Synthesis of MOF and [email protected] NPs.
MIL-100(Fe) NPs were synthesized by the method reported in the literature  and subsequently conjugated to HA to form a multifunctional nanoplatform [email protected] In a typical procedure, firstly, 80 mL of 7.5 mM FeCl3 solution (in methanol) (Sigma-Aldrich, USA) and 80 mL of 7.5 mM BTC solution (in methanol) (Sigma-Aldrich, USA) were mixed under strong stirring for about 10 min. Afterward, the mixture was transferred into a 55°C oil bath and reacted for 4 h. The reaction product was collected by centrifugation and washed 2 times with ethanol and deionized water. Finally, the product was stored after freeze-drying. The [email protected] NPs could be obtained by mixing MOF NPs with HA (Sigma-Aldrich, USA) at weight ratio of 2:1 for 24 h.
The [email protected]@PCA NPs could be obtained by mixing PCA (Aladdin, China) with [email protected] NPs at weight ratio of 2:1 for 24 h. The unloaded free PCA were removed by centrifugation.
The unloaded free PCA in the solution was analyzed by High Performance Liquid Chromatography (HPLC) (Shimadzu, Japan) at the wavelength of 360 nm. The drug loading capacity (LC, %) was calculated by the following formula:
The chemical structure of MOF, [email protected], and [email protected]@PCA NPs were obtained by Fourier transform infrared spectroscopy (FT−IR) (Perkin Elmer, USA). X-ray diffraction (XRD) (Rigaku, Japan) was utilized to characterize the crystalline structure. The size and morphology of nanoparticles were obtained by transmission electron microscopy (TEM) (Bruker, Germany). The size statistics of the MOF NPs and the zeta potential of MOF, [email protected], and [email protected]@PCA NPs were measured by a dynamic light scattering (DLS) (Malvern, UK).
Release of PCA from [email protected]@PCA
The PCA release experiments were performed at pH 5.6 and 7.4, respectively. 10 mg of [email protected]@PCA NPs was immersed in 10 mL PBS of two different pH values. The PBS was collected and replaced with the same PBS at each time point (0, 2, 4, 6, 8, 12, 24 h). The concentration of PCA in the collected PBS were measured by HPLC.
Isolation and culture of chondrocytes
Primary chondrocytes were obtained from 3-day-old Sprague Dawley (SD) rats. The articular cartilage was digested with 0.25 % trypsin-EDTA (Solarbio, China) for 0.5h to remove other tissues. Then, 0.2 % collagenase II (Sigma-Aldrich, China) was used for digestion at 37˚C for 4h. The chondrocytes were collected by centrifugation at 1000 rpm for 5 min and cultured in Dulbecco's Modified Eagle’s Medium-High glucose (DMEM, Gibco, USA) that supplemented with 10 % fetal bovine serum (FBS, Hyclone, USA) and 1 % penicillin/streptomycin (Solarbio, China). The cells were incubated at 37 C in a humidified atmosphere with 5% CO2. The chondrocytes at passage 3 were utilized for subsequent experiments.
Cell cytotoxicity analysis
The cytotoxicity of [email protected] and [email protected]@PCA was evaluated by MTT assay. Cells were cultured in a 96-well plate at a density of 5000 cells/well. Then, the cells were pre-treated with different concentrations of [email protected] (0, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μg/mL) or [email protected]@PCA (0, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μg/mL) for 1 h, and then induced by 10 ng/mL of IL-1β for 24 h. Then, 20μL of MTT was added to each well and the chondrocytes were incubated at 37 °C for 4 h. After the MTT medium was removed, dimethyl sulfoxide (DMSO) was added to dissolve purple triphenylmethylamine crystals produced by living cells. The absorbance was measured at 490 nm using a microplate reader (Thermo Fisher Scientific, USA).
Cell viability assay
The Live/dead cells assay was performed using a live/dead viability assay kit (Invitrogen, USA). The cells were washed by PBS and incubated with the solution containing 2 μM of calcein AM and 4 μM of ethidium homodimer-1 for 30 min at room temperature in the dark. Then the cells were washed with PBS before observed using a fluorescent microscope (Olympus, Japan). As described in the manufacturer's protocol, live cells were stained green and dead cells were stained red.
Treatment of inflamed-chondrocytes induced by IL-1β
Chondrocytes were divided into four groups: (1) Control group: chondrocytes treated with culture medium only; (2) IL-1β group: chondrocytes stimulated with 10 ng/mL IL-1β (Gibco, USA) for 24 h; (3) PCA group: chondrocytes pre-incubated with 6 μg/mL PCA for 1 h followed by stimulating with 10 ng/mL IL-1β for 24 h; (4) [email protected]@PCA group: chondrocytes pre-incubated with [email protected]@PCA (containing 6 μg/mL of PCA) for 1 h followed by stimulating with 10 ng/mL IL-1β for 24 h.
Quantitative real-time polymerase chain reaction (qRT-PCR) analysis
Total RNA was isolated from chondrocytes using an RNA isolation kit (Margen, China) and the qRT-PCR was performed according to the study previously reported . The primer sequences used for qRT-PCR were presented in Table 1.
Safranin O and immunofluorescence staining in vitro
For in vitro study, the cells in all groups were fixed with 95% alcohol for 30 min. After washing with PBS, the cells were stained with safranin O (Sigma, USA) for 10 min and then washed with PBS to remove residual dye.
The cells were washed with PBS and incubated 3% (v/v) hydrogen peroxide H2O2 to block endogenous peroxidase activity for 15 min at room temperature. After blocking with normal goat serum for 20 min at room temperature, primary MMP-13 antibodies were incubated (1:200 dilution, Abcam, USA) at 4 °C overnight. After incubation with fluorescent secondary antibody at room temperature for 1h, the nucleus were stained with 4', 6-diamidino-2-phenylindole (DAPI, Solarbio, China) for 5 min. Images were acquired on a microscope (Olympus, Japan).
A total of 42 male Sprague Dawley (SD) rats (10-week-old, weight 200 ± 10 g) obtained from Animal Research Committee of Guangxi Medical University (Nanning, China) were used in the animal experiments. All procedures were carried out according to the guide for the care and use of laboratory animals.
The OA model was surgically induced by anterior cruciate ligament transection (ACLT) according to previous reports . After the rats were anesthetized by intraperitoneal injection of pentobarbital sodium (30 mg/kg), 36 rats underwent bilateral ACLT on the knee joints to induce OA. Other 6 rats underwent sham operations (sham group), in which the articular cavity was opened and the intact short anterior cruciate ligament was sutured. After surgery for one month, the rats were randomly sorted into three groups: NS group, IA injections of 0.5 mL saline; PCA group, IA injections of 0.5 mL saline with PCA (6 μg/mL); [email protected]@PCA group, IA injections of 0.5 mL saline with [email protected]@PCA (containing 6 μg/mL of PCA). IA injections were performed once a week. The rats in these groups were sacrificed for analysis at 4 and 8 weeks after therapy.
After 4 and 8 weeks of treatment, the rats were sacrificed by intraperitoneal injection of excess pentobarbital. The knee joints were collected and changes of cartilage were assessed and scored by three independent observers who were blinded to the treatment groups. The observers evaluated the depth of lesions of articular cartilage on a scale of 0-4 as described by Pelletier et.al . (0=normal-appearing surface, 1=minimal fibrillation or a slight yellow discoloration of the surface, 2=erosion extending into the superficial or middle layers only, 3=erosion extending into the deep layers and 4=erosion extending to the subchondral bone).
Histological staining in vivo
The rats were sacrificed and the knee joints were collected and fixed in 4% paraformaldehyde for 48 h, then decalcified in 10 % ethylenediaminetetraacetate (EDTA) for 4 weeks. After serial dehydration, the joints were embedded in paraffin and sagittally sectioned at 3 μm thickness. The sections were de-waxed and stained with hematoxylin-eosin (HE, Jian Cheng Biotech, China) and safranin O/fast green (Solarbio, Beijing, China). Then, three independent observers graded the sections based on the scoring criteria reported by Osteoarthritis Research Society International (OARSI) .
The immunohistochemical staining was used to analyze the secretion of MMP-13. The dewaxed sections in the different groups were washed with PBS and exposed to 3% (v/v) hydrogen peroxide H2O2 to block endogenous peroxidase activity for 15 min at room temperature. After blocking with normal goat serum for 20 min at room temperature, primary MMP-13 antibodies were added (1:200 dilution, Abcam, USA) and incubated at 4°C overnight. The sections were incubated with secondary antibody for 15 min and then added with biotin-labeled horse radish peroxidase for 15 min. A 3, 3’-diaminobenzidine tetrahydrochloride (DAB) kit and hematoxylin were used for color development and nuclei dye. Tissues sections were observed and photographed with a microscope.
Statistical analyses were performed by SPSS statistics 22.0. All data were expressed as the mean ± standard deviation (SD), and all independent experiments were repeated at least three times. One-way analysis of variance (ANOVA) was used to assess group differences. P < 0.05 was considered statistically significant.