Chemicals and reagents

N-Isopropyl acrylamide (NIPAM), Sodium dodecyl sulfate (SDS) and potassium persulfate were purchased from Macklin (Shanghai, China). N-Bromoacetamide (NBA), collagenase II, dimethyl sulfoxide (DMSO), ascorbic acid and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St Louis, MO, USA). Acrylic acid (AA), l-glutamine and geraniol were purchased from Aladdin (Shanghai, China). Trypsin–EDTA, penicillin and streptomycin were purchased from Solarbio (Beijing, China). Dulbecco’s Modified Eagle’s Medium–High glucose (DMEM) and interleukin-1 beta (IL-1β) were purchased from Gibco (Gaithersburg, MD, USA). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, Utah, USA). Primary antibodies against Keap1, Nrf2, HO-1, p-p65, p65, MMP13, Col2a1, ADAMTS-5, and aggrecan were purchased from Abcam (Cambridge, UK).

Synthesis of pH/redox-responsive nanogels

The pH/redox-responsive nanogels were synthesized as follows: 1.301 g of NIPAM, 0.169 g of NBA, and 0.058 g of SDS were accurately weighed and dissolved in 200 mL of ultrapure water. The mixture was stirred with nitrogen at 70 °C for 0.5 h to remove residual oxygen. Next, 0.054 g of potassium persulfate initiator was dissolved in 2 mL of ultrapure water and added to the mixed solution. The solution was further stirred with nitrogen for 0.5 h. Then, 10 μL of AA was taken and mixed with 0.016 g of potassium persulfate and 1 mL of ultrapure water. The resulting solution was added to the mixed solution, and the reaction was continued for 5 h under nitrogen at 70 °C. After completion of the reaction, the solution was cooled to room temperature and transferred to a dialysis bag (Spectra/Por® Float-A-Lyzer® G2) with a molecular weight cut-off of 3500 Da to remove surfactants and unreacted raw materials. The mixture was dialyzed in tap water for 24 h and then transferred to ultrapure water for dialysis for 3 days, resulting in the formation of the pH/redox-responsive nanogels.

Characterization

The chemical structure of the pH/redox-responsive nanogels was determined using Fourier transform infrared spectroscopy (FT-IR) (Perkin Elmer, USA), while X-ray diffraction (XRD) (Rigaku, Japan) was employed to analyze their crystalline structure. The size and morphology of the nanoparticles were examined using transmission electron microscopy (TEM) (Bruker, Germany).

Drug loading

The geraniol solution (1 mg/mL) was slowly added dropwise into the nanogel solution (2.5 mg/mL), followed by sonication using an ultrasonic homogenizer (Sonics Vibra-Cell VCX 750) until the solution was completely mixed. The mixture was stirred in a dark room for 24 h to allow the geraniol to be encapsulated within the nanogels. The resulting mixture was subjected to ultrafiltration using a centrifugal filter device (Amicon® Ultra-4 Centrifugal Filter Unit) with a molecular weight cut-off of 10 kDa, and centrifuged at 5000× g for 10 min to remove any free geraniol and other small molecules. The purified nanogels were collected by washing the filter with ultrapure water.

Isolation and culture of chondrocytes

This study was approved by permission from the First Affiliated Hospital of Zhejiang University School of Medicine (No. 2023.567). And the mice were supplied by Zhejiang Experimental Animal Center.

Primary chondrocytes were obtained from 3-day-old C57BL/6 mice. The articular cartilage was digested with 0.25% trypsin–EDTA for 0.5 h to remove other tissues. Then, 0.2% (w/v) collagenase II was used for digestion at 37 °C for 4 h. The chondrocytes were collected by centrifugation at 1000 rpm for 5 min and cultured in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, 1% l-glutamine, and 50 μg/mL of ascorbic acid. The cells were incubated at 37 °C in a humidified atmosphere with 5% carbon dioxide. Chondrocytes at passage 3 were used for subsequent experiments.

Cell cytotoxicity analysis

The cytotoxicity of geraniol and nanogel was assessed using the MTT assay (Sigma-Aldrich, USA). Chondrocytes were seeded at a density of 5000 cells/well in a 96-well plate and treated with varying concentrations of geraniol and nanogel for 24 h. The MTT assay was performed by adding 20 μL of MTT solution (5 mg/mL) to each well, followed by incubation at 37 °C for 4 h. The living cells produced purple triphenylmethylamine crystals which were dissolved using DMSO, and the absorbance was measured at 490 nm using a microplate reader (Thermo Scientific Multiskan FC).

Treatment of inflamed-chondrocytes induced by IL-1β

The chondrocytes were divided into three groups: (1) Control group, which received culture medium only; (2) IL-1β group, which was treated with 10 ng/mL IL-1β for 24 h to stimulate inflammation; (3) geraniol group, which was pre-incubated with 1 μM geraniol for 6 h and then treated with 10 ng/mL IL-1β for 24 h.

Western blot analysis of protein expression by mouse chondrocytes

The protein expression levels of Keap1, Nrf2, HO-1, p-p65, p65, MMP13, Col2a1, ADAMTS-5, and aggrecan were measured using Western blotting. Primary chondrocytes were seeded at a density of 5 × 105 cells/well in 6-well plates and lysed using ice-cold RIPA buffer containing protease and phosphatase inhibitors. After sonication and centrifugation at 12,000 rpm for 15 min at 4 °C, the supernatant was collected and its protein concentration was measured using a BCA protein assay kit (Beyotime, China). SDS-PAGE gels with varying concentrations, depending on the protein of interest, were used to load equal amounts of protein, which were then transferred onto a polyvinylidene fluoride (PVDF) membrane using a transfer apparatus. The PVDF membrane was washed with TBS-T buffer and blocked with 5% BSA at 37 °C for 1 h. Primary antibodies against Keap1, Nrf2, HO-1, p-p65, p65, MMP13, Col2a1, ADAMTS-5, and aggrecan were diluted 1:1000 in 5% BSA and added to the PVDF membrane. The membrane was incubated overnight at 4 °C with the primary antibody, then washed three times with TBS-T buffer for 10 min each time. A secondary antibody was added to the membrane, diluted in TBS-T buffer, and incubated for 1 h at room temperature. The membrane was washed three times with TBS-T buffer for 10 min each time. Protein expression levels were detected using an exposure meter after exposing the membrane to X-ray film and developing and fixing the film.

Immunofluorescence staining

The chondrocytes were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.3% Triton X-100 for 10 min, blocked with 5% BSA for 1 h at room temperature, and then incubated with primary antibodies against p65 at 4 °C overnight. After washing three times with PBS, the cells were incubated with the secondary antibody conjugated for 1 h at room temperature. Finally, the cells were counterstained with DAPI, and images were acquired using a fluorescence microscope.

OA-inducing anterior cruciate ligament transection (ACLT) surgery

This study was approved by permission from the First Affiliated Hospital of Zhejiang University School of Medicine (No. 2023.567). ACLT (anterior cruciate ligament transection) was performed on both knees of 8-week-old male C57BL/6 mice as previously described (Sun et al. 2022). Animals were returned to their respective cages immediately after surgery without joint fixation. Mice in the control group did not receive any treatment. Ten days after ACLT surgery, intra-articular injections of either 20 μL of geraniol in PBS (1 mg/mL) or geraniol@nanogel (equivalent to 1 mg/mL of geraniol) were administered to the mice once per week for a total of 8 weeks. Mice were housed in standard cages under controlled conditions of temperature (22 ± 2 °C) and humidity (50 ± 10%) with a 12 h light/dark cycle. Animals had free access to food and water during the experimental period. The mice were sacrificed by carbon dioxide inhalation at the end of the study, and the knee joints were harvested for further analysis. The sample size was 5 mice in each group.

OARSI scoring of murine cartilage

The methods were conducted as previously stated in Chen et al. 2016, which includes histopathological grading using a modified version of the Chambers scoring system. This system is the standard method for grading cartilage degeneration in mice established by the OARSI Histopathology Initiative. Paraffin sections from each sample were scored after safranin-O staining. Grading was performed in four areas: medial femoral condyle, medial tibial plateau, lateral femoral condyle, and lateral tibial plateau. Three blinded observers graded each section, and the results were averaged to obtain the grades for each sample. The grades from each group of mice were then collated for analysis.

Immunohistochemical staining

Immunohistochemical staining was performed to detect the expression of Keap1, Nrf2, HO-1, MMP13, Col2a1, ADAMTS-5, and aggrecan in cartilage tissues. Briefly, paraffin-embedded sections were deparaffinized in xylene and rehydrated in a graded series of ethanol. Antigen retrieval was performed by incubating the slides in citrate buffer (pH 6.0) at 95 °C for 15 min. The sections were then treated with 3% hydrogen peroxide for 10 min to quench endogenous peroxidase activity. After blocking with 5% bovine serum albumin, the sections were incubated with primary antibodies against Keap1, Nrf2, HO-1, MMP13, Col2a1, ADAMTS-5, and aggrecan overnight at 4 °C. The primary antibodies were detected using a horseradish peroxidase-conjugated secondary antibody and visualized using a DAB substrate kit. Finally, the sections were counterstained with hematoxylin, dehydrated, and mounted.

Statistical analysis

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. Kolmogorov–Smirnov test was used to analyze the ANOVA model residuals to check the normality for all groups. p < 0.05 was considered statistically significant.