Drug Delivery System Based on Near-Infrared Light-Responsive

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Functional Inorganic Materials and Devices

Drug Delivery System Based on Near-infrared Light-responsive Molybdenum Disulfide Nanosheets Controls the High-efficiency Release of Dexamethasone to Inhibit Inflammation and Treat Osteoarthritis Yingyu Zhao, Chunfang Wei, Xu Chen, Jiawei Liu, Qianqian Yu, Yanan Liu, and Jie Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b20372 • Publication Date (Web): 07 Mar 2019 Downloaded from http://pubs.acs.org on March 15, 2019

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ACS Applied Materials & Interfaces

Drug Delivery System Based on Near-infrared Light-responsive Molybdenum Disulfide Nanosheets Controls the High-efficiency Release of Dexamethasone to Inhibit Inflammation and Treat Osteoarthritis Yingyu Zhaoa,†, Chunfang Weia,†, Xu Chena, Jiawei Liua, Qianqian Yua, Yanan Liua,* and Jie Liua,* a Department of Chemistry, Jinan University, Guangzhou 510632, China * Corresponding authors, E-mail: [email protected]. E-mail: [email protected]. Tel/Fax: +86-20-85220223 † These authors contributed equally to the work. ABSTRACT Intra-articular injection has unique advantages in the treatment of osteoarthritis (OA), although it risks rapid clearance of the therapeutic drugs in the joint cavity. Combining therapeutic agents with functionalized nanocarriers may provide an effective solution. Controlling the therapeutic concentration of the drug in the joint cavity through the drug-loading nanosystem can synergistically treat OA. Here, we proposed an intra-articular drug delivery nanosystem MoS2@CS@Dex (MCDs), using the chitosan (CS) modified molybdenum disulfide (MoS2) nanosheets as near-infrared (NIR) photo-responsive carriers, loaed with the anti-inflammatory drug dexamethasone (Dex). The MCDs responded to NIR light both in vitro and in vivo and trigger Dex release through photothermal conversion. This enabled the remote controlled Dex release in the joint cavity by adjusting the radiation behavior of the NIR light. The MCDs prolonged the residence time of Dex in the joint cavity. The intra-articular injection of the MCDs in combination with NIR radiation ensured a significant increase in the therapeutic effect of Dex at low systemic doses, which attenuated the cartilage erosion in the OA caused by the secretion of inflammatory factors including TNF-α and IL-1β. The toxicity and side effects on other internal organs during metabolism were reduced in the body. In addition, the photoacoustic imaging capability of MoS2 nanosheets was used to detect the metabolism of the MCDs in the joint cavity. Our research indicated that MCDs have great potential to treat OA. 1

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Keywords: Photothermal conversion, Controlled release, Inflammation, Osteoarthritis

1. INTRODUCTION Osteoarthritis (OA) is a joint degenerative disease that manifests itself as pathological changes in every structure of the whole joint 1-3, including fibrillation and degradation of articular cartilage, osteophyte formation, and synovial inflammation 3-6. Due to the complex physiological and pathological mechanisms of OA, medication is currently limited to alleviating the symptoms of the disease, and does not offer a cure for OA

3, 7.

The chronic and low-grade inflammation process

1-2

in OA not only

promotes disease symptoms, but also accelerates the disease progression

8-9.

Inflammatory factors (such as TNF-α and IL-1β 10-11) promote OA disease progression by promoting synovitis and altering chondrocyte differentiation, function, and vitality 8

to inhibit matrix synthesis and promote cartilage degradation 4, which plays an

important role in the progression of OA

2-3, 5.

Intervening in the secretion of

inflammatory factors in the synovial membranes of the joint could be an effective method for improving OA progression and treatment. The mode of administration of OA treatment is systemic administration and local injection. Systemic administration can induce systemic toxicity and allergic reactions 12.

Local injection administration increases bioavailability, reducs systemic exposure,

reducs off-target effects, side effects, and overall drug costs 13-15. Corticosteroid drugs and hyaluronate, which are approved by the Food and Drug Administration (FDA), are 14.

The intra-articular

14, 16.

The intra-articular

the mainstream drugs used for joint cavity administration injection of corticosteroids is the mainstay of OA treatment

lymphatic drainage 14, 17 and the rapid diffusion from synovial capillaries 14, 18 result in the drug remaining in the joint cavity for less time. Improvements in nanotherapeutic technologies, particularly nanoscale drug delivery systems, offer new ideas to avoid current treatment deficiencies. Nanocarriers deliver the drug into the joint cavity via slow drug release, such as liposomes 6, 14, 19-21 or polymer nanoparticles 22-23. The drug 2


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delivery system of the triamcinolone acetonide sustained release agent loaded with poly(lactic-co-glycolic acid copolymer) (PLGA) microspheres is approved by the FDA for the treatment of knee OA 24, suggesting that controlling the release behavior of the loaded drug by the nanocarrier is a promising approach for OA treatment. PLGA, as a drug carrier, faces the challenges of sudden drug release, no sensitivity, and the inability to control the efficient release of the drugs. A drug-loaded nanosystem that is sensitive, controls the efficient release of drugs, and detects the retention of drugs in the joint cavity is needed to optimize OA treatment. Near-infrared (NIR) light is commonly used within disease treatment, as it has lower tissue absorption, efficient penetration of tissue, reduced scattering, and minimal autofluorescence 25-26, including cancer photothermal therapy 27 and diagnostic imaging 26.

This is largely due to the special surface-area-to-mass ratio, higher absorption

intensity in the NIR region than graphene and gold nanorods, being soluble in water after stripping, easily purified of exfoliated molybdenum disulfide (MoS2) nanosheets 25,

and the high photothermal conversion efficiency in the NIR region 28. Teng Liu et

al. loaded MoS2 nanosheets with drugs and used their photothermal conversion properties for cancer photothermotherapy and chemotherapy 29. Other studies have used it as a photothermal agent and drug carrier for cancer therapy

27-28, 30-32

as well as for

diagnostic imaging 30, 32 and antimicrobial research 33-34. Photothermal agents are easier to apply to the treatment of OA and the use of MoS2 nanosheets for photoacoustic imaging of animal joints is easier and more efficient. Based on the NIR photothermal conversion property of MoS2, we use the nanosheet as a carrier for triggering drug release, and its photoacoustic imaging capability was applied to evaluate the metabolism of MCDs in the joint cavity. In this paper, an intra-articular drug delivery nanosystem MoS2@CS@Dex (MCDs) was constructed using the chitosan (CS) modified MoS2 nanosheets as NIR photo-responsive carriers and loaded with the anti-inflammatory drug dexamethasone (Dex). Dex demonstrates good results when treating OA with intra-articular injection 35-36,

although it risks being quickly cleared in the joint cavity. Loading the Dex on the 3



CS-modified MoS2 nanosheets can prolong the residence time of Dex in the joint cavity. Scheme 1A describes the synthesis of MCDs and the process by which MCDs respond to NIR light and release the drugs in vitro. The MCDs can respond to the NIR light both in vitro and in vivo, and trigger drug release through photothermal conversion, enabling remote control of Dex efficient release in the joint cavity on demand by adjusting the radiation the behavior of the NIR light. Injecting MCDs into the joint cavity and applying the NIR radiation allows for treatment-site-specific delivery of Dex to treat OA at lower systemic doses and significantly improves the treatment efficiency of Dex, which can down-regulate the secretion level of inflammatory factors TNF-α and IL-1β in activated macrophages. This protects the chondrocytes and improvs the cartilage erosion (scheme 1B). The in vivo experiments demonstrate that MCDs do not cause toxicity to other major internal organs during metabolism in vivo. The functionalized MoS2 nanosheets, as a potential photoacoustic contrast agent, is used for photoacoustic imaging of mouse joints to detect the retention time of MCDs in the joint cavity, assist the NIR source to locate radiation, and reduce damage to local tissue. These results indicate the great potential of MCDs for the treatment of OA by intra-articular injection. Scheme 1. The synthesis process of drug-loaded nanosystem and it releases drugs in response to NIR light in vitro (A). The mechanism of the nanosystem in response to NIR light for the treatment of OA in vivo through intra-articular injection (B).

4


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2. MATERIALS AND METHODS 2.1. Synthesis of MCDs MoS2 nanosheets were obtained by a simple and low-cost ultrasonic stripping method. Firstly, 10 mg of MoS2 powder (99.5% metal standard, Maclean Biochemical Co., Ltd.) was dissolved in 10 ml of NMP solution (AR, Maclean Biochemical Co., Ltd.), mixed, and sonicated at 37 °C in a constant temperature water bath for 24 hours. To separate MoS2 nanosheets and powders, the supernatant containing MoS2 nanosheets was obtained by a low-speed centrifugation, the solvent was removed by high-speed centrifugation, and 10 ml of CS (Maclean Biochemical Co., Ltd.) solution (0.1% wt) previously dissolved in acetic acid was added dropwise under ultrasonic condition. After ultrasonicing at 37 °C in a constant temperature water bath for 4 hours, the obtained MoS2@CS was collected by centrifugation. Finally, MoS2@CS was dissolved in a PBS solution at pH 7.4, and the solution of Dex (Sangon Biotech Co., Ltd.) was added thereto. The mixture was stirred magnetically at room temperature 5



overnight, centrifuged, and washed with PBS multiple times to remove the unloaded Dex. The obtained MCDs were dissolved in PBS and stored at 4 °C until use.

2.2. Characterization and the evaluation of photothermal property The size and morphology of MoS2 nanosheets were obtained by transmission electron microscopy (TEM, Hitachi H-7650), scanning electron microscopy (SEM, Zeiss ULTRA 55), and high-resolution transmission electron microscopy (HRTEM, JEM-2100). The size statistics of the MoS2 sample and the zeta surface potential of the modified product were measured by a nanoparticle size and Zeta potential analyzer (Malvern Instruments Co., Ltd.), and the data obtained were the average of three measurements. The thickness of the MoS2 nanosheets was obtained by Atomic Force Microscopy (AFM, Veeco Metrology Group). The elemental spectrum and energy dispersive X-ray spectrum (EDX) and selected area electron diffraction (SAED) image of the MoS2 nanosheets were measured by HRTEM. Further characterization of MoS2 nanosheets was achieved by powder X-ray diffraction (XRD, MSAL XD-2) and Raman spectroscopy (Raman, LabRAM HR Evolution). The UV−vis (ultraviolet–visible) absorption spectras of MoS2, CS, Dex, MoS2@CS, and MCDs were measured by an UV−vis spectrophotometer (UV, TU1900). The chemical structure and vibration mode of the respective modified products of MoS2 were obtained by Fourier transform infrared spectroscopy (FT−IR). A NIR thermal imaging camera was used to take the photothermal image of MoS2@CS and MoS2 at different times under NIR radiation. To test the photothermal performance of MoS2@CS, a NIR light source with a wavelength 6


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of 808 nm was used to vertically irradiate a sample cell containing 1 mL of MoS2@CS with different concentrations. At the same time, the temperature of the MoS2@CS was recorded by adjusting the power density of the NIR laser. After irradiating the MoS2@CS solution dissolved in PBS with a NIR light source (1 W/cm2) for 10 minutes, the NIR light source was turned off for natural cooling, and the temperature change of the system was measured every 30 s. Similarly, after irradiating for 5 minutes with a NIR source at 1 W/cm2, the NIR source was turned off for 5 minutes, the system was irradiated and cooled again after that, and the cycle was repeated 5 times. The temperature of the system was measured every 30 seconds to determine the photothermal stability of MoS2@CS.

2.3. Drug loading Different concentrations of Dex solution were mixed with MoS2@CS dissolved in PBS at different pH levels (5.4, 6.8, and 8.0) and stirred overnight at room temperature with a magnetic stirrer. The unloaded Dex was collected by centrifugation and multiple washings with PBS, and the Dex content in the supernatant was determined from the absorbance at 291 nm in the UV−vis spectrum of Dex, and the drug loading rate was calculated by the following equation. Drug loading rate (%) =

C0V0 - CV m

, where C0

(mg/mL) and V0 (mL) are the concentration and volume initially added to Dex, C (mg/mL) and V (mL) are the concentration of Dex in the supernatant and the volume of the supernatant, m is the quality of MoS2@CS.

7



2.4. Photothermal triggered drug release At room temperature, the MCDs solution dissolved in PBS at a pH of 6.8 was vertically irradiated with NIR light source of different power densities, respectively. After irradiated for 9 minutes, the MCDs were rapidly centrifuged at a high speed and the absorbance of the Dex in the supernatant was measured using a UV−vis spectrophotometer. The content of the released Dex was calculated based on the UV−vis spectrum. The supernatant after centrifugation was mixed into the precipitate. After standing at room temperature for 51 minutes, the released content of Dex was calculated by the above method. The next step was repeated for the next hour and the process was continuously measured for 8 hours.

2.5. Cellular uptake assessment After the pH of MoS2@CS dissolved in PBS solution was adjusted to 7.4, the FITC solution was added to MoS2@CS and MoS2 solution. The mixed solution was stirred with magnetic stirrer at constant temperature for 4 hours, and the supernatant was discarded after a high-speed centrifugation. Finally, the FITC-labeled MoS2@CS was dissolved in PBS again and stored at 4 °C in the dark. RAW 264.7 cell line were cultured in DMEM (dulbecco's modified eagle medium, GIBCO) containing 10% fetal bovine serum (GIBCO) at 37 °C in a sterile environment with a carbon dioxide concentration of 5%. The cells were seeded at a density of 100,000 cells per well in two 6-well plates labeled with different time gradients (0, 1 hours, 3 hours, and 6 hours) for 24 hours and the serum-free DMEM containing fluorescently labeled MoS2@CS at the 8


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final concentration of 20 μg/mL was then incubated with the cells in place of the medium. At the same time, the other group was cultured with FITC-labeled MoS2 at the same concentration, and they were placed in the incubator for different times. The cell culture wells of this period were washed three times with PBS, and the cells were collected and analyzed using flow cytometer (FCM, BD FACSCanto). The cells were seeded at a density of 100,000 per well in confocal dishes labeled with different time gradients (1 hour, 3 hours, and 6 hours), and the fluorescently labeled MoS2 and MoS2@CS at a final concentration of 20 μg/mL were then respectively co-cultured with RAW 264.7 cell line, and each was placed in an incubator to incubate for different time. Samples were gently washed three times with PBS to wash off the fluorescently labeled MoS2 and MoS2@CS that had not been taken up by the cells and nuclei were labeled with DAPI. The cell uptake was observed using a confocal laser scanning microscope (CLSM, Leica TCS SP5) at excitation wavelengths of 455 nm and 488 nm.

2.6. The mechanism of cellular uptake In order to explore the mechanism of cellular uptake, two groups of cells seeded overnight on culture dishes were incubated with serum-free DMEM containing fluorescently labeled MoS2@CS at a final concentration of 20 μg/mL instead of medium, and placed at different ambient temperatures (4 °C and 37 °C) to culture overnight. And cells in the culture dish were collected and analyzed by flow cytometry. The RAW 264.7 cell line was incubated with fluorescently labeled MoS2@CS for different times, during which, the petri dishes were vertically irradiated with a NIR light 9



source (0.4 W/cm2) for 5 minutes, and the radiation was circulated every hour and then washed three times with PBS. Endosome/lysosomes were stained with lysosomal red probe according to the manufacturer's protocol and the nuclei were stained with DAPI. CLSM was used to observe cellular uptake at different time.

2.7. Assessment of Cellular Activity To evaluate the effect of MCDs on the activity of activated RAW 264.7 cells, RAW 264.7 cell line were first seeded at a density of 5,000 cells per well in a 96-well plate and incubated for 24 hours. Serum-free DMEM containing different final concentrations of MoS2, MoS2@CS and MCDs was used to incubate the cells activated by LPS (Sigma) at the final concentration of 1 μg/mL before 4 hours instead of medium. After cocultured for 24 hours, the media was aspirated and the PBS solution containing MTT reagent at the concentration of 1 mg/mL was added to the wells. After 4 hours, dimethyl sulfoxide (DMSO) was added to dissolve purple triphenylmethylamine crystals produced by living cells. Relative cell viability was measured by comparing the absorbance difference between the control and experimental wells at a wavelength of 580 nm by a microplate reader (Bio-Tek, American). To investigate the effect of NIR radiation on the effects of MCDs on cells, using the above method, activated macrophages were incubated with serum-free medium containing MCDs (20 μg/mL final concentration) and no other components for 4 hours, respectively. After that, the irradiation was continued for 9 minutes using a NIR light source (0.4 W/cm2), and the cell viability was measured at different time. 10


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2.8. Evaluation of the Effects of MCDs on Cells Apoptotic cells were detected using the Annexin V−FITC/PI apoptosis detection kit (Sizhengbai Biotechnology Co., Ltd.) according to the manufacturer's instruction. Macrophages were seeded on confocal dishes at a density of 100,000 cells per well and cultured overnight. After the cells were activated by LPS, the medium was removed and DMEM containing the same concentration of Dex and MCDs was incubated with them. In the MCDs treatment group, one was irradiated with a NIR light source of 808 nm (0.4 W/cm2) for 5 minutes and incubated for an additional 50 minutes without NIR light, and cycling for 8 hours. After incubated overnight, the cells were washed with PBS and stained with Annexin V-FITC for 10 minutes at room temperature. After washing excess dye with PBS, stained with PI for 5 minutes, washed with PBS, and observed the cells with CLSM imaging. Similar to the above procedure, the treated cells were collected and stained with markers according to the manufacturer's protocol, and the treated cells were grouped by flow cytometry (ACEA NovoCyte). Inflammatory factors secreted by macrophages were determined by ELISA. RAW264.7 cells (100000 cells/well) were seeded in two 6-well plates and incubated overnight, and the medium was removed after induction of activation by LPS. DMEM containing the same concentration of Dex, MCDs, and the same volume of DMEM were incubated with the activated macrophages. One of the two plates was irradiated with an 808 nm NIR light source (0.4 W/cm2) for 9 minutes and incubated for an additional 50 minutes without NIR light, and cycling for 8 hours and the other plate 11



was not treated with NIR radiation. After incubated overnight, the cells were dissolved in IP lysis buffer (Sigma) and centrifuged at 10000 rpm for 5 minutes at 4 °C. Supernatants were collected and the levels of the inflammatory factor, including TNFα, IL-1β, and IL-8, were measured using the corresponding ELISA kits, where controls without MCDs and Dex but containing LPS and without LPS were used for comparison. To determine the effect of the secretion of activated macrophages treated with drug on the metabolic activity of human normal chondrocytes, Raw 264.7 cells (100000 cells/well) were first seeded in a 6-well plate overnight, and the medium was removed after induction of activation by LPS. DMEM containing the same concentration of Dex and MCDs were incubated with activated macrophages. The MCDs treatment group was irradiated with an 808 nm NIR light source (0.4 W/cm2) for 9 minutes and then incubated for 50 minutes, and the cycle was continued for 8 hours. The other MCDs treatment group was not treated with NIR radiation. After overnight incubation, the cell supernatants were centrifuged and incubated with human normal chondrocytes (purchased from Millipore) pre-inoculated in a six-well plate under standard condition. After incubation for 24 hours, the cells were washed three times with PBS and collected. The cells were processed according to the protocol of the manufacturer of JC-1 kit (Sizhengbai Biotechnology Co., Ltd.) and flow cytometric analysis was performed.

2.9. Animal Studies Wild-type Kunming mice were purchased from the Guangdong Medical Experimental Animal Center to study the treating effect of MCDs on OA in vivo. All 12


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animal experiments were conducted in compliance with the "Animal Management Regulations" of the Ministry of Health of the People's Republic of China (No. 55 of 2001) and guidelines for the care and use of experimental animals of Jinan University. Mice were divided into 5 groups using a random number table, and five in each group were set. In order to induce arthritis symptoms in 6-10 weeks old Kunming mice other than control group, we injected papain (0.2 mL/Kg, mass concentration 6% (w/v), Sigma) into the knee joint of the mouse. A week later, symptoms of arthritis appeared. Five groups of mice were injected with different drugs for treatment.

2.10.

Photothermography and photoacoustic imaging of mouse knee joint OA model mice were anesthetized with pentobarbital (0.3%) at a dose of 45 mg/kg

and physiological saline containing MoS2@CS and MoS2@CS@Dex were injected into the normal joint cavity at a dose of 1.0 mg/kg. Then, the mouse knee joint was irradiated with an 808 nm NIR light source (0.4 W/cm2) for 12 minutes at a room temperature of 30 °C. The temperature of the mouse joint site was recorded every 4 minutes using a NIR camera. Image acquisition was performed using a photoacoustic enhanced ultrasound Nexus 128 system (ENDRA Life Sciences Inc., Ann Arbor, Michigan USA). The mice in the treatment group at various times after the first intraarticular injection of MCDs were placed in a 3D hemispherical detector, and PA signals were recorded by the apparatus. The joint area was detected by a tunable laser operating at 800 nm wavelength and the experimental data were processed with OsiriX Lite software. 13



2.11.

Study on the therapeutic effect of OA mice Mice with OA symptoms were anesthetized with pentobarbital (0.3%) at a dose of

45 mg/kg, and saline, Dex (1 mg/kg), and MCDs (1 mg/kg) were injected into the joint cavity. In one of the MCDs treatment group, an 808 nm NIR light source (0.4 W/cm2) was used to irradiate the knee joint for 9 minutes, then the NIR light source was turned off, and after 1 hour, the NIR light source was turned on to irradiate for another 9 minutes, and this cycle lasted for 8 hours. The day of application was recorded on the first day, and the drug was administered once every three days until the mice were euthanized for 28 days.

2.12.

Mice joint pathology analysis Mice were euthanized and the obtained mouse joint tissues were further analyzed.

The joints were cleaned, soaked in 4% PFA for 48 hours and decalcified in 10% EDTA for one month. Paraffin-embedded tissue sections (5 μm) were cut and analyzed by hematoxylin and eosin (HE) and safranin-O staining. The expression of inflammatory cytokines (TNF-α, IL-1β and IL-8) was detected in synovial tissue by immunohistochemistry, that is, after the primary antibodies of TNF-α, IL-1β, and IL-8 were incubated with the tissue section at 4 °C overnight, the tissue sections were incubated with the secondary antibodies at room temperature for 2 hours. Mouse joint samples from different treatment groups were homogenized in cold PBS and centrifuged at 4 °C for 20 minutes. The levels of TNF-α, IL-1β, and IL-8 secretion in 14


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the supernatant were determined using ELISA kits. The newly collected mouse blood was coagulated at room temperature for 30 minutes, and the supernatant serum was taken by centrifugation for 10 minutes. The collected serum was dispensed to -80 ° C for further analysis. Serum levels of IL-1β, TNF-α, PEG2, and IL-8 were measured using the Elisa kit according to the instruction. After euthanization of the mice, the right knee joint of the right posterior aspect of the mouse (including the total knee joint and the proximal femur and the proximal tibia) was taken and fixed in a paraformaldehyde solution for 48 hours. The prepared total knee joint specimens were scanned on a micro CT system (Skyscan 1076, Skyscan, Kontich, Belgium): 100 kV voltage, 100 μA current, 275 ms exposure time, and 35 μm isotropic resolution. Select the area you are interested in and image processing was performed using 3D Medical Image Processing and Analyzing System. For the detection of OA pathology and cartilage remodelingrelated proteins (MMP13, ADAMTS5, and aggrecan), we incubated the primary antibodies of MMP13, ADAMTS5, and aggrecan with tissue sections overnight at 4 °C. The tissue sections were incubated with a secondary antibody conjugated with the fluorescent molecule AlexaFluor 488 at a room temperature and the nuclei were stained with DAPI. The number of related protein positive cells in each group of was calculated by counting the number of related protein positive cells in three consecutive sections of the whole joint section of mice in each independent experimental group and the average value was calculated. The obtained mouse joint tissues were fixed in 70% ethanol, and each joint was subjected to a computerized tomography imaging. HE sections of the mouse's main internal organs (heart, liver, spleen, lung, kidney) were stained with the 15



same method. We used the method described in the literature to perform histopathologic scores on arthritis 37. Based on the severity of OA, we used a 0‒6 scoring system to identify. A score of 0 represents normal cartilage, a score of 0.5 represents lack of safranin-O staining and a score of 1 represents surface fibrosis without cartilage loss. A score of 2 means that when the cartilage surface has vertical cracks and when the vertical fracture and erosion covers the joint surface < 25%, 25‒50%, 50‒75%, > 75%, they are 3 points, 4 points, 5 points, and 6 points respectively. The expression of different cytokines in synovial tissue was semi-quantitatively scored using four-point scores: 0 indicates no cytokine expression, 1 indicates mild expression of cytokines, 2 indicates moderate expression of cells, and 3 indicates significant expression of cells 38.

All histological scores were evaluated blindly and preformed independently by three

assessors, and their scores were calculated and averaged.

2.13.

Statistical analysis All data were expressed as the average of three independent experiments ± the

standard deviation of the mean, and Student's T-test or one-way analysis of variance (SPSS statistical software, SPSS Inc., Chicago, IL) was used to determine whether the significant difference between the means of the groups was statistically significant. If ***p < 0.001, **p < 0.01, and *p < 0.05, there was a statistically significant difference between the groups.

3. RESULTS AND DISCUSSION 16


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3.1. Synthesis and characterization of MCDs A simple and low-cost solvent stripping process was introduced to prepare the MoS2 nanosheets, where a specific amount of MoS2 powder was ultrasonically stripped in a solvent via water bath. The stripping effect of the MoS2 powder in three different solvents (N-Vinyl-2-pyrrolidone (NVP)

39,

N-Methyl pyrrolidine (NMP)

40,

and

isopropanol (IPA))41 was compared to obtain high-yield MoS2 nanosheets. The MoS2 powder had the best stripping effect in NVP. Prolonging the ultrasonic time and increasing the ultrasonic power increased the yield of the nanosheets and reduced the size of the nanosheets, which was consistent with previous reports 28, 42. Transmission electron microscopy (TEM), scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HRTEM), and dynamic light scattering analysis (DLS) were used to evaluate the morphology and size of the obtained MoS2 nanosheets. TEM image, SEM image, and HRTEM image revealed the sheet-like structure of MoS2, where the sheets were stacked in a single layer or multiple layers (Fig. 1Aa, Fig. 1Ab, Fig. 1Ac). The transverse dimension of the nanosheets was 78 ± 18 nm, which agreed with the statistical result of the DLS (Fig. S1). The thickness of the MoS2 nanosheets was characterized by atomic force microscopy (AFM). The AFM image showed that the size of the MoS2 nanosheets in the selected range was about 70 nm, with a thickness between 2‒3 nm (Fig. S2). As revealed by the energy dispersive X-ray spectra (EDX), the sample was mainly composed of Mo and S elements, and the Mo element and the S element were distributed together on the nanosheet in the element map (Fig. 1B, Fig. S3). To further characterize the structure of MoS2 nanosheets, the absorption spectra of the exfoliated MoS2 nanosheets sample were analyzed. The sample exhibited characteristic absorption peaks of the stripped 2H-MoS2 at the wavelength of 400 nm, 443 nm, 605 nm, and 669 nm in the Ultraviolet−Visible−Near Infrared (UV−VIS−NIR) spectra (Fig. S4) 40, 43. The X-ray diffraction (XRD) and the Raman spectroscopy were further applied to investigate the crystals of the sample. The (002), (103), and (110) crystallographic planes in the XRD pattern, (100) and (110) crystallographic planes in the selected area electron diffraction (SAED) pattern indicated that the exfoliated MoS2 17



nanosheets were in the 2H crystal phase, and the (002), (103), and (105) crystal planes 44-45.

showed the layered structure of nanosheets (Fig.1Ac, Fig. 1C)

The Raman

spectrum showed right shift peaks at 376 cm-1 and 402 cm-1 corresponding to the inplane (E12g) and out-of-plane (A1g) vibration modes of the layered 2H-MoS2 nanosheets. This indicated the synthesis of MoS2 nanosheets (Fig. 1D) 28, 40, 46. A

a

b

100 nm

B

C

D

500

Cu

500

0

6

12

18

24

energy (kev)

E 1.0 0.8

1500

9

CS MCDs MoS2@CS

6

Dex

0.4 0.2 0.0 500

600

0

30

MoS2

0.6

400

3000

F

1.2

300

4500

Mo Mo

Cu

700

Wavenumber (nm)

10

20

30

40

50

60

Degree (2θ)

70

MoS2 nanosheets

300

1

E 2g

200 100 0

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G

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420-1

Raman shift (cm )

440

CS Dex

MoS2@CS

3

MoS2@CS

0 -3

MoS2@CS@Dex

-6 -9

800 -12

A1g

MoS2 powder

400

(002) (100) (103) (105) (110)

Intensity (a.u.)

1000

Weight% 41.777 4.772 18.69 0.53 0.28

Intensity (a.u.)

Element Mo s Cu O Cu

Zeta potential (mV)

counts (cps)

50 nm

6000

1500 Mo-s

0

c

500 nm

2000

Absorbance (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 46

MoS2@CS@Dex

MoS2

4000

3000

2000

1000

-1

Wavenumber (cm )

Fig. 1. Synthesis and characterization of MCDs. (Aa) TEM image of MoS2 nanosheets. (Ab) SEM image of MoS2 nanosheets. (Ac) HRTEM and SAED image of MoS2 nanosheets. (B) EDS of MoS2 nanosheets. (C) XRD pattern of MoS2 nanosheets. (D) Raman spectrum of MoS2 powders and MoS2 nanosheets. (E) UV−vis spectra of MCDs, MoS2@CS, Dex, CS, and MoS2. (F) The potential changes of MoS2 nanosheets during functionalization. The data represent the average of n (n = 3) and the error bars represent the standard deviation of the mean. (G) FT−IR spectra of CS (black), Dex (red), MoS2@CS (gray), and MCDs (blue).

To determine if each component was successfully modified during the functionalization of MoS2, the physicochemical properties of the modified product 18


Page 19 of 46

were analyzed via Ultraviolet-Visible (UV−vis) spectroscopy, Zeta potential, and Fourier transform infrared (FT−IR) spectroscopy. The UV−vis spectrum had a weaker absorption peak at 390 nm and a strong absorption peak at 292 nm of MoS2@CS, indicating that CS was successfully attached to MoS2. The MoS2 had a strong absorption in the NIR region that was not affected by the modified CS, indicating that MoS2@CS could be used as a NIR light-responsive nanocarrier (Fig. 1E). The change of the zeta potential on the surface of MoS2 indicated that CS and Dex were successfully modified on MoS2 (Fig. 1F). The FT−IR spectroscopy was used to evaluate if CS and Dex were successfully modified on MoS2 (Fig. 1G). MoS2@CS exhibited N-H stretching vibration peak at 3500-3400 cm-1 and C-O bending vibration peak at 13001050 cm-1, which were characteristic absorption peaks of CS. This indicated that the CS was successfully attached to MoS2

28.

The FI−IR diagram of MoS2@CS@Dex

showed the absorption peak of the Dex’s carbon-carbon double bond at 1680-1600 cm-1 (dotted region in blue), demonstrating that Dex was successfully loaded on MoS2@CS. A

B

min 0

30 20 Time (min)

10

D 0 µg/mL 1 µg/mL 5 µg/mL 10 µg/mL 20 μg/mL 40 μg/mL

6 μg/mL 8 μg/mL 10 μg/mL 12 μg/mL 14 μg/mL 16 μg/mL

1.2 0.9 0.6

0.0 300

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"OFF"

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75 45

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Release ratio of Dex (%)

1.5

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32

0

G

Time (s)

38

Zeta potential (mV)

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30 0

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33 30

F

E

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45

0

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36

MoS2@CS

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0.1 W/cm 2 0.2 W/cm 2 0.5 W/cm 2 1 W/cm

39

MoS2

55

Temperature (°C)

39

40

42

Temperature (ºC)

Temperature (°C)

C 42

Temperature (°C)

MoS2 @CS

45

60

max

MoS2

Absorbance (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4

6

Release time (h)

8

MCDs MCDs+NIR

-3 -6 -9 0

2

4

Time (h)

6

8

Fig. 2. Study of the photothermal property of MoS2@CS and the behavior of drug 19



release in response to NIR light after drug loading. (A) Photothermographic images and quantitative statistics of temperature changes of MoS2 nanosheets and MoS2 nanosheets modified with CS (MoS2@CS), the power density of the NIR light source is 1 W/cm2. (B) Quantitative statistics of temperature changes in MoS2 and MoS2@CS in one hour. (C) Temperature changes of different concentrations of MoS2@CS with time gradients, and the power density of the NIR light source is 1 W/cm2. (D) Temperature changes of MoS2@CS at different power densities, the concentration of MoS2@CS is 20 μg/mL. (E) Photothermal stability of MoS2@CS (20 μg/mL) during radiation (1 W/cm2) and natural cooling cycles. (F) UV−vis spectra of Dex at different concentrations. (G) The behavior of MCDs to release drugs in the case of alternately opening and closing NIR light at different power densities. (H) The potential changes of MCDs after treatment with and without NIR radiation.

3.2. Evaluation of the photothermal property and drug release ability of MoS2@CS The release efficiency of the drug triggered by the photothermal conversion of MoS2@CS under NIR radiation impacted the therapeutic effect of the drug. Under the 808 nm NIR light, the photothermal images and temperature changes of MoS2@CS and MoS2 solutions after 1 hour showed that the temperature of MoS2@CS and MoS2 solutions gradually increased to 50 °C and 58.2 °C. As the irradiation time increased, it rose to the highest value at 9 minutes, and eventually levelled out (Fig. 2A, Fig. 2B). The first 9 minutes with the rapid temperature increase was used as the study period for the evaluation of photothermal performance and the radiation duration of the NIR light to trigger drug release. This prevented the temperature of the MoS2 from rising too high, which would result in damaging the organism, even though the photothermal conversion ability of the MoS2@CS was reduced more than the MoS2. The photoacoustic signal intensity of MoS2@CS was centration-dependent (Fig. S5), which was used to evaluate the retention effect of the drug in the joint cavity based on the intensity of the photoacoustic signal. As shown in Fig. 2C, the MoS2@CS had strong 20


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photothermal conversion capability and its temperature change was concentration dependent. The temperature changes of the MoS2@CS solution (20 μg/mL) within the first 9 minutes of NIR light irradiation with different power densities are shown in Fig. 2D. This revealed that the temperature of the MoS2@CS solution increased as the radiation power increased and had a radiation power dependence. To further investigate the photothermal conversion performance of the MoS2@CS, the MoS2@CS (20 μg/mL) solution was irradiated with a NIR light source at 1 W/cm2, and the NIR light source was turned off to cool naturally after 10 minutes (Fig. S7). Roper et al., Yuliang zhao et al., and Junqing Hu et al. 28, 47-48 found that the photothermal conversion rate of the MoS2@CS solution reached 28.6% (supporting information. S9) according to the data analysis (Fig. S6, Fig. S7, Fig. S8), which was higher than the 24.3% previously reported

28.

The MoS2@CS solution (20 μg/mL) was irradiated with NIR light for 5

minutes in an intermittent manner and exhibited excellent photothermal stability (Fig. 2E). Prior to measuring the drug loading of the system and the drug release behavior under NIR light stimulation, the UV−vis spectra of the different concentrations of Dex were determined and linearly fitted (Fig. 2F, Fig. S10). The loading ratios of MoS2@CS to Dex at different pH levels were compared to determine that MoS2@CS exhibited a pH dependence on the drug loading and reached a maximum loading of 63.7% at pH 8.0 (Fig. S11). To investigate the ability of the MCDs to respond to NIR light and release drugs, we calculated the drug release of the MCDs under the continuous alternating opening and closing of the NIR light (Fig. 2G). The drug release rate of 21



MCDs increased significantly after 1 hour and reached 28.1% after being irradiated with NIR radiation at 1 W/cm2 for 9 minutes, which was higher than the MCDs without NIR radiation with only 4.4%. When the NIR light source was turned off, the drug release rate was reduced to about 1%. When the NIR light source was turned on after the first hour, the drug release rate of MCDs increased rapidly by 12.1%, while the release rate without NIR radiation increased by 1.2%, showing NIR light source dependence. After 8 hours of alternating the NIR source with different power densities, the drug release rate of the MCDs increased with the increased power density. The drug release rate of MCDs treated with NIR radiation at 1 W/cm2 was higher than the other groups, reaching 71.2%. The drug release rate of the MCDs treated with NIR radiation at 0.4 W/cm2 reached 44.6%, exhibiting a power density dependence. The changes of the zeta potential of MCDs before and after the application of NIR light were also combined to determine that the drug release could cause surface charge changes of the MCDs (Fig. 2H). Since the MCDs could release drugs in response to NIR light, so the MCDs could be used as an on-demand drug release nanosystem to treat OA. Dex was released on demand by controlling the opening of the NIR light source, and the quantity Dex released was controlled by regulating power density and radiation behavior of the NIR light source to obtain effective Dex concentration for the treatment, promoting the Dex efficacy while maintaining the Dex concentration within a safe range and avoiding toxicity and side effects during the metabolic process.

22


Page 22 of 46

A

C

MoS2@FITC

MoS2@CS@FITC

B

MoS2@CS@FITC MoS2 @CS@FITC DAPI

MoS2@CS@FITC

100

0h 1h 3h 6h

0h 1h 3h 6h

MoS2 @FITC

DAPI

40 20

D

MoS2@FITC Merge

60

Merge

0

1

3

Time (h)

MoS2@FITC

100

80

0

Fluorescence intensity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fluorescence intensity (%)

Page 23 of 46

6

80 60 40 20 0 0

Lysosome MoS2 @CS@FITC DAPI

1

3

Time (h)

6

Merge

1h

1h 2h 3h 4h 6h 8h

Fig. 3. Cellular uptake. (A) Cell flow analysis of cellular uptake after incubating MoS2 and MoS2@CS which are labeled with FITC with the RAW 264.7 macrophage cell line for different time periods. (B) Corresponding to (A), laser confocal imaging was used to detect cellular uptake, and the nuclei were labeled with DAPI (scale bar: 50 μm). (C) Corresponding to (B), the quantification of cellular uptake. (D) The uptake of nanocarriers by cells after incubation of the fluorescently labeled nanocarriers with macrophages for different time periods (scale bar: 20 μm). Red fluorescently labeled lysosomes and blue fluorescently labeled nuclei. The power density of NIR light is 0.4 W/cm2.

3.3. Cellular uptake of MCDs and its mechanism The uptake of MCDs by cells affected their entry into cells, via using the FITC as a model drug marker MoS2@CS and MoS2, which were then co-incubation with RAW 264.7 macrophages to determine the cellular uptake behavior. The power density of NIR radiation was adjusted to 0.4 W/cm2 and radiated via multiple cycles, which reduced the effect of the photothermal effect that the MoS2@CS had on cells and promoted the efficient release of drugs from drug-loaded nanosystems. The flow cytometric analysis revealed the uptake of MoS2@CS by cells in a time gradient, where the cell uptake reached the maximum (78.4%) at 6 hours, exhibiting a time dependence. 23



The modification of CS enhanced the uptake efficiency of the MoS2@CS by cells more than the MoS2 treatment group (Fig. 3A). Confocal laser scanning microscopy (CLSM) imaging further visualized the uptake process (Fig. 3B). The results indicated that the cell uptake of FITC-labeled MoS2@CS reached the highest at 6 hours. The CS modification improved the biocompatibility of MoS2, increased the uptake efficiency of cells to fluorescently labeled MoS2@CS by 21%, and increased its intracellular concentration (Fig. 3C). If the MCDs are a drug delivery system that responds to the NIR light entering cells and function is correlated with complex intracellular kinetics. The cellular internalization pathway was explored by comparing the uptake of the FITC-labeled MoS2@CS by macrophages at a low temperature (4 °C) and a normal temperature (37 °C). As shown in Fig. S12, the uptake of the FITC-labeled MoS2@CS by macrophages was reduced by 60% when the energy-dependent endocytic pathway was shut down at 4 °C, indicating that the internalization of most FITC-labeled MoS2@CS was energy-dependent endocytosis. This result was consistent with the CLSM observations, where the green fluorescence of the FITC-labeled MoS2@CS coincided with the red fluorescence of lysosomal labeling, indicating that the FITC-labeled MoS2@CS was captured by endosomes/lysosomes. The FITC-labeled MoS2@CS partially captured by lysosomes escaped from the lysosomes after 4 hours, and the green fluorescence showed a point-like infiltration into the nucleus as shown in Fig. 3D 49. This might be due to the endosomal/lysosomal destruction controlled by the NIR light radiation, which facilitated the escape of drug-loaded nanosystems from lysosomes and 24


Page 24 of 46

Page 25 of 46

improved the penetration efficiency of the drug into cells 50 (scheme 1B). A

Dex

MCDs

MCDs+NIR

PI

Control

B

Concentration (pg/mg total proteins)

TNF-α

D

–NIR

+NIR

8

**

6 4 2 0

LPS Dex MCDs


C

  



  

Control

  

  

IL-1β

6

– NIR

+ NIR

5

*

4 3 2 1

0 LPS Dex MCDs


Merge

PI

AnnexinV -FITC

Annexin V-FITC

  

  

Dex

  

  

MCDs

PerCP-Cy5.5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


FITC 25


IL-8

6

– NIR

+ NIR

5 *

4 3 2 1 0

LPS Dex MCDs

– – –

+ – –

+ + –

MCDs+NIR

+ – +


Fig. 4. The effect of MCDs on cell metabolism. (A) After co-incubation of Dex, MCDs, and NIR-radiated MCDs with activated macrophages induced by LPS for 24 hours, the quantified flow cytometric analysis for the apoptosis of activated macrophages. (B) Corresponding to (A), the laser confocal imaging detection reflecting the apoptosis of activated macrophages (scale bar: 50 μm). (C) After LPS-induced activation of RAW 264.7 macrophages, the secretion levels of inflammatory cytokines (IL-1β, TNF-α, and IL-8) in each treatment group in the presence or absence of NIR radiation were counted using ELISA, and the data represent the mean of n (n = 3) determinations, error bars represent the standard deviation of the mean, where **p < 0.01, *p < 0.05. (D) After activated macrophages were treated with different groups of drugs, the effect of activated macrophage secretion (macrophage culture supernatant) on chondrocyte metabolism (mitochondrial membrane potential changes) was analyzed by flow cytometry, and the power density of NIR light is 0.4 W/cm2.

3.4. Inhibition of activated macrophages by MCDs combined with NIR light Activated macrophages are involved in the progression of OA 10, and the inhibition of activated macrophages by MCDs could affect the progression of OA. Lipopolysaccharide (LPS), a bacterial toxin, was used to induce macrophage activation. The toxicity of MoS2, MoS2@CS, and MCDs to the activated RAW 264.7 cell line induced by LPS was evaluated using the MTT assay. Fig. S13 shows that the MCDs significantly inhibited the survival rate of the activated macrophages in a concentrationdependent manner. The survival rate of the activated macrophages co-incubated with MoS2@CS was significantly higher than the cells co-incubated with MoS2, indicating that modifying the CS improved the biocompatibility of MoS2 and reduced the toxicity of MoS2 to cells. Fig. S14 shows that inhibiting the activated macrophage survival rate via MCDs treated with NIR light was significantly enhanced in a time-dependent manner, which could be due to the prolongation of irradiation time that promoted the release of drugs on MCDs, thereby enhancing the effect of the drug. The survival rate of the untreated cells was not affected during the irradiation with only NIR light (0.4 26


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Page 27 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


W/cm2) for 9 minutes, indicating that the NIR radiation of this power density did not affect the cells. Since the NIR light irradiation combined with MCDs had an inhibitory effect on the survival rate of activated macrophages, the treated activated macrophages were grouped with Annexin V−FITC/propidium iodide (PI) apoptosis kit. The flow cytometric analysis in Fig. 4A was used to compare the Dex group (3.48% of early apoptotic cells and 2.47% of late apoptotic cells), demonstrating that the apoptosis effect of the activated macrophages in the MCDs group was more pronounced. The apoptosis rate of the MCDs alone was 13.89% (the early apoptotic cells accounted for 9.34% and the late apoptotic cells accounted for 4.55%), while the apoptosis level of the group combined with the NIR light was 39.98% (the early apoptotic cells accounted for 24.03% and the late apoptotic cells accounted for 15.95%). These results were significantly higher than the group that was treated with MCDs alone. The results of laser confocal imaging in Fig. 4B further demonstrated that the MCDs treatment group irradiated by NIR light was the most effective when inducing activated macrophage apoptosis. The results indicated that the MCDs could trigger the apoptosis pathway of activated macrophages and induce apoptosis of activated macrophages, where the MCDs combined with NIR irradiation could significantly enhance the effect that the MCDs had on cells. LPS can induce macrophage secretion of pro-inflammatory cytokines, such as TNF-α and IL-1β, resulting in secondary inflammatory reactions in the tissue 51. The MCDs might inhibit inflammation by regulating the expression of TNF-α and IL-1β. 27



Dex and MCDs were separately incubated with the activated macrophages induced by the LPS with or without NIR radiation, where the effect that each treatment group had on the inflammatory factors of TNF-α, IL-1β, and IL-8 secreted by activated macrophages were determined. As shown in Figure 4C, the three treatment groups reduced the excessive secretion of inflammatory cytokines, including TNF-α, IL-1β, and IL-8 due to the LPS-induced activation macrophages. The MCDs group treated with NIR irradiation had a more pronounced effect on the down-regulation of inflammatory factors secreted by activated macrophages than the group treated with MCDs alone, and the down-regulation effect of IL-Iβ was significant. The MCDs combined with NIR radiation was more effective than MCDs for attenuating the inflammatory response in the activated macrophages induced by LPS. Inflammatory factors alter the metabolism and vitality of chondrocytes and advance the progression of OA. The supernatant of activated macrophages treated with drugs was co-incubated with chondrocytes and the JC-1 probes were used to detect the oxidative stress of the chondrocyte mitochondria. As shown in Fig. 4D, the secretion of the activated macrophages caused the depolarization of the chondrocyte mitochondrial, and different drug treatment groups could reverse mitochondrial depolarization in chondrocytes. This could be due to the drug's ability to regulate the secretion of inflammatory factors in activated macrophages, which attenuated the effects on the chondrocyte metabolism. The MCDs combined with NIR radiation could reverse mitochondrial depolarization. The MCDs combined with NIR radiation attenuated the effect that the activated macrophage secretion had on the mitochondrial 28


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function of chondrocyte and protected the chondrocyte mitochondrial health. The results indicated that the MCDs combined with NIR irradiation could induce apoptosis of activated macrophages induced by LPS and reduce its effects on chondrocyte metabolism while attenuating the inflammatory response of activated macrophages, playing a role in protecting chondrocytes (scheme 1B). A

B Temperature (ºC)

max MCDs

MoS2@CS

52

Control MoS2@CS

48

MoS2@CS@Dex

44 40 36 32

Control min 0

C

0

D

8 4 12 Time (min) 24

48

(h)

PA signal intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0

2

0

10

4

6

8

Time (min)

10

12

5 4 3 2 1 20

30

Time (h)

40

50

Fig. 5. Study on the photothermal effect and retention time of MCDs in mouse joints. (A) After intra-articular injection of equal doses of saline, MoS2@CS, and MCDs (1 mg/kg with final concentration in PBS of 20 μg/mL), the thermal imaging of knee joint and its temperature changes under the 808 nm NIR source radiation, wherein the power density of NIR laser is 0.4 W/cm2. (B) Quantitative statistics of photothermal images of joints. (C) Representative photoacoustic images of mouse knee joints at different times after intra-articular injection of MCDs (1 mg/kg with final concentration in PBS of 20 μg/mL). (D) Quantitative statistics of photoacoustic imaging of joints.

3.5. Study on the photothermal effect of MCDs in mouse joints and the retention of MCDs in the joint cavity The NIR photothermal conversion ability of the MCDs in mice affected the release 29



of the loaded drugs, further affecting the therapeutic effect that the MCDs had on the OA model mice. To study if MCDs respond to NIR light and perform photothermal conversion in the joint cavity of mice, the photothermal effects of the healthy joints of the mice that were injected with MoS2@CS, MCDs, or saline were compared under the NIR irradiation. As shown in Fig. 5A and Fig. 5B, there was a significant temperature increase in the articular area of the mice injected with MCDs in the joint cavity, and the temperature of the joints injected with MCDs increased to 46.3 °C after 12 minutes. The MCDs in the joint cavity could still respond to the NIR light, and convert NIR light energy into heat to trigger the drug release. The retention of MCDs in the joint space of mice was examined based on the photoacoustic imaging capability of MCDs. The photoacoustic images of the mouse joint cavity at different times showed that there was a strong photoacoustic signal after 24 hours of the drug treatment injection, and there was still a small quantity of MCDs in the joint cavity for 48 hours, indicating that MCDs could avoid the rapid clearance of the lymphatic system in the joint cavity and prolong the residence time of the drug in the joint cavity (Fig. 5C, Fig. 5D).

30


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Page 31 of 46

A

C OA

Dex

MCDs

MCDs+NIR Concentration (pg/mL )

Control

TNF-α

Concentration (pg/mL )

IL-1β

IL-8

240

*

**

***

3 2

***

4

**

*

*** *** *** * *

4

1

1

0

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

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Ctrl

OA 

*** ***

**

*

2

1

Ctrl

**

3

3 2

***

Dex MCDs MCDs +NIR

Ctrl

OA

Dex MCDs MCDs +NIR

Concentration ( pg/mL)

***

IL-8

4

IL-1β

B

TNF-α

***

**

180

*** *** *** *** ***

120 60 0 250

Ctrl

OA

Dex MCDs MCDs +NIR 1L-1β

*** *

200

*** *** *** *** ***

150 100 50 0 Ctrl

TNF-α

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


OA

*** 2000

Dex MCDs MCDs +NIR 1L-8

*** *** *** *** *** ***

1500 1000 500 0

Ctrl

OA

Dex MCDs MCDs +NIR



Fig. 6. Study on the regulation of MCDs on inflammatory factors in mouse joints. (A) Immunohistochemical staining of inflammatory factors (TNF-α, IL-1β, and IL-8) in the joint tissue extracted from normal and diseased mice after 28 days of treatment (scale bar: 100 μm). (B) Semi-quantitative analysis of immunohistochemical staining for inflammatory factors (TNF-α, IL-1β, and IL-8). (C) Expression levels of IL-1β, TNFα, and IL-8 in joints after various treatments. The data represent the mean of n (n = 3) determinations and error bars represent the standard deviation of the mean, where ***p < 0.001, **p < 0.01, *p < 0.05.

3.6. Modulation of inflammatory factors by MCDs in vivo Inflammation plays an important role in the progression of OA by promoting disease symptoms and accelerating the disease progression, where inflammatory cytokines secreted by cells in the synovial membrane play an important role 8. To investigate the regulation of the MCDs on inflammatory factors in the synovial membrane of OA mice, the joints of the five groups of mice were taken and the expression of inflammatory factors in the synovial tissue of the joint cavity were 31



analyzed by immunohistochemistry. The expression of inflammatory factors (IL-1β, TNF-α, and IL-8) in the synovial membrane of the OA group was higher, while the expression of inflammatory factors in the synovial membrane of the MCDs+NIR treatment group was lower (Fig. 6A, Fig. 6B). The quantitative statistics of inflammatory factors in the joint cavity, serum, and inflammation-related protein in the serum demonstrated that the MCDs treated with NIR radiation had an excellent regulatory effect on inflammatory factors (Fig. 6C, Fig. S15). The MCDs treated with NIR irradiation was significantly better than the Dex in down-regulating the secretion level of inflammatory cytokines and attenuating the synovial inflammation induced by papain in OA mice. A

Control

OA

Dex

MCDs+NIR

MCDs

Aggrecan

MMP13

120

***

**

***

120

**

90 60 30 0

Ctrl

OA

MMP13 positive cells (%)

B

Dex MCDs MCDs +NIR

*** **

90

*** *** *** * ***

60 30 0

Ctrl

OA

Dex MCDs MCDs +NIR

ADAMTS5 positive cells (%)

ADAMTS5

Aggrecan positive cells (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 46

120

***

90

*** *** *** *** ** **

60 30 0

Ctrl

OA

Dex MCDsMCDs +NIR

Fig. 7. The therapeutic effect of MCDs on OA model mice. (A) After 28 days of treatment in diseased mice, immunofluorescence staining of several proteins (MMP13, 32


Page 33 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ADAMTS5, and aggrecan) associated with OA pathology and cartilage reconstruction in the joint tissue of different treatments. Green fluorescent markers protein, blue fluorescence labels nuclei, and the power density of NIR radiation is 0.4 W/cm2 (scale bar: 50 μm). (B) Quantitative analysis of the number of protein-positive cells in mouse joints in different treatment groups. The data represent the mean of n (n = 3) determinations, and error bars represent the standard deviation of the mean. One-way ANOVA is used to analyze whether the differences between the mean values of the individual groups are statistically significant (***p < 0.001, **p < 0.01, *p < 0.05).

3.7. Therapeutic effect of MCDs on OA model mice Matrix metallopeptidase 13 (MMP13) and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) were involved in cartilage degradation and aggrecan cleavage in the process of OA. The aggrecan provided the ability to resist the compressive load in the cartilage and was an important component of the structure and function of the articular cartilage. To investigative the therapeutic effect that the MCDs had on OA model mice, the expression of MMP13, ADAMTS5, and aggrecan in the articular cartilage after treatment was measured by immunofluorescence analysis of the articular cartilage tissue. As shown in Fig. 7A, MCDs could reduce the expression of proteins involved in the cartilage destruction, including MMP13 and ADAMTS5. The inhibitory effect on MMP13 was more pronounced. The expression of aggrecan in the articular cartilage of the mice increased after treatment. The MCDs combined with NIR radiation was more effective in the treatment of OA model mice. The statistical results of the proportion of the relevant protein-positive cells in the articular cartilage in Fig. 7B conformed these conclusions. The MCDs injected into the joint cavity might exert an anti-inflammatory effect by down-regulating the secretion of inflammatory cytokines, including TNF-α and IL-1β in the synovial membrane, which remotely 33



modulated the expression of MMP13, aggrecan, and ADAMTS5 in the cartilage matrix and treated OA 52. MCDs combined with NIR light at low systemic doses could exert excellent therapeutic effects. This could be because the the NIR radiation caused MoS2@CS to convert light energy into heat energy to trigger the high-efficiency Dex release, while the multiple intervals of radiation enabled sustained release of Dex, thus improving the therapeutic effect that the Dex had. A Control

OA

Dex

MCDs

MCDs+NIR

CT imaging

H&E

Safranin-o

8

***

***

*** **

***

2000

Control OA Dex MCDs MCDs+NIR

1600

**

Distance (cm)

***

6

1200

4 2 0 Ctrl

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800 400 0-10

10-20

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Velocity (cm·s-1)

C

B OARSI score

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Control OA Dex MCDs MCDs+NIR

4 3 2 1 0

0-10

10-20

Time (min)

20-30

Fig. 8. Therapeutic effect of MCDs on OA model mice. (A) High resolution representative three-dimensional imaging of joints and the histopathological analysis of articular cartilage. After 28 days of treatment, CT imaging analysis of knee joints obtained from normal mice and treated mice was performed, and the obtained cartilage tissue was analyzed by H&E staining (with a column size of 100 μm) and safranin-O staining (with a column size of 100 μm). (B) The histogram is a semi-quantitative composite score of OA changes in each experimental group. The data represent the mean of n (n = 3) determinations and the error bars represent the standard deviation of the mean, and the differences between the mean values of each level group were statistically significant using one-way ANOVA (**p < 0.01, ***p < 0.001). (C) The mobility of OA mice after different treatments is evaluated from the movement distance and the movement speed per 10 min. The data represent the mean of n (n = 3) determinations and the error bars represent the standard deviation of the mean. 34


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Micro-computed tomography (micro-CT) was performed to analyze changes in bone morphology in the joint of mice following treatment. The micro-CT imaging of the knee joints of mice showed that the joint surface erosion of the OA mice was worse than the normal mice, and the articular surface erosion in various treatment groups improved to varying degrees. The MCDs+NIR group had the most significant therapeutic effect, where there was no obvious erosion on the articular surface, and the articular surface was smooth and intact, close to the normal joint (Fig. 8A). To further analyze the subtle morphological changes in the articular cartilage surface, the tissue sections of the knee joint were examined and the histopathological changes of the articular cartilage were analyzed. Hematoxylin-eosin (HE) staining of articular cartilage demonstrated that the articular surface of the mouse joints in the MCDs+NIR treatment group was intact and continuous, not damaged by erosion, and the chondrocytes were distributed in order. There were different degrees of articular surface erosion and disordered distribution of the articular chondrocytes in other treatment groups (Fig. 8A). The range of the safranin-O staining in the articular cartilage of MCDs+NIR treatment group was not significantly different than the normal joints, and the glycosaminoglycan content in the joints was nearly normal. The other treatment groups had reduced red staining (the glycosaminoglycan content was reduced) (Fig. 8A). A composite score for osteoarthritic changes (OARSI score) in each group was combined, and the MCDs+NIR treatment group scored lower, indicating that the MCDs+NIR treatment group relieved the articular surface erosion and the chondrocyte distribution disorder caused by arthritis lesions (Fig. 8B). The comparison of the 35



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moving distance, moving speed, and immobility time of the mice with different treatment showed that the MCDs+NIR treatment group had significantly improved mobility defect of the OA mice (Fig. 8C, Fig. S15). Heart

Liver

Spleen

Lung

Kidney

Control (Saline)

MCDs

MCDs +NIR

Fig. 9. Assessment of toxicity in mice. After 28 days of treatment, H&E staining of heart, liver, spleen, lung, and kidney tissue sections extracted from the control group and the drug-administered group, wherein the power density of NIR radiation was 0.4 W/cm2 and the column size was 100 μm.

3.8. Toxicity evaluation in vivo The safety of the nanosystem in vivo affected its further application. To evaluate if the MCDs had toxic side effects on major organs during metabolism in vivo, HE stained sections of the main organs of the mice including heart, liver, spleen, lung, and kidney after 28 days of treatment were analyzed. As shown in Fig. 9, the texture of each organ was clear, and there were no obvious pathological features such as inflammation, edema, and necrosis, indicating that the drug-loaded nanosystems did not produce toxicity or side effects in the metabolism of the body. The sustained-intermittent radiation of the NIR light caused the drug loaded on MCDs to be almost completely 36


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released in the joint cavity. The longer residence time of MCDs in the joint cavity prolonged the action time of the drug and improved the drug efficacy, which maintained the drug concentration within a safe range during metabolism in the body and avoided toxic side effects during the metabolic process. 4.

CONCLUSIONS In a word, an intra-articular drug delivery nanosystem MCDs (MoS2@CS@Dex)

was successfully constructed in this paper. The drug delivery nanosystem responded to NIR light and triggered drug release through photothermal conversion, which enabled the remote control of the high efficent release of Dex in the joint cavity by adjusting the radiation behavior of the NIR light. The intra-articular injection of the MCDs in combination with NIR radiation enabled treatment-site-specific delivery of drugs to treat OA at lower systemic doses. The in vitro experiments proved that the photothermal conversion efficiency of MoS2@CS reached 28.6%. The NIR radiation at 1 W/cm2 could increase the drug release efficiency of the MCDs to 71.2%. The MCDs could prolong the residence time of the Dex in the joint cavity and control the concentration of Dex by NIR radiation to improve the treatment efficiency of Dex, thereby reducing the side effects that the other internal organs could experience during metabolism in the body. The MCDs under NIR radiation could significantly down-regulate the secretion level of the synovial inflammatory factors, including TNF-α and IL-Iβ, which affected the pathological processes, such as cartilage degradation and treated OA. These results have indicated that the MCDs could be used as a highly efficient intra-articular drug delivery nanosystem for the treatment of OA, and has the potential to be used clinically 37



to treat OA. The design idea could be used as a reference for the design of other intraarticular drug delivery nanosystems to achieve a more effective delivery of drugs to the joint cavity for the treatment of OA. ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (21877051, 81803027 and 21371075), the Natural Science Foundation of Guangdong Province (2018A030310628) and the Planned Item of Science and Technology of Guangdong Province (2016A020217011).

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ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 46


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Drug Delivery System Based on Near-Infrared Light-Responsive

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