One-Pot Synthesis of MoS2 Nanoflakes with Desirable Degradability

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One-Pot Synthesis of MoS Nanoflakes with Desirable Degradability for Photothermal Cancer Therapy Liang Chen, Yihan Feng, Xiaojun Zhou, Qianqian Zhang, Wei Nie, Weizhong Wang, Yanzhong Zhang, and Chuanglong He ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 04 May 2017 Downloaded from http://pubs.acs.org on May 4, 2017

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ACS Applied Materials & Interfaces

One-Pot Synthesis of MoS2 Nanoflakes with Desirable Degradability for Photothermal Cancer Therapy Liang Chena,1, Yihan Fenga,1, Xiaojun Zhouc, Qianqian Zhanga, Wei Niea, Weizhong Wanga, Yanzhong Zhanga, Chuanglong He*a,b a

College of Chemistry, Chemical Engineering and Biotechnology, Donghua

University, Shanghai 201620, China. b

State Key Laboratory for Modification of Chemical Fibers and Polymer Materials,

Donghua University, Shanghai 201620, China. c

Shanghai Key Laboratory of Orthopaedic Implants, Shanghai Ninth People’s

Hospital Affiliated Shanghai Jiao Tong University School of Medicine, Shanghai, 200011, China

*Corresponding author.

Professor Chuanglong He

College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, 2999 North Renmin Road, Shanghai 201620, China.

Tel. /fax: +86 021 6779 2742

Email address: [email protected] (C.L He) 1

These authors contributed equally to this work.

ACS Paragon Plus Environment


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ABSTRACT Developing biodegradable photothermal agent holds great significance for potential clinical translation of photothermal therapy. In the current study, one-pot hydrothermal synthesis of MoS2 nanoflakes with desirable degradation capability was presented. The participation of poly (acrylic acid) (PAA) in hydrothermal process could not only facilitate the modification of polyethylene glycol (PEG), but also bestow degradability to the prepared MoS2 nanoflakes. Moreover, the PEGylated hybrid nanoflakes (MoS2-PPEG) also exhibited excellent stability in various medium and outstanding photothermal properties. Interestingly, MoS2-PPEG behaved distinctly different degradation rate in diverse condition. The rapid degradation of MoS2-PPEG was observed in neutral pH solution, while much slower degradation occurred in acidic tumor microenvironment. Furthermore, data indicated that the major degradation product of MoS2-PPEG was water-soluble Mo-based ion. Meanwhile, the good in vitro biocompatibility of MoS2-PPEG was also confirmed in terms of cytotoxicity and hemolysis. With favorable photothermal performance, MoS2-PPEG can efficiently killing cancer cells in vitro and suppress the tumor growth in vivo. More importantly, the gradual decrease content of MoS2-PPEG in organs and detectable Mo element in urine of mice suggested that the degradability of MoS2-PPEG might facilitate its excretion to some degree. Hence, the degradable MoS2 nanoflakes prepared by one-pot hydrothermal routine may provide insight for further biomedical applications of inorganic photothermal agent. Keywords: Degradable; one-pot; inorganic; MoS2 nanoflakes; photothermal therapy


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1. INTRODUCTION Being prompted by the innate shortcomings of conventional therapeutic methods in clinic, the exploration and application of various nanomaterials for realizing improved efficacy and subdued adverse effects in cancer treatment, also known as “nanomedicine”, have attracted widespread attention over the past decade.1,2 Particularly, the intriguing physiochemical properties of inorganic nanoparticles, including physiological stability, versatile components and easy modification, make them stand out as a great promising nanoplatform for cancer therapy.3 Even though a large

variety

of

nanoparticles,

comprising

nanomaterials,4,5

gold

carbon

nanomaterials,6,7 magnetic nanoparticles8,9 and silica-based nanoparticles,10-12 have been demonstrated for efficient cancer theranostic and even achieved encouraging progress in some pre-clinical researches,13 their undesired accumulation and retention in vital organs of the so-called reticuloendothelial system (RES) have provoked increased concerns about the long-term risk of side effects, greatly impeding their further clinical translation.14,15 Generally, one efficient strategy to relieve the harmful retention of inorganic nanoparticles in normal organs is to employ ultrasmall nanoparticles with size below the threshold of kidney filtration that enables their excretion from body through renal-urinary route.16,

17

Nevertheless, the rapid

clearance of ultrasmall nanoparticles at the same time can also impair their sufficient accumulation concentration and retention time in tumor tissue because of relatively short circulation period and weakened enhanced permeability and retention (EPR) effect.18 Accordingly, synthesis of larger biodegradable nanoparticles likely provides a



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hopeful solution for concurrently exerting the advantage of inorganic nanoparticles and facilitating their excretion from body.19-21 As an emerging therapeutic approach, photothermal therapy (PTT) has been intensively developed as one of the most potential alternative modality for cancer therapy at an impressive rate, on account of its non-invasiveness, lower side effects and remote-controllable feature.22-24 By efficiently converting deep penetrated near-infrared (NIR) laser into heat energy, various nanoparticles, such as semiconductor nanomaterials,25-27 noble metal nanomaterials28-30 and carbon nanomaterials,31-33 have been widely utilized for photothermal ablation of tumor. Unfortunately, further application of these inorganic photothermal agents still faces great challenge due to their poor biodegradability and the risk of long-term retention in organs,34 thus always motivating researchers to exploit satisfactory photothermal agents with biodegradability as well as favorable photothermal performance.35 Recently, Song and co-workers synthesized a new type of degradable molybdenum oxide (MoOx) nanosheets for photothermal tumor ablation.18 Apart from the rapid clearance from mice and prominent tumor homing ability, the attractive pH-dependent degradation behavior of the obtained MoOx-PEG makes it a fascinating inorganic agent for in vivo tumor ablation given that the significant difference of pH value between normal organs and tumor tissue. After that, to overcome the rapid renal excretion and performance regression of ultrasmall black phosphorus quantum dots (BPQDs), Shao et al has fabricated BPQDs-loaded poly (lactic-co-glycolic acid) (BPQDs/PLGA) nanospheres to accomplish superior efficacy and desirable


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biodegradability.36 The well-recognized biodegradable and biocompatible PLGA could not only protect BPQDs from rapid excretion and premature degradation to maintain the photothermal therapeutic effect in a rational period, but also favor the prolonged circulation and enhanced tumor accumulation of BPQDs/PLGA nanospheres with an optimal size (~100 nm). Being from the same two-dimensional (2D) graphene-like layered family with BP,37 molybdenum disulfide (MoS2) with good biocompatibility38 has been studied earlier for efficient photothermal therapy,39 combined therapy40-42 and multifunctional cancer theranostic.43,44 Nonetheless, the synthesis of degradable MoS2 for photothermal therapy has rarely been reported. More recently, Liu’s group systematically investigated the long-term biodistribution, excretion and toxicology of three types of transition-metal dichalcogenides.45 Encouragingly, the results suggested that MoS2 was the only biodegradable material that can be ultimately excreted from body. Moreover, the enzymatic biodegradation of exfoliated MoS2 under biological conditions has also been confirmed in another latest work,46 probably boosting further biomedical application of MoS2. However, irremissible obstacle still lies on the uncontrollable size and morphology of “top-down” exfoliated strategy47,48 and relatively time-consuming degradation time of chemical-exfoliated MoS2. In consequence, it is exceedingly worthwhile to develop a facile approach to prepare degradable MoS2 with satisfactory degradability for cancer therapy. Aiming at that, one-pot hydrothermal method was employed to synthesis MoS2 nanoflakes with desirable degradability in this work. Despite the “bottom-up”



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synthesis of MoS2 nanomaterials for biomedical application has been presented in several literatures,49-51 the degradable MoS2 nanoflakes prepared by hydrothermal routine have not been proposed yet to the best of our knowledge. With the participation of poly (acrylic acid) (PAA) during hydrothermal reaction, polyethylene glycol (PEG) was conjugated onto the as-prepared PAA-capped MoS2 (MoS2-PAA) to obtain PEGylated hybrid nanoflakes (MoS2-PPEG) via amide bond. The physicochemical properties of MoS2-PPEG were fully investigated using a series of characterization techniques. Other than photothermal performance, the degradable capability of MoS2-PPEG was also inspected in detail. Furthermore, the in vitro biocompatibility of MoS2-PPEG was assessed in terms of cytotoxicity and hemolysis. In view of high NIR absorbance of MoS2, the photothermal anti-tumor efficacy of MoS2 was also evaluated both in vitro and in vivo. Besides, the biodistribution, potential excretion and histological assessment of the degradable MoS2-PPEG in body were preliminarily studied.

2. MATERIALS AND METHODS 2.1. Materials Ammonium

molybdate

tetrahydrate

(H24Mo7N6O24·4H2O,

N-(3-Dimethylaminopropyl)-3-ethylcarbodiimide

hydrochloride

99%) (EDC)

and were

purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Thioacetamide (TAA, 99%), PAA (MW ~3000) and N-Hydroxy succinimide (NHS) were obtained from Aladdin Industrial Inc (Shanghai, China). Amine-terminated polyethylene glycol (PEG-NH2, Mw ~5000) was provided by Shanghai Yarebio Co.,


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Ltd. (Shanghai, China). Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), penicillin-streptomycin and trypsin were purchased from Gibco Life Technologies Co. (Grand Island, USA). Cell Counting Kit-8 (CCK-8) was obtained from Beyotime Institute of Biotechnology (China). Live-dead cell staining kit, i.e. Calcein-AM and propidium iodide (PI), was obtained from Jiangsu KeyGEN Bio Tech Corp., Ltd. (Nanjing, China). The ultrapure water was produced by our lab and used throughout the experiment. All other reagents were of analytic grade and used without further purification. 2.2. Synthesis of Degradable MoS2 Nanoflakes Degradable MoS2 nanoflakes were synthesized by a previously reported hydrothermal

method

with

some

modifications.52

In

brief,

88

mg

of

H24Mo7N6O24·4H2O was dissolved in 9 mL of water, into which 1 g of PAA was added. Then 75 mg of TAA dissolved in 5 mL of water was added into the mixture under vigorously stirring. After continuously stirring for 0.5 h, the solution was transferred into stainless steel kettle and sealed. The hydrothermal process was maintained at 180 ºC for 18 h. The resulting black precipitates, denoted as MoS2-PAA, were purified by repeatedly centrifugation and dispersed in ultrapure water for further use. For PEG functionalization, 10 mg of MoS2-PAA was dispersed in 20 mL of water, into which 5 mg of EDC and 4 mg of NHS were added to activate the carboxyl group of PAA. After reacting for 1 h, 5 mg of PEG-NH2 was introduced and reacted for another 24 h. The mixture was centrifuged with high speed to collect hybrid MoS2



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nanoflakes (MoS2-PPEG). 2.3. Characterization The morphology of prepared nanoflakes was observed by transmission electron microscopy (TEM) under accelerated voltage of 200 kV (TEM-2100, JEOL Ltd., Japan). Element mapping and energy dispersive X-ray spectroscope (EDS) were performed using field emission scanning electron microscopy (FESEM, Hitachi S-4800, Japan). The sample was analyzed by X-ray photoelectron spectroscopy (XPS, ESCALab250, USA). X-ray diffraction (XRD) of sample was recorded on a D/max-2550 PC X-ray diffractometer (XRD; Rigaku, Japan). Raman spectrum was recorded by inVia-Reflex micro-raman spectroscopy system (Renishaw, UK) with laser wavelength of 633 nm. Fourier transform infrared (FTIR) spectrum was measured by a Nicolet 6700 (Thermo Fisher, USA) spectrometer using KBr pellets. Thermogravimetric analysis (TGA) was carried out with the temperature ranging from 25 to 600 °C under nitrogen flow using a thermal analyzer (TG 209 F1, Germany). Size distribution of sample was determined by dynamic light scattering (DLS) method using a BI-200SM multiangle dynamic/static laser scattering instrument (Brookhaven, U.S.). UV-vis-NIR spectrum was recorded on a Lambda 35 UV-vis spectrophotometer (PerkinElmer, USA) under ambient conditions. Zeta potential measurement was conducted on Zetasizer Nano ZS apparatus (Malvern, UK). The concentration of Mo element was analyzed by a Leeman Prodigy inductively coupled plasma-atomic emission spectroscopy (ICP-AES) system (Hudson, NH03051, USA). 2.4. Photothermal Performance of MoS2-PPEG


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To determine the photothermal effect of hybrid nanoflakes, 0.2 mL of MoS2-PPEG aqueous solutions with different concentrations (12.5, 25, 50, 100, 200 µg/mL) were put in a 0.25 mL Eppendorf tube, which was subjected to a continuous 808 nm near infrared (NIR) laser (SFOLT Co., Ltd., Shanghai, China) with power density of 1 W/cm2 for 10 min. The temperature variation of MoS2-PPEG aqueous dispersion was monitored by thermocouple thermometer (DT-8891E, Shenzhen Everbest Machinery Industry Co., Ltd, China). The temperature of aqueous dispersion with concentration of 200 µg/mL irradiated by different power density of NIR laser was also recorded. Moreover, the photostability of MoS2-PPEG was evaluated using on-off cycles of NIR laser irradiation. 2.5. Degradation Behavior of MoS2-PPEG The degradation behavior of as-prepared hybrid nanoflakes was investigated by incubating MoS2-PPEG with phosphate buffer solution (PBS) at 37 °C in shaker. In detail, 2 mL of MoS2-PPEG PBS dispersions with different pH values (5.0 and 7.4) were sealed in Eppendorf tube and maintained at 37 °C shaker with shaking speed of 100 rpm. At specific time points, photographs of dispersions were captured by a digital camera. Also, two kinds of MoS2-PPEG colloidal dispersions were diluted by ethanol and dropped on copper grid for TEM observation. Meanwhile, the UV-vis spectrum of sample was recorded at certain time points. To measure the released content of Mo element, 1 mL of MoS2-PPEG in different PBS was sealed in dialysis bag (cut off Mw ~3500) and immersed in 9 mL of corresponding PBS in centrifuge tube. At determined time intervals, the released



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media was withdrawn for ICP measurement and fresh PBS was supplemented. The cumulative released content of Mo was expressed as percentage of the total Mo content in MoS2-PPEG dispersions. 2.6. Cell Lines and Culture Murine breast cancer cell line (4T1 cells) and mouse leukemic monocyte macrophage cell line (RAW 264.7 cells) were provided by cell bank of Chinese academy of science (Shanghai, China). Both two kinds of cells were cultured in standard RPMI 1640 medium supplemented with 10% (v/v) FBS, 100 mg mL-1 streptomycin and 100 U mL-1 penicillin at 37 °C in a humidified incubator with 5% CO2. 2.7. In Vitro Biocompatibility of MoS2-PPEG The cytotoxicity of MoS2-PPEG was evaluated by CCK-8 assay. 4T1 or RAW 264.7 cells were detached from culture flask and seeded into 96-well plate at a density of 104 cells per well. After fully adhesion and spread of cells, the medium was replaced with free medium containing different concentrations of MoS2-PPEG (200, 100, 50, 25, 12.5, 6.25 µg/mL), followed by incubating for another 24 h. Then the cells were repeatedly rinsed with PBS and incubated with serum-free medium containing 10% CCK-8 solution for 2 h. Finally, the cells in 96-well plate were directly subjected to microplate reader to measure absorption value at 450 nm. Cells treated with complete medium without MoS2-PPEG were used as control group. Four parallel experiments were conducted for each group. Hemolysis assay was also performed to evaluate the biocompatibility of


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MoS2-PPEG. According to our previously reported protocol,53 the red blood cells (RBCs) were isolated from fresh blood of male 8 weeks old ICR mice, purified and diluted by PBS. Subsequently, 0.3 mL of MoS2-PPEG dispersed in PBS with different concentrations were co-incubated with 1.2 mL of RBCs suspension for 3 h. The mixture was centrifuged and imaged by a digital camera. The supernatant of sample was measured by microplate reader at wavelength of 541 nm. Hemolysis percentage was determined by the same equation described previously. 2.8. In Vitro Photothermal Therapy of Cancer Cells To assess the in vitro photothermal therapeutic efficacy of MoS2-PPEG against cancer cells, 4T1 cells were seeded into 96-well plate and allowed to fully adhere overnight. MoS2-PPEG dispersed in fresh medium with different concentrations was added into the wells and co-cultured with cells for 4 h. Thereafter, the cells were irradiated by an 808 nm NIR laser at a power density of 1 W/cm2 for 10 min, followed by removing the medium. Then the cells were washed with PBS and incubated for another 2 h. Cell viability was evaluated by CCK-8 assay. The cells without any treatments were set as control. Besides, the power density of NIR laser was varied to investigate the power density-dependent photothermal therapeutic efficacy. For qualitatively assessment, the live-dead staining was conducted after photothermal treatment. The photothermal treated cells were washed and stained with calcein-AM and PI for 15 min. The cells treated with MoS2-PPEG or NIR laser alone were also stained for control. Subsequently, the fluorescent images were captured by inverted microscopy equipped with a CCD photographic system.



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2.9. In Vivo Photothermal Therapy All animal experiments were performed in accordance with guidelines of Institutional Animal Care and Use Committee (IACUC). 6-8 weeks old male ICR mice and female Balb/c mice were both purchased from Slac Laboratory Animal Co. Ltd. (Shanghai, China). 4T1 tumor model was established on Balb/c mice through subcutaneously injection of 4T1 cells (2 × 106 cells per mouse). Once the tumor volume was reached around ~50 mm3, tumor-bearing mice were randomly divided into 4 groups (4 mice for each group): PBS, MoS2-PPEG alone, NIR laser alone and MoS2-PPEG plus NIR laser. The mice were anesthetized by pentobarbital before treatments. Then 50 µL of PBS or MoS2-PPEG suspension (1 mg/mL) was intratumorally injected into tumor-bearing mice. NIR laser at density of 1 W/cm2 was applied to irradiate the tumor site for 10 min. During irradiation process, the temperature variation of tumor site was monitored and imaged by infrared thermal imaging system (GX-A300, Shanghai Guixin Corporation). Tumor volume, which was calculated as a*b2/2 (a and b represented the length and width of tumor, respectively), was recorded in the process of treatments. The relative tumor volume was calculated by normalizing the measured tumor volume (V) to initial volume (V0). Body weight of tumor-bearing mice was also monitored. Moreover, the tumor was excised for histologic evaluation after the treatments. 2.10. Biodistribution of MoS2-PPEG Typically, MoS2-PPEG dispersed in PBS (10 mg/kg) was intravenously injected


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into male ICR mice. At different time intervals post-injection (2 h, 6 h, 12 h, 24 h, 24 h, 48 h, 7 d, three mice for each time point), the mice were sacrificed by injecting over-dosed pentobarbital. Then the major organs of mice, including heart, liver, spleen, lung, kidney, were excised, lyophilized, weighted and digested by aqua fortis for ICP measurement to determine the content of Mo element in those organs. Additionally, three mice injected with MoS2-PPEG were fed and housed in metabolism cages to daily collect urine and feces of mouse within 1 week. Then the major organs of mice were harvested, fixed in formalin, embedded in paraffin and sectioned for hematoxylin and eosin (H&E) staining. The healthy mice were used as control. 2.11. Statistical Analysis Three independent groups were performed throughout experiment. Significant difference between different groups was analyzed by one-way analysis of variance (ANOVA) and Scheffe’s post hoc test. The criterion was expressed as *P < 0.05 and **P < 0.01.

3. RESULTS AND DISCUSSION 3.1. Synthesis of Degradable MoS2-PPEG Synthetic procedure of degradable MoS2-PPEG is schematically depicted in Scheme 1. First, one-pot hydrothermal approach was applied to prepare degradable MoS2 nanoflakes using ammonium molybdate tetrahydrate and thioacetamide as Mo and S source, respectively. Unlike previously reported method,52 the introduction of PAA in hydrothermal reaction renders the obtained MoS2 nanoflakes good degradable



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capability in the present study. Taking advantage of active carboxyl groups in PAA, the PEGylated MoS2 nanoflakes (MoS2-PPEG) was subsequently prepared by conjugating amino-terminated PEG onto MoS2-PAA via amide reaction. As seen in TEM image (Figure 1A), the as-prepared MoS2-PPEG possessed similar sheet-like morphology with previous report47 and individual nanoflake displayed clearly wrinkle appearance with diameter around 90 nm (Figure 1B). Also, FESEM image (Figure 1C) clearly shows the well dispersed MoS2-PPEG nanoflakes with curving layered structure. The co-existence of Mo and S elements was observed in element mappings of MoS2-PPEG (Figure 1D), within which C and O elements derived from polymer on surface of nanoflakes were presented, indicating the successful formation of functionalized nanoflakes. Moreover, typical broad diffraction peaks were also observed in XRD results (Figure S1), which was consistent with previous report.52 Further, chemical composition and covalent states of as-prepared nanoflakes were investigated by XPS. As shown in Figure 1E, the emergence of two typical absorption peaks at ~229 (Mo 3d5/2) and 233 eV (Mo 3d3/2) in XPS of both MoS2-PAA and MoS2-PPEG confirmed that the as-prepared nanoflakes mainly consist of MoS2 regardless of different surface polymers. Meanwhile, the peak around 163 eV was ascribed to S2- according to previous report,50 which further suggested the successful formation of MoS2 phase (Figure S2). On the other hand, surface grafted polymers could confer the as-prepared nanoflakes good dispersity and stability in different medium. As shown in Figure 1F, the dispersed MoS2-PPEG displayed no appreciable aggregation in both water and PBS as well as cell culture medium (inset photograph).


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DLS results also proved the stability of MoS2-PPEG in various medium as no significant alteration of hydrodynamic diameter in all medium was observed, partially owing to the shielding effect of PEG.54 The data also indicated that the size of MoS2-PAA was smaller than prepared MoS2-PPEG (Figure S3). Besides, the zeta potential of MoS2-PAA and MoS2-PPEG was determinded to be -43.6 and -36.8 mV, respectively. The highly charged surface may also be conducive to its good colliod stabiliy. Next,

other physicochemical properties of as-prepared nanoflakes were

systematically inspected. Apparently, Raman spectra of MoS2-PAA and MoS2-PPEG all shows two broaden peaks at ~374 and 401 cm–1, which could be assigned to in-plane E12g and out-of-plane A1g modes, respectively (Figure 2A). The broadening and red-shifting of Raman peaks demonstrated the nanoscale lateral dimensions of layers and the curving multilayer structure of the prepared MoS2 nanoflakes.55,56 Further, the successful modification of nanoflakes was verified by FTIR spectra and TG analysis. As depicted in Figure 2B, peaks at 2852 and 2956 cm–1 belonged to stretching vibration of C-H bond in polymer. Specially, FTIR spectrum of MoS2-PAA exhibited strong absorption at ~1730 cm–1 corresponding to carboxyl group of PAA, which was obviously weakened and red-shifted in spectrum of MoS2-PPEG, indicative of successful conjugation of PEG onto surface of nanoflakes. Besides, the weight loss of nanoflakes was increased from 81.52% for MoS2-PAA to 67.8% for MoS2-PPEG on the basis of TGA results, which further confirmed the presence of PEG in hybrid nanoflakes. Meanwhile, as the identical NIR absorption of MoS2-PAA



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and MoS2-PPEG were testified by UV-vis spectra in Figure 2D, it can be deduced that the modification of PEG on MoS2 nanoflakes would not exert negative influence on their photothermal effect. 3.2. Photothermal Performance of MoS2-PPEG Owing to their strong broad absorption in NIR region, MoS2-PPEG was expected to exhibit excellent photothermal performance. So the temperature elevation of MoS2-PPEG aqueous dispersion under NIR laser irradiation was detected to validate the photothermal effect of hybrid nanoflakes. Not surprisingly, the temperature of MoS2-PPEG dispersion was rapidly increased upon laser irradiation (Figure 3A). It was noteworthy that the temperature of MoS2-PPEG dispersion at concentration of 200 µg/mL was increased from 25.8 to 51.4 °C after 300 s irradiation of NIR laser, while the temperature change for water was only 1.3 °C, thereby confirming the favorable photothermal performance of as-prepared MoS2-PPEG. Moreover, by separately altering the concentration of MoS2-PPEG dispersion and power density of NIR laser, it can be clearly observed that the effect of temperature elevation was both concentration and power dependent (Figure 3B). Another prerequisite of photothermal agent is photostability under laser irradiation. To validate the photostability of hybrid nanoflakes, MoS2-PPEG dispersion was also irradiated by continuous NIR laser at power density of 1 W/cm2. Not surprisingly, the temperature variation was almost constant during switching cycles of laser irradiation, (Figure 3C). Additionally, it was observed that MoS2-PPEG dispersions before and after irradiation exhibited similar absorption in UV-vis spectra with steady absorption


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value at 808 nm under cycles of irradiation, further suggesting the satisfactory photostability of the obtained MoS2-PPEG. 3.3. Degradability of MoS2-PPEG Since the reluctant biodegradability of inorganic nanomaterials severely clouds their biosafety and biomedical applications, the biodegradable capability of nanoparticles appears particular prominent.3 So the degradability of MoS2-PPEG was fully investigated by subjecting it to imitative physiological and acidic pH buffer solutions. Very inspiringly, MoS2-PPEG exhibited different degradation behavior in those two conditions. Compared with the hybrid nanoflakes immersed in pH 5.0, obvious faster degradation rate at pH 7.4 was observed. The black color of MoS2-PPEG dispersed in PBS with pH 7.4 distinctly faded after 1 d, while the variation was visible until 7 d for that of 5.0 (Figure 4A). Especially, the dispersion with pH 7.4 was colorless after one week incubation at 37 °C, indicative of its nearly complete degradation. Meanwhile, a fact particularly worthy of mention was that both dispersion at pH 7.4 and 5.0 appeared no precipitates even after degradation for 7 d, indicating that the degradation products of MoS2-PPEG was highly soluble in buffer solution. According to a recent study of Liu’s group, the MoS2 nanosheets prepared by chemical exfoliation method could be oxidized into water-soluble MoVI-oxide species (e.g., MoO2–4) after a relative long period (one month).45 Therefore, we speculated that MoS2-PPEG prepared by hydrothermal method in this study could also be degraded into water-soluble Mo-based ions. On the other hand, UV-vis spectroscopy was applied to monitor the absorption value at 808 nm of different MoS2-PPEG



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dispersions. As expected, the reduction in UV-vis spectra presented faster rate in physiological pH than that of tumor pH condition. Specifically, the absorption value clearly decreased from 0.34 to 0.07 for MoS2-PPEG dispersed in pH 7.4 after 1 d, while the decline of value from 0.35 to 0.08 consumed 7 days for acidic pH condition (Figure 4B, C), which was in good agreement with above tendency. The results suggested that pH value turns out to be a crucial factor affecting the degradation rate of as-prepared MoS2-PPEG, which was likely resulted from the important role of OH– concentration in its degradation process.57 Considering the acidic microenvironment of tumor tissue, this discrepancy in degradation rate for MoS2-PPEG may be beneficial to its application in cancer therapy.3,18 To further confirm its degradability, MoS2-PPEG dispersions at different pH conditions were sealed in dialysis bag and placed in shaker (37 °C) to determine the released content of Mo element using ICP-AES. Evidently, a burst releasing behavior at first 3 days was observed for pH 7.4, with around 80% content of Mo element was decomposed from MoS2-PPEG. By contrast, the released Mo content of MoS2-PPEG dispersed in pH 5.0 was only 48% at 3 d and eventually achieved 82% after 9 d, displaying sustained releasing behavior with much slower rate than neutral pH (Figure 4D). Subsequently, the morphology evolution of MoS2-PPEG in the process of degradation was directly surveyed using TEM (Figure 4E). It can be intuitively observed that the morphology of MoS2-PPEG in neutral buffer solution undergone destructive change after 1 d, resulting in ultrasmall and irregularly ruptured clusters. When the pH of soaking solution was 5.0, the structural decomposition of


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MoS2-PPEG also occurred somewhat slowly. The edges of nanoflakes turned indistinct and finally evolved to collapsed structure with eroded framework after 7 d. Last, to shed more light on its degradation, XPS was utilized to study the valence state of Mo element in degradation products under physiological condition (Figure S4). The data revealed that the dominating MoIV in MoS2-PPEG was eventually oxidized to MoVI, which was evidenced by the newly emerged peak at 236.8 eV on the basis of previous research.45,46 Therefore, not only the aforementioned pH-dependent degradation rate would favor its application for cancer therapy, we also expected that the soluble degradation products of MoS2-PPEG could alleviate its unwanted retention and toxicity in vivo, thus making it more promising for biomedical application. 3.4. In Vitro Biocompatibility of MoS2-PPEG In

addition

to

desirable

photothermal

performance

and

degradability,

biocompatibility of MoS2-PPEG also needs to be carefully assessed before its photothermal application. So the cytotoxicity of MoS2-PPEG was evaluated by CCK-8 assay. Figure 5A shows the cell viability of 4T1 and RAW 264.7 cells after treated with different concentrations of MoS2-PPEG for 24 h. Clearly, no appreciable cytotoxicity was detected as evidenced by the high cell viability of treated cells, which was still as high as 87.11% and 89.04% for 4T1 and RAW 264.7 cells after exposed to 200 µg/mL of MoS2-PPEG, respectively. Furthermore, the impact of MoS2-PPEG on hemolytic behavior of RBCs was also evaluated by hemolysis assay.58 As presented in Figure 5B, after incubated with



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different MoS2-PPEG for 3 h, negligible hemolysis of RBCs was observed. Specifically, the hemolytic percentage of MoS2-PPEG at a concentration of 400 µg/mL was calculated to be 3.8%, implying the good hemocompatibility of MoS2-PPEG in tested concentrations. Combining with above results, it can be predicted that the favorable biocompatibility of prepared MoS2-PPEG would benefits its further biomedical applications. 3.5. In Vitro Photothermal Killing of Cancer Cells After clarifying its favorable biocompatibility, the photothermal killing efficacy of MoS2-PPEG against cancer cells was investigated. Benefiting from admirable photothermal performance of MoS2-PPEG, the relative cell viability of 4T1 cells was obviously decreased with the increased concentration of MoS2-PPEG under laser irradiation, indicating the enhanced photothermal killing efficacy with higher concentration of MoS2-PPEG (Figure 6A). Specifically, the viability of cells treated with 100 µg/mL MoS2-PPEG was calculated to be 39.21% and the cell mortality rate reached up to 81.36%, implying the favorable photothermal efficacy of MoS2-PPEG. In addition, the power density of NIR laser also affected the therapeutic efficacy of MoS2-PPEG. As depicted in Figure 6B, more 4T1 cells were destroyed with the elevated power density of NIR laser, while NIR laser irradiation alone apparently could not kill cancer cells in the presence of MoS2-PPEG. To provide deeper insight of in vitro photothermal killing effect, live/dead staining was also performed after different treatments. Figure 6C shows the fluorescence images of treated cells, in which live and dead cells displayed green (calcein AM) and red (PI) fluorescence,


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respectively. As with the control group, no red fluorescence was found for cells treated with MoS2-PPEG or laser irradiation alone, suggesting their negligible influence on cancer cells. Conversely, substantial cell death was observed in photothermal group (MoS2-PPEG + laser irradiation). Thus, above results declared that prepared MoS2-PPEG can act as an efficient photothermal agent for cancer therapy. 3.6. In Vivo Photothermal Therapy Motivated by the excellent in vitro photothermal killing effect of MoS2-PPEG on 4T1 cells, in vivo photothermal antitumor efficacy of the hybrid nanoflakes was inspected using subcutaneously xenografted 4T1 tumor on Balb/c mice. Prior to evaluate the therapeutic efficacy, the in vivo photothermal effect of MoS2-PPEG was investigated. After intratumorally injection of MoS2-PPEG dispersion (50 µL, 1 mg/mL), it was clearly observed that temperature of tumor site rapidly elevated upon NIR laser irradiation as compared with control mouse injected with PBS. Concretely, the temperature reached up to 52.1 °C for MoS2-PPEG, while the increment was just ~1 °C for PBS, indicative of equally favorable in vivo photothermal conversion of MoS2-PPEG (Figure 7A, B). Afterward, once tumor size grown to ~50 mm3, those tumor-bearing mice were randomly divided into four groups (4 mice per group), including control, MoS2-PPEG, NIR laser irradiation and MoS2-PPEG plus NIR laser irradiation. After being subjected to corresponding treatment, the tumor size and body weight of mice were monitored to evaluate the therapeutic efficacy. As shown in Figure 7C, similar with



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untreated control mice, the tumor volume of mice treated with MoS2-PPEG or NIR laser irradiation alone continuously increased during the detection period, which further confirmed that laser irradiation or hybrid nanoflakes alone cannot inhibit the tumor growth as descried before. In sharp contrast, the photothermal therapy of MoS2-PPEG effectively suppressed the tumor growth as the tumor volume dramatically decreased after treatment. Meanwhile, the body weight of mice showed no significant variation in treatment duration, indicating that the mice were well tolerated with no adverse effects toward photothermal therapy (Figure 7D). In addition, the therapeutic efficacy was also immediately analyzed by H&E staining of the tumors slices after treatments (Figure 7E). Predictably, the cancer cells of photothermal group displayed typical signs of cell damage, including cell shrinkage, nuclear necrosis and loss of contact, while the same densely-packed cells and regular cell morphology were observed in two other groups and control group. Besides, the flat right hind of photothermal treated mouse (inset photograph in Figure 7E) was also apparently discriminated with the severely humpy tumor of other three groups at the end of treatments, thus offering intuitive evidence of excellent photothermal therapeutic efficacy. Taken together, our results manifested that the as-prepared MoS2-PPEG holds potential for in vivo photothermal therapy of tumor with low adverse effects. 3.7. Biodistribution and Histological Evaluation Although the good biocompatibility and favorable photothermal therapeutic efficacy of hybrid nanoflakes were validated above, attentions should also be devoted


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to the in vivo behavior of MoS2-PPEG. Therefore, the biodistribution of MoS2-PPEG, i.e. the Mo content in major organ such as heart, liver, spleen, lung and kidney, was determined by ICP-AES at seven different time points. Surprisingly, unexpected highest concentration was detected in lung, a major organ of RES.59 Even though the reason of higher accumulation of MoS2-PPEG in lung remains uncertain, similar tendency about biodistribution of nanomaterials was also obtained in a recent study, which ascribed this result to the particle size and size distribution of the nanocarriers.60 Moreover, the abundant content of MoS2 nanosheets prepared by bottom-up approach in lung was also previously reported.47 Definitely, high Mo level was also detected in another two major RES organs, liver and spleen as presented in Figure 8A. In addition, the retention content of MoS2-PPEG in liver and spleen slowly increased within 1 d post-injection. Clearly, it also can be noticed that the Mo content in lung dramatically decreased from initial 473.89 µg/g to 175.83 µg/g at 7 d post-injection. Also, the relative decrease of Mo in those organs was observed after 7 d in comparison to that of 1 d, suggesting the gradual clearance of MoS2-PPEG.52 To further validate this, the excretion of mice injected with MoS2-PPEG was analyzed in 1 week. Inspiringly, we indeed detected Mo from the urines of treated mice, which was especially obvious in the first day after administration of MoS2-PPEG (Figure 8B). By comparison, negligible content of Mo element was found in feces throughout the experimental period, displaying some consistency with former study.61 Hence, despite more complicated physiological environment and in vivo fate of nanomaterials hampered the inquiry of in vivo degradable property of hybrid nanoflakes, we



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deduced that the degradability of as-prepared MoS2-PPEG would partly facilitate its clearance from body by taking the advantage of dissolvable degraded products. Following the biodistribution, histological examination was performed to preliminarily evaluate the in vivo toxicity of MoS2-PPEG. Figure 8C shows the H&E stained slices of major organs (heart, liver, spleen, lung and kidney) of control and treated mice. The images demonstrated that no appreciable tissue damage and abnormal inflammatory response were discovered at 7 d post-injection. So the almost identical histological patterns of injected group with untreated group affirmed the negligible toxicity of MoS2-PPEG at the experimental condition, suggesting its good applicability for in vivo photothermal therapy.

4. CONCLUSION In this work, degradable MoS2-PPEG has been successfully developed as a photothermal nanoplatform for in vivo tumor ablation. A facile hydrothermal approach was employed to synthesis the pristine nanoflakes by using PAA as organic ligand, which unexpectedly endowed the nanoflakes with degradable capability. After modifying as-prepared nanoflakes with PEG via amidation reaction, the obtained MoS2-PPEG possessed excellent colloidal stability without undesirable aggregations. Furthermore, the negligible cytotoxicity and hemolysis were also demonstrated in vitro. Most importantly, unlike previously reported MoS2 nanomaterials prepared by hydrothermal procedure, the synthesized MoS2-PPEG in this work exhibited attractive degradability, which behaved significant different under imitative normal and acidic conditions. It was found that MoS2-PPEG degraded much faster under physiological


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condition (pH 7.4) than that of acidic condition (pH 5.0). Additionally, the data suggested that MoS2-PPEG could eventually degrade into water-soluble ions MoO2–4, somewhat highlighting its superiority than other non-degradable inorganic photothermal agent. Moreover, the favorable photothermal performance of MoS2-PPEG was not only able to efficiently kill cancer cells in vitro, but can also significantly suppress the growth of subcutaneously xenografted 4T1 tumor in vivo. Besides, in spite of the unexpected high pulmonary accumulation of MoS2-PPEG was obtained from biodistribution result, no appreciable organ damage was observed on basis of histological examination. Also, the detectable Mo in urine of mice injected with MoS2-PPEG partially affirmed its possibility of gradual in vivo clearance considering its water-soluble degradation products. Overall, we anticipate that this study could provide an insight for further biomedical applications of degradable MoS2 and attract more interest to develop degradable inorganic nanomaterials for nanomedicine.

ACKNOWLEDGEMENTS This study was financially supported by the National Natural Science Foundation of China (31570984, 31271028), International Cooperation Fund of the Science and Technology Commission of Shanghai Municipality (15540723400), the Open Foundation of State Key Laboratory for Modification of Chemical Fibers and Polymer

Materials

(LK1416)

and

Chinese

Universities

(CUSF-DH-D-2015043).


Scientific

Fund


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ASSOCIATED CONTENT Supporting information The XRD data of prepared MoS2 nanoflakes and MoS2-PPEG; The S 3p XPS of MoS2-PAA and MoS2-PPEG; Zeta potential of MoS2-PAA and MoS2-PPEG; Size distribution of MoS2-PAA dispersed in water measured by DLS; The Mo XPS results of MoS2-PPEG after degraded in 7.4 for 1 week.

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FIGURES

Scheme 1. The schematic diagram of preparation of MoS2-PPEG with desirable degradability for in vivo photothermal cancer therapy.



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Figure 1. Physicochemical properties of MoS2 nanoflakes. (A) TEM image and (B) high magnified TEM image of MoS2-PPEG; (C) FESEM image and (D) elemental mapping images (Mo, S, C, O) of MoS2-PPEG; (E) Mo 3d XPS of as-prepared MoS2 nanoflakes; (F) Size distribution of MoS2-PPEG in water, PBS and cell culture medium measured by DLS, inset photograph presented corresponding MoS2-PPEG dispersions in different medium.


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Figure 2. (A) Raman spectra; (B) FTIR spectra; (C) Thermogravimetric analysis and (D) UV-vis spectra of MoS2 nanoflakes.



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Figure 3. Photothermal performance of MoS2-PPEG. (A) Temperature variation of MoS2-PPEG aqueous dispersions with different concentrations irradiated by 808 nm NIR laser at power density of 1 W/cm2; (B) Temperature variation of MoS2-PPEG with a concentration of 200 µg/mL under 808 nm NIR laser irradiation (1 W/cm2); (C) Temperature variation of MoS2-PPEG dispersion (200 µg/mL) under “on-off” cycles of NIR laser irradiation; (D) UV-vis spectra of MoS2-PPEG dispersion before and after NIR laser irradiation, the 808 nm absorption value of dispersion during cycles of irradiation were expressed by blue coordinate axis (right and up), inset pictures represented the well-dispersed MoS2-PPEG solution before and after irradiation.


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Figure 4. The degradation behavior of MoS2-PPEG. (A) Photographs of MoS2-PPEG dispersed in PBS (pH 5.0 and 7.4) after incubation at 37 °C for determined time intervals; UV-vis spectra of MoS2-PPEG dispersions with (B) pH 5.0 and (C) pH 7.4 at different degradation time, right and up axis expressed the 808 nm absorption values at corresponding time points; (D) The cumulative release Mo content from MoS2-PPEG dispersions at pH 5.0 and 7.4; (E) TEM observation of MoS2-PPEG after degraded in pH 5.0 and 7.4 for 1 d, 3 d and 7 d, respectively.



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Figure 5. In vitro biocompatibility of MoS2-PPEG. (A) Cell viability of 4T1 cells treated with MoS2-PPEG at different concentrations (6.25, 12.5, 25, 50, 100, 200 µg/mL) for 24 h; (B) Hemolytic percentage of RBCs after incubated with MoS2-PPEG at different concentrations for 3 h, inset image was results of hemolysis assay.


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Figure 6. In vitro photothermal killing of cancer cells. (A) Cell viability of 4T1 cells exposed to various concentrations of MoS2-PPEG and irradiated by 808 nm laser for 10 min; (B) Cell viability of 4T1 cells treated with (W) or without (W/O) MoS2-PPEG at a concentration of 200 µg/mL and irradiated by 808 nm laser at different power density; (C) Live/dead staining of 4T1 cells after different treatments: control, MoS2-PPEG,NIR laser, and MoS2-PPEG plus NIR laser, color: green (Calcium AM) and red (PI).



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Figure 7. In vivo photothermal therapy of MoS2-PPEG. (A) Thermal images of tumor site of mice injected with PBS or MoS2-PPEG under irradiation of 808 nm NIR laser; (B) Corresponding temperature variation of tumor site based on thermal images; (C) Relative tumor volume and (D) body weight of mice under different treatments; (E) Representative H&E stained slices of mice under different treatments, inset images showed typical tumor-bearing mice at the end of treatment.


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Figure 8. In vivo evaluation of MoS2-PPEG. (A) Mo content in major organs of mice injected with MoS2-PPEG at different time points; (B) Mo content in urine and feces collected from mice injected with MoS2-PPEG; (C) Representative H&E stained organ slices of mice 7 d post-injection of MoS2-PPEG.



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Table of Content


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One-Pot Synthesis of MoS2 Nanoflakes with Desirable Degradability

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