A nanocomposite hydrogel for treating osteoarthritis and a preparation method and application thereof

Abstract

The application discloses a kind of nanocomposite hydrogel for treating osteoarthritis and its preparation method and application, the nanocomposite hydrogel includes cerium dioxide nanoparticles, the cerium dioxide nanoparticles are modified with hyaluronic acid;Ergothioneine is loaded on hyaluronic acid modified cerium dioxide nanoparticles to form nanocomposite EGT-CeO2@HA;And temperature-sensitive hydrogel carrier, the nanocomposite EGT-CeO2@HA is uniformly dispersed in the temperature-sensitive hydrogel carrier.This composite hydrogel can activate ATF4-dependent pro-survival autophagy pathway, reduce cartilage cell inflammatory damage, and improve cartilage degeneration.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a nanocomposite hydrogel for treating osteoarthritis, its preparation method, and its application. Background Technology

[0002] Osteoarthritis (OA) is a common, slowly progressive, and continuously deteriorating degenerative joint disease characterized by progressive loss of articular cartilage, subchondral bone remodeling, synovitis, and osteophytes, severely impacting patients' quality of life. The prevalence of OA increases significantly with age, becoming one of the leading causes of pain and disability in middle-aged and elderly individuals, placing a heavy burden on the healthcare system. Currently, OA treatments mainly include nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, physical therapy, and surgical intervention. However, because cartilage tissue lacks vascular and nerve innervation, systemic drug administration struggles to maintain effective concentrations at the lesion site, while repeated intra-articular injections pose risks of infection and patient compliance issues. Most of these methods only relieve symptoms and cannot fundamentally reverse the degenerative process of cartilage. Therefore, developing a drug delivery system capable of sustained release and maintaining local therapeutic effects at the lesion site is crucial for developing novel OA treatment strategies.

[0003] In recent years, hyaluronic acid (HA)-based hydrogels have become ideal carriers for local drug delivery in osteoarthritis (OA) due to their good biocompatibility, cartilage tissue affinity, and sustained drug release properties. HA hydrogels are often loaded with anti-inflammatory drugs, growth factors, or stem cells for OA treatment; however, they have key limitations: First, they mostly focus on inhibiting inflammatory pathways or promoting matrix synthesis, without intervening in deep repair mechanisms such as the regulation of chondrocyte autophagy homeostasis, making it difficult to reverse cartilage degeneration; second, ordinary HA hydrogels lack active targeted modification, allowing drugs to easily diffuse rapidly from the joint cavity to the whole body, resulting in low drug concentrations at the lesion site and a narrow therapeutic window; existing loaded drugs are mostly chemically synthesized drugs or recombinant proteins, which have problems such as immunogenicity and poor stability, and the development of highly effective chondrocyte protectants from natural sources urgently needs to be strengthened. Summary of the Invention

[0004] To address the aforementioned problems, this invention proposes a nanocomposite hydrogel for treating osteoarthritis, its preparation method, and its application, thereby overcoming the limitations of existing OA treatment methods.

[0005] To achieve the above objectives, embodiments of the present invention, in a first aspect, provide a nanocomposite hydrogel for treating osteoarthritis, comprising: Cerium dioxide nanoparticles, wherein the cerium dioxide nanoparticles are modified with hyaluronic acid; Ergothioneine, loaded onto hyaluronic acid-modified cerium dioxide nanoparticles to form the nanocomposite EGT-CeO2@HA; and A thermosensitive hydrogel carrier in which the nanocomposite EGT-CeO2@HA is uniformly dispersed.

[0006] According to embodiments of the present invention, the nanocomposite hydrogel contains EGT, which acts as a potent natural antioxidant and anti-inflammatory agent, directly scavenging ROS and inhibiting inflammatory pathways, thereby reducing chondrocyte damage. CeO2 nanoparticles possess SOD and CAT mimicry enzyme activities, providing long-lasting ROS scavenging capabilities and synergistically enhancing antioxidant effects with EGT. HA modification significantly enhances the enrichment and retention of nanoparticles in cartilage tissue, achieving efficient delivery. After intra-articular injection, the thermosensitive hydrogel carrier gels in situ at body temperature, effectively prolonging the drug's retention time in the joint cavity and providing a controllable sustained-release microenvironment. The nanocomposite hydrogel exhibits significant articular cartilage protection at both cellular and animal levels. Through multi-omics analysis and functional validation, the nanocomposite hydrogel can activate the ATF4-dependent pro-survival autophagy pathway, restore the damaged autophagy homeostasis under inflammatory conditions, and reshape the metabolic homeostasis of chondrocytes, thereby alleviating cartilage degeneration in OA rats.

[0007] In a second aspect, embodiments of the present invention provide a method for preparing a nanocomposite hydrogel for treating osteoarthritis, comprising the following steps: Preparation of hyaluronic acid-modified cerium dioxide nanoparticles: Cerium dioxide nanoparticles were dispersed in a hyaluronic acid solution, stirred and reacted, then centrifuged, washed and dried; Ergothioneine loading: Hyaluronic acid-modified cerium dioxide nanoparticles were dispersed in an ergothioneine solution, stirred for adsorption, centrifuged, washed, and dried to obtain the nanocomposite EGT-CeO2@HA; Preparation of thermosensitive hydrogel carrier: Dissolve the thermosensitive hydrogel carrier in water and stir until completely dissolved; Preparation of nanocomposite hydrogel: The nanocomposite EGT-CeO2@HA was dispersed in the thermosensitive hydrogel carrier and stirred evenly to obtain the nanocomposite hydrogel.

[0008] According to the preparation method of this invention, EGT, CeO2 nanoparticles, and HA hydrogel are organically combined to achieve long-term targeted delivery of EGT, improving the retention time and bioavailability of the drug in the joint cavity. The SOD and CAT-like activities of CeO2 nanoparticles synergistically interact with the antioxidant activity of EGT, enabling more effective scavenging of ROS and reducing oxidative stress damage. The prepared composite hydrogel can activate the ATF4-dependent survival-promoting autophagy pathway, restore chondrocyte autophagy homeostasis, reduce inflammatory damage, and improve cartilage degeneration, providing a new molecular mechanism for OA treatment. Thermosensitive hydrogel carriers have good injectability and biocompatibility, and can gel in situ within the joint cavity, providing a stable microenvironment for drug release.

[0009] The embodiments of the present invention, in a third aspect, propose the application of the above-described nanocomposite hydrogel or the nanocomposite hydrogel prepared by the above-described preparation method in the preparation of a medicament for treating osteoarthritis.

[0010] In a fourth aspect, embodiments of the present invention provide a drug delivery system for treating osteoarthritis, comprising the above-described nanocomposite hydrogel or the nanocomposite hydrogel prepared by the above-described preparation method.

[0011] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0012] Figure 1To illustrate the construction and characterization of EGT-CeO2@HA hydrogel according to embodiments of the present invention, wherein: A: hydrodynamic diameter of different nanoparticles; B: surface potential of different nanoparticles; C: polydispersity index (PDI) of different nanoparticles; D: transmission electron microscopy (TEM) images of CeO2@HA (left) and EGT-CeO2@HA (right) nanoparticles; E: TEM elemental distribution map of EGT-CeO2@HA nanoparticles: oxygen (O), nitrogen (N), cerium (Ce), and sulfur (S) elements were detected; F: X-ray diffraction (XRD) pattern of EGT-CeO2@HA nanoparticles; G: X-ray photoelectron spectroscopy (XPS) spectrum of EGT-CeO2@HA nanoparticles; H, I: ABTS radical scavenging curves and quantitative analysis of EGT-CeO2@HA nanoparticles at different concentrations; J: EGT-CeO2@HA nanoparticles at pH 7.2 EGT release curves in solution; K: Rheological measurements of storage modulus (G') and loss modulus (G") of blank hydrogel over time; L: Rheological measurements of G' and G" of blank hydrogel over temperature; M: Rheological measurements of G' and G" of EGT-CeO2@HA hydrogel over time; N: Rheological measurements of G' and G" of EGT-CeO2@HA hydrogel over temperature; O: Release curves of EGT-CeO2@HA nanoparticles from the hydrogel; P: Scanning electron microscopy (SEM) elemental distribution map of the hydrogel: O, N, and Ce elements were detected; Figure 2 To prepare and characterize the EGT-CeO2@HA hydrogel according to embodiments of the present invention, wherein: A: TEM images of different nanoparticles at different magnifications; B: SEM images of different hydrogels at different magnifications; C: Particle size, Zeta potential, and PDI of EGT-CeO2@HA nanoparticles during a one-week storage period; D: TEM images of EGT-CeO2@HA nanoparticles at different magnifications after one week of storage; E: Loading efficiency of EGT-CeO2@HA nanoparticles at different EGT concentrations, and the stability of its loading efficiency during a one-week storage period; F: EGT release curve of EGT-CeO2@HA nanoparticles in pH 6.0 solution; G: Photographs of different hydrogels at different temperatures; Figure 3To explore drug dosage in ATDC5 cell line and human chondrocytes according to embodiments of the present invention, wherein: A: human chondrocytes were identified by COL-II immunofluorescence staining; B: the effect of ergothioneine on the activity of ATDC5 cell line was detected by CCK-8 assay; C: the effect of CeO2@HA Hydrogel on the activity of ATDC5 cell line was detected by CCK-8 assay; D: the effect of ergothioneine on the activity of human chondrocytes was detected by CCK-8 assay; E: the effect of CeO2@HA Hydrogel on the activity of human chondrocytes was detected by CCK-8 assay; F: the effect of ATF4 knockdown on ATF4 mRNA expression in ATDC5 cell line was detected by RT-qPCR; G: the effect of ATF4 knockdown on ATF4 protein expression in ATDC5 cell line was detected by RT-qPCR, n=3, *P<0.05, **P<0.01; Figure 4 To investigate the effects of cerium dioxide hydrogel loaded with ergothioneine on inflammatory chondrocytes in the ATDC5 cell line and human chondrocytes according to embodiments of the present invention, wherein: A: the effect of cerium dioxide hydrogel loaded with ergothioneine on inflammatory chondrocytes in the ATDC5 cell line was detected by RT-qPCR; B: the effect of cerium dioxide hydrogel loaded with ergothioneine on inflammatory chondrocytes in human chondrocytes was detected by RT-qPCR; CD: the effect of cerium dioxide hydrogel loaded with ergothioneine on inflammatory chondrocytes in the ATDC5 cell line was detected by Western blotting; EF: the effect of cerium dioxide hydrogel loaded with ergothioneine on inflammatory chondrocytes in human chondrocytes was detected by Western blotting, n=3, *P<0.05, **P<0.01; Figure 5 To investigate the effects of cerium dioxide hydrogel loaded with ergothioneine on inflammatory chondrocytes in the ATDC5 cell line and human chondrocytes according to embodiments of the present invention, wherein: A: the effect of cerium dioxide hydrogel loaded with ergothioneine on ROS accumulation in inflammatory chondrocytes in the ATDC5 cell line was detected using a ROS kit; B: the effect of cerium dioxide hydrogel loaded with ergothioneine on mitochondrial membrane potential in inflammatory chondrocytes in the ATDC5 cell line was detected using a JC-1 kit; C: the effect of cerium dioxide hydrogel loaded with ergothioneine on ROS accumulation in inflammatory chondrocytes in human chondrocytes was detected using a ROS kit; D: the effect of cerium dioxide hydrogel loaded with ergothioneine on mitochondrial membrane potential in inflammatory chondrocytes in human chondrocytes was detected using a JC-1 kit, n=3; Figure 6To reveal the effects of ergothioneine-loaded cerium dioxide hydrogel on IL-1β-induced ATDC5 cell line using multi-omics analysis according to embodiments of the present invention, wherein: A: Schematic diagram of multi-omics analysis; B: Heatmap analysis of differentially expressed genes in transcriptomics revealing the effects of ergothioneine-loaded cerium dioxide hydrogel on IL-1β-induced ATDC5 cell line; C: Heatmap analysis of differentially expressed proteins in proteomics revealing the effects of ergothioneine-loaded cerium dioxide hydrogel on IL-1β-induced ATDC5 cell line; D: Protein network interaction (PPI) diagram of the intersection of differentially expressed genes / proteins in transcriptomics and proteomics; E: Differentially expressed genes in transcriptomics... F: GO enrichment analysis of ergothioneine-loaded cerium dioxide hydrogels revealed the effect of ergothioneine-loaded cerium dioxide hydrogels on IL-1β-induced ATDC5 cell lines; G: KEGG enrichment analysis of transcriptomic differentially expressed genes revealed the effect of ergothioneine-loaded cerium dioxide hydrogels on IL-1β-induced ATDC5 cell lines; H: KEGG enrichment analysis of proteomic differentially expressed proteins revealed the effect of ergothioneine-loaded cerium dioxide hydrogels on IL-1β-induced ATDC5 cell lines, n=3. Figure 7 To conduct a multi-omics analysis of the effects of ergothioneine-loaded cerium dioxide hydrogel on IL-1β-induced ATDC5 cell lines according to embodiments of the present invention, the following methods were used: A: Volcano plot of differentially expressed genes in transcriptomics revealing the effects of ergothioneine-loaded cerium dioxide hydrogel on IL-1β-induced ATDC5 cell lines; B: Volcano plot of differentially expressed proteins in proteomics revealing the effects of ergothioneine-loaded cerium dioxide hydrogel on IL-1β-induced ATDC5 cell lines; C: Venn diagram showing the intersection of differentially expressed genes in transcriptomics and differentially expressed proteins in proteomics; D: PCA analysis of transcriptomics; E: PCA analysis of proteomics; F: Correlation analysis of global expression in transcriptomics and proteomics; G: Correlation analysis of logFC differential expression in transcriptomics and proteomics, n=3. Figure 8 To demonstrate the protective effect of ergothionein-loaded cerium dioxide hydrogel on IL-1β-induced ATDC5 cell line by partially reversing ATF4 knockdown according to embodiments of the present invention, wherein: A: Immunofluorescence staining of ATF4 protein in ATDC5 cell line; B: RT-qPCR detection of autophagy-related gene expression in ATDC5 cell line; CD: Western blot detection of chondrocyte metabolism and autophagy-related protein expression in ATDC5 cell line; E: Immunofluorescence detection of ROS accumulation in ATDC5 cell line; F: Immunofluorescence detection of mitochondrial membrane potential level in ATDC5 cell line, n=3, *P<0.05, **P<0.01; Figure 9For in vivo imaging of CY7-labeled cerium dioxide hydrogel loaded with ergothioneine according to an embodiment of the present invention, wherein, AC: in vivo imaging at different time points after injection of cerium dioxide hydrogel loaded with ergothioneine into the knee joint; D: in vivo imaging of an isolated knee joint with intact joint capsule after injection of cerium dioxide hydrogel loaded with ergothioneine; E: in vivo imaging of the distal femoral cartilage layer and proximal tibial cartilage layer after transection of the knee joint after injection of cerium dioxide hydrogel loaded with ergothioneine. Figure 10 To alleviate ACLT-induced OA pain in rats using cerium dioxide hydrogel loaded with ergothioneine according to embodiments of the present invention, wherein A: rat weight change; B: ACLT surgery diagram; CD: gait changes in rats assessed by Catwalk gait analysis system, n=8, *P<0.05, **P<0.01; Figure 11 To improve ACLT-induced subchondral bone remodeling in OA rats using cerium dioxide hydrogel loaded with ergothioneine according to embodiments of the present invention, wherein AC: 2D / 3D reconstruction images of subchondral bone of rat femur and tibia; D: changes in bone morphometric parameters of subchondral bone of rat femur; E: changes in bone morphometric parameters of subchondral bone of rat tibia, n=8, *P<0.05, **P<0.01; Figure 12 The visceral safety of cerium dioxide hydrogel loaded with ergothioneine according to embodiments of the present invention was evaluated by HE staining. Figure 13 To alleviate ACLT-induced OA cartilage degeneration in rats using cerium dioxide hydrogel loaded with ergothioneine according to embodiments of the present invention, wherein: A: HE staining of rat knee joint tissue; B: Safranin O and Fast Green staining of rat knee joint tissue; CD: Immunohistochemical staining to detect the expression of COL-II and MMP13 proteins in rat knee joint tissue; EF: Mankin score and OARSI score based on HE staining and Safranin O and Fast Green staining of rat knee joint tissue, respectively; GH: Statistical analysis results of COL-II and MMP13 protein expression in rat knee joint tissue; n=8, *P<0.05, **P<0.01; Figure 14 To activate ACLT-induced chondrogenesis in OA rats using cerium dioxide hydrogel loaded with ergothioneine according to embodiments of the present invention, wherein: A: Immunohistochemical staining to detect ATF4 protein expression in rat knee joint tissue; B: Immunofluorescence staining to detect Beclin1 and LC3B protein expression in rat knee joint tissue; CD: Statistical analysis results of ATF4, Beclin1 and LC3B protein expression in rat knee joint tissue, n=8, *P<0.05, **P<0.01; Figure 15This is a schematic diagram of the mechanism according to an embodiment of the present invention. Detailed Implementation

[0013] The technical solution of the present invention is illustrated below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or defining the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0014] This application is based on the inventors' considerations regarding the following: Ergothioneine (EGT), a natural sulfur-containing amino acid antioxidant, is widely found in foods such as mushrooms and possesses potent antioxidant and anti-inflammatory activities. In a metabolic dysfunction-associated fatty liver disease model, EGT significantly upregulates the expression of Beclin1 and Atg5 while reducing p62 levels, thereby restoring impaired autophagic flux. In an aging model, EGT improves mitochondrial function, alleviates oxidative stress, and promotes autophagy through the AMPK / SIRT1 / PGC-1 α pathway. Furthermore, EGT has shown protective effects in various age-related diseases, including metabolic syndrome, with mechanisms involving the regulation of oxidative stress, inhibition of inflammatory pathways, and maintenance of intracellular homeostasis. Despite EGT's considerable therapeutic potential, as a small molecule, it is rapidly cleared from the joint cavity and lacks targeting specificity for diseased cartilage tissue, severely limiting its direct application in the treatment of osteoarthritis (OA).

[0015] Therefore, to improve the local delivery efficiency and retention time of EGT within the joint cavity, this application aims to construct a novel nanocomposite hydrogel delivery system for EGT loading, exploring a new strategy for intra-articular injection therapy of osteoarthritis (OA), namely, combining EGT with cerium dioxide (CeO2) nanoparticles with enzyme-mimicking catalytic activity. CeO2 nanoparticles exhibit significant dual superoxide dismutase (SOD)-like and catalase (CAT)-like activities, capable of continuously scavenging excess reactive oxygen species (ROS) in the joint microenvironment, creating favorable conditions for EGT to exert its chondrogenic protective effect. However, the catalytic performance and tissue targeting of CeO2 nanoparticles under pathological conditions still need improvement. Hyaluronic acid (HA) is a natural polysaccharide synthesized and degraded by various cells, which can specifically recognize and bind to the CD44 receptor highly expressed on the surface of chondrocytes. Based on the above considerations, such as... Figure 15As shown, this application constructs an injectable delivery platform: firstly, CeO2 nanoparticles are modified with HA to enhance their cartilage affinity; then, the antioxidant amino acid EGT is loaded to form a nanocomposite EGT-CeO2@HA; finally, this nanocomposite is uniformly dispersed in a thermosensitive hydrogel to construct an integrated injectable EGT-CeO2@HA hydrogel system. In this EGT-CeO2@HA hydrogel system, EGT acts as a potent natural antioxidant and anti-inflammatory agent, directly scavenging ROS and inhibiting inflammatory pathways, thus reducing chondrocyte damage. CeO2 nanoparticles possess SOD and CAT mimicry enzyme activities, providing long-lasting ROS scavenging capabilities and synergistically enhancing antioxidant effects with EGT. HA modification significantly enhances the enrichment and retention of nanoparticles in cartilage tissue, achieving efficient delivery. After intra-articular injection, the thermosensitive hydrogel carrier gels in situ at body temperature, effectively prolonging the drug's retention time in the joint cavity and providing a controllable sustained-release microenvironment.

[0016] To better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

[0017] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0018] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0019] Example 1: EGT-CeO2@HA hydrogel 1. Preparation of HA-modified CeO2 nanoparticles 100 mg of CeO2 nanoparticles (125 nm in diameter) were dispersed in 100 mL of HA solution with a concentration of 4 mg / mL, and the mixture was stirred at 37 °C for 12 h. After the reaction was completed, the precipitate was collected by centrifugation at 10,000 rpm for 20 min, washed three times with deionized water, and freeze-dried to obtain HA-modified CeO2 nanoparticles.

[0020] 2. Preparation of EGT-CeO2@HA nanocomposite 100 mg of HA-modified CeO2 nanoparticles were dispersed in 20 mL of EGT solution with a concentration of 2 mg / mL and adsorbed by stirring at 25 °C for 8 h.

[0021] 3. Preparation of thermosensitive hydrogel carriers Pluronic F127 and hyaluronic acid were added to ultrapure water in a certain proportion and gently stirred at 4°C until completely dissolved to obtain a homogeneous composite solution; the total concentration of Pluronic F127 and hyaluronic acid in the composite solution was 24% (w / v).

[0022] 4. Preparation of nanocomposite hydrogels 10 mg of EGT-CeO2@HA nanocomposite was dispersed in 10 mL of thermosensitive hydrogel carrier and stirred until homogeneous to obtain EGT-CeO2@HA hydrogel.

[0023] 5. Characterization of EGT-CeO2@HA hydrogel like Figure 1 As shown and Figure 2 As shown, Figure 1 A: Hydrodynamic diameter of different nanoparticles; B: Surface potential of different nanoparticles; C: Polydispersion index of different nanoparticles; D: Transmission electron microscopy images of CeO2@HA (left) and EGT-CeO2@HA (right) nanoparticles; E: Elemental distribution map of EGT-CeO2@HA nanoparticles under transmission electron microscopy: oxygen, nitrogen, cerium, and sulfur were detected; F: X-ray diffraction pattern of EGT-CeO2@HA nanoparticles; G: X-ray photoelectron spectroscopy of EGT-CeO2@HA nanoparticles; HI: ABTS radical scavenging curves and quantitative analysis of EGT-CeO2@HA nanoparticles at different concentrations; J: EGT-CeO2@HA nanoparticles at pH 7.2 EGT release curves in solution; K: Rheological measurements of storage modulus and loss modulus of blank hydrogel over time; L: Rheological measurements of storage modulus and loss modulus of blank hydrogel over temperature; M: Rheological measurements of storage modulus and loss modulus of EGT-CeO2@HA hydrogel over time; N: Rheological measurements of storage modulus and loss modulus of EGT-CeO2@HA hydrogel over temperature; O: Release curves of EGT-CeO2@HA nanoparticles from the hydrogel; P: Scanning electron microscopy elemental distribution map of the hydrogel: oxygen, nitrogen, and cerium were detected. Figure 2A: Transmission electron microscope images of different nanoparticles at different magnifications; B: Scanning electron microscope images of different hydrogels at different magnifications; C: Particle size, Zeta potential, and polydispersity index of EGT-CeO2@HA nanoparticles after one week of storage; D: Transmission electron microscope images of EGT-CeO2@HA nanoparticles at different magnifications after one week of storage; E: Loading efficiency of EGT-CeO2@HA nanoparticles at different EGT concentrations, and the stability of its loading efficiency after one week of storage; F: EGT release curve of EGT-CeO2@HA nanoparticles in pH 6.0 solution; G: Macroscopic photographs of different hydrogels at different temperatures.

[0024] Example 2 Cell Experiment ATDC5 cells and human chondrocytes, purchased from Cybio (Shanghai) Biotechnology Co., Ltd., were used. The safety and antioxidant properties of the EGT-CeO2@HA hydrogel obtained in Example 1 were evaluated by CCK-8, RT-qPCR, WB, and ROS / JC-1 experiments.

[0025] 1. Immunofluorescence staining Once cells reached 80% confluence, the complete culture medium in the six-well plate was aspirated, and the cells were washed three times with 1× pre-chilled PBS. The PBS was discarded, and the cells were digested with 0.25% trypsin. After digestion, twice the volume of complete culture medium was added to stop the digestion, and the cell suspension was transferred to a 15 mL centrifuge tube. The cells were centrifuged at 800 rpm for 5 min at room temperature. The supernatant was discarded, and the cell pellet was retained. After cell counting, the cells were seeded into a plate. When the cells reached approximately 50% confluence, 10 ng / mL IL-1β and other drugs were added. Immunofluorescence staining was performed 24 h after intervention. After aspirating the complete culture medium, the cells to be tested were washed three times with 1× pre-chilled PBS. The cells were fixed with 4% paraformaldehyde for 10 min. After aspirating the 4% paraformaldehyde, the cells were washed three times again with 1× pre-chilled PBS. Subsequently, the cells were treated sequentially with 0.1% Triton X-100 (incubated at room temperature for 15 minutes), 10% goat serum (incubated at room temperature for 30 minutes), primary antibody (incubated overnight at 4°C), secondary antibody (incubated at room temperature in the dark for 1.5 hours), and DAPI (incubated at room temperature in the dark for 5 minutes). Finally, the cells were observed and imaged under a fluorescence microscope.

[0026] 2. CCK-8 Experiment Once the cells reached 80% confluence, the complete culture medium in the six-well plate was aspirated, and the cells were washed three times with 1× pre-cooled PBS. The PBS was discarded, and the cells were digested with 0.25% trypsin. After digestion, twice the volume of complete culture medium was added to stop the digestion, and the cell suspension was transferred to a 15ml centrifuge tube. The cells were centrifuged at 800rpm for 5 minutes at room temperature. The supernatant was discarded, and the cell pellet was retained. After cell counting, the cells were seeded into plates. When the cells reached approximately 50% confluence, different concentrations of ergothioneine and CeO2@HA Hydrogel were added. The CCK-8 assay was performed 24 hours after drug intervention. The complete culture medium in each well was aspirated, and 90 μL of fresh complete culture medium and 10 μL of CCK-8 reagent were added. After incubation in a cell culture incubator for 1.5 hours (adjusting the time appropriately according to the cell colorimetric intensity), the absorbance at 450 nm was measured using a microplate reader.

[0027] 3. Real-time quantitative PCR (RT-qPCR) Total RNA was extracted from cells using the Trizol method. The simplified procedure is as follows: After treatment with IL-1β, EGT, CeO2@HAHydrogel, and EGT-CeO2@HA Hydrogel for 24 h, the complete culture medium in the six-well plates was discarded, and the cells were washed three times with 1× pre-chilled PBS. 1 mL of Trizol was added to each well to lyse the cells, and the lysis buffer was transferred to a 1.5 mL centrifuge tube. Then, 200 μL of chloroform was added to each tube, and the mixture was vigorously vortexed. After incubation at room temperature for 10 min, the cells were centrifuged at 1200 rpm for 15 min at 4 °C. After centrifugation, approximately 300 μL of the upper aqueous phase was collected and mixed with an equal volume of isopropanol. The tube was inverted several times to mix thoroughly, and the mixture was incubated at room temperature for 10 min. Then, the cells were centrifuged at 1200 rpm for 15 min at 4 °C. The supernatant was discarded, and the RNA precipitate was washed with 1 mL of 75% ethanol (freshly prepared with DEPC-treated water). After mixing, centrifuge at 7500 rpm for 10 minutes at 4°C. Discard the supernatant and air-dry the tubes containing the RNA precipitate in a fume hood. Then, add 20 μL of DEPC-treated water to each tube to dissolve the RNA and determine the RNA concentration. Following the kit instructions, synthesize cDNA from the RNA using a reverse transcription kit. Finally, quantitatively detect the expression level of the target gene using the SYBR Green fluorescent dye assay. ΔΔCT The method is to conduct data analysis.

[0028] 4. Western blot (WB) of proteins Total cellular protein was extracted using RIPA lysis buffer containing a 1% protease inhibitor mixture. The simplified procedure was as follows: After treatment with IL-1β, EGT, CeO2@HA Hydrogel, and EGT-CeO2@HA Hydrogel for 24 h, respectively, the complete culture medium in the six-well plates was discarded, and the cells were washed three times with 1× pre-chilled PBS. After discarding the PBS, 100 μL of RIPA lysis buffer was added to each well, and the plate was incubated on ice with gentle shaking for 20 min. Adherent cells were scraped off using a cell scraper and transferred to 1.5 mL centrifuge tubes. The lysis buffer was briefly sonicated using a cell sonicator, followed by centrifugation at 12000 rpm for 10 min at 4 °C. The supernatant was collected, mixed with 5× loading buffer, and boiled in a metal bath for 6 min. Subsequently, standard protein immunoblotting experiments were performed, including vertical electrophoresis to separate proteins, wet transfer to a PVDF membrane, blocking with 5% skim milk at room temperature for 1.5 hours, incubation with primary antibody at 4°C overnight, incubation with secondary antibody at room temperature for 1.5 hours, and finally detection using a chemiluminescence imaging system.

[0029] Table 1 Antibody Information

[0030] 5. ROS detection experiment Once cells reached 80% confluence, the complete culture medium in the six-well plates was aspirated, and the cells were washed three times with 1× pre-chilled PBS. The PBS was discarded, and the cells were digested with 0.25% trypsin. After digestion, twice the volume of complete culture medium was added to stop the digestion, and the cell suspension was transferred to a 15 ml centrifuge tube. The cells were centrifuged at 800 rpm for 5 min at room temperature. The supernatant was discarded, and the cell pellet was retained. After cell counting, the cells were seeded into 24-well plates. When the cell confluence reached approximately 50%, drug treatment was initiated. After treatment with IL-1β, EGT, CeO2@HAHydrogel, and EGT-CeO2@HA Hydrogel for 24 h, the intracellular reactive oxygen species (ROS) levels in chondrocytes were measured using a ROS detection kit (Beyotime, S0033S). First, 50 μg / mL Rosup reagent was added to the positive control wells and incubated in a cell culture incubator for 30 min. After incubation, the complete culture medium in all wells (including the positive control wells) was aspirated, and the cells were washed once with 1× PBS. Subsequently, 200 μL of prepared DCFH-DA working solution (diluted to 10 μM with basal medium) was added to each well, and the cells were incubated in a cell culture incubator for 30 minutes. After incubation, the cells were washed three times with 1×PBS, and 400 μL of basal medium was added to each well. Fluorescence intensity was measured at excitation / emission wavelengths of 488 / 525 nm. The cells were observed and photographed using a fluorescence microscope.

[0031] 6. JC-1 Experiment Cells were treated in 24-well plates according to the ROS assay method. After treatment with IL-1β, EGT, CeO2@HAHydrogel, and EGT-CeO2@HA Hydrogel for 24 h, changes in mitochondrial membrane potential (MMP) of chondrocytes were assessed using the JC-1 assay kit (UElandy, J6004S). Before MMP detection, the JC-1 kit was removed from the -20°C freezer and allowed to equilibrate to room temperature. First, 50 mM carbonyl cyanide m-chlorophenylhydrazone (CCCP) reagent was added to the positive control wells and incubated in a cell culture incubator for 20 min. After incubation, the complete culture medium in all wells (including the positive control wells) was aspirated, and the cells were washed once with 1×PBS. Then, 200 μL of fresh complete culture medium was added to each well. Next, 200 μL of prepared JC-1 working solution (diluted with PBS and 1× assay buffer according to the kit instructions) was added to each well and incubated in a cell culture incubator for 20 min. After incubation, wash cells three times with 1× detection buffer, and add 400 μL of basal culture medium to each well. Detect red fluorescence at excitation / emission wavelengths of 550 / 600 nm and green fluorescence at excitation / emission wavelengths of 485 / 535 nm. Observe and photograph using a fluorescence microscope.

[0032] 7. Transcriptome and proteome sequencing Chondrocytes were seeded in 10 cm cell culture dishes. After drug treatment, the cells were washed three times with 1× pre-chilled PBS. After centrifugation at 3000 rpm for 5 minutes, the cell pellet was divided into two aliquots: one for transcriptome sequencing and the other for proteome sequencing. For transcriptome sequencing, the samples were lysed in Trizol reagent, flash-frozen in liquid nitrogen, and then sent for analysis. The results were based on transcriptome and proteome expression matrices after quality control (QC), alignment, and quantification. Subsequent data analysis was performed using R (v4.4.3). A linear model was constructed using the limma package for differential expression analysis. The screening criteria were as follows: for transcriptome data, |log2FC|>1 and P-value <0.05; for proteome data, |log2FC|>0.585 and P-value <0.05. Differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were identified. The intersection of DEGs and DEPs was extracted to obtain co-expressed differentially expressed molecules. These co-expressed molecules were mapped to the STRING database (https: / / string-db.org / , version 12.0) to construct a protein-protein interaction (PPI) network. By analyzing the network topology, key nodes (hub genes) were identified to clarify the core interaction factors. Finally, the clusterProfiler package was used to perform gene ontology (GO) annotation analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on the selected DEGs and DEPs, respectively. A p-value < 0.05 was used as the threshold for significant enrichment.

[0033] The results are as follows Figures 3-5 As shown, ATDC5 cells and human chondrocytes were used to investigate whether EGT-CeO2@HA hydrogel could alleviate the inflammatory response of chondrocytes. Human chondrocytes were successfully identified by immunofluorescence detection of COL-II protein expression. Figure 3 A). The safe dose ranges of EGT and CeO2@HA in ATDC5 cells and human chondrocytes were determined using the CCK-8 assay. Figure 3 B-3C). Furthermore, RT-qPCR results showed that in ATDC5 cells, both EGT and EGT-CeO2@HA hydrogels significantly promoted the expression of the anabolic marker gene COL2A1, while inhibiting the expression of catabolic marker genes MMP9 and MMP13, as well as inflammatory genes TNF-α and IL-6. Figure 4 A). Similarly, in human chondrocytes, EGT and EGT-CeO2@HA hydrogels also promote anabolism while inhibiting catabolism and the expression of inflammatory factors. Figure 4B). These results indicate that EGT and EGT-CeO2@HA hydrogels may partially reverse IL-1β-induced chondrocyte inflammatory responses. Western blot analysis, by detecting the protein expression of COL-II and MMP13 in ATDC5 cells and human chondrocytes, further confirmed these results. Figure 4 C-4D). Therefore, ROS levels and MMPs in ATDC5 cells and human chondrocytes were detected. The results showed that both EGT and CeO2@HA significantly inhibited ROS accumulation in chondrocytes (C-4D). Figure 5 A and 5C), and reduce MMP ( Figure 5 (B and 5D). Importantly, the EGT-CeO2@HA hydrogel exhibited the strongest antioxidant properties. In summary, these results preliminarily confirm that EGT and EGT-CeO2@HA hydrogel may partially reverse IL-1β-induced chondrocyte inflammatory responses by inhibiting oxidative stress.

[0034] To further explore the molecular mechanism by which EGT-CeO2@HA hydrogel alleviates chondrocyte inflammatory response, transcriptomic and proteomic sequencing analyses were performed. Figure 6 A-6H and Figure 7 A-7G). Transcriptome sequencing results showed that, compared with the IL-1β-treated group, the EGT-CeO2@HA hydrogel-treated group had 254 upregulated genes and 333 downregulated genes. Figure 6 B and Figure 7 A). Meanwhile, proteomic results showed that 137 proteins were upregulated and 89 proteins were downregulated (A). Figure 6 C and Figure 7 B). Intersection analysis of DEGs and DEPs yielded 36 genes / proteins with consistent expression changes at both the transcriptional and protein levels. Figure 7 C). Subsequently, DEGs were analyzed using the GO and KEGG databases. Figure 6 E and 6G) ​​and DEPs ( Figure 6 Signal pathway enrichment analysis was performed using F and 6H. The results showed that these changes were mainly enriched in oxidative stress, apoptosis, and autophagy-related pathways. Notably, in co-expressed DEGs / DEPs, a key autophagy regulatory gene, ATF4, was found to be upregulated at both mRNA and protein levels in the EGT-CeO2@HA hydrogel group. Figure 6 B-6D and Figure 7 C). Therefore, a preliminary research hypothesis is proposed: EGT-CeO2@HA hydrogel may alleviate IL-1β-induced chondrocyte inflammatory damage by activating ATF4-dependent pro-survival autophagy.

[0035] To verify this hypothesis, siRNA-ATF4 was constructed. Preliminary RT-qPCR and WB analysis confirmed that the si-ATF4-3 sequence significantly inhibited the expression of the ATF4 gene and protein in chondrocytes. Figure 4 A-4F). Upon activation, ATF4 can directly bind to and initiate the transcription of autophagy-related genes in the cell nucleus, such as LC3B and ATG5, thereby promoting autophagy. Immunofluorescence staining shows that ATF4 is mainly located in the nuclei of chondrocytes (A-4F). Figure 8 A). IL-1β treatment inhibited ATF4 protein expression, while ATF4 knockdown partially reversed the activation of ATF4 by EGT-CeO2@HA hydrogel. Figure 8 A). RT-qPCR results further confirmed that EGT-CeO2@HA hydrogel can activate the transcriptional expression of autophagy-related genes (including LC3B and ATG5) by promoting ATF4 expression in chondrocytes. Figure 8 B), indicating that EGT-CeO2@HA hydrogel may promote chondrocyte autophagy. Further research showed that EGT-CeO2@HA hydrogel enhanced the expression of autophagy marker proteins such as ATF4, Beclin1, and LC3B II / I, indicating that chondrocyte autophagy was activated (B). Figure 8 C-8D). However, ATF4 knockdown partially reversed EGT-CeO2@HA hydrogel-induced chondrocyte autophagy activation (C-8D). Figure 8 C-8D). ROS ( Figure 8 E) and MMP ( Figure 8 F) Detection showed that EGT-CeO2@HA hydrogel significantly inhibited ROS accumulation and MMP loss in chondrocytes, while ATF4 knockdown partially reversed these changes. These results suggest that EGT-CeO2@HA hydrogel may promote chondrocyte autophagy by activating ATF4 nuclear translocation and initiating the transcription of key autophagy-related proteins, thereby ultimately alleviating IL-1β-induced chondrocyte inflammatory damage.

[0036] Example 3 Animal Experiment Forty 10-week-old male Sprague-Dawley rats were purchased from Vital River Laboratory Animal Technology Co., Ltd. All rats were housed in a specific pathogen-free (SPF) grade animal facility at Xiamen University. The animals were allowed free movement under controlled environmental conditions, with a constant temperature of 22±2℃ and relative humidity of 55-75%. After one week of acclimatization, the rats were randomly divided into five groups: sham surgery group (Sham group, n=8), anterior cruciate ligament (ACLT) induced OA model group (ACLT group, n=8), ACLT + intra-articular ergothioneine injection group (EGT group), ACLT + intra-articular hyaluronic acid-modified cerium dioxide hydrogel injection group (CeO2@HA hydrogel group), and ACLT + intra-articular ergothioneine-loaded hyaluronic acid-modified cerium dioxide hydrogel group (EGT-CeO2@HA hydrogel group). The rat OA model was established via ACLT surgery. The simplified procedure is as follows: After isoflurane anesthesia, the skin on the medial side of the knee joint was incised with a scalpel. Using the medial collateral ligament as a surface anatomical landmark, the joint capsule was opened with ophthalmic surgical scissors. The patella was dislocated laterally to expose the anterior cruciate ligament, which was then severed with the tip of ophthalmic scissors. The wound was then sutured in layers. Antibiotics were administered intramuscularly for three consecutive days postoperatively. Starting one week postoperatively, the drug was injected into the knee joint weekly. The EGT group received ergothioneine (5 mg / kg), the CeO2@HA hydrogel group received hyaluronic acid-modified cerium dioxide hydrogel (0.5 mg / kg), and the EGT-CeO2@HA hydrogel group received ergothioneine-loaded cerium dioxide hydrogel. Eight weeks postoperatively, all rats underwent behavioral assessment using the Catwalk automated gait analysis system. Rats were then weighed under isoflurane anesthesia and euthanized by cervical dislocation. All experimental procedures were approved by the Animal Ethics Committee of the Experimental Animal Center of Xiamen University (Approval No.: XMULAC20250225) and the Medical Ethics Committee of Zhengzhou Orthopedic Hospital (Approval No.: 2025KY04602).

[0037] 1. Catwalk gait analysis The day before the experiment, all rats were allowed to move freely in the Catwalk gait analysis system (Catwalk XT, Noldus) tunnel to acclimatize to the environment. All rats were fasted for 12-24 hours before the formal test. On the day of the experiment, the rats were placed in the behavioral testing room 3 hours in advance for environmental acclimatization. The room was kept dimly lit and quiet as much as possible. Before the experiment, the tunnel width and camera position were adjusted. The light sources (red and green) in the small animal tunnel were turned on. At the start of the formal experiment, each rat was placed at the beginning of the tunnel (a dark box containing food particles was placed at the other end to attract the rats to move freely through the tunnel). When a rat entered the camera's field of view, the high-speed camera automatically recorded video (recording could also be started manually) to capture the rat's paw prints and gait. Videos meeting the gait analysis quality standards were automatically saved and automatically gaited (manual correction of incorrectly identified paw prints was possible). All paw print and gait data were automatically analyzed and exported by the system software. Each time a rat urinates or defecates, its passageway is immediately wiped with 75% ethanol and lint-free paper to avoid interfering with the high-speed camera capturing fluorescent paw prints on the glass plate. The experiment ends after three successful walking videos of each rat.

[0038] 2. Micro-CT scan After powering on the Skyscan scanner (SkyScan 1272, Bruker), machine warm-up and bright / dark field calibration were performed sequentially. The Skyscan scanner's scanning resolution was set to 13 μm for scanning the rat knee joint. After scanning, NRecon, DataViewer, CTvol, and CTVox software were used sequentially for angle adjustment, region of interest (ROI) selection, and 3D reconstruction of the knee joint. The subchondral bone of the femur and tibia was reconstructed and analyzed separately. Bone morphometric parameters were obtained, including bone volume fraction (BV / TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N).

[0039] 3. Hematoxylin-eosin (HE) staining and safranin O-fast green staining Following micro-CT scanning, the knee joint was placed in a 10% EDTA solution and decalcified on a shaker at room temperature. The 10% EDTA solution was changed every two days. Decalcification was considered complete when the injection needle could penetrate the femur. The sample was then subjected to routine dehydration, clearing, and paraffin embedding. Subsequently, knee joint tissue sections (4 μm thick) were prepared using a paraffin microtome. The sections were flattened in a 40°C water bath and then dried in a 60°C oven. The paraffin-embedded knee joint sections were dewaxed and rehydrated to distilled water using a gradient of ethanol solutions. For HE staining, the procedure involved staining with hematoxylin solution, differentiation with 1% acidic ethanol, blue resizing with 1% dilute ammonia, and counterstaining with eosin solution. The procedure for safranin O-fast green staining was similar to HE staining, except that the HE solution was replaced with safranin O-fast green solution. Finally, after routine dehydration, the slides were mounted with neutral resin and observed and imaged under an inverted microscope.

[0040] 4. Immunohistochemistry (IHC) / Immunofluorescence (IF) The paraffin-embedded knee joint sections were dewaxed and rehydrated to distilled water. The sections were placed in antigen retrieval buffer and incubated overnight at 60°C for antigen retrieval. After antigen retrieval, incubation with 3% hydrogen peroxide solution was performed to block endogenous peroxidase activity. A hydrophobic barrier was drawn around the tissue sections using an IHC pen, followed by blocking with 3% bovine serum albumin (BSA) solution at room temperature for 30 minutes. Subsequently, primary antibody was incubated overnight at 4°C, and the corresponding secondary antibody was incubated at room temperature for 1 hour. For IHC, 3,3'-diaminobenzidine (DAB) substrate was used for staining, and the cell nuclei were counterstained with hematoxylin (same as HE staining). Finally, the sections were dehydrated, mounted, and observed and imaged under an inverted microscope. For IF staining, fluorescently labeled secondary antibody was used for incubation in the dark, and the cell nuclei were stained with 4',6-diamidinyl-2-phenylindole (DAPI). Additionally, the sections were mounted with anti-fluorescence quenching mounting medium and observed and imaged under a fluorescence microscope; antibody times are shown in Table 1.

[0041] The results are as follows Figures 9-14 As shown, the antioxidant amino acid taurine can alleviate ACLT-induced cartilage degeneration in OA rats. In this example, the potential of EGT in alleviating OA cartilage degeneration was further explored. A HA-modified CeO2 hydrogel delivery system was used to achieve sustained release of EGT, thereby effectively enhancing its antioxidant efficacy through intra-articular injection. Figure 9 A-9C). Figure 9 D-9E demonstrates in vitro fluorescence imaging of the rat knee joint after intra-articular injection of CY7-labeled EGT-CeO2@HA or CY7-labeled EGT-CeO2@HA hydrogel. The results show that the CY7-labeled EGT-CeO2@HA hydrogel is absorbed by the distal femoral and proximal tibial cartilage. Therefore, EGT-CeO2@HA hydrogel was used in subsequent animal experiments.

[0042] like Figure 10 As shown in Figure A, there were no significant differences in the preoperative and postoperative body weights of rats in each group. Figure 10 Image B shows representative images of the surgical field before and after anterior cruciate ligament transection surgery in rats. Gait analysis was performed on all rats before sacrifice. Figure 10 C-10D). Results showed that ACLT-induced OA rats exhibited significantly reduced mean walking speed, maximum contact intensity, paw print length and width, and significantly increased swing time, indicating a typical pain-related gait. However, these parameters were partially reversed in the EGT-CeO2@HA hydrogel treatment group, indicating relief of the pain-related gait in this group. Furthermore, periarticular osteophyte formation was closely associated with OA-related pain. Three-dimensional reconstruction of the rat knee joint was performed using Micro-CT (…). Figure 11 A-11E). Results showed a significant increase in periarticular osteophytes in the ACLT group, while osteophyte formation was significantly reduced in the EGT treatment group and the EGT-CeO2@HA hydrogel treatment group. These results indicate that EGT-CeO2@HA hydrogel can significantly alleviate knee pain in OA rats. HE staining of internal organs in all groups showed normal tissue structure (…). Figure 12 This indicates that intra-articular injection of EGT-CeO2@HA hydrogel is safe.

[0043] Furthermore, HE and Safranin O-Fixed Green staining of the knee joints of different groups of rats showed that, compared with the ACLT group, the articular cartilage tissue in the EGT treatment group and the EGT-CeO2@HA hydrogel treatment group was smoother, with no significant chondrocyte loss or fibrosis. Figure 13 A-13B). Mankin and OARSI scores further confirmed that EGT-CeO2@HA hydrogel treatment partially reversed ACLT-induced cartilage degeneration and damage in rats (A-13B). Figure 13 E-13F). Furthermore, immunohistochemical staining showed that, compared to the Sham group, the ACLT group exhibited significantly decreased COL-II protein expression and significantly increased MMP13 protein expression in cartilage, indicating cartilage metabolic disorder. However, EGT-CeO2@HA hydrogel treatment significantly reversed the expression of these proteins (E-13F). Figure 13 C-13D and 13G-13H). These results indicate that EGT-CeO2@HA hydrogel alleviates ACLT-induced OA cartilage degeneration in rats. Furthermore, in vitro experiments suggest that ATF4-mediated pro-survival autophagy may be a potential mechanism by which EGT-CeO2@HA hydrogel alleviates IL-1β-induced chondrocyte damage. At the animal level, ATF4 expression in rat cartilage tissue was assessed by immunohistochemistry. Compared to the Sham group, ATF4 protein expression was significantly reduced in the ACLT group. EGT administration significantly promoted ATF4 protein expression, with a particularly significant upregulation in the EGT-CeO2@HA hydrogel group (C-13D and 13G-13H). Figure 14A). Importantly, consistent with in vitro results, EGT-CeO2@HA hydrogel treatment significantly reversed ACLT-induced expression of LC3B and Beclin1 proteins in rat cartilage ( Figure 14 These results indicate that the EGT-CeO2@HA hydrogel activates ATF4-mediated pro-survival autophagy, thereby alleviating ACLT-induced cartilage degeneration in rats.

[0044] In summary, according to embodiments of the present invention, an injectable EGT-CeO2@HA hydrogel was successfully developed based on the synergistic construction of HA-modified CeO2 nanoparticles and a thermosensitive hydrogel, achieving long-term targeted delivery of EGT within the joint cavity. This hydrogel exhibits highly efficient ROS scavenging and cartilage inflammation inhibition properties, demonstrating significant articular cartilage protection at both cellular and animal levels. Through multi-omics analysis and functional validation, it was revealed that the EGT-CeO2@HA hydrogel can activate the ATF4-dependent pro-survival autophagy pathway, restore the damaged autophagy homeostasis under inflammatory conditions, and reshape the metabolic homeostasis of chondrocytes, thereby alleviating cartilage degeneration in OA rats.

[0045] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. The illustrative expressions of the above terms in this specification should not be construed as necessarily referring to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.

[0046] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A nanocomposite hydrogel for treating osteoarthritis, characterized in that, include: Cerium dioxide nanoparticles, wherein the cerium dioxide nanoparticles are modified with hyaluronic acid; Ergothioneine, loaded onto hyaluronic acid-modified cerium dioxide nanoparticles to form the nanocomposite EGT-CeO2@HA; and A thermosensitive hydrogel carrier in which the nanocomposite EGT-CeO2@HA is uniformly dispersed.

2. The nanocomposite hydrogel as described in claim 1, characterized in that, In hyaluronic acid-modified cerium dioxide nanoparticles, the mass ratio of hyaluronic acid to cerium dioxide is 4:

1.

3. The nanocomposite hydrogel as described in claim 1, characterized in that, The loading of ergothionein is 22.3% of the mass of the hyaluronic acid-modified cerium dioxide nanoparticles.

4. The nanocomposite hydrogel as described in claim 1, characterized in that, The thermosensitive hydrogel carrier is PF127 / HA hydrogel.

5. A method for preparing a nanocomposite hydrogel for treating osteoarthritis, characterized in that, Includes the following steps: Preparation of hyaluronic acid-modified cerium dioxide nanoparticles: Cerium dioxide nanoparticles were dispersed in a hyaluronic acid solution, stirred and reacted, then centrifuged, washed and dried; Ergothioneine loading: Hyaluronic acid-modified cerium dioxide nanoparticles were dispersed in an ergothioneine solution, stirred for adsorption, centrifuged, washed, and dried to obtain the nanocomposite EGT-CeO2@HA; Preparation of thermosensitive hydrogel carrier: Dissolve the thermosensitive hydrogel carrier in water and stir until completely dissolved; Preparation of nanocomposite hydrogel: The nanocomposite EGT-CeO2@HA was dispersed in the thermosensitive hydrogel carrier and stirred evenly to obtain the nanocomposite hydrogel.

6. The use of the nanocomposite hydrogel according to any one of claims 1-4 or the nanocomposite hydrogel prepared by the preparation method of claim 5 in the preparation of a medicament for treating osteoarthritis.

7. The application as described in claim 6, characterized in that, The drug is an injectable preparation.

8. The application as described in claim 7, characterized in that, The drug is a preparation for intra-articular injection.

9. The application as described in claim 6, characterized in that, The drug reduces chondrocyte inflammatory damage and improves cartilage degeneration by activating the ATF4-dependent pro-survival autophagy pathway.

10. A drug delivery system for treating osteoarthritis, characterized in that, The nanocomposite hydrogel includes any one of claims 1-4 or the nanocomposite hydrogel prepared by the preparation method of claim 5.

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