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A Multifunctional Nanosystem for Synergistic Tumor Therapy Delivered by Two Dimensional MoS

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Mengyun Peng, Diwei Zheng, Shi-Bo Wang, Si-Xue Cheng, and Xian-Zheng Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b03276 • Publication Date (Web): 05 Apr 2017 Downloaded from http://pubs.acs.org on April 11, 2017

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ACS Applied Materials & Interfaces

A Multifunctional Nanosystem for Synergistic Tumor Therapy Delivered by Two Dimensional MoS2 Meng-Yun Peng,† Di-Wei Zheng,† Shi-Bo Wang,‡ Si-Xue Cheng† and Xian-Zheng Zhang*,†,‡

†

Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry ‡

The Institute for Advanced Studies, Wuhan University, Wuhan 430072, China

* Corresponding Author: [email protected] Keywords: MoS2 nanoflake, photothermal therapy, photodynamic therapy, charge conversion, TBO release

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ABSTRACT: A multifunctional nanosystem based on two dimensional molybdenum disulfide (MoS2) was developed for synergistic tumor therapy. MoS2 was stabilized with lipoic acid modified PEG and modified with pH-responsive charge convertible peptide (LA-K11(DMA)). Then a positively charged photosensitizer, toluidine blue O (TBO) was loaded on MoS2 via physical absorption. The negatively charged LA-K11(DMA) peptide was converted into positively charged one under acid conditions. Charge conversion of peptide could reduce binding force between positively charged TBO and MoS2, leading to TBO release. Furthermore, positively charged nanosystem was easy to be endocytosed by cells. Photo-induced hyperthermia of MoS2 in tumor areas could promote TBO release and exhibited photothermal therapy. In vitro and in vivo results demonstrated that fluorescence and photo-induced ROS generation of TBO were severely decreased by MoS2 in normal condition. While in acid condition, the pHresponsive nanosystem exhibited highly specific and efficient antitumor effect with TBO release and photo-induced reactive oxygen species (ROS) generation, suggesting a promising accessory for synergistic tumor therapy.

1. INTRODUCTION Photodynamic therapy (PDT) is a treatment triggered by light with photosensitizer (PS).1 Under optical irradiation, PS is excited into an excited singlet state. As many fluorescence molecules do, excited PS falls back to ground state and exhibits fluorescence. On the other hand, energy of the excited PS is also transferred to surrounding oxygen molecules to form reactive oxygen species (ROS). ROS is highly reactive which can oxidize biomolecules including lipid, protein and DNA, thus finally induce cell death.2,3 Compared with wildly used chemotherapy and radiotherapy, PDT has no cumulative toxicity in tumor cells, which is not inclined to induce


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drug resistance.4-6 Additionally, Hamblin et al. have discussed that PDT is able to induce tumor immunity, which is helpful for long-term tumour control, in contrast to conventional treatments that are mostly immunosuppressive.7 Furthermore, commonly used photosensitizers are fluorescent and can also serve as fluorescence imaging agents, which can play an important role in diagnostic imaging applications.8,9 Despite PDT shows promising applications in tumor therapy, therapeutic effect of single PDT treatment is generally unsatisfactory. First, ROS generated by PSs is extremely unstable with a less than 3.5 µs lifetime, and the diffusion range of ROS is only about 20 nm. As a result, ROS damage only can be displayed near PS molecules, which severely decreased the PDT therapeutic efficiency.10,11 Secondly, PDT therapeutic efficiency depends on the oxygen concentration and is decreased by hypoxic environment in tumor tissues.12,13 Moreover, inefficient PS cellular internalization of tumor cells limits PDT efficiency, and nonspecific PS internalization by normal cells usually leads to serious side effects.14 Therefore, it is urgent to develop an effective solution for highly efficient and specific PDT against tumor. Recently, two dimensional MoS2 has been widely used as drug carriers and photothermal agents due to its high surface-to-volume ratio, good biocompatibility, ease of functionalization and photothermal effect properties, etc.15-17 Photothermal therapy (PTT) is triggered by nearinfrared (NIR) light and energy absorbed by photothermal agents is transferred into heat.18 Photo-heating of tumor areas could increase blood flow, raise tumor oxygen supply and consequently enhance PDT efficiency.19 Furthermore, in many investigations, it has been reported that heating ranging between 40 °C and 45 °C was mild for normal tissues and unlikely to induce inflammatory disease and tumor metastasis.20 Zhao et. al proposed the combination of peroxidase-like catalytic activity and PTT to avoid skin damage caused by long-term exposure


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under high power density of NIR laser.21 Moreover, the photothermal effect of MoS2 could help drug release. For example, Kim et al. developed a photothermally controllable drug delivery system, which released loaded DOX into the aqueous environment with photothermal effect of MoS2.22 In this concept, a pH-responsive multifunctional nanosystem based on two dimensional molybdenum disulfide (MoS2) was developed. In this study, two dimensional MoS2 was first modified with a pH-responsive charge convertible peptide (LA-K11(DMA)). Then a positively charged photosensitizer toluidine blue O (TBO) was loaded on MoS2 via physical absorption process. As illustrated in Scheme 1, under physiological conditions, TBO was absorbed on MoS2 and negatively charged peptide stretched out of MoS2, leading to a prolonged circulation time. Fluorescence and photo-induced ROS generation of TBO were largely decreased by two dimensional MoS2 due to an instantaneous Förster resonance energy transfer (FRET).23 However, under acid conditions, such as tumor extracellular matrix (ECM, pH~6.8) or endo/lysosome (pH~5.0), the lysyl succinyl amides of LA-K11(DMA) were hydrolyzed and positively charged amino groups were exposed.24 Charge-converted nanosystem under tumor ECM obtained higher affinity toward negative cell membranes and are easy for cell internalization.25 What’s more, the positively charged peptide was expected to serve as a “spy”, which could reduce interactions between the absorbed TBO and two dimensional MoS2, and eventually led to TBO release. PTT of MoS2 caused hyperthermia in tumor areas, which could damage cancer cells,26 and promote therapeutic efficiency of the followed PDT.27 At the same time, photo-heating of carrier helped realize further TBO release from carrier by reducing the binding force between TBO and MoS2.17,20 The pH-responsive multifunctional nanosystem


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enhanced specific cell internalization of materials and exhibited effective PDT therapeutic effect under irradiation, suggesting a promising application for further synergistic tumor therapy. 2. EXPERIMENTAL SECTION 2.1. Synthesis of LA-PEG. 0.1 g lipoic acid (0.5 mmol, Heowns Biochemical Technology Co., Ltd. Tianjing, China) was dissolved in 5 mL DMF in a 25 mL flask. Another 5 mL DMF containing 0.115 g 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide·HCl (EDC·HCl, 0.6 mmol, 1.2 eq) and 0.07 g N-hydroxysuccinimide (NHS, 0.6 mmol, 1.2 eq) was added into the flask by drop. Another 5 ml DMF containing 0.5 g PEG-NH2 (Mw 5000, 0.1 mmol, Yare Biotech, Inc., Shanghai, China) was obtained and added into the mixture. Reactions were allowed for whole night with stirring. The solution was dialyzed with dialysis membrane (cellulose ester, MWCO of 2 kDa) against DMF to remove the excess lipoic acid and other impurities and then dialyzed against DI water to replace the solvent. Production was obtained after freeze-drying and characterized by FT-IR (Fourier Transform Infrared Spectroscopy) (Figure S1). 2.2. Peptide Synthesis. LA-GKKKKKKKKKKK-NH2 (LA-K11) were prepared stepwise according to the Fmoc solid-phase peptide synthesis method. Fmoc protection of the Rink Amide-AM resin and amide acids was removed by 20% (V/V) piperidine/DMF and provided amino group to react with the next amide acid. Amidation process was carried out in a DMF solution containing benzotriazole-N,N,N’,N’-tetramethyluroniumhexafluorophosphate (HBTU), N-hydroxybenzotriazole (HOBt), and N,N-diisopropylethylamine (DIEA) (GL Biochem Ltd., Shanghai). 10% ninhydrin in methanol was used for the coupling efficacy monitoring. LA was linked to N-terminus of Gly in DMF solution with HOBt and N, N-diisopropylcarbodiimide (DIC) at the end of peptide synthesis and stayed reaction overnight. Product was cleaved from the resin utilizing a recipe of Trifluoroacetic acid (TFA)/triisopropylsilane (TIS)/H2O/ethylene


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dithioglycol (EDT) (95/1/2/2, V/V/V/V) and precipitated out in the cold ether. Peptide was further characterized by ESI-MS (Electrospray Ionization Mass Spectrometry) (Figure S2). 2.3. Synthesis of LA-K11(DMA). 66 mg LA-K11 peptide (40 µmol) was dissolved in DI water, and then excessive Dimethylmaleic anhydride (DMA, 330 mg, 8 mmol, 20 eq) (Aladdin Reagent Co. Ltd., Shanghai) was added to the solution. The pH of the reaction solution was adjusted to 89 using 1 mol/L NaOH solution and kept over the reaction process. The reaction was allowed to continue overnight at room temperature after the pH was constant. Product was obtained after dialyzed against water at pH 8-9 for 24 h and freeze-dried. 1H NMR (Proton Nuclear Magnetic Resonance) spectrum was performed for the characterization (Figure S3). 2.4. Two Dimensional MoS2 Nanoflakes Synthesis. Two dimensional MoS2 nanoflakes were synthesized using ultrasonic exfoliation. 0.5 g MoS2 crystal powder (Aladdin Reagent Co. Ltd., Shanghai) was encapsulated in a 25 mL flask and then removing air via nitrogen atmosphere. 5 mL of 1.6 M n-butyllithium solution in hexane was injected for MoS2 crystal immersion. The mixture was stirred for 3 days under nitrogen atmosphere to ensure adequately component interaction and lithium intercalation into the MoS2 crystal. Hexane was used to wash the reactant and remove unreacted lithium and other organic residues by suction filtration. After immediately adding DI water and ultrasonicated for 3 hours, the suspension was centrifuged under 3000 rpm for 10 min to ensure unexfoliated bulk MoS2 was removed. Supernate was collected and freeze dried. MoS2-PEG was subsequently synthesized by reacting of MoS2 and LA-PEG under stirring for 5 h. MoS2-PEG was dialyzed with dialysis membrane (cellulose ester, MWCO of 14 kDa) against DI water to remove the excess LA-PEG then freeze dried and analyzed by TGA (Thermogravimetric Analysis) under nitrogen atmosphere. Characterizations of MoS2 and MoS2-


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PEG were performed via transmission electron microscope (TEM), powder X-Ray Diffraction (PXRD) (Figure S4) and atomic force microscope (AFM) (Figure S5). 2.5. Fluorescence Quenching and Recovery of TBO. Freeze-dried MoS2-PEG was dispersed in PBS buffer solution (pH 7.4) at different concentration and incubated with TBO (5 µM) for 20 min, fluorescence of MoS2/TBO (designed as MT) was then analyzed using RF-530/PC spectrofluorophotometer to detect the fluorescence emission spectra of TBO, the emission spectra were collected from 600 nm to 800 nm. To detect the pH-sensitive fluorescence recovery of TBO from the MoS2 nanoflake, solutions with three different pH buffer (pH 6.8, 7.4 phosphate buffer and pH 5.0 acetate buffer) containing 0.3 mg/mL MoS2-PEG were prepared and ultrasonicated for 20 min. Then 5 µM TBO and 0.3, 0.6, 1.0 mg/mL LA-K11(DMA) peptide were added into the suspensions subsequently and ultrasonicated for another 30 min. After incubated for 1 h, fluorescence of MoS2-K11(DMA)/TBO (designed as MKT) solutions was then analyzed using RF-530/PC spectrofluorophotometer to detect the fluorescence emission spectra (600-800 nm) of TBO. 0.3 mg/mL MoS2-PEG with 0.6 mg/mL LA-K11(DMA) was the optimal ratio for TBO release. MT and MKT were analyzed by TGA (Thermogravimetric Analysis) under N2 atmosphere. 2.6. MKT Stability Detection. MKT (0.3 mg/mL MoS2-PEG with 0.6 mg/mL LA-K11(DMA) and 10 µM TBO) was dispersed in DI water and PBS buffer (pH 7.4), respectively. Suspensions were stood at room temperature. Pictures of the nanomaterial suspension along with time and the hydrodynamic size of the nanomaterial in water and PBS were collected at different time (Figure S6). 2.7. The pH-Sensitive Charge Conversion. Materials were combined with the dosage of 0.3 mg/mL MoS2-PEG, 0.6 mg/mL LA-K11(DMA) peptide and 5 µM TBO were applied. Samples


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were incubated in a water bath at 37 °C for 1 hour to ensure complete charge conversion the LAK11(DMA) peptide. Zeta potential of materials at three pH was determined using the ZetaNanosizer (Figure 2F and Figure S7). 2.8. TBO Loading and Release. 0.3 mg/mL MoS2-PEG and 0.6 mg/mL LA-K11(DMA) peptide incubated with 5 µM, 10 µM and 33 µM TBO for 1 h, respectively. Then MT solution and MKT solution were ultracentrifugated at 40000 rpm/min for 30 min. The supernatant was tested by RF-530/PC spectrofluorophotometer to detect the fluorescence emission spectra (600800 nm) of TBO. Unloaded TBO was calculated with a standardization curve of TBO (Figure S8). TBO loading efficiency was calculated with the whole TBO as denominator. MKT TBO release with 0.3 mg/mL MoS2-PEG, 0.6 mg/mL LA-K11(DMA) peptide and 10 µM TBO was carried out in a dialysis tube (MWCO 500-1000 Da) under 37 oC. 1 mL MKT solution was dialyzed against 4 mL pH 7.4 buffer solution, pH 6.8 buffer solution and pH 5.0 buffer solution, respectively. At each period the dialysate was collected at different time, and dialysate was replaced with 4 mL fresh buffer solution. Group with laser irradiation was irradiated at 2 h and 20 h with 808 nm laser (1.5 W/cm2, 2 min), Released TBO was calculated with the standardization curve of TBO. 2.9. Intracellular Fluorescence Recovery and ROS Generation. In order to explore the in vitro antitumor effect in detail, confocal laser scanning microscopy (CLSM, Nikon C1-si, BD Laser) and flow cytometry (FCM, BD FACSAriaTM III) were employed for fluorescence recovery and ROS generation in cells. SCC-7 cell (squamous cell carcinoma) and COS-7 cell (African green monkey kidney lines) were chosen as tumorous cell and the normal control. Cells were seeded at the density of 1 × 105 cells/well in glass-bottom dish for CLSM, and for FCM, cells were seeded at the density of 5 × 104 cells/well in 6-well plates. Cells were cultured with 1


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mL DMEM for 24 h. After cell adherence, medium was replaced with 1 mL DMEM containing TBO (10 µM), MT (0.3 mg/mL MoS2, 10 µM TBO) or MKT (0.3 mg/mL MoS2, 0.6 mg/mL LAK11(DMA), 10 µM TBO) and cells were further incubated at 37 °C. To detect the TBO fluorescence recovery in cells, media containing materials were removed after incubating for 4 h and the cells were washed three times with PBS (pH 7.4). Cells of MKT PTT group were further illuminated with 808 nm laser (1.5 W/cm2, 2 min) for photo-heating. After that, cells in glass-bottom dish could be observed by CLSM immediately. In flow cytometry treatment, all the cells were digested by trypsin and collected by centrifugation (1000 rpm × 4 min). Supernatant was removed and the bottom cells were washed twice with PBS. Then the suspended cells were filtrated and examined by flow cytometry. Excitation of TBO was performed with laser at 543 nm and emission spectra were collected using a wavelength range of 570-620 nm. Intracellular ROS generation was measured using 2',7'-dichlorofluorescin diacetate (DCFHDA, Beyotime Institute of Biotechnology, China) as the fluorescence sensor. After incubating with cells for 4 h, media containing materials were removed and DCFH-DA was added (final concentration 1 × 10−5 M) and incubated for 20 min. After that, the cells were further illuminated with 808 nm laser (1.5 W/cm2, 2 min) for photo-heating and 630 nm laser (30 mW/cm2, 5 min) for photodynamic therapy, respectively. Cells were then examined by flow cytometry. The fluorescence scan was performed with 1 × 104 cells. Emission of DCFH was collected using a wavelength range of 510-540 nm. 2.10. MoS2-PEG Hemolysis Test. To evaluate the preliminary biocompatibility of MoS2PEG, 1 mL of blood samples (obtained from New Zealand rabbit), which were stabilized by ethylenediaminetetracetic acid, was added to 2 mL of PBS. Red blood cells were separated from


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serum by centrifugation (2000 rpm × 10 min), and then diluted into 10 mL of PBS. Then, 0.2 mL of diluted RBC suspension was mixed with 0.8 mL sample solutions. PBS and deionized water were used as a negative and positive control, respectively. MoS2-PEG dispersions concentration ranged from 0 to 1.2 mg/mL. After that, all the mixtures were kept for 4 h at room temperature. After centrifugation at 12000 rpm for 5 min. The optical density (OD) of mixtures and relevant concentrations of MoS2-PEG in PBS was measured at 541 nm using a microplate reader (Model 550; Bio-Rad, Hercules, CA, USA). The hemolysis percent of RBCs was calculated using the equation: Hemolysis persent %

sample !"

× 100% ,

where

ODsample,

ODMoS2, ODnegative and ODpositive are the absorbance of samples, MoS2-PEG, the negative control and positive control, respectively. 2.11. In Vitro Cytotoxicity. In vitro cytotoxicity assay was performed with MTT assay following the literature procedures. SCC-7 and COS-7 cells were seeded in 96-well plates at a density of approx. 1.2 × 104 cells/well and cultured in 100 µL DMEM, which was supplemented with 1% antibiotics (10,000 U/mL penicillin/streptomycin) and 10% FBS. Cells were cultured in a humidified atmosphere containing 5% CO2 at 37 °C for 24 h before adding materials. Following the initial cell culture, DMEM was removed and 100 µL fresh medium (pH 6.8 DMEM for SCC-7 cells and pH 7.4 DMEM for COS-7 cells) containing TBO (10 µM), MoS2 (0.3 mg/mL), MT (0.3 mg/mL MoS2, 10 µM TBO) and MKT (0.3 mg/mL MoS2, 0.6 mg/mL LA-K11(DMA), 10 µM TBO) were added respectively. After incubating for 4 h, another 100 µL fresh medium was introduced to remove extra materials, and then cells were treated with laser irradiation respectively. For mild PTT, cells were irradiated with 808 nm (1.5 W/cm2) for 2 min, for PDT, cells were irradiated with 630 nm (30 mW/cm2) for 5 min, for synergistic PTT and PDT, cells were irradiated with 808 nm (1.5 W/cm2) for 2 min and then with 630 nm (30


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mW/cm2) for 5 min. Cells were cultured for another 24 h and 10 µL MTT solution (5 mg/mL) was added to each well. After incubating for 4 h, the medium was replaced with 150 µL DMSO. The optical density (OD) was measured at 570 nm using a microplate reader (Model 550; BioRad, Hercules, CA, USA). Relative cell viability of the materials was calculated according to the formula: Cell viability %

sample )*+

× 100% .

2.12. Live/Dead Cell Staining Assay and Detection of Apoptotic Cells. SCC-7 and COS-7 cells were seeded in 6-well plates at a density of 5 × 105 cells per well, respectively. After incubated in 2 mL DMEM containing 10% FBS and 1% antibiotics for 24 h, the cells were treated with materials (TBO: 10 µM; MoS2: 0.3 mg/mL; MT: 0.3 mg/mL MoS2, 10 µM TBO; MKT: 0.3 mg/mL MoS2, 0.6 mg/mL LA-K11(DMA), 10 µM TBO). After co-incubation for 4 h, the culture medium was replaced with fresh medium. Cells were irradiated with 808 nm laser (1.5 W/cm2, 2 min) and 630 nm laser (1.0 W/cm2, 5 min) for PTT, PDT and PTT/PDT therapy, respectively. After incubating for another 10 h, all cells were stained with Calcein-AM (4 × 10−6 M) and PI solutions (4 × 10−6 M) in PBS buffer solution and incubated for 30 min at 37 °C with 5% CO2. Finally, the cells were washed with PBS thrice and visualized by CLSM. Excitation of the Calcein-AM, and PI were performed with lasers at 488 and 543 nm. The corresponding emission spectra were collected using two different ranges of wavelength at 510-540 nm (green) and 570-620 nm (red), respectively. The levels of apoptotic cells were measured by flow cytometry. SCC-7 cells were seeded at the density of 5 × 104 cells/well in 6-well plates. After the same treatment with the Live/Dead staining assay, cells were stained with Annexin V-FITC and propidium iodide (PI) (Beyotime Institute of Biotechnology, China) for 15 min. After that, cells were collected and examined using FCM.


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2.13. In Vivo Biodistribution and Pharmacokinetics. BALB/c nude mice (5-week old) were bought from Wuhan University Animal Biosafety Level III Lab and used for animal experiments. All animal experiments were agreed with institutional animal use and care regulations from Wuhan University. The tumor-bearing mice were obtained by subcutaneously injecting SCC-7 cells (1 × 107 cells) into the female mice on the right back of hind leg region. After tumor volume grew into 300-400 mm3, mice were divided into three groups and intravenously injected with TBO (0.1 mg/kg), MT (3 mg/kg MoS2, 0.1 mg/kg TBO) and MKT (3 mg/kg MoS2, 6 mg/kg LA-K11(DMA), 0.1 mg/kg TBO) respectively. After 24 h post injection, fluorescence distribution of mice were imaged using an in vivo imaging system (Maestro; CRi Inc.,Woburn, MA, USA, excitation: 640 nm, emission: 650-750 nm). Sacrificed tumors and organs fluorescence distribution were then imaged in the same way. In vivo pharmacokinetics of materials were imaged with a living image IVIS® spectrum (Perkin-Elmer) (excitation: 640 nm, emission: 680 nm). Fluorescence distribution of the tumor tissues was measured at different time points after TBO, MT and MKT intravenous injection. And total fluorescence of tumor area at every time point was quantified into a curve. 2.14. In Vivo Photothermal Imaging. BALB/c nude mice with tumor on its back leg were injected with MKT (3 mg/kg MoS2, 6 mg/kg LA-K11(DMA), 0.1 mg/kg TBO) intravenously. After 24 h, infrared thermal images were recorded by using a FLIR A × 5 camera under irradiation with an 808 nm laser at a power density of 1.5 W/cm2 and the temperature was quantified by BM_IR software. 2.15. In Vivo Antitumor Effect. Tumor-bearing animals were randomly divided into five groups (5 mice in each group) with similar starting weight (about 20 g). When the volume of tumors had grown to 300-400 mm3, each group were respectively received an equivalent volume


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(100 µL) of PBS, free MoS2 (3 mg/kg), free TBO (0.1 mg/kg), MT (3 mg/kg MoS2, 0.1 mg/kg TBO) and MKT (3 mg/kg MoS2, 6 mg/kg LA-K11(DMA), 0.1 mg/kg TBO) via intravenous injection. PBS group as negative control only received irradiation treatment. MoS2 group received 808 nm irradiation (1.5 W/cm2, 2 min) and TBO group received 630 nm irradiation (1.0 W/cm2) for 5 min at 4 h after injection. MT and MKT were irradiated with 808 nm (1.5 W/cm2) for 2 min at 4 h after injection, and then irradiated with 630 nm (1.0 W/cm2) for 5 min after 30 min of the first irradiation. The body weight and the subcutaneous tumor size (estimated by the formula 1/2 × L × W2) were recorded throughout the experimental period. Tumor growth ratio was defined as V/V0 (where V0 is the initial tumor volume without any treatment). The antitumor activity was evaluated by tumor growth inhibition analysis. The mice were sacrificed upon completion of the treatment. Tumors and organs were extracted and immersed in a buffered solution of 4% paraformaldehyde. For hematoxylin and eosin (H&E) staining assay, the specimen was embedded in paraffin and sectioned serially at 4 mm thickness and stained with H&E. These histological sections were evaluated by optical microscopy. 3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of MoS2 Nanoflakes. MoS2 nanoflakes were prepared according to Liu’s group has reported.28 Briefly, layer gaps of bulk MoS2 crystal were inserted by lithium cation, then swelled after combining with adding water and ultrasonically exfoliated. MoS2 nanoflakes were then stabilized with lipoic acid modified PEG (LA-PEG). Characterizations of LA-PEG was shown in Figure S1 (Supporting Information). TEM image of LA-PEG modified MoS2 (MoS2-PEG) were displayed in Figure 1A. Irregular several layered nanoflakes were presented in TEM images, and this morphology of MoS2 provided an applicative nanoplatform for cargo loading. Size distribution determined through DLS of the


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synthesized MoS2-PEG nanoflakes was in the range of 50-150 nm (Figure 1B), which was an optimal scale for body circulation testified in previously reports.29 The AFM images and height profiles of MoS2, MoS2-PEG and MKT were shown in Figure S5. MoS2 was stabilized with LAPEG and the MoS2-PEG did exhibit better dispersity than MoS2. After further modified with LAK11(DMA), the height of MKT was about 20 nm, which was higher than MoS2-PEG, indicating the successful modification of LA-K11(DMA). Figure 1C showed the hemolytic test of MoS2-PEG with red blood cells (RBCs) to tentatively evaluate its biocompatibility.17 Deionized water (+) and PBS (-) incubated with RBCs were used as positive and negative controls. It was found that different concentrations of MoS2-PEG (0.011.2 mg/mL) in PBS did not induce fearful hemolysis, indicating good biocompatibility of the two dimensional MoS2-PEG. Another outstanding property of two dimensional MoS2 was the photothermal effect under NIR laser irradiation.30 For further verification, 0.3, 0.4, 0.5 mg/mL MoS2-PEG and deionized water were treated with 808 nm laser (1.5 W/cm2). From temperature variation curves in Figure 1D, it was obvious that with irradiation time increasing, MoS2-PEG could reach thirty degree temperature increment. However, severe hyperthermia treatment (eg., over 48 °C) has been questioned about triggering detrimental inflammatory responses,31 which suggested that high temperature PTT may be not suitable for tumor therapy. Therefore, in this study, 0.3 mg/mL MoS2-PEG with 2 min irradiation was selected for photothermal therapy. 3.2. Fluorescence Quenching and Recovery of TBO. To test the fluorescence quenching of TBO by the MoS2, TBO was co-incubated with MoS2-PEG at different concentrations (Figure 2A). Upon the MoS2-PEG addition, fluorescence of TBO was quenched immediately. With MoS2-PEG concentration increasing, fluorescence of TBO was almost quenched (Figure 2B). This fast absorption and quenching process should attribute to the electrostatic attraction


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interactions between TBO and MoS2.32,33 In order to explore pH-sensitive peptide triggered TBO release from MoS2-PEG nanoflakes, The TBO fluorescence recovery under different pH values with and without the pH-responsive LA-K11(DMA) peptide was studied. As shown in Figure 2C, LA-K11(DMA) modified and TBO loaded MoS2-PEG (designated as MKT) exhibited remarkable fluorescence recovery at acid pH conditions as excepted, fluorescence recovery of MKT with 0.6 mg/mL LA-K11(DMA) was up to 55% (Figure 2D). In contrast, MoS2-PEG loading with TBO alone (designated as MT) didn’t show much response towards pH value change and no more than 10 % fluorescence was recovered at pH 5.0 (Figure 2D). 3.3. Thermogravimetric Analysis and Charge Conversion of Nanosystem. TGA (Thermogravimetric Analysis) of MoS2-PEG, MT and MKT under N2 atmosphere were displayed in Figure 2E. MoS2-PEG, MT and MKT were first ultracentrifugated (40000 rpm/min) for 30 min to remove unloaded components and then freeze dried. It could be observed that MoS2 was modified with 29.82 % (w/w) PEG and MT weight loss before 100 oC should due to the remaining water. MKT didn’t show a flat curve until 800 oC and nearly 80 % LA-K11(DMA) peptide was successfully modified on MoS2. Further exploration of nanosystems’ charge conversion were shown in Figure 2F. Zeta potential of LA-K11(DMA) peptide modified MoS2PEG (designated as MK) was more negative than individual MoS2-PEG at pH 7.4 (-26 mV), but converted into positive at pH 5.0 (9 mV) due to the pH-sensitive peptide. And non-sensitive peptide (LA-K11-NH2) modified MoS2-PEG (designated as MK11) was also explored for further determination of MK charge conversion. There was no significant difference between zeta potential of MK and MK11 at pH 5.0, indicating successful hydrolysis of the lysyl succinyl amides in LA-K11(DMA) peptide, namely charge conversion. Additionally, behaviors of MKT and MT at three pH values were further studied and shown in Figure 2F and Figure S5


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(supporting information). It could be found that under acid conditions, zeta potential of MKT (4 mV at pH 6.8, 9 mV at pH 5.0) was more positive than in normal pH condition (pH 7.4, -13 mV). MT didn’t show satisfactory results (-0.1 mV at pH 6.8 and 2.7 mV at pH 5.0) with only TBO protonation under acid conditions. As demonstrated in previous researches, positively charged materials showed higher affinity toward the negatively charged plasma membrane.25 Positively charged MKT at acid condition was more likely to be internalized by tumor cells. 3.4. TBO Loading and Release. TBO loading efficiency of different concentration TBO were shown in Figure 3A. MT and MKT mixtures were ultracentrifugated (40000 rpm/min) for 30 min and the supernatant was tested. Both MT and MKT showed highly TBO loading efficiency. But a slightly decrease was observed with the increase of TBO concentration, which should due to the oversaturation of TBO. TBO release of MKT at different pH values was performed. As shown in Figure 3B, under acid conditions, MKT with pH-sensitive peptide showed higher TBO release ration than normal condition. Additionally, MKT under 808 nm laser irradiation developed more release due to the photo-heating effect of MoS2. Further efforts were made in pH triggered fluorescence and ROS generation recovery explorations on cells. Confocal laser scanning microscopy (CLSM) was applied for TBO cell fluorescence imaging. Images of tumorous SCC-7 were shown in Figure 3C. It was obvious that SCC-7 cell with MKT under 808 nm irradiation presented the highest TBO fluorescence. While MT didn’t show observable TBO fluorescence. These differences should come from charge conversion of MKT. That is, MKT was converted into positively charged under acid condition and displayed TBO release. Quantified results were measured using flow cytometry (Figure 3D, 3E). As described in previous reports, fluorogenic reagent 2,7-Dichlorofluorescein diacetate (DCF-DA) was used for the intracellular ROS detection.34 The non-fluorescent DCF-DA


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permeated into live cells and emitted bright green fluorescence after ROS oxidation. Fluorescence and ROS generation of MT were obviously quenched by MoS2. However, fluorescence intensity of MKT combined PTT (MKT PTT) was nearly as strong as free TBO. In Figure 3E, ROS production of MKT PTT was 2.0-fold of free TBO and 3.5-fold of quenched MT. In normal control COS-7 cells, fluorescence intensity of MKT was only a little higher than quenched MT, perhaps because of cellular uptake resistance resulted from negative potential of MKT and MT under normal conditions (pH 7.4) (Figure S9). The two-dimensional MoS2 worked as carrier to enhance materials cellular internalization, but quenched fluorescence and photoinduced ROS generation of photosensitizer simultaneously. The pH-sensitive peptide modified MKT realized ROS generation recovery in specific cells, which was critical in enhancing PDT therapeutic efficiency. 3.5. In Vitro Cytotoxicity Assay. PTT and PDT of materials were first performed in in vitro experiments. In tumorous SCC-7 cells, MKT combined PTT and PDT (designated as MKT PTT+PDT) showed efficient antitumor effect compared with PDT alone of MKT (Figure 4A). MKT TBO release was enhanced by MoS2 mild photothermal heating and exhibited PDT effect under NIR irradiation. However, Photo-induced ROS generation of MT was severely quenched, which decreased therapeutic effect of PDT treatment and synergistic PTT and PDT treatment. Cytotoxicity against normal COS-7 cell was shown in Figure 4C. Photothermal heating of MoS2 worked mild on COS-7 cell and photo-induced ROS generation of MT was quenched by MoS2. However, MKT exhibited PDT toxicity under laser due to TBO release in endo/lysosome condition (pH~5.0). Dark toxicity of materials against SCC-7 cells at pH 6.8 and COS-7 cells at pH 7.4 were shown in Figure 4C and 4D respectively. Acceptable results demonstrated the feasibility of materials for potential bio-application.


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3.6. Live/Dead Cell Staining and Cell Apoptosis Assay. Antitumor efficiency of MKT was further investigated via Live/Dead cell staining method.35,36 Results were shown directly in Figure 5A, live and dead cells were dyed with calcein (green) and PI (red) respectively. PTT treatment of MoS2 and PDT treatment of MT didn’t cause any noticeable death both in COS-7 and SCC-7 cells. While synergistic therapy of MKT against SCC-7 cells caused more cell death than free TBO and PDT treatment of MKT. On the one hand, charge conversion of this nanosystem was helpful for cellular internalization, and on the other hand, mild PTT of MoS2 also promoted TBO release, which ensured maximum and specific tumor cell photosensitizer release. In Figure 5B, flow cytometry analysis of SCC-7 cells were classified into four categories based on dye uptake: viable cells (negative for Annexin V-FITC and propidium iodide (PI)), early apoptotic cells (positive for FITC only), late apoptotic cells (positive for FITC and PI), and necrotic cells (positive for PI only). MT with no pH-sensitive peptide was unable to induce severe cell apoptosis. However, MKT treated with PDT alone or PTT combined PDT largely decreased the viability of SCC-7 cells, indicating the improved antitumor efficiency of MKT. 3.7. In Vivo Biodistribution of MKT and Photothermal Imaging. In vivo experiments were employed for further antitumor evaluation of materials. SCC-7 tumor bearing mice were injected with MKT and the in vivo distribution of MKT, MT and free TBO was firstly investigated. After intravenous injection of materials for 24 h, mice and their subsequently sacrificed organs and tumors were imaged with an in vivo imaging system (Figure 6A). MKT displayed specific tumor accumulation and emitted strong fluorescence in tumor, suggesting the specific internalization of MKT in tumor cells and effective TBO release. Without pH-responsive peptide, MT and free TBO were unsatisfactory on tumor targeting performance, instead exhibited other organs


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retention. Nonspecific materials circulating in the body were easily captured by metabolic organs such as liver and kidney and leading to organic accumulation. In vivo pharmacokinetic behavior of MKT was displayed with a living image IVIS® spectrum (Perkin-Elmer).37 After intravenous injection of MKT, images of mice at different time points were acquired and displayed in Figure 6B. It was noticed that at 4 h after injection, fluorescence of MKT appeared in tumor area. As time passed, more extensive and stronger fluorescence was observed in tumor area. Total fluorescence of the tumor area was quantified and summarized into a graph (Figure 6B). From the insert graph of Figure 6B, it can be found that tumor fluorescence intensity increased before 30 h, after 30 h fluorescence begun to decrease. It indicated that MKT accumulated in tumor area and released TBO slowly. MKT was promising for in vivo antitumor therapy with specific accumulation in tumor and prolonged retention time in tumor area for cargo molecules release. In vivo photothermal effect of MKT was explored with a thermal imaging camera. Mice were injected with MKT and PBS, respectively. After 4h, mice were irradiated under NIR laser (808 nm, 1.5 W/cm2) for 3 min. Thermal imaging of mice were recorded every minute. In Figure 6C, it was found that tumor temperature of MKT injected mice increased to 49.2 oC after 3 min irradiation, while tumor temperature of PBS injected mice only slightly increased (41.4 oC). MKT showed satisfactory photothermal effect and created hyperthermia in tumor areas, which was promising for synergistic therapy. 3.8. In Vivo Antitumor Effect of MKT. Furthermore, to perform a long period treatment evaluation, another 25 tumor-bearing nude mice were divided into five groups (five in each group). PBS group served as the negative control, materials were injected intravenously at the first day (red arrows in Figure 7A and 7B). Then photothermal therapy and photodynamic


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therapy were performed on each group as described in experiment section. Weight and tumor volume of each mouse were recorded every day during the treatment (Figure 7A and 7B). As all the materials were low toxicity to normal tissues and only once injection was displayed all through the treatment, physical activity of animals didn’t receive excess influence. Mice weight of five groups were increasing in the similar tendency and no animals died from the treatment. However, tumor increase ratio reflected that tumor of the MKT group was effective suppressed, due to the specific tumor accumulation and successful release of photosensitizer in target cells. MKT maintained normal physical activities of mice and provided prolonged living time for further control of tumor. On the contrary, neither MoS2, free TBO nor MT suppressed tumor growth successfully (Figure 7B and 7C). Figure 7C showed the picture of the sacrificed tumor after treatment. Negative controlled PBS group showed continuous increase in tumor volume and eventually formed huge solid tumors. Tumor volumes of MoS2, free TBO and MT were larger than MKT group, indicating ineffective tumor suppression. H&E staining method was displayed on sacrificed tumor slices of five groups (Figure 7D).38 Compared with negative controlled PBS group, apparent karyolysis and tumor necrosis were found in MKT group, indicating that tumor of mice injected with MKT was effectively suppressed without any additional operation after the first day treatment. Other H&E staining images of major organs such as liver, heart, lung, kidney and spleen were displayed in Figure S10. No obvious damages were found in normal tissues, suggesting well biocompatibility of MoS2 and TBO. 4. CONCLUSIONS In summary, a pH-responsive nanosystem MKT was constructed for enhanced synergistic PTT and PDT treatment of tumor. MKT was demonstrated to obtain enhanced synergistic therapy


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against tumor. After accumulated in tumor tissues and triggered by acidic microenvironment, photosensitizer TBO was released from MKT with recovered fluorescence and photo-induced ROS generation. Combined with the PTT of two dimensional MoS2 nanoflakes, MKT exhibited highly specific and efficient antitumor efficiency, showing great potential in synergistic tumor therapy.


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the website at http://pubs.acs.org. Detailed characterization of IR spectrum, TEM images, ESI-MS spectrum, 1H NMR spectrum, Zeta potential analysis, standardization curve of TBO, confocal images, flow cytometry analysis and H&E staining images (PDF) AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (51233003 and 51533006) and the Fundamental Research Funds for the Central Universities.


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Scheme 1. Illustration of the pH-responsive ROS generation and synergist therapy of MKT against tumor. Fluorescence and ROS generation of TBO were quenched by MoS2 in normal conditions. After arriving at the tumor tissues, TBO was released from MoS2 nanoflakes due to pH-responsive charge conversion of LA-K11(DMA). MKT exhibited synergistic therapy under 808 nm and 630 nm laser irradiation against tumor.


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Figure 1. (A) Transmission Electron Microscopy (TEM) image of exfoliated MoS2-PEG nanoflakes. (B) Size distribution by number of MoS2-PEG determined through dynamic light scattering (DLS) (Peak: 80.69 nm, PDI: 0.432). (C) Hemolytic percent of RBCs incubated with MoS2-PEG at various concentrations. Inset: Photographs of hemolysis in centrifuge tubes. (D) Photothermal effect of MoS2-PEG at different concentrations in DI water.


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Figure 2. (A) Fluorescence quenching spectrum of TBO and (B) fluorescence intensity of quenched TBO at 650 nm. (C) TBO fluorescence recovery of MT and MKT at different pH values, and (D) fluorescence recovery ratio of TBO at 650 nm. (E) TGA curves of MKT, MT and MoS2-PEG under N2 atmosphere. (F) Zeta potential of MoS2, MK11, MK and MKT at different pH values.


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Figure 3. (A) TBO loading efficiency of MT and MKT at pH 7.4. (B) TBO release of MKT under different conditions. Each group was performed with three duplicate samples. (C) Confocal laser scanning microscopy (CLSM) images of intracellular TBO fluorescence (red) against SCC-7 cells under pH 6.8. Mean fluorescennoce intensity (MFI) of cellular fluorescence measured via flow cytometry: (D) TBO fluorescence and (E) ROS generation in SCC-7 cells.


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Figure 4. PTT and PDT cytotoxicity of materials against (A) SCC-7 cells under pH 6.8 and (C) COS-7 cells at pH 7.4. Dark cytotoxicity of materials against (B) SCC-7 cells at pH 6.8 and (D) COS-7 cells at pH 7.4.


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Figure 5. (A) Live/Dead Cell Staining Assay. Live cells were stained with Calcein-AM (green), and dead cells were stained with PI (red). (B) Representative PI/Annexin V-FITC flow cytometry plots of SCC-7 cells.


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Figure 6. (A) In vivo distribution of TBO fluorescence at 24 h after intravenous injection of MKT, MT and TBO. Relevant sacrificed organs and tumor images were also presented in right pictures. (B) In vivo pharmacokinetic of MKT. Fluorescence distribution images of TBO at different time points were arranged by time, the insert graph was the total fluorescence change of the tumor area. (C) Photothermal imaging of MKT and PBS injected mice under 808 nm laser irradiation at different time.


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Figure 7. In vivo antitumor evaluation of materials. (A) Weight and (B) tumor volume change tendency of mice recorded every day. (C) Image of five groups sacrificed tumor after treatment. (D) H&E stain images of tumor slices, scale bar: 50 µm.


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ToC image


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Multifunctional Nanosystem for Synergistic Tumor ... - ACS Publications

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