbing agent, MoS2 has potent absorbance in NIR region (even higher than gold nanorods and graphene).21 MoS2 nanosheets with atomically-thin 2-dimentional structure and hence huge surface-area-to-mass ratio can efficiently carry therapeutic molecules. Therefore, MoS2 has high potential for anti-cancer photothermal therapy, chemotherapy, or their combination.9, 19-20 However, the poor dispersibility and stability of MoS2 in aqueous solutions limit its further applications.22 The disadvantages of MoS2 can be overcome by surface modification via physical adsorption based on its enormous surface area and strong adsorption capacity. For example, Wang et al. synthesized PEGylated MoS2 nanosheets, which displayed excellent colloidal and photothermal stability, good biosafety, and significant anti-tumor effect.19 Yin et al. modified MoS2 with chitosan, and the modified MoS2 as a photo absorbing agent was also used to deliver a



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chemotherapeutic drug in combined chemo-photothermal therapy, and achieved a better therapeutic effectiveness both in vitro and in vivo.20 Up to now, photo absorbing agents are enriched in tumor site mainly by passive accumulation effect in photothermal therapy.19, 23-24 In this way, to prolong the blood circulation time of photo absorbing agents is an important means to achieve better passive targeting to tumors. As reported, linear polymers are more rapidly cleared from blood circulation than their branched counterparts.25 In this work, hyperbranched polyglycidyl (HPG) was used for the surface modification of the photo absorbing agent MoS2. HPG has excellent water solubility and biocompatibility, and more importantly long blood circulation time in vivo.25-27 Moreover, functional modifications can be achieved on the abundant hydroxyl groups in HPG molecules. Specifically, MoS2 was synthesized and functionally modified with HPG. Then, the physicochemical dispersibility and stability, drug delivery efficacy, biocompatibility, and anti-tumor effect of the HPG-functionalized MoS2 (MoS2-HPG) nanosheets were systematically studied for the combined chemo-photothermal therapy.

2. EXPERIMENTAL 2.1. Materials (NH4)2MoS4 was obtained from Sigma-Aldrich (Shanghai, China). Glycidol and adriamycin hydrochloride were obtained from Aladdin Industrial Corporation (Shanghai, China). Cell counting kit-8 (CCK-8) was obtained from Beyotime Institute of Biotechnology (Shanghai, China). BALB/c nude mice (5-7 weeks old, female) were provided by Beijing HFK Bio-Tech Company (Beijing, China). Fresh blood was donated by healthy consented volunteers into the anticoagulant sodium citrate tubes. The animal and human blood tests were carried out in compliance with the protocol of Animal Center and ethical rules of Jinan University. HPG was synthesized and characterized, as described in the supporting information. 2.2. Preparation of MoS2 nanosheets MoS2 nanosheets were fabricated according to the reported hydrothermal method.19 Typically, (NH4)2MoS4 (300 mg) was added into 60 mL ultrapure water under stirring. The obtained homogenous solution was put into a 100 mL autoclave, and the reaction was carried out at 220°C for 12 h. Then, the autoclave was cooled naturally. The final product was obtained by centrifuging


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at 5000 r/min for 10 min, and rinsed with anhydrous ethanol and deionized water, respectively. The washed product was dialyzed for 2 d against deionized water using cellulose acetate dialysis tubing (MWCO =3000, USA), frozen at -20°C and lyophilized in a lyophilizer (ALPHA1-2 LD, CHRiST, Germany). 2.3. Preparation of MoS2 -HPG MoS2-HPG nanosheets were prepared by the adsorption of HPG on the surface of MoS2 nanosheets. In brief, MoS2 nanosheets (50 mg) were dispersed in 25 mL ultrapure water. Then, 100 mg HPG was dissolved into the MoS2 suspension. The suspension was ultrasonic treated for half an hour, and stirred for 24 h. The product MoS2-HPG was obtained by centrifuging at 7500 r/min for 10 min, and rinsed with deionized water, and lyophilized. 2.4. Characterization of MoS2 and MoS2-HPG The morphology of MoS2 and MoS2-HPG nanosheets was observed with an atomic force microscope (AFM, Multimode, Bruker, Germany) and a transmission electron microscope (TEM, tecnai 12, Philips, Netherlands), respectively. MoS2 or MoS2-HPG nanosheets dispersed in pure water (100 µg/mL) was sonicated, and the hydrodynamic diameters and zeta potentials of MoS2 and MoS2-HPG nanosheets were measured with a zeta potential analyzer (Zetasizer Nano ZS, Malvern, UK). The fourier transform infrared (FTIR) spectra of MoS2 and MoS2-HPG were measured with a FTIR spectrometer (VERTEX70, Bruker, Germany). The weight loss curves of MoS2 and MoS2-HPG were obtained with a thermogravimetric analyzer (TG209F3-ASC, NETZSCH, Germany) at a rate of 10°C/min with N2 protection. The UV-visible spectra of MoS2 and MoS2-HPG were measured with a UV-visible spectrophotometer (uv-2550, Suzhou Shimadzu Corporation, China) at room temperature. 2.5. Photothermal performance of MoS2-HPG nanosheets The photothermal performance of MoS2-HPG nanosheets (2 mL suspension) was measured by irradiation with an 808 nm NIR laser device (8D02FN-8W, BWT Beijing LTD, China). The temperature was recorded with a thermometer (TES-1310, TES Electrical Electronic Corp, Taiwan). The photothermal conversion efficiency of MoS2-HPG nanosheets was calculated referring to the literature.28-29 2.6. In vitro cytotoxicity of MoS2 and MoS2-HPG nanosheets The in vitro cytotoxicity of MoS2 and MoS2-HPG nanosheets was measured by using three



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types of cells (normal mouse fibroblast 3T3, human cervical carcinoma HeLa, or melanoma cells B16). The cells (1×104 cells/well) were cultured in 96-well plates, and cultured for 1 day. After removing the medium, MoS2 or MoS2-HPG nanosheets dispersed in DMEM incomplete medium (Gibco, USA) were co-cultured with the cells for 24 h and removed. After that, DMEM complete medium (100 µL) including 10% v/v CCK-8 reagent was incubated with the cells for 1 h. The optical density (OD) of each well was read at 450 nm on a microplate reader (MULTISKAN MK3, Thermo Fisher Scientific, USA). The cell viability was obtained in accordance with the equation (1):

Cell viability (%) =

OD3 − OD2 × 100% OD1 − OD0

(1)

Where OD0 is the optical density of the well only containing CCK-8 reagent complete medium; OD1 is the optical density of the well containing the cells and CCK-8 reagent complete medium; OD2 is the optical density of the materials-treated well containing CCK-8 reagent complete medium; OD3 is the optical density of the materials-treated well containing the cells and CCK-8 reagent complete medium. 2.7. In vitro hemocompatibility evaluation The in vitro hemocompatibility of MoS2-HPG nanosheets was evaluated by studying the MoS2-HPG-induced alterations of the morphology and lysis of human red blood cells (RBCs), as well as whole blood coagulation in vitro. First, the MoS2-HPG-induced alteration of RBC morphology was observed according to our previously reported method.30 Briefly, RBCs were obtained from the fresh whole blood by centrifugation at 2000 r/min for 5 min and washing with phosphate buffer solution (PBS). The washed RBCs were kept for 10 min with different concentrations of MoS2-HPG suspensions in PBS. The washed RBCs kept for 10 min in PBS were used as a control. After that, the RBCs were separated by centrifuging, and rinsed. Then, the washed RBCs were fixed for 2 h in 4% paraformaldehyde, subjected to a gradient dehydration with ethanol solutions (75, 85, 95, and 100%, v/v), air-dried, and observed with a scanning electron microscope (SEM, Ultra 55, Zeiss, Germany). In hemolysis assay, RBCs were obtained from the fresh whole blood by centrifugation at 2000 r/min for 5 min and rinsed with PBS. The washed RBCs were dispersed in PBS to obtain 16 % v/v


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RBC suspension. The RBC suspension (0.2 mL) was kept at 37°C for 24 h with 0.8 mL of PBS, deionized water or MoS2-HPG suspensions in PBS. Then, the RBC suspensions were centrifuged at 10,000 r/min for 5 min, and the supernatants were obtained. The OD of the supernatants was read at 540 nm with the microplate reader. The hemolysis was obtained according to the equation (2): Hemolysis percentage (%) =

OD sample − OD negative OD positive − OD negative

× 100 %

(2)

Where ODsample, ODnegative, and ODpositive are the OD values of the samples, PBS (negative control) and deionized water (positive control), respectively. Thromboelastography (TEG) was applied to detect the MoS2-HPG-induced interference in the whole blood coagulation process in vitro. Briefly, 900 µL of the sodium citrate-anticoagulated whole blood and 100 µL of MoS2-HPG suspensions in PBS were added to the tube containing kaolin and mixed. Then, 340 µL of the mixture was sampled and added to CaCl2 solution (20 µL, 0.2 M) in a cup, and the testing was conducted on a TEG analyzer (TEG 5000, Haemoscope Corporation, USA). 2.8. In vitro photothermal therapy of MoS2-HPG Hela or B16 cells were cultured in 96-well plates (1×104 cells/well) for 24 h in DMEM complete medium. Then, the complete media in the wells were replaced with MoS2-HPG suspensions in DMEM incomplete medium. After incubation for 6 h, the culture media were discarded and the cells rinsed with PBS thrice. Then, fresh complete medium was supplemented to the wells. The cells were treated with an 808 nm laser for 10 min with 2 W/cm2. After that, the cells were cultured for 24 h and detected by using the CCK-8 assay. 2.9. DOX loading and release MoS2-HPG nanosheets (50 mg) were dispersed into 50 mL PBS, and 25 mg DOX was dissolved into the suspension. The suspension was stirred for 24 h in dark. Free DOX was separated by centrifuging and repeated rinsing with PBS. The product MoS2-HPG-DOX was lyophilized. All the supernatants were collected, and the absorbance of DOX in the supernatants was tested at 480 nm by using the UV-vis spectrophotometer. DOX loading amount was calculated by using the following equation:



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Loading amount =

m1 − 1000 Μ Vc × 100% m2

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(3)

Where V (L) is the supernatant volume, c (mol/L) is the DOX concentration in the supernatants, M (g/mol) is the molar mass of DOX, and m1 (g) and m2 (g) are the mass of DOX and MoS2-HPG, respectively. To test the release of DOX from MoS2-HPG-DOX, 2 mg of MoS2-HPG-DOX was dispersed into pH5 PBS or pH7.4 PBS, and shaken at 37°C. After different intervals, the suspensions were centrifuged, and 1 mL of the supernatants was sampled. Fresh PBS (1 mL, pH5 or pH7.4) was added to the MoS2-HPG-DOX suspensions. The absorbance of DOX in the supernatants was tested at 480 nm by using the UV-vis spectrophotometer.

2.10. In vitro anti-tumor effect of MoS2-HPG-DOX HeLa or B16 cells were cultured in 96-well plates (1×104 cells/well) for 24 h. Then, the cells were co-cultured with different materials for 4 h. After removing the materials, the cells were rinsed three times, and cultured in fresh complete medium. Some of the cells were subject to irradiation for 10 min under 808 nm laser (2W/cm2). After that, all the cells were incubated for 24 h and tested with the CCK-8 assay.

2.11. Combined chemo-photothermal therapy against in vivo tumor BALB/c nude mice (5-7 weeks old, female) were inoculated subcutaneously with B16 cells (2×106, 40 µL) in the side of the backside near the right forelimb, and the size of the formed tumor was measured periodically using a vernier caliper. When the diameter of the tumors was close to about 10 mm, the mice were randomly divided to five groups (4/each). The mice were intratumorally administered with 40 µL of formulations: (1) PBS+NIR, (2) MoS2-HPG (2 mg/kg), (3) DOX (0.625 mg/kg), (4) MoS2-HPG+NIR (2 mg/kg), (5) MoS2-HPG-DOX+NIR (MoS2-HPG ~2 mg/kg, DOX ~0.625 mg/kg). The mice in the groups (1), (4) and (5) were anesthetized with 1.5% pentobarbital (45 mg/kg), and were subject to irradiation for 10 min with 808 nm NIR light at 1 W/cm2. After that, the tumor size and mice body weights were measured daily. After 15 days post injection, the mice were sacrificed and the tumors were removed and weighed. Tumor volumes were obtained in accordance with the following equation:

V=

ab2 2

(4)


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Where V (mm3) is the volume of the tumor, and a (mm) and b (mm) are the length and width of the tumors, respectively. Relative tumor volumes were the ratios of the tumor volumes at different treatment times to the initial tumor volumes before the treatment. Relative tumor growth ratio (G) was calculated as follows:

G(%) =

Vtest − V0 ×100% Vnegative − V0

(5)

Where V0 is the tumor volume before treatment, Vtest and Vnegative are the tumor volumes after treatment with the tested materials and the negative control, respectively.

2.12. Statistical analysis All data were expressed as mean ± standard deviation. Student’s t-test for independent means was conducted to distinguish the significant difference of the data (* meaning p < 0.05, ** meaning p < 0.01, and *** meaning p < 0.001).

3. RESULTS AND DISCUSSION 3.1. Preparation and characterization In this study, MoS2 was prepared by the safe and simple hydrothermal method as previously reported.19 To improve the dispersibility and stability of MoS2 in water, HPG with chemical structure shown in Figure 1a was used to modify MoS2 via physical adsorption. The presence of HPG on the MoS2-HPG nanosheets was tested by FTIR analysis, as shown in Figure 1b. The peak at 3375 cm-1 was owing to the O-H stretching. The peaks from 2877 to 2927 cm-1 were owing to the C-H stretching from CH3 and CH2. The peaks at 1463 and 1100 cm-1 were the typical peaks of CH2 and C-O-C, respectively. This indicates that HPG was introduced on the MoS2 surface. The UV-vis spectra of MoS2 and MoS2-HPG are shown in Figure 1c. Both MoS2 and MoS2-HPG had strong absorption from 900 to 300 nm, but MoS2-HPG had stronger absorption than MoS2. This should be due to the better dispersibility and stability of MoS2-HPG in water. Further, thermogravimetry (TG) was performed to analyze whether MoS2 was modified by HPG. As displayed in Figure 1d, the weight loss of MoS2-HPG was 16.2% from 200 to 400°C, but the weight loss of MoS2 was only 1.6%. It is clear that HPG was introduced on the surface of MoS2, and the difference of 14.6% in



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weight loss could be attributed to the degradation of HPG. The TEM images of MoS2 and MoS2-HPG nanosheets were shown in Figure 1e and Figure 1f, respectively. MoS2 and MoS2-HPG were dispersed to water or PBS to evaluate the effect of HPG modification on the dispersibility and stability of MoS2, as shown in Figure 2a. As expected, HPG-modified MoS2 had better dispersibility and stability in water and PBS. The stability of MoS2-HPG was further studied by measuring their hydrodynamic sizes in different solutions or with different dilutions by using the zeta potential analyzer. Figure S3a shows the hydrodynamic sizes of MoS2-HPG nanoparticles dispersed in different solutions (H2O, PBS, RPMI-1640 and RPMI-1640+10%FBS). It indicates that the MoS2-HPG nanosheets were well dispersed in the different solutions. In addition, Figure S3b shows the hydrodynamic sizes of MoS2-HPG nanosheets dispersed in RPMI-1640 with different dilutions. It indicates that the size of MoS2-HPG nanosheets did not varied a lot along with the dilutions, suggesting that the MoS2-HPG nanosheets had good stability. In addition, both MoS2 and MoS2-HPG had net negative charge (Figure 2b), and the particle sizes of MoS2 and MoS2-HPG were ~110 and ~90 nm, respectively (Figures 2c&d), which is in good agreement with the TEM observation. MoS2 nanosheets were prone to aggregation and the adsorption of HPG on MoS2 surface improved the dispersibility of the MoS2-HPG nanosheets. Therefore, the particle size of MoS2-HPG nanosheets was smaller than that of MoS2 nanosheets. From Figure 3, the thicknesses of MoS2 and MoS2-HPG were 3.5 and 3 nm, respectively.

3.2. In vitro photothermal performance of MoS2-HPG The photothermal performance of MoS2-HPG was tested in vitro. From Figure 4a&b, MoS2-HPG could effectively convert light energy into heat, and the temperature of irradiated MoS2-HPG suspension increased along with increased irradiation time, MoS2-HPG concentration, and irradiation power density. The highest increment temperature up to 34°C was obtained in the 180 µg/mL of MoS2-HPG suspension for 10 min irradiation at 2 W/cm2. The temperature increased by about 15°C, which is sufficient to irreversibly damage cancer cells according to the report.31 Therefore, MoS2-HPG has great potential as a photo absorbing agent used in photothermal therapy. From Figure 4c&d, the photothermal conversion efficiency of MoS2-HPG was calculated to be 29.4% according to the literatures.28-29 This indicates that MoS2-HPG nanosheets had high photothermal conversion efficiency, which should be attributed to the improved dispersibility and colloidal stability of MoS2-HPG in water.


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3.3. In vitro cytotoxicity Biocompatibility is critical for biomedical materials used in clinic. In this work, cytotoxicity of MoS2-HPG and MoS2 was tested in vitro by using CCK-8 assay and three different types of cells (3T3, B16, and HeLa), as shown in Figure 5. It is clear that the cell viabilities in the presence of MoS2-HPG were higher than those in the presence of MoS2. The cell viability decreased obviously with the increased concentrations of MoS2, but did not change much with the increased concentrations of MoS2-HPG. The cell viability was still above 80% even in the presence of 200 µg/mL of MoS2-HPG at 24 h. The results indicate that the modification with HPG improved the biocompatibility of MoS2.

3.4. Blood compatibility At present, photo absorbing agents are administered mainly via intravenous injection, transported in blood circulation, and then enriched in tumor sites by the EPR effect. In this way, the photo absorbing agents administered would unavoidably encounter various blood components, which could affect the blood components and even the metabolism of the whole body. Hence, the blood compatibility of photo absorbing agents should be clearly elucidated.

3.4.1. Morphology of RBCs RBCs the most abundant blood cells are often applied to study the interaction between biomaterials and cell membranes.30 The typical biconcave disk shape of normal RBCs would be altered by membrane-active substances, which may interact with RBC membrane and alter the RBC morphology. In this work, the effect of MoS2-HPG on RBC morphology was observed by using SEM. As shown in Figure 6, MoS2-HPG at the studied concentration range did not change the typical RBC morphology compared with the control group. This suggests that MoS2-HPG was biocompatible with RBC membrane.

3.4.2. Hemolysis Hemolysis indicates the interaction of materials with the RBC membrane, and has been widely used in the biosafety evaluations of various biomedical materials. Figure 7 shows the RBC hemolysis in the presence of MoS2 or MoS2-HPG with different concentrations. It can be found that the hemolysis caused by MoS2-HPG was much lower than that by MoS2, and the hemolysis in the presence of 180 µg/mL of MoS2-HPG was lower than 4%. The results indicate that the hemolysis caused by MoS2-HPG was negligible in the concentration range studied.



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3.4.3. TEG assay The effect of biomedical materials on blood clotting is an important hemocompatibility parameter. TEG can dynamically monitor the whole blood clotting process, and has been widely applied in clinical and basic research. TEG assay includes four key parameters: (1) R, represents the time from the addition of calcium chloride to the formation of the first fibrin; (2) K, represents the dynamic thrombotic time; (3) α, represents the rate of fibrin crosslinking into a clot; (4) MA, refers to the clot strength. In this work, the effect of MoS2-HPG on blood coagulation was studied in vitro by using TEG assay. Figure 8 shows the traces of the coagulation process of the whole blood containing MoS2-HPG, with the corresponding parameters listed in Table 1. From Figure 8, it seems that the TEG traces of 10-500 µg/mL of MoS2-HPG did not deform visibly, compared to the PBS control. The four parameters were all within the normal range in the presence of ≤ 250 µg/mL of MoS2-HPG. However, 500 µg/mL of MoS2-HPG caused lower R value than the normal range, displaying a procoagulant activity. The results suggest that MoS2-HPG ≤ 250 µg/mL could be safe for the blood clotting function.

3.5. In vitro photothermal therapy Further, the photothermal treatment of MoS2-HPG was evaluated in vitro against cancer cells HeLa and B16 cells. Figure 9 shows the cell viability after treated by MoS2-HPG with or without irradiation. In Figure 9a, the photothermal treatment of MoS2-HPG ≥ 15 µg/mL caused significantly lower B16 cell viability than the same concentration of MoS2-HPG without irradiation. In Figure 9b, the photothermal treatment of MoS2-HPG ≥ 30 µg/mL caused significantly lower HeLa cell viability than the same concentration of MoS2-HPG without irradiation. In addition, it is also found that higher MoS2-HPG concentration and higher irradiation power both contributed to higher cancer cell death, as shown in Figure S5 of the supporting information. The results imply that the photothermal treatment of MoS2-HPG could effectively fight cancer cells in vitro.

3.6. DOX loading and release Currently, combination therapy has caught more and more attention, and a variety of combined treatments

often

display

extraordinary

efficacy,

such

as

gene-drug

co-therapy,5

photothermal-photodynamic co-therapy,31 photothermal-gene co-therapy,7 photothermal-immune co-therapy,8 and photothermal-drug co-therapy.6 MoS2 as drug carrier has been proven to efficiently


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load therapeutic molecules, due to the enormous surface-area-to-mass ratio of MoS2 nanosheets. As reported,9 MoS2 could carry a hydrophobic drug DOX with a drug loading amount (weight ratio between the drug and MoS2) up to ~239%. In this work, DOX was loaded to MoS2-HPG, and the release of DOX from the carriers was measured in vitro. The DOX loading amount was measured and calculated to be ~32.1%. From Figure 10a, only ~10% DOX was released from MoS2-HPG-DOX at 24 h in pH7.4 PBS, while ~46% DOX was released in pH5.0 PBS. The enhanced DOX release in the weakly acidic medium should be due to the protonation of DOX under acidic conditions and as a result increased hydrophilicity of DOX.9 This indicates that MoS2-HPG-DOX could better keep DOX in normal tissues with pH7.4, but could faster release DOX in tumor sites with weakly acidic pH, which could attenuate unexpected side-effects and better target tumor sites.

3.7. In vitro combined chemo-photothermal therapy Further, the combined chemical and photothermal therapy was studied in vitro on B16 or HeLa cells, as shown in Figures 10 b&c, respectively. The cell viability of the MoS2-HPG group treated with NIR was significantly lower than that not exposed to NIR. Moreover, the cell viability of the MoS2-HPG-DOX group treated with NIR was significantly lower than that not exposed to NIR. Under the NIR treatment, the viability of the cells in the MoS2-HPG-DOX group was significantly lower than other groups. In short, the cell viability in the group of MoS2-HPG-DOX with NIR irradiation was the lowest in all the groups. Subsequently, in order to visually observe the chemo-photothermal therapy effect in vitro, B16 cells were treated with different formulations and co-stained with Calcein-AM and PI, as shown in Figure S6 of the supporting information. Irradiation alone did not cause cell death. MoS2-HPG treatment alone caused a small amount of the cells to die. By contrast, the photothermal treatment with MoS2-HPG caused a large amount of the cells to die. Further, combination of the photothermal treatment with MoS2-HPG and the chemical therapy with DOX caused all the cells to die. The results indicate that the combined chemo-photothermal therapy of MoS2-HPG-DOX achieved the best cancer cells-killing effect in vitro.

3.8. In vivo combined chemo-photothermal therapy Melanoma was selected as a tumor model to study the therapeutic effect in vivo. In Figure 11a, compared with the PBS control, the MoS2-HPG had no anti-cancer effect in the absence of NIR, but



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DOX, MoS2-HPG+NIR and MoS2-HPG-DOX+NIR groups all had significant anti-cancer effect to different extents. The relative tumor volume in the MoS2-HPG-DOX+NIR group at day 15 was lower than those of other groups. In Figure 11b, the relative tumor growth ratio in the MoS2-HPG-DOX+NIR group was significantly lower than other groups. As shown in Figure 11e, the tumor in one of the mice in the MoS2-HPG-DOX+NIR group was cured on the 15th day. The results suggest that the MoS2-HPG-DOX+NIR group displayed the strongest anti-cancer effect via the combined chemo-photothermal therapy. The tumor photographs and mean tumor weight in each group were shown in Figure S9a of the supporting information and Figure 11c, respectively. The tumor weight in the MoS2-HPG-DOX+NIR group at day 15 was significantly lower than those of other groups, further indicating that MoS2-HPG-DOX could effectively inhibit tumor growth under 808 nm NIR irradiation. From Figure 11d, the weights of all the test mice had no obvious difference, indicating that the materials had no detectable toxicity on the mice. Histological examinations of the tumors collected from each group were shown in Figure S9b of the supporting information. Most of tumor tissues died in MoS2-HPG-DOX+NIR group, suggesting a good therapeutic effect of the formulation.

4. CONCLUSION In this work, MoS2 nanosheets were synthesized by hydrothermal method and modified by surface adsorption with HPG. The results show that the dispersion and stability in aqueous solutions, and the biocompatibility of MoS2-HPG were all improved after the modification. MoS2-HPG had good photothermal performance with the photothermal conversion efficiency of 29.4%. MoS2-HPG could efficiently kill cancer cells in vitro through its photothermal effect. Furthermore, MoS2-HPG could also be used as drug carriers to deliver anti-tumor drug DOX. The formed MoS2-HPG-DOX displayed excellent anti-tumor effect through the combined chemo-photothermal therapy.

ACKNOWLEDGMENTS The experiments were financially funded by Guangdong Provincial Key Laboratory of Construction and Detection in Tissue Engineering, the Natural Science Foundation of Guangdong Province (2015A030313314).


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:. Additional experimental methods and results about preparation and characterization of HPG, stability of MoS2-HPG nanosheets, flow cytometry analysis of the viability of B16 cells after photothermal treatment, fluorescent images of viable/dead B16 cells after photothermal treatment, preparation and characterization of MoS2-PEG nanosheets, in vivo blood circulation retention time, and photographs of the tumors and H&E stained slices of the tumors.

AUTHOR INFORMATION Corresponding Authors * Zonghua Liu * Sha Li

E-mail: [email protected]

E-mail: [email protected]

ORCID: 0000-0001-8019-4180

Notes The authors declare no competing financial interest.

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Table 1. Clotting kinetics values of human whole blood containing the MoS2-HPG nanosheets. Samples

R (min)

K (min)

α (deg)

MA (mm)

5–10

1–3

53–72

50–70

PBS control

5.2

1.7

66.3

60.7

10 µg/mL MoS2-HPG

5.2

1.6

67

62

50 µg/mL MoS2-HPG

5.1

1.4

69.3

60.7

250 µg/mL MoS2-HPG

4.9

1.6

67.2

59.5

500 µg/mL MoS2-HPG

3.2 ↓

2.5

58.9

53.1

Normal range

The sign ↓ indicates a low value and ↑ a high value compared with the normal range provided by the TEG analyzer.


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For Table of Contents Use Only Combined

chemo-photothermal

anti-tumor

therapy

disulfide modified with hyperbranched polyglycidyl

Kewei Wang, Qianqian Chen, Wei Xue, Sha Li, Zonghua Liu


using

molybdenum


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Figure 1. (a) Chemical structure of HPG. (b) FTIR spectra of MoS2, HPG and MoS2-HPG. (c) UV-vis absorption spectra of 80 µg/mL of MoS2 or MoS2-HPG suspension. (d) TG curves of MoS2 and MoS2-HPG. (e) TEM image of MoS2. (f) TEM image of MoS2-HPG. 152x147mm (300 x 300 DPI)


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Figure 2. (a) MoS2-HPG (1 mg/mL) and MoS2 nanosheets (1 mg/mL) dispersed in water and PBS for 1 week at 4°C. (b) Zeta potentials of MoS2 and MoS2-HPG in water. (c) The hydrodynamic diameter of MoS2 in water. (d) The hydrodynamic diameter of MoS2-HPG in water. 559x440mm (300 x 300 DPI)



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Figure 3. AFM images of MoS2 and MoS2-HPG. 187x137mm (300 x 300 DPI)


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Figure 4. (a) Temperature profiles of MoS2-HPG suspensions irradiated by 808 nm laser for 10 min at a power density of 2 W/cm2. (b) Temperature profiles of 80 µg/mL MoS2-HPG suspension irradiated by different power densities of 808 nm laser for 10 min. (c) Temperature profiles of 80 µg/mL MoS2-HPG suspension irradiated for 600 s by 808 nm laser at a power density of 2 W/cm2 but not irradiated later. (d) Plot of cooling time (after 600 s) versus negative natural logarithm of the driving force temperature obtained from cooling stage as shown in (c). 410x309mm (300 x 300 DPI)



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Figure 5. Viabilities of 3T3 (a), B16 (b), and HeLa cells (c) incubated with MoS2 and MoS2-HPG for 24 h. 199x454mm (300 x 300 DPI)


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Figure 6. Morphology and aggregation of the RBCs in the presence of different concentrations of the MoS2HPG nanosheets as observed with SEM. 338x254mm (300 x 300 DPI)



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Figure 7. Hemolysis of the RBCs incubated for 24 h with different concentrations of MoS2 and MoS2-HPG at room temperature. 288x201mm (300 x 300 DPI)


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Figure 8. TEG traces of in vitro whole blood coagulation in the presence of the MoS2-HPG. 169x127mm (300 x 300 DPI)



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Figure 9. Cell viability of B16 (a) and HeLa cells (b) after exposed to MoS2-HPG with or without the irradiation of NIR 808 nm laser (2 W/cm2, 10 min). 577x201mm (300 x 300 DPI)


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Figure 10. (a) Release profile of DOX from MoS2-HPG-DOX in PBS (pH5.0 or pH7.4). Viability of B16 (b) and HeLa cells (c) in the presence of PBS, MoS2-HPG (80 µg/mL), DOX (25 µg/mL) and MoS2-HPG-DOX (80 µg/mL) with or without NIR 808nm laser irradiation (2 W/cm2, 10 min). 288x602mm (300 x 300 DPI)



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Figure 11. (a) Tumor growth curves of the mice with the different treatments. (b) Relative tumor growth ratio of the mice with the different treatments. (c) Tumor weights of the mice with the different treatments. (d) Body weights of the mice with the different treatments. (e) The mouse with the tumor cured on the 15th day in the group of MoS2-HPG-DOX+NIR. 420x454mm (300 x 300 DPI)


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Combined Chemo-photothermal Antitumor Therapy Using

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