Ultrasmall MoS2 Nanodots-Doped Biodegradable SiO2 Nanoparticles

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Biological and Medical Applications of Materials and Interfaces

Ultrasmall MoS2 Nanodots Doped Biodegradable SiO2 Nanoparticles for Clearable FL/CT/MSOT Imaging Guided PTT/PDT Combination Tumor therapy Peishan Li, Li Liu, Qianglan Lu, Shan Yang, Lifang Yang, Yu Cheng, Yidan Wang, Siyu Wang, Yilin Song, Fengping Tan, and Nan Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b18924 • Publication Date (Web): 17 Jan 2019 Downloaded from http://pubs.acs.org on January 18, 2019

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ACS Applied Materials & Interfaces

Ultrasmall MoS2 Nanodots Doped Biodegradable SiO2 Nanoparticles for Clearable FL/CT/MSOT Imaging Guided PTT/PDT Combination Tumor Therapy

Peishan Li, Li Liu, Qianglan Lu, Shan Yang, Lifang Yang, Yu Cheng, YiDan Wang, SiYu Wang, YiLin Song, Fengping Tan * and Nan Li* Tianjin Key Laboratory of Drug Delivery & High-Efficiency, School of Pharmaceutical Science and Technology, Tianjin University, 300072, Tianjin, PR China.

Tel.: +86-022-27404986 *Corresponding author: [email protected] *Co-corresponding author: [email protected]

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Abstract Recently, we developed ultrasmall molybdenum disulfide (MoS2) quantum dots for computed tomography (CT) and multispectral optoacoustic tomography (MSOT) image-guided phuotothermal therapy (PTT). But, due to the rapid body elimination and the limited blood circulation time, the tumor uptake of the dots is low. In our study, this problem was solved via designing an amino modified biodegradable nanomaterial based on MoS2 quantum dots doped disulfide-based SiO2 nanoparticles (denoted MoS2@ss-SiO2) for multimodal application. By integrating the MoS2 quantum dots into clearable SiO2 nanoparticles, this nanoplatform with an appropriate particle size can not only degrade and excrete in a reasonable period induced by the redox-responsive with the glutathione (GSH), but also exhibits a high tumor uptake due to the longer blood circulation time. Moreover, hyaluronic acid (HA) and chlorin e6 (Ce6) were adsorbed on the outer shell for the tumor targeting effect and photodynamic therapy, respectively. So this biodegradable and clearable theranostic nanocomposite applicable in integrated fluorescence(FL)/CT/MSOT imaging guided combined photothermal therapy (PTT) and photodynamic therapy (PDT) is very optimistic in biomedical applications in the future. KEYWORDS: ultrasmall MoS2 quantum dots, biodegradable, clearable, multi-modal imaging, combination tumor therapy


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1． Introduction Mesoporous silica nanoparticles (NPs) are attracting more and more attention in the innovative nanomedical systems because they meet the necessary requirements of non-toxicity, effective cellular uptake, drug loading possibility1, and controllable drug release. Thus, they can often be used as diverse therapeutic agents by combining some other compounds, for example photosensitizers, chemotherapy drugs, gene and so on2. Because of the versatile preparation method of NPs, we can adjust the size of the mesoporous flexibly, and structures of the NPs is adaptable for various guest molecules3. However, in spite of the verified silica, the issue that if the NPs can completely and safely remove from the biological system has not yet been well addressed4. So this problem remains one major obstacle for the clinical translation. It would be of great importance to design a kind of biodegradable silica nanoparticle for drug delivery, and after degradation, the products could be eliminated from the body5. Disulfide bond (-S-S-) is a multifunctional group which has good reactivity and biological activities6-7. Importantly, because of the disulfide band rupture reactions triggered by the glutathione (GSH), an abundant thiol-containing small molecule, this band is rarely founding inside cells. GSH is a tripeptide whose thiol group is very active, and whose concentration in the tumor cell is much larger than that in the plasma8. As a result, GSH is able to penetrate in the mesopores and break up the disulfide bond, thus facilitating nanoparticles degrade into smaller size nanoparticles and excrete from body5. The structure of two-dimensional transition metal dichalcogenides (TMDCs) is 3 ACS Paragon Plus Environment


similar to graphene, and attracting more research interest nanomedicine9-11. Among them, MoS2 is a typical dichalcogenides which is widely studied and applied12. Furthermore, MoS2 is suitable for photothermal conversion because the high NIR absorbance13, which has been used for the photothermal therapy (PTT) of cancers14. Furthermore, because of the strong X-ray attenuation it has, Mo can be used for CT/MSOT imaging15. It has been already synthetized to nano-size clusters16, nanosheets, nanodots17-18 ，nanoflowers19 and three-dimensional spherical shape20 of the MoS2. Among these, the nanoscaled MoS2 nanodots are attracting more attention by many researchers. However, the ultrasmall diameter nanomaterials has outstanding renal clearance properties, they have a low efficiency for tumor targeting, thus leading to a reduced cell-uptake efficiency and circulation halflife21-24. Of particular interest is the fact that amino modified ultrasmall MoS2-doped disulfide-bond mesoporous silica (MoS2@ss-SiO2) can overcome the above-mentioned limitations. In this work, we synthetized MoS2 QDs whose size is around 5 nm. By integrating MoS2 QDs to the clearable mesoporous silica, not only the size of the prepared MoS2@ss-SiO2 nanoparticle became lager, making it more suitable for the tumor targeting25, but also the multiple imaging functions such as CT and MSOT can be combined into one nanocomposite. In addition, hyaluronic acid (HA) and chlorin e6 (Ce6) were adsorbed on the outer shell for tumor targeting and PDT, respectively. As a result, we developed a highly integrated HA and Ce6 conjugated nanohybrid based on MoS2-doped disulfide-bond mesoporous silica (MoS2@ss-SiO2-Ce6/HA) for the multiple FL/CT/MSOT imaging 4 ACS Paragon Plus Environment

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guided dual-modal PTT/PDT strategy for tumor therapy with some unique advantages. (1) Ultrasmall MoS2 QDs are synthesized by up-bottom method from the bulk MoS2 at a mild temperature which has low cost. (2) The design of MoS2@ss-SiO2 can not only enlarge the diameter of the MoS2 QDs for tumor targeting, but also reduce the cytotoxicity of MoS2 nanocomposites; (3) Our design has two novel feature, one is the clearable MoS2 dots and another is the biodegradable silica nanoparticles, after degradation, both of this two parts can be cleared by the renal. So, this nanocomposite would be rapidly broken and cleaned in the body and then avoid the long-term toxicity. (4) HA can enhance the nanoparticles’ targeting effect and the physiological stability. This nanoplatform concurrently combined strong tumor uptake ability and biodegradable as well as good renal-clearable property, which could be used in the future clinical applications. 2. Experimental 2.1. Materials. MoS2 powder was obtained from Alfa Aesar. Photosensitizer Ce6 was obtained from J&K Scientific Ltd (China). Hexadecyltrimethylammonium bromide (CTAB, 99%) and NaOH were obtained from Yuanli Chemical (Tianjin, China). Bis[3-(triethoxysilyl)propyl]disulfide (BTSPD) was bought from HWRK Chem

Co.

Ltd

(Beijing,

China).

(3-aminopropyl)-triethoxysilane

(APTES),

tetraethylorthosilicate (TEOS) were purchased from Jiangtian Chemical (Tianjin, China). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-Htetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), 1,3-diphenyl isobenzofuran (DPBF), propidium iodide (PI), calcein acetoxymethyl ester (calcein AM), annexin V-fluorescein isothiocyanate 5 ACS Paragon Plus Environment


(FITC)/PI apoptosis detection agents, 2’,7’-dichlorodihydro-fluoresceinn diacetate (DCFH-DA) and 4’, 6-diamidino-2-phenylindole (DAPI) were purchased from SigmaAldrich (USA). Fetal bovine serum (FBS), serum, 4T1 cells and penicillin/streptomycin were obtained from Procell (China). 2.2. Synthesis of MoS2@ss-SiO2 Hybrids. Bulk MoS2 powder was prepared to nanodots using the solvent exfoliation method26. Briefly, 100 mL of DMF and 1 g of MoS2 powder were kept sonicating for 6 h in a 150 mL serum bottle to exfoliate MoS2 powder (250 W). Then, the supernatant was put into the flask and stirred for 6 h (140 ℃). After that, discard the precipitate after centrifuged (10 min, 8000 rpm). The MoS2 quantum dots were presented in the supernatant. Evaporating the supernatant under vacuum at 70℃ to except the excessive solvent and then washed three times by ethanol, then stored after lyophilization for further usage. The disulfide doping silica nanoparticles (ss-NPs) were synthesized by the modified base-catalyzed Stöber process involving two silanes: One is the TEOS and the other is BTSPD. According to the Si source, the molar ratio of this two substance is 70 : 307. Under intense stirring, the dried powder (50 mg) was put in 5 mL of 55 mM CTAB solution. And then NaOH (0.3 mL, 2 M) and 45 mL of H2O was added into this solution. When the temperature heated to 70°C, 400 μL of TEOS, 103 μL of BTSPD, and 3 mL of ethyl acetate were put into the solution respectively. For growth of HA and Ce6 on the outer of ss-NPs, 60μL of APTES was added to form the amine groups. After that, keep the solution vigorous stirring for 3h. By centrifugation (10000 rpm, 10 min), crude MoS2@ss-SiO2 nanoparticle were obtained. Then resuspended the precipitation 6 ACS Paragon Plus Environment

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in 50 mL of ethanol, and refluxed overnight at 60 °C under vigorously stirring for removing the CTAB. In the end, MoS2@ss-SiO2 nanoparticle was got by centrifugation, stored after lyophilization for further use27. 2.3. Preparation of the MoS2@ss-SiO2-HA Nanosystem. Firstly, we used the NHS and EDC to activated the carboxyl group of HA. Then the amine group on the MoS2@ss-SiO2 surface was combined with the activated carboxyl group of HA. Specifically, EDC (12.8 mg) and NHS (8 mg) was mixed with the HA solution (52 mg, 10 mL water), and keep stirring vigorously for 3 h. After that, added the MoS2@ssSiO2 (2 mL, 2 mg/mL) solution under magnetic stirring28. 2.4 Preparation of the MoS2@ss-SiO2-Ce6/HA Nanosystem. After modifying the amino group, the remaining amino group were also utilized for reacting with highly aminoreactive carboxyl group of Ce629. Specifically, 1 mg of EDC and1.2 mg of NHS were mixed with 8 mL of Ce6 (DMSO solution, 0.25 mg/mL), then stirred for 60 min. After that, 2 mL of MoS2@ss-SiO2-HA was added in to the system and kept stirring vigorously for 24 h. The MoS2@ss-SiO2-Ce6/HA nanocomposite was obtained and then stored in 20 ml DI water, after centrifugation (9000 rpm, 10 min).



Scheme 1. The synthesis and mechanism of MoS2@ss-SiO2-Ce6/HA nanocomposites and subsequent GSH triggered biodegradable phenomenon and renal clearance.

2.5 Characterization. The characterization of samples was determined by Zetasizer Nano-ZS (Malvern Instruments, UK), and transmission electron microscopy (TEM) (FEI Tecnai G2 F30，X-Max Oxford). The characteristic spectra of samples were characterized by UV-vis spectra (Agilent, Santa Clara, USA), SC300 infrared camera (Fluke TiR, USA), fluorescence spectra (Hengping Technology, Shanghai, China) respectively. The metal concentration in the samples was tested by inductively coupled plasma atomic emission spectroscopy (ICP-AES) (7700x, Agilent). IVIS Lumina imaging system (Caliper, USA) was used for the fluorescence imaging and biodistribution. The CT imaging was detected by the micro CT scanner (Quantum FX, PerkinElmer, Hopkinton, MA, USA). The photothermal and photodynamic effects was 8 ACS Paragon Plus Environment

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detected by excitation source of 808 nm or 660 nm (diode-pumped solid-state laser system, LASERGLOW Technologies, China).

Fig 1. Size of MoS2 Quantum dots (a), MoS2@ss-SiO2 nanoparticles (b), and MoS2@ss-SiO2-Ce6/HA nanoparticles (c) determined by TEM. (d) HRTEM images of MoS2@ss-SiO2. (e) The element maps shown the distribution of O (red), Si (Orange), S (yellow) and Mo (green).

2.6 Photothermal Effect and Singlet Oxygen Generation Detection. Different concentrations of MoS2@ss-SiO2-Ce6/HA (0.5 mL) were put into the centrifuge tubes with 808 nm laser radiation (5 min, 1.5 W/cm2). Simultaneously, the temperature was recorded every 30 seconds. Besides, we also used the infrared thermal camera to get the thermal images of the different formulations. The photothermal conversion efficiency (η) was evaluated by detecting the temperature changes of MoS2@ss-SiO2-Ce6/HA solution which were trigger by NIR laser (808 nm, 1.5 W/cm2). The η value was tested by previously reported formula30-31.



The formation of 1O2 was determined by the method which has been reported by Wöhrle et al32. DPBF was employed to detect the generation capability of 1O2 from different formulations after laser irradiation (660 nm, 1.0 W/cm2). Firstly, 1 mL of H2O, free Ce6 (10 μg/mL) in DMSO, MoS2@ss-SiO2-HA (200 μg/mL), and MoS2@ss-SiO2Ce6/HA (200 μg/mL for MoS2@ss-SiO2, 10 μg/mL for free Ce6) were added in DPBF (100 μL, 0.5 mg/mL), respectively. Then system was expanded to 3 mL by adding acetonitrile under dark condition and exposed to 660 nm laser (1.0 W/cm2), the generation of 1O2 was monitored at 410 nm wavelength by UV–vis spectrophotometer33.


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Fig 2. (a) Fluorescence spectra of free Ce6, MoS2@ss-SiO2-HA and MoS2@ss-SiO2-Ce6/HA . (b) UVvis adsorption spectra of Ce6 in DMSO and MoS2@ss-SiO2-HA, MoS2@ss-SiO2-Ce6/HA in H2O. (c) The absorbance of DPBF in various solutions at 410 nm. (d) Temperature change curves of different MoS2@ss-SiO2-Ce6/HA concentrations under 808 nm radiation (5 min, 1.5 W/cm2). (e) Temperature change curves of MoS2@ss-SiO2-Ce6/HA (1000 μg/mL) over Laser on and Laser off cycles under 808 nm laser irradiation. (f) Photothermal images of PBS, MoS2@ss-SiO2 and MoS2@ss-SiO2-Ce6/HA formulation under 808 nm laser (1.5 W/cm2, 10 min). (g) Photothermal effect of the aqueous solution of MoS2@ss-SiO2-Ce6/HA under 808 nm laser (1.5 W/cm2) for 1000s then turn off. (h) Plot of linear time data versus -ln(θ) which was got from the cooling stage (g).

2.7 Cellular Experiment. In this study, 4T1 cell were cultivated by DMEM at 37℃ in the incubator which contains 5% CO2, the culture medium contained 10% FBS, 100 U/mL penicillin and streptomycin (concentration: 100 mL/mL). 2.8 In vitro Cytotoxicity Evaluation. In this study, 4T1 cells was used for detecting the efficacy of killing cancer cells of the different formulation by MTT assay. For dark cytotoxicity, MoS2@ss-SiO2-Ce6/HA with different concentration were put 11 ACS Paragon Plus Environment


into each well and incubated for about 24 h. For phototoxicity, the cells were radiated by NIR laser for 5 min (808 nm, 1.5W/cm2, and/or 660 nm 1.0 W/cm2) after incubated with different MoS2@ss-SiO2-Ce6/HA concentration. After 18 h incubation, we added the 20 μL of MTT solution at 37℃ for further 4 h incubation. After separating, DMSO (500 μL) was put in and the absorbance was examined at 490 nm by microplate reader. Live/dead cell costaining experiment was applied for testing the tumor cell-killing ability. 4T1 cells were pre-seeded into CLSM dishes with different formulations (1000 μg/mL of MoS2@ss-SiO2-Ce6/HA and 50 μg/mL of Ce6) under different treatment. The experimental method is based on the previously reported34. The hemolysis assay according to the previous method, using MoS2@ss-SiO2Ce6/HA at different concentrations (250-1000 μg/mL)35. 2.9 In vivo cellular experiment. The saline, free Ce6, MoS2@ss-SiO2-HA and MoS2@ss-SiO2-Ce6/HA were diluted to different concentrations (1000 μg/mL of MoS2@ss-SiO2-Ce6/HA and 50 μg/mL of Ce6) by serum-free medium respectively. After dealt with those four different formulations for 6h, the 4T1 cells was washed and stained with DAPI (10 mg/mL, 1 mL) then detected by CLSM (DAPI, 345/455 nm; Ce6: 406/660 nm). The DCFH-DA was used for detecting the ROS generation. The 4T1 cell seeding methods are the same as the previous one and incubated overnight. After the different treatments (1000 μg/mL of MoS2@ss-SiO2-Ce6/HA and 50 μg/mL of Ce6), all those four groups were incubated with DCFH-DA (10 μM, 37℃, 20 min). After washing for three times, the ROS images was got by CLSM (DCF:488/525 nm). 12 ACS Paragon Plus Environment

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2.10 Animals Models. All the animal was obtained from Huafukang Biological Technology Co. Ltd (Beijing, China). The nude mice were used for the imaging detection, and the Kunming mice were used for different therapeutic detection. The animal experimental procedures were followed the standard protocol of Tianjin University, Tianjin, China. The 4T1 cells (100mL, 3 × 106) were implanted in oxter parts of the mice. We divided the mice into four groups (n = 10) when the tumor volume reached around 100 mm3. 2.11 FL/ CT /MSOT Imaging. For in vivo FL detection, the mice were intravenously injected with free Ce6 or MoS2@ss-SiO2-Ce6/HA (2 mg/mL, 200 μL). After that, the FL images were detected by the IVIS imaging system at the predesigned time. The major organs and tumor were also taken to analyze in the same way. To test the CT contrast efficiency, the mice were injected with MoS2@ss-SiO2Ce6/HA nanoparticle (8 mg/mL, 200 μL) through the tail vein. And the image was scanned in the micro CT scanner at 0 h, 4 h, 8 h, 12 h, and 24 h respectively to detected the biodistribution in vivo. Image analysis software: Analyze 12.0(AnalyzeDirect, Overland Park, KS, USA). Imaging parameters were in the following: Voltage:90kV， Current:180μA，field of view (FOV):73mm, Scan Time:4.5min. For in vivo MSOT images, the mice were injected with MoS2@ss-SiO2-Ce6/HA nanocomposite (PBS, 4 mg/mL, 200 μL) through tail vein. The MSOT images at different times were detected and the results were analyzed by ImageJ. 2.12 The in vivo Thermal Imaging. After injection with 200 μL of PBS or MoS2@ss-SiO2-Ce6/HA (2mg/mL) through tail-vein for 12 h, the center of the tumor 13 ACS Paragon Plus Environment


area was exposed to NIR laser (808nm, 1.5 W/cm2). The thermal images were captured at designed time points by IR camera (TiS55, Fluke, USA) 2.13 Structural Breakdown and Excretion study. Firstly, the rupture of the S-S bonds of ss-NPs was studied using the dithiothreitol (DTT). DTT is a reducing agent which can oxidized the S-S bonds to a to a cyclic one, and the oxidation state presenting a characteristic absorption at 290 nm. We simulated the extracellular and intracellular environment to cleavage the disulfide bond and then degrade the NPs40-41. The ss-NPs in PBS (0.1 mg/mL) was subsequently added with DTT (5 mM, PBS) and flowed the nitrogen to avoid oxidation of DTT at 37 °C condition. Then, 1.5 mL of the solution were extracted at each designed time for the absorbance detection. Furthermore, we also used the GSH to detect the structural break-down process of nanoparticle. GSH, a thiol-containing tripeptide, whose concentration in cancer cells is higher than that in the normal cells, plasma and other body fluids, can activate the breakdown process in the cytoplasm of cancer cells by specific reacting with the disulfide bonds. The ss-NPs (0.1 mg/mL) in PBS were mixed with the reduced GSH (10 mM, PBS) solution to a final volume of 15 mL and stirred at 37℃. Every 0.5 mL of the solution was taken out at predesigned time (from 0 to 168 hours) for TEM test to observe the cracking condition. After injection of MoS2@ss-SiO2-Ce6/HA, major organs were tested for ICP-AES analysis36. For the excretion condition, after intravenous injection with MoS2@ss-SiO2Ce6/HA, the mice were kept in metabolic cages for collecting feces and urine. Then the Mo levels was tested by ICP-AES. And after 8 days the tumor and major organs were 14 ACS Paragon Plus Environment

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taken for mental detection. 2.14 In vivo Synergistic Antitumor Efficacy. For the synergistic antitumor efficacy, the 4T1 tumor-bearing mice were randomly divided into four groups. When the tumor volume reached to 100mm3, the mice were treated with saline + 808 nm, free Ce6 + 660 nm, MoS2@ss-SiO2-HA + 808 nm and MoS2@ss-SiO2-Ce6/HA + 808/660 nm respectively. The weight change and tumor volume change of the mice were detected every three days by an electronic balance and caliper, respectively. We used the followed equation to calculated the tumor volume: (volume = width2 × length/2)37. Meanwhile, the survival percentage of the mice were recorded. After experiment, tumors and major organs were isolated for the H&E staining studies. 2.15. Statistical Analysis. All data in this article were used as mean result standard deviation (SD), the difference between different groups was tested by Student's t-test. When **p < 0.01, *p < 0.05, it was regarded as significant. 3. Results and discussion 3.1.

Preparation

and

Characterization

of

MoS2@ss-SiO2-Ce6/HA

Nanocomposites. MoS2 QDs were synthesised by the previous method26, as shown in Scheme 1. Firstly, in an alkaline environment, MoS2 quantum dot was mixed with CTAB, TEOS and APTES under reflux condensation. The S-S cleavable bonds in BTSPD were introduced to perform the function of self-destruction trigger in the media. CTAB was removed to get MoS2 doped mesoporous silicon. NHS, and EDC were used to activate the HA and Ce6. In the end, HA and Ce6 were linked with the amine group of the MoS2@ss-SiO2 nanoparticle. 15 ACS Paragon Plus Environment


The morphologies and structures of the MoS2 dots, MoS2@ss-SiO2 and MoS2@ssSiO2-Ce6/HA were measured by TEM. Figure 1a showed the particle size distribution of MoS2 quantum dots was around 5 nm. Moreover, it demonstrated that the structure was approximately ordered from the HRTEM results of the MoS2@ss-SiO2 nanocomposites (Figure 1d). The diameter of MoS2@ss-SiO2 nanocomposite is around 100 nm (Figure 1b), and it was consistent with the Malvern Master-sizer results (99.7 nm) (Figure S3). Furthermore, the element mapping images showed that the Mo, O, and S distribution is matched with the distribution of Si map, confirming the uniformly MoS2 distribution in the nanocomposites (Figure 1e). Furthermore, synthesis process of MoS2@ss-SiO2-Ce6/HA (HA and Ce6 conjugation) could be confirmed by the zeta potential and the hydrodynamic size change (Figure S4). Since the amino modified MoS2@ss-SiO2 nanoparticles prepared without HA is positive (+8.5 mV), and the MoS2@ss-SiO2-HA nanoparticles were negative (-28 mV). It indicated that the HA has linked with of MoS2@ss-SiO2. From Figure 1c, we can see that the MoS2@ss-SiO2-Ce6/HA has a spherical morphology. The diameter of the MoS2@ss-SiO2-Ce6/HA nanoparticles tested by dynamic light scattering was about 110 nm, which was a suitable size for highly effective endocytosis and tissue penetration38. Furthermore, from the Fig.S8, we can see the physiological stability of the nanocomposites is well, we can apply is for the further experiments. In order to confirm that Ce6 has been linked with MoS2@ss-SiO2-HA, the FL intensities of Ce6 (2.5 μg/mL) in DMSO, MoS2@ss-SiO2-HA (50 μg/mL) and 16 ACS Paragon Plus Environment

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MoS2@ss-SiO2-Ce6/HA (50 μg/mL) in H2O was measured respectively. Obviously, Ce6 showed strong fluorescence compared with no fluorescence intensity of MoS2@ssSiO2-HA. In addition, the fluorescence intensities of the attached Ce6' dropped significantly (Figure 2a). From the UV–Vis absorption spectra (Figure 2b), there was no characteristic peak of MoS2@ss-SiO2-HA at 404 nm. But, both of free Ce6 and MoS2@ss-SiO2-Ce6/HA had obviously absorption at 404 nm, so it indicated that Ce6 had been linked with the carrier. Due to the standard absorption curve of Ce6 (Figure S1, and S2), the concentration of Ce6 molecules loaded on the MoS2@ss-SiO2-HA nanosystem was calculated. Percentage of Ce6 loaded on the nanohybrids was measured to about be 5% (w/w), measuring by removing unreacted Ce6 in the nanoparticle. 3.2. MoS2@ss-SiO2-Ce6/HA Stability Study The stability of MoS2@ss-SiO2Ce6/HA nanocomposite was detected by incubating with the Deionized water, PBS, DMEM and PBS and stored for one week at 25°C. The size distribution was detected at different time points (2h, 6h, 12h, 24h, 7d). As presented in the Figure S8, the size of the MoS2@ss-SiO2-Ce6/HA was almost unchanged at around 110 nm (Figure S8). In addition, the inset photographs of Figure S8 showed that there was no aggregation in the system after storage for 7 days, indicating the good stability of prepared nanoparticles, which was promising for further experiments. 3.3. In vitro Photothermal Effect and ROS Detection. The photothermal effect of MoS2@ss-SiO2-Ce6/HA nanocomposite with different concentrations (0, 250, 500, 750 and 1000 μg/mL) was calculated by the temperature change in 5 min with NIR 17 ACS Paragon Plus Environment


radiation (808 nm, 1.5 W/cm2). The temperature of MoS2@ss-SiO2-Ce6/HA nanoparticles (1000 μg/mL) has increased from 25 °C to 56.8 °C （Figure 2d ）. In marked contrast, there was negligible temperature change for PBS (Figure 2f). When temperatures higher than 55℃, rapid cellular damage would occur due to the protein coagulation inducting by instantaneous.


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Fig.3 Viability of 4T1 cells treated with different concentrations of free Ce6, MoS2@ss-SiO2-HA and MoS2@ss-SiO2-Ce6/HA without (a) or with NIR (b) (n = 3) **p < 0.01, *p < 0.05. (c) Hemolysis of red blood cells after incubation with MoS2@ss-SiO2-Ce6/HA at different concentrations (0 -1000 μg/mL) for 2 h. Inset: Hemolysis photo after centrifugation. (d) CLSM images of 4T1 cells treated with saline + 808/660 nm, free Ce6 + 660 nm, MoS2@ss-SiO2-Ce6/HA + 808 nm and MoS2@ss-SiO2-Ce6/HA + 808/660 nm (1.5 W/cm2, 808 nm, 5 min). Scale bar indicated 25 μm. (e) CLSM images of 4T1 cells cultivated with saline, free Ce6 + 660 nm, MoS2@ss-SiO2-HA + 808 nm and MoS2@ss-SiO2-Ce6/HA + 660/808 nm. Scale bar indicated 100 μm.

To test the photothermal conversion efficiency, we used the methods of previous report38. With continuous irradiation, the sample temperature change curve was recorded. From the Figure 2g and Figure 2h, the photothermal conversion efficiency of MoS2@ss-SiO2-Ce6/HA we calculated was 22.34%. In addition, it was also found that the temperature rising of MoS2@ss-SiO2-Ce6/HA was almost stable after several 19 ACS Paragon Plus Environment


cycles (Figure 2e), suggesting the good photothermal conversion stability. So all this could let MoS2@ss-SiO2-Ce6/HA nanocomposites be a promising PTT agent in future clinical treatment. The singlet oxygen generation is very important for the PDT effect. In this part, we used DPBF to detect singlet oxygen generation of MoS2@ss-SiO2-Ce6/HA nanocomposite. With production of 1O2 increasing, the absorbance of DPBF (410 nm) would decrease. We can see from the Figure 2c, the absorbance of DPBF + Ce6 group nearly decreased to zero. Besides, under the same conditions the MoS2@ss-SiO2Ce6/HA generated more ROS than free Ce6 in 5 min, which might because the less effective energy absorption of the MoS2@ss-SiO2-HA. In both pure DPBF and MoS2@ss-SiO2-HA treated group, the decrease of DPBF was negligible, suggesting no ROS generation of MoS2@ss-SiO2-HA. 3.4. In vivo cellular experiment After incubate with free Ce6, MoS2@ss-SiO2-HA and MoS2@ss-SiO2-Ce6/HA at different concentrations for 24h, the cell viabilities still keep high. It indicated that there was no obvious cytotoxicity in MoS2@ss-SiO2Ce6/HA even at highest concentration (1000 μg/mL of MoS2@ss-SiO2-HA and 50 μg/mL of Ce6) (Figure 3a). It indicated that the MoS2@ss-SiO2-Ce6/HA nanocomposites had good biocompatibility which was good for using in the future clinical treatment. Moreover, as we can see from the Figure 3b, the cancer cell killing effect of MoS2@ss-SiO2-Ce6/HA +808/660 nm is dose-dependent. For the phototoxicity, after incubation with different three agents, the 4T1 cells were treated with 660 nm and/or 808 nm laser, cell viability of the optimal formulation decreased to 20 ACS Paragon Plus Environment

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40% at the highest concentration, which was lower than other treatment groups at the same concentration. In addition, the hemolysis ratio we tested was shown in Figure 3c, and it was lower than 3% at the highest concentration (1000 μg/mL). It confirmed that MoS2@ss-SiO2Ce6/HA nanocomposite had excellent hemocompatible and could be used for intravenous injection. What’s more, calcein-AM/PI costaining was performed in our experiment for observing the live (green) and dead (red) cells. The combination therapy group treated with MoS2@ss-SiO2-Ce6/HA + 808/660 nm showed the brightest red compared with other monotherapy group (Figure 3e), confirming the best antitumor efficacy of this formulation. The cellular uptake behavior and intracellular ROS generation was detected by 4T1 cells in CLSM. Comparing with free Ce6 treatment cells, red fluorescence was observed in the MoS2@ss-SiO2-Ce6/HA treatment group, indicating the efficient cellular uptake of the MoS2@ss-SiO2-Ce6/HA nanoparticle (Figure S5). The red fluorescence became much brighter because more Ce6 was released after exposing to 808 nm laser (1.5 W/cm2, 5 min). After that, the DCFH-DA was performed to test the ROS generation. In Figure 3d, green fluorescence in the MoS2@ss-SiO2-Ce6/HA +808/660 nm group was much brighter than that in the free Ce6 + 660 nm group. It might because of poor uptake and self-aggregation of Ce6. All these results proved that MoS2@ss-SiO2-Ce6/HA nanomaterials could be internalized and induce ROS generation with low cytotoxicity. 21 ACS Paragon Plus Environment


3.5. In vivo FL/ CT /MSOT Imaging. The ideal preparation should accumulate at the tumor site at a specific time and then excreted from the body to avoid the long-term toxicity. The fluorescence signal producing by the Ce6 was recorded at pre-designed time (0, 4, 8, 12, and 24 h), after intravenous injection of free Ce6 or MoS2@ss-SiO2Ce6/HA nanoparticles. With time increasing, the fluorescence signal changes in the body can be observed obviously (Figure 4a, top panel). From the MoS2@ss-SiO2Ce6/HA treatment group, we can see a highest fluorescence signals of the tumor at 12 h and gradually decreasing after that, suggesting that the nanohybrids can accumulate in the tumor site and degrade in the end. In contrast, fluorescence images of free Ce6 injected mice had no tumor targeting. Comparing the two sets of results, it confirmed that MoS2@ss-SiO2-Ce6/HA nanocomposite had a good targeting effects and selfdegrading ability. After 24 h, major organ and tumors in the MoS2@ss-SiO2-Ce6/HA treatment group were excised to test the biodistribution of the nanocomposites. As we can see from the picture, the MoS2@ss-SiO2-Ce6/HA nanoparticles were mainly accumulated in the tumor sites and the free Ce6 was primarily accumulated in the liver (Figure 4a, bottom panel) which was consistent with the semi-quantitative analyses of biodistribution in tissues. It was also proved that the tumor accumulation of MoS2@ss-SiO2-Ce6/HA nanoparticles was high than other organs (Figure 4b).


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Fig. 4 (a) In vivo fluorescence images of mice after treated with free Ce6 or MoS2@ss-SiO2-Ce6/HA at various time points: the bottom panel shows the fluorescence signal in major organs after experiment. (b) FL signal of tumors and major organs after the in vivo test (n = 10), *p < 0.05, **p < 0.01. (c) MSOT images at different time points after injection with MoS2@ss-SiO2-Ce6/HA nanocomposites intravenously. (d) Corresponding MSOT signal intensity of MoS2@ss-SiO2-Ce6/HA nanoparticles at different concentration (3.2, 1.6, 0.8, 0.4mg/mL). Inset: MSOT imaging intensity of various concentrations of MoS2@ss-SiO2-Ce6/HA. (e) CT images at 0 h, 4 h, 8 h, 12 h and 24 h post i.v. injection of MoS2@ss-SiO2-Ce6/HA (8 mg/mL, 200 μL). (The red color represents tumor, the blue color represents different organs.) (f) Corresponding HU value of MoS2@ss-SiO2-Ce6/HA nanoparticle in the tumor before injection and 12 h after injection. *p < 0.05, **p < 0.01.



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In order to detect the CT contrast performance, we reconstructed images of other organs and found that the image of mice post i.v. injection via tail vein revealed an increasing signals of tumor over time and reached maximum at 12h (Figure 4e). The numbers of Hounsfield units (HU) increased from 119.3 ± 24 to 225.2 ± 41.2 (Figure 4f), suggesting that the MoS2@ss-SiO2-Ce6/HA nanoparticle had superior CT imaging ability. In addition, the signal disappeared at 24 hours which was consistent with conclusion discovered by the FL. Thus, MoS2@ss-SiO2-Ce6/HA nanoparticle showed a remarkable CT imaging capabilities and obvious tumor-targeting effects and good body clearance abilities. As for the in vivo MSOT imaging ability, we can see the MSOT signal in the tumor region increased over time comparing with the signal before injection indicating the effective tumor retention in the living body of the MoS2@ss-SiO2-Ce6/HA nanoparticle (Figure 4c). The MSOT signal reached the highest value in the tumor site at 12 h, and disappeared after 24 h which was consistent with the FL results. Furthermore, MSOT images of MoS2@ss-SiO2-Ce6/HA with different concentrations and the quantitative analysis of different concentrations of MoS2@ss-SiO2-Ce6/HA (1 to 8 mg/mL) displayed a good linear correlation between MSOT signal intensity and the formulation concentration-dependent profile (Figure 4d). These

results

demonstrating

the

MoS2@ss-SiO2-Ce6/HA

nanoparticles

experienced notable tri-modal FL/CT/MSOT imaging abilities and have obvious tumor targeting ability. 3.6. Structural Breakdown and Excretion Study. Firstly, we use dithiothreitol 24 ACS Paragon Plus Environment

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(DTT) to test the reaction of the -S-S- bonds in the breakable particles. As shown in Figure 5c, there was an obviously increasing absorbance of ox-DTT, which confirmed that the DTT can get into the framework and break the S-S bonds of the nanoparticle. In addition, as we can see from the Figure 5a, with persistent exposure to the GSH, the degradation of the framework is becoming more and more obvious, which was not detected in the 37℃ in the absence of GSH (figure 5b). So those confirmed that the nanoparticles had a good breakability and could be try for the clinical treatment. 3.7. In vivo Photothermal Imaging. We used the photothermal imaging to detected the PTT effect of MoS2@ss-SiO2-Ce6/HA. In this part, we measured the in vivo PTT performance. In the PBS-injected mice, the finally tumor temperature had little change within 5min. However, the temperatures of tumor site in the MoS2@ssSiO2-Ce6/HA treatment group rapidly increased from 25℃ to 55.2℃ within 5 min (Figure 6a), which was capable to lead to the tumor suppression. This results confirmed that our nanoparticle can perform a good PTT effect.



Fig. 5 (a) TEM analysis of the MoS2@ss-SiO2 (0.1 mg/mL) with GSH (10 mM) in different time at 37 ℃; (b) TEM analysis of the MoS2@ss-SiO2 (0.1 mg/mL) with PBS reduction for 7 days at 37 ℃; (c) The UV-vis absorption of DTT (5 mM) when exposed to of MoS2@ss-SiO2 (0.1 mg/mL, PBS) with different time; (d) Time dependent biodistribution of MoS2@ss-SiO2-Ce6/HA nanocomposite in major organs. (n = 10) *p < 0.05, **p < 0.01.

At selected time points, major organs were taken for ICP-AES analysis (Figure 5d). From the picture, we can see obviously a large value in the liver and spleen at 1 day after injection. However, Si had more accumulation in those organs, meaning slower degradation in the living body (Figure S6). But, both Si and Mo concentrations in the MPS organs decreased obviously after 7 d, indicating a possible metabolism of the nanoparticle. In addition, high amounts of Mo could be tested in feces and urine after injecting MoS2@ss-SiO2-Ce6/HA (Figure S7), suggesting that the liver play a main role in the clearance of Mo via the biliary pathways, and the molybdenum content in tumors and organs is significantly reduced (Figure S9). So, we hypothesized that the ultrasmall MoS2 QDs in the living body can degrade and excrete from body within a certain period time. 26 ACS Paragon Plus Environment

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Fig.6 (a) Thermal images of tumor-bearing mice under 808 nm laser (1.5 W/cm2) at different time points after injection with saline or MoS2@ss-SiO2-Ce6/HA. (b) The tumors of mice in different treatment groups after experiment. (c) Weight changes of various treatment groups. (n = 10) *p < 0.05, **p < 0.01. (d) Tumor volume changes of various treatment groups. (n = 10) *p < 0.05, **p < 0.01. (e) Percent survival ratio of tumor-bearing mice in various treatment groups. (n = 10) *p < 0.05, **p < 0.01. (f) H&E staining images of major organs in different treatment group after 35 days.



3.8. Therapeutic Efficacy Study. In this study, the body weight, tumor volume and survival rate of different group of the mice was measured as an indicator of the therapy efficacy. 4T1 tumor-bearing mice with different treatment were treated with: (1) saline, (2) free Ce6 + 660 nm, (3) MoS2@ss-SiO2-HA + 808 nm, (4) MoS2@ssSiO2-Ce6/HA + 808/660 nm. The MoS2@ss-SiO2-Ce6/HA + 808/660 nm treatment group showed an insignificant weight loss at 21 days and presented obviously tumor inhibitory efficiency, suggesting a little side effects (Figure 6c). The tumor volume changes of the 4T1 tumor bearing mice indicated the antitumor ability of the nanoparticle (Figure 6d), and there was an efficient tumor suppression in MoS2@ssSiO2-Ce6/HA + 808/660 nm treated group after 21day treatment, however there was still tumor growth for the saline group and free Ce6 group and MoS2@ss-SiO2-HA treated groups. Moreover, the survival rate of the MoS2@ss-SiO2-Ce6/HA + 808/660 nm treated mice group was approximately 100% after 35 days in (Figure 6e), suggesting the higher antitumor efficacy when combine PTT and PDT. After treatment with various samples for 35 days, the volume of the tumor were tested after sacrificed the mice. The tumor volume of the MoS2@ss-SiO2-Ce6/HA treatment group was the smallest in the four groups, which confirmed that combination therapy was much better than other single treatment groups (Figure 6b). Moreover, as we can see from the H&E staining organ tissue slices, there were no significant necrotic cell in major organs of the MoS2@ss-SiO2-Ce6/HA nanoparticle treatment group (Figure 6f). But the tumor tissues had more necrosis and apoptosis in combined therapy (PTT/PDT) for the control group. In general, it demonstrated that the synergistic 28 ACS Paragon Plus Environment

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therapy of MoS2@ss-SiO2-Ce6/HA nanocomposites with 808/660 nm laser could be used as a promising agent for PTT/PDT in the clinical trials in the future. 4. CONCLUSION Overall, we have synthesised an innovative nanosystem based on a biodegradable nanocomposite to use for the multimodal cancer imaging and treatment. The -S-S- bond doping MoS2@ss-SiO2 can avoid the long-term toxicity because of the rapid degradation and excretion in living body so. Because of the longer blood circulation time,

the as-synthesized nanocomposites achieved a higher tumor uptake. The particle

size enlarged to about 110 nm after the silica and functional groups were introduced in, thus enhancing the tumor uptake and the therapeutic efficiency. Furthermore, the MoS2@ss-SiO2-Ce6/HA nanosystem performed excellent tri-modal FL/CT/MSOT imaging ability as well as combined PTT and PDT in vivo. We anticipate that the ultrasmall MoS2 nanodots doped biodegradable SiO2 nanoparticles for Clearable FL/CT/MSOT Imaging Guided PTT/PDT Combination Tumor therapy is promising for the clinical practice in the future. ACKNOWLEDGMENTS This work was supported by the National Basic Research Project (973 Program) of China (2014CB932200), National Natural Science Foundation of China (81503016, 81771880, 81401453), Application Foundation and Cutting-edge Technologies Research Project of Tianjin (Young Program) (15JCQNJC13800). ASSOCIATED CONTENT Supporting Information available: [description of content.] 29 ACS Paragon Plus Environment


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Ultrasmall MoS2 Nanodots-Doped Biodegradable SiO2 Nanoparticles

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