Materials, reagents and antibodies
MSN with cyanine-5 fluorescence (XFNANO Materials Tech. Co. Ltd., Nanjing, China); 3-mercaptopropionic acid, anhydrous acetone, N-hydroxysuccinimide (NHS), 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), (Sigma-Aldrich, St Louis, USA); acetonitrile, ammonium acetate and tween-80 (Aladdin Industrial Corporation, Shanghai, China); rat recombinant IL-1β (PeproTech EC, London, UK); oltipraz (Abmole Bioscience Incorporation, Shanghai, China); phalloidin-iFlour 488, primary antibodies against Nrf2, aggrecan, collagen type II and MMP13, NQO1, HO-1 (Abcam, Cambrige, UK); primary antibodies against β-Actin, Bcl-2, Bax and Caspase-3 (Abcam, Cambrige, UK); primary antibodies against Lamin B1 and MMP9 (Cell Signalling Technology Inc., Danvers, USA); horseradish-peroxidase (HRP)-labeled anti-rabbit IgG, the Annexin V-FITC/PI apoptosis detection kit, DCFH-DA, Cell Counting Kit-8 (CCK-8) and JC-1 (Beyotime Institute of Biotechnology, Shanghai, China); Dulbecco’s Modified Eagle’s Medium (DMEM), foetal bovine serum (FBS), phosphate-buffered saline (PBS), collagenase II and 0.25% trypsin (Gibco; Grand Island, NY, USA).
Synthesis of ROS-cleavable thioketal (TK)-containing linker
A blend of 3-mercaptopropionic acid (3-MPA, 5.2 g, 49.1 mmol) and anhydrous acetone (5.8 g, 98.2 mmol) was saturated with dry hydrogen chloride. The mixture was stirred well for 4 h at room temperature. The mixture was then placed on ice to quench the reaction. After washed with hexane, the TK-containing linker was obtained.
Synthesis and characterization of mPEG-TK
To synthesize mPEG-TK-COOH polymer, a mixture of mPEG-NH2 (1 g, 0.5 mmol), TK-containing linker (1.26 g, 5 mmol), EDC.HCl (1.15 g, 6 mmol) and NHS (0.69 g, 6 mmol) were dissolved in 20 mL of DCM and stirred well for 4 h at room temperature. Rotating evaporation was used to remove the solvent and cold ether was added to precipitate the product. After vacuum drying, the product was dissolved in DMF and dialyzed against distilled water using an MWCO: 3500 Da dialysis membrane for three days. After freeze-drying, the expected mPEG-TK-COOH was collected as a white powder. After that, the 1H-Nuclear magnetic resonance (NMR) spectra were obtained on a VNMRS-400 (VARIAN, USA) spectrometer at 400 MHz by using CDCl3 as the solvent with TMS as an internal standard.
Preparation of MSN-OL
15 mg MSN and 5 mg oltipraz were fully dissolved in 166μL DMSO by the aid of sonication and incubated overnight at 37 °C to promote OL completely adsorbed into MSN. The mixture was centrifuged at 18,000 rpm for 20 min to separate the supernatant and the sediment. The supernatant is retained for the subsequent synthesis process and measurement of drug loading and encapsulation efficiency. 1.5 mg mPEG-TK was dissolved in 10 μL supernatant and then added it to the previous precipitate with 0.3 mg EDC for 3 h reaction. The nanoparticles were separated by centrifugation at 18,000 rpm and washed with pure water three times, re-dispersed in ultrapure water at a concentration of 10 mg/mL and stored in the dark at 4 °C for subsequent study.
Drug loading efficiency (LE) and encapsulation efficiency (EE%) determination: The OL in the supernatant mentioned above were quantified using high-performance liquid chromatography (HPLC, Shimadzu, Japan) according to the pre-established standard curve. The LE and EE% were calculated according to the following formulas:
LE = weight of encapsulated OL/weight of OL-loaded nanoparticles.
EE% = weight of encapsulated OL/weight of the total OL × 100%
Characterization of nanoparticles
The nanoparticles were observed by transmission electron microscopy (TEM, JEM-200CX, JEOL, Akishima, Tokyo, Japan) and scanning electron microscopy (SEM, ULTRA plus; Zeiss, Zurich, Switzerland). Dynamic light scattering (DLS) was performed to determine particle size and zeta potential on a Zetasizer Nano ZS90 (Malvern, Worcestershire, UK). Nanoparticles were observed under a confocal microscopy (FV3000, Olympus, Tokyo, Japan).
Cell isolation and culture
All animal experiments were approved by the Ethics Committee on Animal Experiments of Shanghai Sixth People’s Hospital (No. 2021-0194). Six 4-week-old male Sprague Dawley (SD) rats (Shanghai SLAC laboratory animal CO.LTD) were euthanized using CO2 inhalation. Death was confirmed by cardiac and respiratory arrest. The cartilages were harvested from the rat joints and minced into small pieces. Cartilages were digested with a 0.25% trypsin solution for 30 min and subsequently digested with 0.1% collagenase II solution for 12 h. The chondrocytes were collected and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% foetal bovine serum (FBS) at 37 °C and 5% CO2.
Nanoparticles cellular uptake determination in vitro
After treatment for 24 h, chondrocytes were washed with PBS and then fixed with 4% paraformaldehyde for 20 min at room temperature. Chondrocytes were stained with Alexa Flour 488-conjugated phalloidin (1:500) for 45 min followed with hoechst for 5 min. Chondrocytes were observed under confocal microscopy (magnification, × 200). Additionally, chondrocytes were collected and washed with PBS 3 times to remove the supernatant. Chondrocytes were re-suspended in 500 μL of PBS and analyzed using flow cytometry to quantify the cellular uptake of the nanoparticles at the APC channel.
Quantification of released OL
1 mL of MSN-OL suspension was transferred to a dialysis tube, and the dialysis tube was immersed in 10 mL of PBS with or without 0.1% hydrogen peroxid. The tube was stirred at 100 rpm at 37 °C. At predetermined time intervals (6, 12, 24, 48 and 72 h), the release medium was collected and the fresh PBS was then added. Concentrations in the release medium were determined by UV–visible spectra with a UV–visible spectrophotometer (FlexStation 3, Molecular Devices, United States).
Cell viability assay
Chondrocyte were seeded in 96-well plates at a dense of 10,000 cells/well. Cells were treated with different concentrations of MSN-OL (30, 60, 120, 240, 480 μg/mL) for 24 h and 48 h. Chondrocytes were incubated with serum-free DMEM containing 10% Cell Counting Kit-8 (CCK-8) solution at 37 °C for 2 h. The absorption was evaluated at 450 nm using a spectrophotometer (FlexStation 3, Molecular Devices, United States).
ROS levels evaluation
ROS levels in chondrocytes were evaluated using the ROS sensitive dye 2′,7′-dichlorofluorescein diacetate (DCFH-DA). Briefly, chondrocytes were stained with 10 μM DCF-DA for 30 min and then washed with serum-free DMEM three time to remove the residual extracellular DCFH-DA. Flow cytometry analysis were conducted using a BD Accuri C6 plus flow cytometer (BD Biosciences, Vianen, The Netherlands).
Mitochondrial membrane potential determination
The mitochondrial membrane potential of chondrocytes was assessed using JC-1 staining. Chondrocytes were collected and stained with JC-1 (5 µg/ml) for 25 min at 37 °C. Cells were washed with PBS 3 times again to remove the residual JC-1. Flow cytometry analysis were conducted using a BD Accuri C6 plus flow cytometer.
Cell apoptosis evaluation
Apoptosis analysis was conducted Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. Chondrocytes were collected and stained with 300 μl binding buffer containing 5 μL Annexin V followed with 200 μL binding buffer containing 5 μL PI for 5 min at 37 ℃ in the dark. Flow cytometry analysis were conducted using a BD Accuri C6 plus flow cytometer within 30 min.
Western blotting
Western blotting was performed to evaluate protein expression. Total protein was extracted using radioimmunoprecipitation assay buffer while nuclear protein was extracted using a nuclear protein extraction kit. Western blot analysis was performed as previously described. The blots were visualized using enhanced chemiluminescence on an imaging system (Tanon, Shanghai, China) while integrated fluorescence intensity was analyzed using ImageJ software (version 1.8.0; National Institutes of Health, USA).
Bioinformatics analysis
Cartilages from the joints of rats were used for ex-vivo cartilage explants culture. Ex-vivo cartilage explants were divided into four groups: control group, IL-1β group, IL-1β + OL group and IL-1β + MSN-OL group. The concentrations of IL-1β, OL and MSN-OL were 10 ng/mL, 40 µM and 0.27 mg/mL, respectively. After 3 days, total RNA was extracted and the transcriptome was sequenced o using the Illumina sequencing platform. Differentially expressed genes (DEGs) were analyzed with the DESeq2 package (1.34.0). Gene ontologies were analyzed to identify different biological process.
Rat OA model
In vivo study was performed using 90 eight-week-old SD rats with six rats in each group. Anterior cruciate ligament transection (ACLT) was performed to establish rat osteoarthritis model as previously described [42]. The right knee joint is the side of the operation. Rats were randomized to sham group, ACLT group, ACLT + OL group, ACLT + MSN-NC group and ACLT + MSN-OL group. Each group had three time points of 4, 8 and 12 weeks.
Nanoparticles absorption and degradation in vivo
OA rats received an IA injection of 100 μL nanoparticles (7 mg/mL). Fluorescence images were taken using the In Vivo Imaging System (IVIS) Spectrum (Perkin Elmer, Santa Clara, CA) and IVIS imaging software (Perkin Elmer, Santa Clara, CA) at day 0 (after IA injection immediately), day3, day7, day14 and day21. The cartilages were used to make frozen sections. The sections were stained with DAPI and scanned using an Olympus BX51 microscope (Olympus, Tokyo, Japan).
Behavioral evaluation (gait analysis)
Gait analysis was performed using the Catwalk automated gait analysis system (Noldus Information Technology, The Netherlands). Each rat walked freely on a glass lit by green light. Paw prints were then digitized. The whole process was recorded by the video camera. Data was collected and analyzed using catwalk program software (Noldus, CatWalk XT version 10.6.608).
Radiographic analysis
Micro-computed tomography (microCT) scans of the joints were preformed using SCANCO 50 (Switzerland) at week 12. Three-dimensional reconstructed images of the knee joints were generated. The BV/TV were analyzed.
Histopathology analysis
Organs including heart, liver, kidney and knee joint were harvested and fixed in 10% formalin at week 4, week 8 and week 12. As to the knee joints, after decalcified in 10% ethylenediamine tetraacetic acid (EDTA) for 4 weeks, tissues were dehydrated, embedded in paraffin and cut into 5 μm thick slices with an ultrathin semiautomatic microtome (RM2016, Leica, Germany). The sections were stained with H&E, Safranin O/Fast Green and toluidine blue. Immunohistochemistry (IHC) of collagen type II, HO-1 and NQO1 were also performed. The sections were scanned using a microscope. OARSI score, Mankin score and expression levels of collagen type II, HO-1 and NQO1 were evaluated [43, 44].
Statistical analysis
Each experiment was independently performed three times. Data are presented as the mean ± standard deviation. Students t-test was used to compare the two groups. Statistical comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) followed by Tukey multiple comparison test or a Mann–Whitney test (nonparametric, OARSI score and Mankin score). P values < 0.05 were considered statistically significant. All statistical analyses were performed using SPSS software (version 22.0; IBM Corp.).