CH@SHP099-nanoparticle composite hydrogel, and preparation method and application thereof

Abstract

This invention discloses a CH@SHP099-NPs composite hydrogel, its preparation method, and its applications, belonging to the field of biomedical technology. Addressing the technical challenge of achieving long-term, safe, and precise metabolic intervention with SHP2, this invention constructs a hydrogel system composed of chondroitin sulfate and hyaluronic acid. This system can retain SHP099-loaded nanoparticles within the joint cavity, forming a long-acting intrasynovial drug library, thereby continuously inhibiting the SHP2-AMPK signaling axis. The CH@SHP099-NPs composite hydrogel prepared in this invention can effectively reprogram the metabolic state of macrophages, reduce glycolysis flux, restore synovial immune metabolic homeostasis, and significantly improve synovial inflammation, chondrocyte activity, and cartilage structural integrity in vivo. This invention proposes a mechanism-guided local delivery metabolic intervention strategy, which is expected to provide a new interventional approach for the treatment of osteoarthritis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to the preparation of a composite hydrogel and its application in a drug delivery system. Background Technology

[0002] Osteoarthritis (OA) is the most common chronic degenerative joint disease worldwide, with an incidence rate that increases significantly with age, severely impairing the quality of life of middle-aged and elderly people and imposing a heavy social and medical burden. Traditional understanding and treatment of OA have focused primarily on the mechanical wear and degenerative changes of articular cartilage. Clinical interventions mainly aim to relieve pain and improve joint function, such as nonsteroidal anti-inflammatory drugs (NSAIDs) and intra-articular injections of sodium hyaluronate. However, these methods are mostly symptomatic treatments and are unlikely to fundamentally slow down or reverse the disease progression of OA. For example, sodium hyaluronate injections can relieve symptoms by improving joint lubrication and temporarily blocking pain receptors, but its retention time in the joint cavity is short (approximately 72 hours), making it impossible to maintain long-term efficacy, and it lacks a substantial promoting effect on cartilage regeneration.

[0003] With in-depth research, our understanding of the pathological mechanisms of osteoarthritis (OA) has undergone significant changes. Increasing evidence suggests that chronic low-grade inflammation of the synovial tissue is not a passive secondary phenomenon of OA, but rather a persistent and key pathological factor driving joint tissue damage throughout the disease's development and progression. Dysregulation of the synovial immune microenvironment, particularly the abnormal inflammatory response mediated by macrophages, directly accelerates cartilage matrix degradation and inhibits its self-repair capacity by releasing large amounts of inflammatory factors and proteases. This suggests that the nature of OA has transcended simple mechanical wear and tear, involving a complex dysregulation of the immune-inflammatory network. Therefore, targeting and regulating synovial immune inflammation, especially the functional state of macrophages, provides new insights for developing modulatory therapies for OA.

[0004] Among synovial immune cells, macrophages, as core innate immune cells, are closely related to the inflammatory process of osteoarthritis (OA). Traditionally, macrophages are statically classified into pro-inflammatory M1 and anti-inflammatory / repairing M2 types. However, this phenotypic classification fails to fully explain the persistence of chronic inflammation in OA. Recent immunometabolic studies have revealed that macrophage function is closely coupled with its energy metabolism program: pro-inflammatory activation is usually accompanied by enhanced glycolysis, while anti-inflammatory and tissue repair functions are more dependent on oxidative phosphorylation. Therefore, the aberrant reprogramming of synovial macrophage metabolic patterns, i.e., the shift towards glycolysis, may be the core intrinsic mechanism driving their persistent pro-inflammatory state and accelerating joint destruction. Identifying and intervening in key upstream nodes regulating this metabolic shift is of great significance for developing novel OA treatment strategies.

[0005] Protein tyrosine phosphatase SHP2 is a key signaling molecule regulating cell growth, differentiation, and inflammatory responses. Recent studies have found that the SHP2 allosteric inhibitor SHP099 shows potential therapeutic value in osteoarthritis (OA) models. For example, patent publication CN112823796A discloses the application of SHP2 inhibitors in the preparation of drugs for treating osteoarthritis, demonstrating that SHP099 can significantly improve cartilage damage in surgically induced and naturally aging mice with OA, inhibit the expression of matrix metalloproteinases (such as MMP13 and MMP3) in chondrocytes, and simultaneously promote the expression of cartilage synthesis-related genes (such as Aggrecan and Col2a1), showing a dual regulatory effect on cartilage metabolism. These studies suggest that targeting SHP2 may intervene in the OA process by influencing downstream signaling pathways (such as the AMPK pathway, which is closely related to energy metabolism homeostasis) to regulate the metabolism and function of cells (including macrophages).

[0006] However, despite the potential of SHP2 inhibitors in basic research, their clinical translation into OA treatment faces significant challenges. The pathological processes within the joint cavity are highly localized, and systemic administration (such as intraperitoneal injection) struggles to maintain effective and sustained drug concentrations within the joint cavity. Furthermore, systemic exposure can lead to off-target effects and potential side effects, hindering precise and long-term intervention in the synovial local immune microenvironment. Therefore, developing an advanced delivery strategy that adapts to the joint cavity environment and achieves long-term local drug retention and controlled release is a crucial prerequisite for overcoming these translational obstacles and transforming novel intervention concepts such as SHP2-targeted therapy into safe and effective treatments.

[0007] In the field of local joint delivery, natural polymeric materials have attracted widespread attention due to their excellent biocompatibility, biodegradability, and natural affinity for joint tissues. For example, hyaluronic acid and chondroitin sulfate, as natural components of articular cartilage and synovial fluid, have been studied for use in constructing intra-articular injection formulations. Patent CN1585645A discloses an attempt to directly inject a mixture of sodium hyaluronate and sodium chondroitin sulfate into the joint for the treatment of osteoarthritis (OA), aiming to utilize their viscoelasticity to provide lubrication and protection, and potentially provide a temporary matrix environment for chondrocytes. However, such formulations primarily function as physical fillers or lubricants, with relatively simple mechanisms of action and limited ability to deeply regulate disease progression (such as immune metabolic reprogramming). Furthermore, the loading and controlled release of drugs (such as small molecule inhibitors) are not their original design objectives.

[0008] In summary, current treatments for osteoarthritis (OA) still lack effective methods to fundamentally intervene in synovial immune metabolic abnormalities and achieve precise, long-lasting local drug delivery to the joint. Existing small-molecule targeted drugs (such as SHP2 inhibitors) lack suitable local delivery systems, while existing intra-articular injection materials (such as hyaluronic acid / chondroitin sulfate mixtures) are insufficient to meet the requirements for intelligent controlled release and targeted delivery of active drug molecules. Therefore, there is an urgent need to develop a novel composite therapeutic system that combines specific immunomodulatory agents with biocompatible locally controlled-release carriers to achieve precise intervention in the synovial immune microenvironment of OA, providing a new solution for disease-modifying treatment of OA. Summary of the Invention

[0009] This invention addresses the technical problem that existing systemic inhibition of SHP2 is easily limited by pharmacokinetics and may cause widespread off-target effects, making it difficult to achieve long-term, safe and precise metabolic intervention. It proposes a CH@SHP099-NPs composite hydrogel, its preparation method and application.

[0010] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0011] This invention provides a method for preparing CH@SHP099-NPs composite hydrogel, comprising the following steps:

[0012] (1) Dissolve sodium chondroitin sulfate and sodium hyaluronate separately in deionized water and mix them evenly to obtain CH hydrogel precursor solution;

[0013] (2) After dissolving the thiol-containing cLABL cyclic peptide in a buffer solution, it was reacted with PLGA-PEG-MAL (polylactic acid-glycolic acid copolymer-polyethylene glycol-maleimide) in the dark. After dialysis and freeze-drying, PLGA-PEG-cLABL block copolymer was obtained.

[0014] (3) Dissolve PLGA-PEG-cLABL block copolymer and SHP099 together in an organic solvent, then add dropwise to deionized water and ultrasonically emulsify. After evaporating the organic solvent, SHP099 NPs are obtained.

[0015] (4) Disperse SHP099 NPs in the CH hydrogel precursor solution prepared in step (1), then add Genipin and stir, heat and react, and wash to obtain CH@SHP099-NPs composite hydrogel.

[0016] In step (1), the mass ratio of sodium chondroitin sulfate and sodium hyaluronate is 1-3:1-3, the concentration of sodium chondroitin sulfate is 8-12 mg / mL, and the concentration of sodium hyaluronate is 8-12 mg / mL.

[0017] In step (2), the mass ratio of cLABL cyclic peptide to PLGA-PEG-MAL is 0.12-0.2:1-2, the initial concentration of cLABL cyclic peptide is 0.5-10 mg / mL, the buffer solution is PBS buffer solution with pH 7.2-7.6, and the reaction time is 8-14 h in the dark.

[0018] In step (3), the mass ratio of PLGA-PEG-cLABL block copolymer to SHP099 is 10:1, and the concentration of SHP099 in the organic solvent is 0.5-2.0 mg / mL; the organic solvent is a mixture of dichloromethane and acetonitrile with a volume ratio of 1:1.

[0019] In step (4), the final concentration of SHP099 NPs is 0.8-1.2 mg / mL; the final concentration of Genipin is 0.3-0.7 mg / mL; the heating reaction temperature is 35-39℃ and the time is 4-8 h.

[0020] This invention provides a CH@SHP099-NPs composite hydrogel prepared using the above-described preparation method.

[0021] The present invention also provides the application of the CH@SHP099-NPs composite hydrogel in the preparation of drugs for relieving or treating osteoarthritis.

[0022] The beneficial effects of this invention are:

[0023] 1. This invention systematically reveals the key role of the SHP2-AMPK immune metabolic axis in driving pro-inflammatory reprogramming of synovial macrophages and the progression of osteoarthritis. Continuous regulation of the SHP2-AMPK axis is crucial for reversing the immune metabolic state of macrophages. Mechanistic studies reveal that SHP2 locks the macrophage metabolic state onto a pro-inflammatory track dominated by glycolysis by inhibiting AMPK signaling. This invention proposes a precise intervention strategy based on engineered delivery, thus providing a new theoretical framework for understanding and treating OA.

[0024] 2. This invention constructs an injectable hydrogel system (CH) composed of chondroitin sulfate (CS) and hyaluronic acid (HA) as an intra-articular in-situ delivery platform. This hydrogel forms a stable cross-linked network under mild conditions mediated by Genipin, providing a reliable material basis for drug loading and long-term local retention. This invention further constructs a PLGA-PEG-cLABL nanocarrier with macrophage targeting capability for SHP099 delivery, embedding it in the CH hydrogel to form a composite delivery system to achieve local and sustained intra-articular release of SHP099-loaded nanoparticles, and systematically evaluates its in vivo intervention effects on the synovial inflammatory microenvironment and cartilage degeneration.

[0025] 3. This invention successfully constructed an engineered composite delivery system with stability, injectability, and sustained-release properties, exhibiting good system safety. Its safety at the cellular level and macrophage uptake capacity were verified. In vivo experiments showed that CH@SHP099-NPs, through engineered methods, achieved sustained intervention on the SHP2-AMPK immune metabolic axis in vivo, effectively alleviating structural joint damage in OA mice, significantly protecting the cartilage matrix, and delaying disease progression. This not only expands our understanding of the pathogenesis of OA but also provides new theoretical and experimental basis for developing local joint treatment strategies targeting immune metabolism. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1This study aims to construct a single-cell atlas of synovial tissue in mice with osteoarthritis and analyze its cell communication network. A shows a schematic diagram of the single-cell analysis process for synovial tissue in mice with osteoarthritis. B shows a UMAP clustering diagram illustrating the distribution of 25 cell subpopulations in the synovial tissues of the OA and sham groups. C shows a UMAP diagram with cell subpopulation annotations illustrating the distribution of different cell types in two-dimensional space. D shows a stacked bar chart illustrating the proportion of each cell type in different samples. E shows a global communication network diagram of different cell types in the synovial tissues of the sham group (top) and the OA group (bottom) generated using CellChat analysis. The left image shows the communication strength between each cell pair (interaction weights strength), and the right image shows the number of interactions between each cell pair. Each node represents a cell type, and the thickness of the edges indicates the signal transduction strength or number of interactions between the corresponding cells. F compares the number of inferred interactions and interaction strength between cells in the synovial tissues of different treatment groups. The left image shows the number of inferred interactions inferred by CellChat, and the right image shows the total communication strength (interaction weights strength). (strength); G is a bar chart showing the information flow of different signaling pathways and their changes between the two groups; H is a heatmap showing the amount of communication and interaction intensity between different cell pairs; the samples were taken from the synovial tissue of C57BL / 6J mice, n=2.

[0028] Figure 2 This study included quality control and principal component analysis (PCA) of single-cell transcriptomes from synovial tissues in osteoarthritis and control groups. A is a violin plot showing the number of transcripts (nCount_RNA), the number of detected genes (nFeature_RNA), and the proportion of mitochondrial genes (percent.mt) in each cell of the OA group (OA1, OA2) and the control group (sham1, sham2). B is a scatter plot showing the correlation between nCount_RNA and percent.mt, and between nCount_RNA and nFeature_RNA. C is a comparison of group distribution and PC1 scores before PCA. D is an embedding plot after Harmony batch correction showing the consistency of cell distribution across groups and plotting the Harmony_1 axis scores. E is a heatmap of hypervariable genes on the first six PC dimensions of PCA showing expression differences among different samples. Samples were obtained from synovial tissue of the knee joint of C57BL / 6J mice, n=2.

[0029] Figure 3This study elucidated the cell communication network of the TNF and FGF signaling pathways in OA synovial tissue. AB represents the cell communication network of the TNF and FGF signaling pathways in OA synovial tissue analyzed using CellChat, where Sender, Receiver, Mediator, and Influencer represent the signal sender, receiver, mediator, and key regulator, respectively. CD shows the distribution of communication roles of the TNF and FGF signaling pathways in sham synovial tissue. EF represents the source-to-target unidirectional communication structure of each signaling pathway across different cell types, with node size representing cell involvement and line color derived from the sending cell. G represents the global intercellular communication network of the TNF signaling pathway in both the sham and OA groups. H represents the intercellular network of the FGF signaling pathway in both the sham and OA groups. Each group was derived from synovial tissue of C57BL / 6J mice, n=2.

[0030] Figure 4 Transcriptomic characteristics and SHP2 expression levels of synovial macrophage subsets in osteoarthritis were analyzed. A shows the distribution of macrophage populations in synovial tissue using UMAP clustering based on scRNA-seq data; B shows a DotPlot plot of typical marker genes used to identify macrophage subsets; C divides macrophages into three subsets based on functional status: M1-like, M2-like, and Intermediate (n=2); D shows the UMAP plot of Ptpn11 (SHP2 encoding gene) expression in macrophages (n=2); EF shows that RT-qPCR and Western blot results indicate significantly increased SHP2 expression levels in the synovial tissue of the OA group (P<0.0001); G shows the co-localization of SHP2 protein in F4 / 80 positive macrophages (bar: 25 μm) using immunofluorescence staining (P<0.0001).

[0031] Figure 5 Transcriptomic features of synovial macrophage subsets in osteoarthritis are presented. Among them, A is a UMAP plot showing the expression characteristics of inflammation-related genes Syne1, Ebf1, and Il1b in macrophages, n=2; B is a volcano plot showing the distribution of differentially expressed genes in macrophages of the OA group and the sham group; CF is a GO enrichment analysis showing the major enriched entries of differentially expressed genes in biological processes (BP), molecular functions (MF), cellular components (CC), and the KEGG pathway, n=2.

[0032] Figure 6This study aimed to alleviate cartilage destruction and synovial inflammation in osteoarthritis induced by osteoarthritis (OA) by macrophage-specific SHP2 knockout. A shows a schematic diagram of the OA-induced mouse model construction and key indicator detection experiments. B shows micro-CT imaging, H&E staining, and Safranin O-Fast Green staining to observe joint bone structure and cartilage degeneration (bar: 100 μm). C shows the OARSI scores of mice with different genotypes after surgery. DE shows immunohistochemistry (IHC) detection of TNF-α, IL-1β, IL-6, and IL-10 expression levels in synovial tissue (bar: 25 μm). F shows immunofluorescence double staining analysis of the distribution of iNOS⁺F4 / 80⁺ and Arg-1⁺F4 / 80⁺ macrophages in synovial tissue (bar: 25 μm). μm; G represents the expression intensity (MFI) of MHC-II in different genotype BMDMs after LPS / IFN-γ stimulation by flow cytometry; H represents the expression intensity (MFI) of CD206 in different genotype BMDMs after IL-4 stimulation by flow cytometry; I represents the mRNA expression of Tnf, Il1b, and Il6 under M1-induced conditions by RT-qPCR; J represents the mRNA expression of Il10, Tgfb, and Cd206 under M2-induced conditions by RT-qPCR; K represents the expression level of iNOS protein under M1-induced conditions by Western blot; L represents the expression level of CD206 protein under M2-induced conditions by Western blot; n=6 animals per group in animal experiments, ns indicates no significant difference between the two groups, ****P<0.0001.

[0033] Figure 7 This study reprogrammed SHP2 to regulate macrophage metabolism via the AMPK signaling axis; where A is the experimental flowchart including LPS stimulation, SHP2 intervention, metabolic flux analysis, and AMPK signaling activation / inhibition steps; B is the Western spectral density curve. Blot analysis of protein expression of GLUT3, HK1, LDHA and p-AMPK in macrophages after LPS, siSHP2 and SHP099 intervention; C: Western blot quantitative analysis of changes in various metabolic enzymes and AMPK signal; D: RT-qPCR detection of GLUT3 and LDHA mRNA levels; E: Seahorse analysis of glycolysis flux (ECAR) under different treatment conditions; F: Seahorse analysis of oxidative phosphorylation capacity (OCR); G: Lactate concentration in cell culture supernatant; H: ATP concentration in each group of cells detected by ATP kit; I: Schematic diagram of the molecular mechanism of SHP2 regulating macrophage metabolic reprogramming through AMPK; The experiment was repeated three times, ns indicates no significant difference between the two groups, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0034] Figure 8 Intervention on the SHP2 / AMPK signaling axis can effectively inhibit M1 polarization in macrophages. A is a schematic diagram of the in vitro experimental procedure, showing the construction of M0 BMDMs cells, SHP2 overexpression, and SHP099 / AICAR treatment. B shows the mRNA expression levels of pro-inflammatory factors IL-1β, IL-6, TNF-α, and iNOS in different treatment groups detected by RT-qPCR. C shows the protein expression of IL-1β, IL-6, TNF-α, and iNOS detected by Western blot. D shows the expression of CD86 and iNOS M1 markers analyzed by immunofluorescence staining (bar: 25 μm). E shows the proportion of CD86 macrophages detected by flow cytometry and the positive expression rate. F is a schematic diagram of the molecular mechanism by which the SHP2 / AMPK signaling axis regulates M1 polarization. Cell experiments were repeated three times. *** indicates P < 0.001 between two groups, **** indicates P < 0.0001.

[0035] Figure 9 This study describes the preparation and physicochemical characterization of CH injectable hydrogels. A shows a schematic diagram of the chemical structure of CS and HA forming an injectable hydrogel (CH) under Genipin crosslinking conditions. B shows Fourier transform infrared spectroscopy (FTIR) detection of functional group changes in CS, HA, Genipin, and their crosslinking to form the hydrogel. C shows a time sweep rheological test to detect the stability of the storage modulus (G') and loss modulus (G'') of hydrogels with different ratios over time. D shows a frequency sweep rheological test to detect the mechanical response of hydrogels with different ratios under frequency variations. E shows the inverted tube method to evaluate the gelation state and flowability of CH hydrogels with different ratios. F shows scanning electron microscopy (SEM) observation of the micropore structure of CH hydrogels with different ratios (bar: 200 μm). G shows an injection feasibility test demonstrating the injectability of the hydrogels with different ratios via a 23 G needle. The experiment was repeated three times.

[0036] Figure 10This paper describes the preparation and physicochemical characterization of CH@SHP099-NPs composite hydrogels loaded with SHP099 nanoparticles. A shows a schematic diagram of the preparation of chondroitin sulfate-based hydrogels loaded with SHP099 nanoparticles; B shows the particle size distribution of PLGA-PEG-cLABL nanoparticles detected by dynamic light scattering (DLS); C shows the particle size distribution of SHP099-NPs after SHP099 drug loading detected by dynamic light scattering (DLS); D shows the morphology of SHP099-NPs observed by transmission electron microscopy (TEM), bar: 100 nm; E shows the drug release curve of CH@SHP099-NPs composite hydrogels in simulated slippery fluid medium detected by high performance liquid chromatography (HPLC); and F shows the evaluation of CH@SHP099 using an inverted bottle test. G represents the gelation state of the NPs composite hydrogel; G is the injection test to evaluate the injection flowability of the composite hydrogel through a 23G needle; H is a schematic diagram of the sustained-release experiment of CH@SHP099-NPs in a simulated synovial fluid environment; I is the distribution dynamics of DiR-labeled CH@SHP099-NPs and free SHP099 in the mouse knee joint monitored by the IVIS imaging system; J is the quantitative analysis of joint fluorescence signal intensity at different time points; K is the distribution of fluorescently labeled CH@SHP099-NPs in synovial tissue observed by confocal microscopy, bar: 25 μm; L is the statistical analysis of the time-series changes in fluorescence penetration depth in synovial tissue; the experiment was independently repeated three times, with n=6 mice in each group, **** indicates P<0.0001 between the two groups.

[0037] Figure 11 To prepare and characterize the physicochemical properties of CH@SHP099-NPs composite hydrogels loaded with SHP099 nanoparticles; where A is the change of functional groups of PLGA-PEG, cLABL and their coupling product PLGA-PEG-cLABL detected by Fourier transform infrared spectroscopy (FTIR); B is the microscopic distribution of SHP099-NPs after being composited in the hydrogel observed by scanning electron microscopy (SEM), bar: 200 μm.

[0038] Figure 12This study assesses the cell compatibility of CH@SHP099-NPs composite hydrogels. A is a schematic diagram of the experimental design for the cytotoxicity and uptake experiments of CH@SHP099-NPs; B shows the CCK-8 assay for the proliferation activity of macrophages treated with different concentrations of SHP099-NPs and CH@SHP099-NPs; C shows the CCK-8 assay for the proliferation activity of chondrocytes treated with different concentrations of SHP099-NPs and CH@SHP099-NPs; D shows the Live / Dead staining assay for the survival status of macrophages and chondrocytes treated with CH@SHP099-NPs, with green representing live cells and red representing dead cells (bar: 50 μm); E shows confocal laser microscopy observation of the uptake capacity of Cy5-SHP099-NPs in macrophages at time points of 1 h, 2 h, 4 h, and 8 h, with red representing Cy5 labeling signal, blue representing DAPI labeling of the nucleus, and green representing cell membrane labeling (bar: 25 μm). μm; the experiment was repeated three times, ns indicates no significant difference between the two groups, ***P<0.001, ****P<0.0001.

[0039] Figure 13 This study assesses the in vivo biocompatibility of CH@SHP099-NPs composite hydrogel. A represents the schematic diagram of the in vivo biocompatibility study of CH@SHP099-NPs; BE represents the detection of serum biochemical indicators ALT, AST, Cr, and BUN to assess liver and kidney function; FG represents the detection of blood lipid levels TG and CHOL to assess lipid metabolism; HI represents the analysis of relative mass changes in the liver and kidneys using organ index analysis; J represents H&E staining to observe the histological morphology of major organs (heart, liver, spleen, lung, and kidney), bar: 100 μm; n=6 mice per group, ns indicates no significant difference between the two groups.

[0040] Figure 14This study aimed to regulate macrophage metabolic reprogramming and inhibit M1 polarization using CH@SHP099-NPs composite hydrogels. A is the experimental design flowchart, showing different treatment groups and detection index settings; B is the RT-qPCR assay for the mRNA expression levels of key glycolytic enzymes (GLUT1, HK2, PKM2, LDHA) in each group; C is the Western blot assay for the expression of oxidative phosphorylation-related proteins ATP5A and COXIV; D is the lactate assay for the lactate release level in cell supernatant; E is the RT-qPCR assay for the mRNA expression of M1 polarization markers (iNOS, CD86, TNF-α, IL-1β, MCP-1) in each group; F is the ELISA assay for the protein levels of TNF-α, IL-1β, and MCP-1 in cell supernatant; G is the Western blot assay for the expression of M1 polarization markers (iNOS, CD86, TNF-α, IL-1β, MCP-1). blot analysis of iNOS and CD86 protein expression; H represents flow cytometry analysis of CD86⁺ macrophage ratio; cell experiments were repeated three times, * indicates comparison between two groups, P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0041] Figure 15 CH@SHP099-NPs composite hydrogel improves chondrocyte activity and inhibits cartilage matrix degradation by regulating macrophage polarization. A shows the flowchart of the establishment and intervention of the co-culture system of M1 polarized macrophages and chondrocytes; B shows the changes in chondrocyte activity in different treatment groups detected by CCK-8 assay; C shows the TUNEL staining to assess chondrocyte apoptosis level (bar: 50 μm); D shows the Western blot detection of Bax and Bcl-2 expression levels; E shows the immunofluorescence detection of the expression level of cartilage matrix protein Collagen II (bar: 25 μm); F shows the RT-qPCR detection of the expression levels of cartilage synthesis and degradation-related genes COL2A1, ACAN, MMP13, and ADAMTS5; G shows the Western blot detection of MMP13 and ADAMTS5 protein expression. Cell experiments were repeated three times. ** indicates P < 0.01 between two groups, *** P < 0.001, **** P < 0.0001.

[0042] Figure 16This study aimed to investigate the in vivo therapeutic effect of CH@SHP099-NPs composite hydrogel on alleviating joint damage and behavioral disorders in osteoarthritis (OA) mice. A is a flowchart showing the construction of the DMM-induced OA model and the intervention time points for each group; B is micro-computed tomography (Micro-CT) observation of bone structure changes in the femoral-tibial articular surface; C is the assessment of cartilage structure damage using the OARSI scoring system; D is Safranin O-Fast Green staining to detect the morphology and glycosaminoglycan (GAG) distribution of articular cartilage tissue (bar: 200 μm (top row), 50 μm (bottom row); EI is immunohistochemical (IHC) staining to detect the protein expression levels of COL2A1, ACAN, MMP13, and ADAMTS-5 in articular cartilage tissue (bar: 50 μm). Six animals were included in each group. ** indicates P < 0.01 between the two groups, *** P < 0.001, **** P < 0.0001.

[0043] Figure 17 This study aimed to target and regulate the inflammatory microenvironment of OA synovial membrane using CH@SHP099-NPs composite hydrogel. A shows the experimental workflow; B shows RT-qPCR detection of mRNA expression levels of inflammatory factors Il-1β, TNF-α, Il-6, and Ccl2 in synovial tissue; C shows ELISA detection of expression levels of inflammatory factors TNF-α, IL-1β, IL-6, and IL-10 in synovial fluid; D shows H&E staining to assess synovial hyperplasia and inflammation (bar: 100 μm), where C: Cartilage, S: Synovium, M: Meniscus; E shows immunohistochemistry (IHC) detection of protein expression of TNF-α, IL-1β, IL-6, and IL-10 in synovial tissue (bar: 25 μm); F shows immunofluorescence staining to detect iNOS and Arg-1 expression in synovial tissue to assess macrophage polarization (bar: 25 μm); G shows immunofluorescence staining to detect p-AMPK expression in synovial tissue (bar: 25 μm). μm; 6 animals per group, ns indicates no significant difference between the two groups, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Detailed Implementation

[0044] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] The experiment strictly followed national regulations for the use of laboratory animals. Male C57BL / 6J mice (6-8 weeks old, weighing 18-22g, #219) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Prior to the experiment, they were housed in an SPF-grade animal facility for one week at an ambient temperature of 22 ± 2℃, relative humidity of 50-60%, and a 12 h / 12 ​​h light / dark cycle. They were fed sterile pelleted feed and drinking water.

[0046] This invention redefines the regulatory logic of synovial inflammation in osteoarthritis (OA) from the perspective of immune metabolic reprogramming, proposing the SHP2–AMPK axis as a "checkpoint" for the transition of the immune metabolic state of synovial macrophages. Its continuous activation can maintain pro-inflammatory polarization and amplify the synovial inflammatory response. In order to explore the core role of the SHP2–AMPK axis in driving synovial macrophage dysfunction and joint degeneration, the following preliminary research experiments were conducted:

[0047] I. Single-cell atlas identification of SHP2 as a key signaling hub in inflammatory macrophages of synovial membrane in osteoarthritis

[0048] First, a mouse model of osteoarthritis was constructed, as follows:

[0049] An osteoarthritis (OA) model was established in male C57BL / 6J mice via medial meniscus instability repair (DMM surgery). Mice were anesthetized preoperatively with sodium pentobarbital (100 mg / kg, intraperitoneal injection) to ensure a pain-free state. The right knee joint was exposed aseptically, the medial knee joint capsule was incised, the tibial plateau was exposed, and the medial collateral ligament was transcribed under a microscope to disrupt the stability of the medial meniscus. In the control group (Sham surgery group), only the joint capsule was opened, without ligament disruption. Postoperatively, the joint capsule was sutured to the skin with atraumatic sutures, and the surgical area was disinfected with povidone-iodine and erythromycin eye ointment was applied topically to prevent infection. After surgery, mice were placed on a 37°C heated pad for recovery, and their weight and activity level were monitored daily.

[0050] To systematically characterize the immune microenvironment of synovial tissue in the context of osteoarthritis (OA), we performed single-cell transcriptome sequencing analysis on synovial tissue from the OA and sham groups of C57BL / 6J mice (n=2 per group). The flowchart is shown below. Figure 1 As shown in Figure A. Figure 2 AD was used for quality control and principal component analysis of single-cell transcriptomes of synovial tissues in osteoarthritis and control groups. After quality control and batch correction were completed, the integrated analysis obtained stable and consistent cell distribution characteristics, laying the foundation for subsequent analysis of cell composition and functional status.

[0051] Depend on Figure 1According to BC, based on unsupervised clustering and classical marker gene annotation, a variety of cell types were identified, including chondrocytes, macrophages, monocytes, T cells, B cells, endothelial cells, osteoblasts, and fibroblast-like stromal cells. Figure 1 The D-scan showed that the proportion of cells in different groups was significantly expanded in the synovial tissue of OA, with macrophages and monocytes being the most prominent, while the proportion of chondrocytes and some precursor cells was relatively decreased. This result suggests that the synovial microenvironment of OA is dominated by immune cells, especially myeloid cells, which may play a core role in disease progression.

[0052] To further evaluate the functional interactions between different cell types at the systemic level, we used CellChat to analyze the intercellular communication network in synovial tissue. Figure 1 EF showed that, compared with the sham group, the overall cell communication quantity and signal intensity in the synovial tissue of the OA group were significantly enhanced, suggesting that intercellular signal communication was amplified in the OA microenvironment.

[0053] Depend on Figure 1 GH analysis revealed that pathway-level information flow analysis further demonstrated that inflammation-related signaling pathways (such as TNF, CXCL, and CCL) were significantly enhanced in the OA group, while some growth factor and repair-related pathways (such as FGF, IGF, and PDGF) also showed abnormal activation. At the cellular level, the main senders of inflammatory signals were monocytes and macrophages, while structural cells such as chondrocytes and endothelial cells primarily acted as signal receivers, suggesting that myeloid immune cells occupy a central position in the inflammatory communication network of OA synovial tissue.

[0054] Figure 3 AH analysis revealed that further analysis of representative inflammation and repair signaling pathways showed that under OA conditions, TNF signaling was mainly transmitted from monocytes and macrophages to chondrocytes and endothelial cells, while FGF signaling also exhibited an abnormal directional distribution dominated by immune cells. These results support the formation of an "immune-driven" synovial microenvironment in OA at the cellular communication level.

[0055] Given the significant expansion of macrophages in OA synovial tissue and their pivotal role in cell communication, we further analyzed the subpopulations of the synovial macrophage population. Figure 4 AC analysis revealed that macrophages were classified into different functional states, including M1-like, M2-like, and intermediate, based on the expression of representative marker genes. Among them, the proportion of M1-like inflammatory macrophages was significantly increased under OA conditions.

[0056] Depend on Figure 5As shown in Figure A, the expression projection of key inflammation and metabolism-related genes revealed that pro-inflammatory genes such as Il1b, Ebf1, and Syne1 were highly expressed in M1-like macrophages, suggesting that OA synovial macrophages were in a highly activated inflammatory state. Figure 5 B. Further differential expression analysis revealed that multiple inflammation- and immune-related genes were significantly upregulated in macrophages of the OA group. Figure 5 CF analysis revealed that functional enrichment analysis showed these differentially expressed genes were mainly concentrated in key immune processes such as cytokine-mediated signaling pathways, inflammatory cytokine production, and antigen processing and presentation, while also involving cellular components related to energy metabolism and protein synthesis. These results suggest that O synovial macrophages are not only highly pro-inflammatory in function, but their inflammatory phenotype may also be closely related to systemic remodeling of metabolic programs.

[0057] SHP2 (PTPN11), a key regulator of multiple signaling pathways, plays a central role in inflammation and tissue remodeling. Recent studies have shown that SHP2 participates in macrophage activation and the production of inflammatory factors, and affects the function of chondrocytes and osteoblasts; therefore, it may play an important role in the imbalance of the inflammatory microenvironment and osteochondral degeneration in osteoarthritis. Figure 4 As shown in D, the expression distribution analysis at the single-cell level revealed that the high expression of Ptpn1 was mainly concentrated in the M1-like inflammatory macrophage subset and highly overlapped with the expression characteristics of various pro-inflammatory genes.

[0058] To validate the single-cell analysis results, we further examined SHP2 expression changes at the whole synovial tissue level. The RT-qPCR and Western blot results are as follows: Figure 4 EF showed that the levels of SHP2 mRNA and protein in the synovial tissue of the OA group were significantly higher than those of the sham group. Figure 4 Immunofluorescence staining in G further confirmed that SHP2 signaling was mainly located in F4 / 80-positive synovial macrophages and was significantly enhanced under OA conditions. These multi-level pieces of evidence consistently indicate that SHP2 is abnormally activated in OA synovial macrophages and may act as a key signaling hub involved in the sustained amplification of the inflammatory process.

[0059] In summary, single-cell transcriptome analysis revealed the characteristics of immune microenvironment remodeling centered on macrophages in OA synovial tissue, indicating that inflammatory and dysfunctional macrophages dominate the cell communication network. The significant upregulation of SHP2 in this cell population suggests that it may act as a key regulatory factor in maintaining and amplifying the inflammatory microenvironment of OA synovial tissue, providing important clues for subsequent mechanistic research and the exploration of intervention strategies.

[0060] II. Macrophage-specific SHP2 knockout alleviates OA cartilage damage and synovial inflammation

[0061] 1. Construction of SHP2 knockout mouse model (Shp2^fl / fl-Lyz2^Cre)

[0062] In single-cell analysis, we identified SHP2 as abnormally upregulated in inflammatory macrophages of the synovial membrane in OA and its location as a cell communication hub. To verify the functional role of SHP2 in the development and progression of OA from a causal perspective, we constructed macrophage-specific SHP2 knockout mice (Shp2^fl / fl-Lyz2^Cre) and induced an OA model through DMM surgery, the specific procedure of which is as follows: Figure 6 As shown in Figure A, compared with the control group, Shp2^fl / fl-Lyz2^Cre mice showed significantly reduced joint structural damage after surgery. Figure 6 B. Micro-CT imaging, H&E staining, and Safranin O-Fast Green staining were used to observe the joint bone structure and cartilage degeneration. Micro-CT analysis showed that SHP2 deficiency significantly alleviated subchondral bone structure destruction, and the trabecular bone arrangement was more complete and continuous. Histological analysis further supported the above findings. H&E and Safranin O staining results showed that the articular cartilage surface of Shp2^fl / fl-Lyz2^Cre mice was smoother, the proteoglycan staining intensity was significantly enhanced, and the cartilage thickness was better maintained. Correspondingly, by Figure 6 C indicates that during the entire postoperative observation period, the degree of knee joint swelling in SHP2-deficient mice was significantly reduced, and their OARSI scores were significantly lower than those of the control group, suggesting that the absence of SHP2 in macrophages significantly alleviated the degenerative changes of OA at the tissue structure level.

[0063] Given that synovial inflammation is an important driver of OA pathological progression, we further evaluated the local inflammatory response in the joint. Figure 6 DE represents the results of immunohistochemical staining. It can be seen that, compared with the control group, the expression of pro-inflammatory factors TNF-α, IL-1β and IL-6 in the synovial tissue of Shp2^fl / fl-Lyz2^Cre mice was significantly reduced, while the expression of anti-inflammatory factor IL-10 was significantly increased, suggesting that the absence of SHP2 in macrophages can systematically alleviate the inflammatory microenvironment of OA synovial membrane.

[0064] To further analyze whether the aforementioned improvement in inflammation stemmed from changes in macrophage function, we analyzed the polarization profile of synovial macrophages using immunofluorescence double staining. The results are as follows: Figure 6 F showed that in the synovial tissue of the control group, iNOS⁺F4 / 80⁺ macrophages accumulated in large quantities, while Arg-1... + F4 / 80 +The proportion of macrophages was relatively low; in contrast, iNOS in Shp2^fl / fl-Lyz2^Cre mice was higher. + F4 / 80 + Type I macrophages were significantly reduced, while Arg-1 macrophages were significantly reduced. + F4 / 80 + The proportion of macrophages was significantly increased. This result indicates that the loss of myeloid SHP2 can significantly inhibit pro-inflammatory polarization and promote the reconstruction of the anti-inflammatory phenotype, thereby reshaping the synovial immune microenvironment under OA conditions.

[0065] To validate the above in vivo observations, we further performed in vitro functional analyses on bone marrow-derived macrophages (BMDMs) of different genotypes. Figure 6 GH studies showed that under LPS / IFN-γ induction, MHC-II expression in BMDMs derived from Shp2^fl / fl-Lyz2^Cre mice was significantly lower than that in the control group; while under IL-4 induction, CD206 expression was significantly higher than that in the control group. Figure 6 IJ was analyzed by RT-qPCR. The results showed that under M1 induction conditions, the expression of Tnf, Il1b, and Il6 was significantly decreased in the Shp2^fl / fl-Lyz2^Cre group; while under M2 induction conditions, the expression of Il10, Tgfb, and Cd206 was significantly upregulated. Figure 6 According to KL, protein level detection results also showed that SHP2 deficiency significantly reduced iNOS expression and enhanced CD206 expression, further verifying the key role of SHP2 in regulating macrophage inflammatory phenotype.

[0066] In summary, macrophage-specific SHP2 deficiency significantly alleviated OA-related synovial inflammation and cartilage damage in vivo, and consistently drove macrophages from a pro-inflammatory state to an anti-inflammatory, reparative phenotype in both in vivo and in vitro models. These results establish SHP2 as a key causal factor driving OA immune imbalance and tissue damage at the genetic level, providing direct evidence for its potential value as an immunometabolic intervention target.

[0067] III. SHP2 drives macrophage metabolic fate reprogramming via the AMPK axis as an immune metabolic checkpoint.

[0068] To elucidate the molecular mechanism of SHP2 in macrophage immune metabolic reprogramming, we systematically constructed an experimental system encompassing inflammatory stimulation, SHP2 functional regulation, and metabolic function assessment. The experimental procedure is as follows: Figure 7 As shown in Figure A, the aim is to determine whether SHP2 determines the energy metabolism fate of macrophages through a specific metabolic signaling axis.

[0069] Depend on Figure 7According to BC, under LPS stimulation, Western blot results showed that multiple key glycolysis-related enzymes, including GLUT3, HK1, and LDHA, were significantly upregulated, accompanied by significant inhibition of AMPK signaling, manifested as a significant decrease in p-AMPK levels. This result suggests that macrophages rapidly shift from an oxidative metabolic state to a glycolysis-dependent state under inflammatory stimulation. Notably, when SHP2 was knocked down via siRNA or treated with the SHP2-specific inhibitor SHP099, the abnormal increase in the aforementioned glycolytic enzymes was significantly inhibited, while p-AMPK levels were significantly restored, suggesting that SHP2 is a key regulatory node connecting inflammatory stimulation and AMPK inhibition.

[0070] Depend on Figure 7 As shown in D, at the transcriptional level, RT-qPCR analysis further confirmed that SHP2 knockdown or SHP099 treatment significantly reduced the mRNA expression of GLUT3 and LDHA, indicating that the regulation of glycolysis by SHP2 is not a transient change in metabolic enzyme activity, but a systemic remodeling of the metabolic program at the transcriptional level.

[0071] Given that AMPK is a core energy sensor for maintaining cellular metabolic homeostasis, we further verified its functional role in SHP2-mediated metabolic reprogramming through pharmacological methods. Figure 7 EF analysis showed that, in addition to LPS treatment, the addition of the AMPK agonist AICAR or the inhibitor Compound C, Seahorse metabolic flux analysis revealed that SHP099 treatment significantly reduced glycolytic flux (ECAR) while enhancing oxidative phosphorylation (OCR). This metabolic shift was further amplified under AICAR co-treatment, while the AMPK inhibitor significantly reversed the SHP099-induced metabolic reprogramming effect, clearly demonstrating that AMPK is a key mediator in SHP2 regulation of macrophage metabolic fate.

[0072] Figure 7 G showed that, consistent with changes in metabolic flux, SHP2 inhibition significantly reduced the accumulation of lactate, the end product of glycolysis, in the culture supernatant, further validating its inhibitory effect on pro-inflammatory metabolic pathways. Meanwhile, intracellular ATP content measurements showed... Figure 7 H showed that compared with the LPS stimulation group alone, SHP099 treatment significantly increased ATP levels, and the ATP content further increased under AICAR combined treatment, suggesting that SHP2 inhibition effectively improved the energy utilization efficiency of macrophages by restoring AMPK activity.

[0073] In summary, these results consistently demonstrate at multiple levels—molecular, transcriptional, and metabolic—that SHP2 forcibly locks macrophages into a pro-inflammatory metabolic state dominated by glycolysis by inhibiting AMPK activity. Genetic or pharmacological intervention on SHP2 can remove this metabolic restriction, restoring the AMPK-mediated oxidative metabolic program, thereby achieving a fundamental shift in the immunometabolic fate of macrophages. A schematic diagram of the specific molecular mechanism is shown below. Figure 7 As shown in Figure I, this SHP2-AMPK immune metabolic axis provides a direct mechanistic basis for explaining the protective effect of SHP2 deficiency in alleviating OA inflammation and tissue damage in vivo.

[0074] IV. The SHP2 / AMPK axis drives macrophage pro-inflammatory polarization by reshaping metabolic state.

[0075] To verify whether SHP2-AMPK-mediated metabolic reprogramming directly determines the pro-inflammatory output of macrophages, we constructed a stable SHP2-overexpressing unpolarized macrophage model (oe-SHP2) in vitro and intervened with either the SHP2 inhibitor SHP099 or the AMPK agonist AICAR. The experimental flowchart is shown below. Figure 8 As shown in Figure A.

[0076] RT-qPCR results are as follows Figure 8 B showed that oe-SHP2 treatment significantly induced increased transcriptional levels of typical pro-inflammatory genes such as Il1b, Il6, Tnf, and iNOS, while SHP099 or AICAR treatment significantly inhibited the abnormal upregulation of these genes. (See attached image.) Figure 8 As shown in C, at the protein level, oe-SHP2 also significantly enhances the expression of iNOS, IL-1β, TNF-α and IL-6, while SHP2 inhibition or AMPK activation can effectively weaken its pro-inflammatory protein output.

[0077] Depend on Figure 8 DE analysis revealed that, at the cell population level, immunofluorescence and flow cytometry analyses showed that oe-SHP2 treatment significantly increased CD86. + / iNOS + The proportion of macrophages suggests that pro-inflammatory polarization processes are systematically activated; conversely, SHP099 or AICAR intervention can significantly reverse this polarization trend.

[0078] In summary, SHP2 activation stabilizes altered metabolic states and sustains pro-inflammatory polarization by inhibiting AMPK signaling. Both SHP2 inhibition and AMPK pathway activation can effectively block this process at the functional level, further establishing the SHP2-AMPK axis as a key checkpoint regulating macrophage immune metabolism and inflammatory polarization. The specific molecular mechanisms are detailed in the appendix. Figure 8As shown in F.

[0079] The foregoing results indicate that sustained regulation of the SHP2-AMPK axis is crucial for reversing the macrophage immune metabolic state. This invention further focuses on how to achieve local, stable, and sustainable delivery of SHP2 inhibitors within the joint cavity. This invention constructs an injectable CH@SHP099-NPs composite delivery system, the specific implementation of which is as follows:

[0080] Example 1

[0081] A method for preparing CH injection-type hydrogel, the chemical structure of which is shown in the figure below. Figure 9 As shown in A, the specific steps are as follows:

[0082] Sodium chondroitin sulfate (CS) and sodium hyaluronate (HA) were used as base materials, and Genipin was used as a natural cross-linking agent. Dissolve 0.05 g, 0.0667 g, 0.075 g, 0.0333 g, and 0.025 g of CS in 5 mL, 6.67 mL, 7.5 mL, 3.33 mL, and 2.5 mL of sterile deionized water, respectively. Then, dissolve 0.05 g, 0.0333 g, 0.025 g, 0.0667 g, and 0.075 g of HA in 5 mL, 6.67 mL, 7.5 mL, 3.33 mL, and 2.5 mL of sterile deionized water, respectively, at a concentration of 10 mg / mL. At 25 °C, mix CS and HA at mass ratios of 1:1, 2:1, 3:1, 1:2, and 1:3 to obtain mixed solutions. Add 1 mL of 0.5 mg / mL Genipin to the mixed solutions and stir slowly for 30 minutes to promote dissolution. Subsequently, the mixture was reacted at 37°C for 6 hours to form a cross-linked network, ultimately yielding CH injection-type hydrogels with different ratios.

[0083] This hydrogel forms a stable cross-linked network under mild conditions mediated by Genipin, providing a reliable material basis for drug loading and long-term local retention. Figure 9 As can be seen from B, spectroscopic analysis verified the effective cross-linking between CS and HA. Scanning electron microscopy (SEM) observed the micropore structure of CH hydrogels with different ratios. Figure 9 As can be seen from F, the optimized CH hydrogel has a uniform porous structure.

[0084] A method for preparing CH@SHP099-NPs composite hydrogel, the preparation process is as follows: Figure 10 As shown in A, the specific steps are as follows:

[0085] (1) Dissolve 0.0667g sodium chondroitin sulfate and 0.0333g sodium hyaluronate in 6.67mL and 3.33mL of deionized water respectively, and mix them evenly to obtain CH hydrogel precursor solution;

[0086] (2) 0.012 g of thiol-containing cLABL cyclic peptide (synthetic sequence: cyclo(1,12)Pen-ITDGEATDSG-NH2, containing free thiol groups) was dissolved in 6 mL of PBS buffer solution (pH=7.4), and then reacted with 0.1 g of PLGA-PEG-MAL (polylactic acid-glycolic acid copolymer-polyethylene glycol-maleimide) under light-protected conditions for 12 h. cLABL was coupled to the end of the PEG chain by maleimide-thiol click reaction. After removing unreacted cLABL by dialysis, the product was lyophilized to obtain PLGA-PEG-cLABL block copolymer.

[0087] (3) 10 mg of PLGA-PEG-cLABL block copolymer and 1 mg of SHP099 were dissolved together in 1 mL of anhydrous dichloromethane and acetonitrile (1:1, v / v) mixed solvent. After being sonicated and dissolved evenly, the mixture was slowly added dropwise to pre-cooled deionized water (5 mL). At the same time, the mixture was intermittently sonicated using a probe-type sonicator (20 kHz, 100 W) (sonication for 5 s / pause for 5 s, for 2 min). The organic solvent was then evaporated for 4 h under magnetic stirring to obtain SHP099 NPs.

[0088] (4) SHP099 NPs were dispersed in the CH hydrogel precursor solution, and the final drug loading concentration was set to 1 mg / mL. Then, Genipin crosslinking agent with a final concentration of 0.5 mg / mL was added, and the mixture was gently stirred for 30 min to promote uniform distribution. The mixture was reacted at 37 °C for 6 h. During this period, Genipin molecules crosslinked with the amino groups of the polysaccharide chain to form a stable three-dimensional network structure, thus successfully constructing the drug-loaded CH@SHP099-NPs composite hydrogel. The obtained hydrogel was washed with PBS to remove unencapsulated free drug and residual Genipin, and finally, CH@SHP099-NPs composite hydrogel with macrophage targeting and controllable release capabilities was obtained.

[0089] The CH@SHP099-NPs composite hydrogel prepared in this embodiment was characterized, and the results are as follows: Figure 10 and Figure 11 As shown.

[0090] The functional group changes of CS, HA, Genipin, cross-linked hydrogel, PLGA-PEG, cLABL, and the coupling product PLGA-PEG-cLABL were detected using Fourier transform infrared spectroscopy. Samples were freeze-dried for 48 hours, ground into powder, and prepared for testing using the KBr pellet method. Each spectrum was recorded within the range of 4000–500 cm⁻¹. -1 The resolution is 4 cm. -1 The number of scans was 32. The results are as follows: Figure 11 As shown in Figure A, spectroscopic analysis confirmed the successful coupling between cLABL and PLGA-PEG.

[0091] Transmission electron microscopy (TEM) is used for morphological observation, while dynamic light scattering (DLS) is used to detect particle size distribution. Figure 10 As can be seen from BD, the physicochemical characterization results further show that the nanoparticles maintain a uniform and stable particle size distribution before and after drug loading, and exhibit a regular spherical structure, indicating that they have good structural integrity.

[0092] The microstructure of SHP099-NPs composited in hydrogel was observed using scanning electron microscopy (TEM). The gel sample was frozen at -80°C for 6 hours and then freeze-dried for 24 hours. The sample was cut into small pieces using a blade and fixed onto a metal stage, then sputter-coated with gold for 20 seconds. SEM images were acquired using a field emission scanning electron microscope (FET) with an accelerating voltage of 5 kV and a magnification of 500×-1000×. The pore size and connectivity of the pore structure were determined using ImageJ v1.53h. Results are presented by [the journal / organization / etc.]. Figure 11 As shown in B, after loading nanoparticles onto the hydrogel network, no obvious aggregation or phase separation phenomenon occurred in the composite system, indicating that the nanoparticles can be stably distributed inside the hydrogel.

[0093] Example 2

[0094] A method for preparing CH@SHP099-NPs composite hydrogel, the specific steps of which are as follows:

[0095] (1) Dissolve 0.04g sodium chondroitin sulfate and 0.02g sodium hyaluronate in 5mL and 2.5mL of deionized water respectively, and mix them evenly to obtain CH hydrogel precursor solution;

[0096] (2) 0.018 g of thiol-containing cLABL cyclic peptide (synthetic sequence: cyclo(1,12)Pen-ITDGEATDSG-NH2, containing free thiol groups) was dissolved in 36 mL of PBS buffer solution (pH=7.2), and then reacted with 0.15 g of PLGA-PEG-MAL under light-protected conditions for 10 h. cLABL was coupled to the end of the PEG chain by maleimide-thiol click reaction. After removing unreacted cLABL by dialysis, the product was lyophilized to obtain PLGA-PEG-cLABL block copolymer.

[0097] (3) 10 mg of PLGA-PEG-cLABL block copolymer and 1 mg of SHP099 were dissolved together in 0.5 mL of anhydrous dichloromethane and acetonitrile (1:1, v / v) mixed solvent. After being sonicated and dissolved evenly, the mixture was slowly added dropwise to pre-cooled deionized water (5 mL). At the same time, the mixture was intermittently sonicated using a probe-type sonicator (20 kHz, 100 W) (sonication for 5 s / pause for 5 s, for 2 min). The organic solvent was then evaporated for 4 h under magnetic stirring to obtain SHP099 NPs.

[0098] (4) SHP099 NPs were dispersed in the CH hydrogel precursor solution, and the final drug loading concentration was set to 0.8 mg / mL. Then, Genipin crosslinking agent with a final concentration of 0.3 mg / mL was added, and the mixture was gently stirred for 30 min to promote uniform distribution. The mixture was reacted at 35 °C for 8 h. During this period, Genipin molecules crosslinked with the amino groups of the polysaccharide chain to form a stable three-dimensional network structure, thus successfully constructing the drug-loaded CH@SHP099-NPs composite hydrogel. The obtained hydrogel was washed with PBS to remove unencapsulated free drug and residual Genipin, and finally CH@SHP099-NPs composite hydrogel with macrophage targeting and controllable release capabilities was obtained.

[0099] Example 3

[0100] A method for preparing CH@SHP099-NPs composite hydrogel, the specific steps of which are as follows:

[0101] (1) Dissolve 0.09g sodium chondroitin sulfate and 0.03g sodium hyaluronate in 7.5mL and 2.5mL of deionized water respectively, and mix them evenly to obtain CH hydrogel precursor solution;

[0102] (2) 0.02 g of thiol-containing cLABL cyclic peptide (synthetic sequence: cyclo(1,12)Pen-ITDGEATDSG-NH2, containing free thiol groups) was dissolved in 2 mL of PBS buffer solution (pH=7.6), and then reacted with 0.2 g of PLGA-PEG-MAL under light-protected conditions for 14 h. cLABL was coupled to the end of the PEG chain by maleimide-thiol click reaction. After removing unreacted cLABL by dialysis, the product was lyophilized to obtain PLGA-PEG-cLABL block copolymer.

[0103] (3) 10 mg of PLGA-PEG-cLABL block copolymer and 1 mg of SHP099 were dissolved together in 2 mL of anhydrous dichloromethane and acetonitrile (1:1, v / v) mixed solvent. After being sonicated and dissolved evenly, the mixture was slowly added dropwise to pre-cooled deionized water (5 mL). At the same time, the mixture was intermittently sonicated using a probe-type sonicator (20 kHz, 100 W) (sonication for 5 s / pause for 5 s, for a total of 2 min). The organic solvent was then evaporated for 4 h under magnetic stirring to obtain SHP099 NPs.

[0104] (4) SHP099 NPs were dispersed in the CH hydrogel precursor solution, and the final drug loading concentration was set to 1.2 mg / mL. Then, Genipin crosslinking agent with a final concentration of 0.7 mg / mL was added, and the mixture was gently stirred for 30 min to promote uniform distribution. The mixture was reacted at 39 °C for 4 h. During this period, Genipin molecules crosslinked with the amino groups of the polysaccharide chain to form a stable three-dimensional network structure, thus successfully constructing the drug-loaded CH@SHP099-NPs composite hydrogel. The obtained hydrogel was washed with PBS to remove unencapsulated free drug and residual Genipin, and finally, CH@SHP099-NPs composite hydrogel with macrophage targeting and controllable release capabilities was obtained.

[0105] Example of implementation effect 1

[0106] The performance of the CH injection-type hydrogel and the CH@SHP099-NPs composite hydrogel prepared in Example 1 were tested and their cell compatibility was evaluated.

[0107] Depend on Figure 9 As shown in CD, rheological tests indicate that the optimized CH hydrogel possesses stable elastic-dominant mechanical properties; Figure 9 E and Figure 10 As can be seen from F, the optimized CH injection hydrogel and CH@SHP099-NPs composite hydrogel can be rapidly gelled at room temperature. Figure 9 G and Figure 10G shows that the optimized CH injectable hydrogel and CH@SHP099-NPs composite hydrogel can rapidly gel at room temperature and can be successfully injected through a 23G needle, verifying its feasibility for intra-articular drug delivery.

[0108] Drug release results as follows Figure 10 As shown in Figure E, in vitro release experiments demonstrated that CH@SHP099-NPs exhibited sustained and controllable release behavior in a simulated synovial fluid environment. Further evaluation of the biosafety and uptake characteristics of CH@SHP099-NPs at the cellular level was conducted, following the experimental procedure described below. Figure 12 As shown in A, the results are as follows: Figure 12 As shown in Figure BD, within the tested concentration range, SHP099-NPs and CH@SHP099-NPs did not induce significant cytotoxicity in macrophages and chondrocytes, demonstrating good biocompatibility. The results of the fluorescent labeling experiment are as follows... Figure 12 As shown in Figure E, it is further demonstrated that SHP099-NPs can be effectively internalized by macrophages, and the intracellular fluorescence signal gradually increases with the extension of incubation time, suggesting that the delivery system has good cellular uptake capacity and can achieve intracellular delivery.

[0109] In summary, this study successfully constructed an engineered composite delivery system with stability, injectability, and sustained-release properties, and verified its safety and macrophage uptake capacity at the cellular level, laying a reliable technical foundation for subsequent joint local immune metabolic intervention and in vivo functional studies.

[0110] Example of implementation effect 2

[0111] Verification of the CH@SHP099-NPs composite hydrogel prepared in Example 1: It can achieve safe joint retention and synovial tissue delivery.

[0112] To evaluate the biosafety and joint local delivery characteristics of the CH@SHP099-NPs composite hydrogel in vivo, a systematic analysis was conducted in healthy C57BL / 6 mice. The specific experimental procedure is as follows: Figure 13 As shown in Figure A.

[0113] To systematically evaluate the in vivo safety of the CH@SHP099-NPs composite hydrogel system, 8-week-old male C57BL / 6J mice were used for intra-articular administration to a single knee joint. Groups were defined as blank PBS, blank CH hydrogel (drug-free), SHP099-NPs, and CH@SHP099-NPs groups. Injections were administered three times every two weeks at a dose of 10 mg / kg. Blood and major organs were collected at the end of the experiment for biochemical and histological evaluation.

[0114] Depend on Figure 13According to BG, after continuous administration, serum biochemical indicators showed that the liver and kidney function-related indicators (ALT, AST, Cr, BUN) and blood lipid levels (TG, CHOL) in the CH@SHP099-NPs treated group mice were not significantly different from those in the control group, suggesting that the delivery system did not cause significant liver and kidney toxicity or metabolic abnormalities; Figure 13 The H&E staining results of HJ showed that the organ indices of the major organs did not change significantly. The H&E staining results showed that the tissue structures of the heart, liver, spleen, lungs, kidneys and other organs were intact, and no obvious inflammatory infiltration or tissue damage was observed, which further verified its good biocompatibility in vivo.

[0115] Based on this, we further evaluated the sustained-release properties of the CH@SHP099-NPs composite hydrogel in the joint cavity and its distribution and retention characteristics in the synovial tissue. The experimental procedure is as follows: Figure 10 As shown in H.

[0116] Animal in vivo imaging results as follows Figure 10 As shown in IJ, in vivo imaging results showed that the fluorescently labeled CH@SHP099-NPs rapidly accumulated in the joint area after intra-articular injection and maintained a stable signal for a long time, which was significantly better than that of free drugs, suggesting that it can form a long-lasting drug reservoir in the joint. Figure 10 Confocal imaging of KL's synovial tissue sections further demonstrated that the composite hydrogel system not only accumulates on the synovial surface, but also effectively penetrates the synovial barrier and distributes in the subsynovial region. Even after a long drug administration time, a significant signal can still be detected, showing good tissue penetration and spatial distribution characteristics.

[0117] In summary, the CH@SHP099-NPs composite hydrogel exhibits good systemic safety in vivo and can achieve long-term retention and effective synovial tissue penetration within the joint cavity, providing a reliable safety and delivery basis for its continuous local treatment in disease models such as osteoarthritis.

[0118] Example of implementation effect 3

[0119] Verification of the CH@SHP099-NPs composite hydrogel prepared in Example 1: inhibiting macrophage pro-inflammatory polarization by remodeling immune metabolic state.

[0120] To verify whether the engineered delivery system can functionally reproduce the aforementioned SHP2-AMPK-mediated immune metabolic reprogramming process and further inhibit macrophage pro-inflammatory polarization, we constructed multiple control systems in vitro, including an M0 control group, an M1 polarization group (LPS / IFN-γ treatment), a free SHP099 treatment group, and a CH@SHP099-NPs treatment group. Figure 14As shown in Figure A, changes in metabolic state and polarization phenotype were systematically evaluated.

[0121] Figure 14 B shows the mRNA expression levels of key glycolytic enzymes (GLUT1, HK2, PKM2, LDHA) in each group detected by RT-qPCR. At the metabolic level, M1 polarization stimulation significantly induced a typical glycolytic shift in macrophages, with a significant upregulation of the expression of multiple key glycolytic-related enzymes, including GLUT1, HK2, PKM2, and LDHA. In contrast, treatment with CH@SHP099-NPs significantly inhibited the abnormal increase of the above-mentioned glycolytic enzymes. Figure 14 C shows the expression of oxidative phosphorylation-related proteins ATP5A and COXIV detected by Western blot. It can be seen that the expression of ATP5A and COXIV is significantly enhanced with CH@SHP099-NPs treatment, suggesting that the engineered delivery system can effectively promote the transformation of macrophages from a pro-inflammatory metabolic state dominated by glycolysis to a mitochondrial oxidative metabolic state. Figure 14 As indicated by D, consistent with the aforementioned molecular changes, lactate release assays showed that treatment with the CH@SHP099-NPs composite hydrogel significantly reduced lactate production levels under M1 polarization conditions, further validating its inhibitory effect on glycolysis at the level of metabolic end products. These results demonstrate that the CH@SHP099-NPs composite hydrogel can stably remodel the metabolic state of macrophages in vitro, achieving an effective switch from "pro-inflammatory glycolysis" to "immune homeostatic oxidative metabolism."

[0122] Building on this, we further evaluated whether the aforementioned metabolic reprogramming could translate into inhibition of pro-inflammatory function output. RT-qPCR results are as follows: Figure 14 As shown in E, CH@SHP099-NPs treatment significantly downregulated the transcriptional levels of multiple M1 polarization-related genes, including iNOS, CD86, TNF-α, IL-1β, and MCP-1.

[0123] Cytokine levels were detected using a mouse TNF-α, IL-1β, IL-6, and MCP-1 ELISA kit. The results are as follows: Figure 14 As shown in Figure F, consistent with changes in transcription levels, ELISA analysis revealed a significant decrease in the secretion levels of pro-inflammatory factors such as MCP-1, TNF-α, and IL-1β. Figure 14 Western blot results for G further confirmed that CH@SHP099-NPs treatment effectively inhibited the protein expression of iNOS and CD86. Flow cytometry analysis further quantified the polarization state at the cell population level. Results are as follows... Figure 14As shown in Figure H, compared with the M1 polarization group, CH@SHP099-NPs treatment significantly reduced the proportion of CD86⁺ macrophages, suggesting that the engineered delivery system effectively inhibited the establishment of pro-inflammatory polarization programs at the cell population level.

[0124] In summary, these results demonstrate that the CH@SHP099-NPs composite hydrogel can not only stably deliver SHP2 inhibitors via engineered methods, but also accurately reproduce the SHP2-AMPK-mediated immunometabolic reprogramming process in vitro, effectively translating metabolic remodeling into the inhibition of pro-inflammatory polarization. This provides direct functional evidence for subsequent in vivo validation of the regulatory role of the CH@SHP099-NPs composite hydrogel in the synovial immune microenvironment and OA pathological progression.

[0125] Example of implementation effect 4

[0126] Regulating macrophage immune polarization can significantly improve chondrocyte fate and maintain matrix homeostasis.

[0127] Based on the aforementioned results, the CH@SHP099-NPs composite hydrogel can inhibit the pro-inflammatory polarization of macrophages by remodeling their immune metabolic state. This invention further explores whether this immune regulation can be translated into a protective effect on chondrocyte function and matrix homeostasis through intercellular interactions. To this end, a macrophage-chondrocyte co-culture model was used to systematically evaluate relevant functional indicators. The flowchart for the establishment and intervention of the M1 polarized macrophage-chondrocyte co-culture system is shown below. Figure 15 As shown in Figure A.

[0128] Depend on Figure 15 According to BC, functional analysis showed that M1 macrophages significantly inhibited chondrocyte proliferation and induced apoptosis; SHP099 intervention could partially alleviate the above-mentioned damage, while CH@SHP099-NPs treatment further significantly restored chondrocyte proliferation activity and reduced the proportion of TUNEL positive cells. Figure 15 D represents the expression levels of Bax and Bcl-2 detected by Western blot. Consistent with this, CH@SHP099-NPs significantly downregulated the pro-apoptotic protein BAX and upregulated the anti-apoptotic protein Bcl-2, suggesting that they effectively resist inflammation-induced chondrocyte apoptosis at a functional level. Immunohistochemical staining results are as follows: Figure 15 As shown in Figure E, it can be seen that, in terms of matrix homeostasis, CH@SHP099-NPs treatment significantly enhances the immunofluorescence signal of COL2A1 in chondrocytes.

[0129] Figure 15FG represent the results of RT-qPCR detection of the expression levels of cartilage synthesis and degradation-related genes COL2A1, ACAN, MMP13, and ADAMTS5, and Western blot detection of MMP13 and ADAMTS5 protein expression, respectively. It can be seen that CH@SHP099-NPs treatment upregulated the expression of COL2A1 and ACAN at both the transcriptional and protein levels, while significantly inhibiting the expression of MMP13 and ADAMTS5, indicating that it can simultaneously promote matrix synthesis and inhibit matrix degradation.

[0130] In summary, the CH@SHP099-NPs composite hydrogel effectively weakens the damage to chondrocytes caused by the pro-inflammatory microenvironment by reshaping the immune polarization state of macrophages, and transforms the immunomodulatory effect into substantial protection for chondrocyte survival, function and matrix homeostasis, revealing the key role of immunometabolic intervention in maintaining cartilage tissue homeostasis through intercellular communication.

[0131] Example of implementation effect 5

[0132] Verification of the CH@SHP099-NPs composite hydrogel prepared in Example 1 in alleviating joint damage in OA mice

[0133] To systematically evaluate the therapeutic potential of CH@SHP099-NPs composite hydrogel in alleviating osteoarthritis (OA) joint damage and motor dysfunction in vivo, we constructed a DMM-induced OA mouse model and set up different treatment groups for intervention. The experimental procedure is as follows: Figure 16 As shown in Figure A, the construction of the DMM-induced OA model and the intervention time points for each group are illustrated.

[0134] An osteoarthritis (OA) model was established in male C57BL / 6J mice via medial meniscus instability repair (DMM surgery). Mice were anesthetized preoperatively with sodium pentobarbital (100 mg / kg, intraperitoneal injection) to ensure a pain-free state. The right knee joint was exposed aseptically, the medial knee joint capsule was incised, the tibial plateau was exposed, and the medial collateral ligament was transcribed under a microscope to disrupt the stability of the medial meniscus. In the control group (Sham surgery group), only the joint capsule was opened, without ligament disruption. Postoperatively, the joint capsule was sutured to the skin with atraumatic sutures, and the surgical area was disinfected with povidone-iodine and erythromycin eye ointment was applied topically to prevent infection. After surgery, mice were placed on a 37°C heated pad for recovery, and their weight and activity level were monitored daily.

[0135] Eight-week-old C57BL / 6J mice underwent DMM surgery one week prior, and each sample of equal volume was injected intra-articularly into the joint cavity. Animals were randomly divided into five groups (n=6 per group): Sham group, OA group, OA+SHP099 group, OA+SHP099-NPs group, and OA+CH@SHP099-NPs group. The drug was injected intra-articularly at weeks 2 and 4, with a dose of 10 mg / kg. Mice were sacrificed at week 6, and knee joints were obtained for further evaluation.

[0136] Mice were sacrificed on postoperative day 28. The right knee joint was dissected and fixed with 10% neutral buffered formaldehyde for 48 hours. The knee joint was scanned using a high-resolution Micro-CT system (70kV, 200μA, 10μm resolution). Three-dimensional reconstruction and bone tissue parameter analysis were performed using CTAn and CTVol software. The results of micro-CT imaging analysis of bone and joint structures are as follows: Figure 16 As shown in Figure B, the knee joints of mice in the OA group showed significant swelling and severe bone destruction on the articular surface. The SHP099 and CH@SHP099-NPs treatment groups improved the bone structure destruction to some extent, with the CH@SHP099-NPs group showing the most significant effect.

[0137] Figure 16 Histological analysis of D revealed the protective effect of CH@SHP099-NPs on articular cartilage in osteoarthritis (OA). SO staining results showed that the cartilage layer in the OA group was significantly thinned and the tidal line structure was disrupted, while both the SHP099 and SHP099-NPs groups partially alleviated cartilage damage. The CH@SHP099-NPs group showed a smoother cartilage surface and a more intact tidal line structure. Figure 16 As shown in C, the OARSI score results further confirmed this trend, with the OA group score significantly increased and the CH@SHP099-NPs group score significantly decreased, suggesting that it has a significant delaying effect on OA cartilage degeneration.

[0138] Finally, to further analyze the effect of CH@SHP099-NPs on cartilage matrix homeostasis, we examined the expression of cartilage-related matrix proteins. Immunohistochemical results are shown below. Figure 16 EI results showed that COL2A1 and ACAN were significantly downregulated in the OA group, while MMP13 and Adamts4 were significantly upregulated, indicating reduced matrix synthesis and enhanced degradation. After intervention with SHP099 and SHP099-NPs, the levels of COL2A1 and ACAN were partially restored, while the expression of MMP13 and Adamts4 was reduced. The CH@SHP099-NPs group showed the most significant recovery effect, and matrix synthesis and degradation were maintained in a relatively balanced state.

[0139] In summary, the CH@SHP099-NPs composite hydrogel achieved sustained intervention on the SHP2-AMPK immune metabolic axis in vivo through an engineered approach, effectively alleviating structural joint damage in OA mice, significantly protecting the cartilage matrix, and delaying disease progression.

[0140] Example of implementation effect 6

[0141] The CH@SHP099-NPs composite hydrogel prepared in Example 1 was verified to significantly improve the inflammatory microenvironment of OA synovial membrane through immune metabolic regulation.

[0142] To systematically evaluate the anti-inflammatory and immunometabolic regulatory effects of CH@SHP099-NPs in the immune microenvironment of OA synovial membrane, molecular, tissue, and immunostaining techniques were used to comprehensively analyze the inflammatory state of synovial tissue and macrophage polarization. The experimental flowchart is shown below. Figure 17 As shown in Figure A.

[0143] First, the levels of inflammatory factor mRNA in synovial tissue were detected by RT-qPCR, and the results are as follows: Figure 17 B shows that the mRNA expression levels of inflammatory factors IL-1β, TNF-α, IL-6 and CCL2 in the synovial tissue of the OA group were significantly increased, while the expression of inflammatory factors decreased after drug intervention. Among them, the SHP099-NPs group was more effective than free SHP099, and the CH@SHP099-NPs group had the most significant inhibitory effect. Figure 17 C was determined by ELISA to detect the expression levels of inflammatory factors TNF-α, IL-1β, IL-6, and IL-10 in synovial fluid, confirming this trend. In the OA group, the protein levels of TNF-α, IL-1β, and IL-6 in synovial fluid were significantly increased, while the IL-10 level decreased. All three interventions could alleviate the abnormal expression, and the CH@SHP099-NPs group had the strongest effect in reducing pro-inflammatory factors and restoring IL-10 levels.

[0144] Secondly, histological analysis further revealed the protective effect of CH@SHP099-NPs. HE staining results are as follows... Figure 17 As shown in Figure D, the synovium in the OA group was significantly thickened and accompanied by inflammatory cell infiltration. Both the SHP099 and SHP099-NPs groups partially improved the pathological state of the synovium, with SHP099-NPs showing better results. The CH@SHP099-NPs group exhibited significantly reduced synovial hyperplasia and inflammatory infiltration, and its tissue structure was most similar to that of the Sham group. Figure 17 Immunohistochemical results showed that TNF-α, IL-1β and IL-6 proteins were significantly upregulated and IL-10 was downregulated in the synovial tissue of the OA group. After drug intervention, the expression of pro-inflammatory factors decreased and the level of IL-10 increased, and the CH@SHP099-NPs group had the most significant effect in regulating inflammatory factors.

[0145] Immunofluorescence assay

[0146] Synovial tissues from different treatment groups were fixed in 4% paraformaldehyde for 24 h, then dehydrated with 30% sucrose solution, embedded in OCT composite, and prepared into 5-8 μm thick frozen sections. After air-drying at room temperature, the sections were permeabilized with 0.1% Triton X-100 for 10 min and blocked in 5% BSA at room temperature for 1 h. Cells were seeded in 24-well slide plates and fixed with 4% paraformaldehyde for 15 min after drug treatment. They were then permeabilized with 0.5% Triton X-100 for 5 min and blocked in 5% BSA at room temperature for 1 h. The blocked tissue sections or cells were incubated overnight at 4°C with the following primary antibodies: anti-CD86, anti-iNOS, anti-ARG1, anti-CD206, anti-p-AMPK, anti-Collagen II, anti-MMP13, and anti-ADAMTS5. The next day, the cells were washed three times with PBS for 5 min each time. Next, secondary antibodies labeled with Alexa Fluor® 488-conjugated goat anti-rabbit IgG and Alexa Fluor® 594-conjugated goat anti-mouse IgG were added, and the cells were incubated at room temperature in the dark for 1 hour. After secondary antibody incubation, the cells were washed three times with PBS. Cell nuclei were stained using mounting media containing DAPI, incubated in the dark for 5 minutes, washed, and mounted. Images were acquired using confocal laser scanning microscopy, and fluorescence intensity was analyzed using ImageJ v1.53h. Immunofluorescence staining was used to further assess changes in macrophage polarization and metabolic pathways. Figure 17 F indicates that the M1 marker iNOS was significantly enhanced and the M2 marker Arg-1 was decreased in the OA group, suggesting that macrophage polarization is biased towards a pro-inflammatory phenotype. Drug interventions can partially reverse the polarization trend, with the SHP099-NPs group showing better results than SHP099, while the CH@SHP099-NPs group significantly promoted M2 polarization. Furthermore, Figure 17 G showed that the p-AMPK level in the synovial tissue of the OA group was significantly decreased, while all three drug interventions could increase p-AMPK expression, with the CH@SHP099-NPs group showing the most significant recovery effect.

[0147] In summary, the CH@SHP099-NPs composite hydrogel achieves a systematic remodeling of the inflammatory microenvironment of the OA synovial membrane by continuously modulating the SHP2-AMPK immune metabolic axis in vivo, synergistically inhibiting the production of inflammatory factors, reshaping macrophage polarization, and restoring metabolic homeostasis of synovial tissue. These results are highly consistent with the aforementioned in vitro mechanism studies, further solidifying the potential value of targeted immune metabolic regulation in the treatment of osteoarthritis.

[0148] In summary, this invention redefines the regulatory logic of synovial inflammation in osteoarthritis (OA) from the perspective of immunometabolic reprogramming. It systematically reveals the core role of the SHP2-AMPK axis in driving synovial macrophage dysfunction and joint degeneration, and achieves precise in vivo intervention of this axis through an engineered delivery strategy. This research not only expands our understanding of the pathogenesis of OA but also provides new theoretical and experimental basis for developing local joint treatment strategies targeting immunometabolic processes.

[0149] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing CH@SHP099-NPs composite hydrogel, characterized in that, Includes the following steps: (1) Dissolve sodium chondroitin sulfate and sodium hyaluronate separately in deionized water and mix them evenly to obtain CH hydrogel precursor solution; (2) After dissolving the thiol-containing cLABL cyclic peptide in a buffer solution, it was reacted with PLGA-PEG-MAL in the dark, and after dialysis and freeze-drying, PLGA-PEG-cLABL block copolymer was obtained. (3) Dissolve PLGA-PEG-cLABL block copolymer and SHP099 together in an organic solvent, then add dropwise to deionized water and ultrasonically emulsify. After evaporating the organic solvent, SHP099 NPs are obtained. (4) Disperse SHP099 NPs in the CH hydrogel precursor solution prepared in step (1), then add Genipin and stir, heat and react, and wash to obtain CH@SHP099-NPs composite hydrogel.

2. The preparation method of CH@SHP099-NPs composite hydrogel according to claim 1, characterized in that: In step (1), the mass ratio of sodium chondroitin sulfate and sodium hyaluronate is 1-3:1-3, the concentration of sodium chondroitin sulfate is 8-12 mg / mL, and the concentration of sodium hyaluronate is 8-12 mg / mL.

3. The method for preparing CH@SHP099-NPs composite hydrogel according to claim 2, characterized in that: In step (2), the mass ratio of cLABL cyclic peptide to PLGA-PEG-MAL is 0.12-0.2:1-2, and the initial concentration of cLABL cyclic peptide is 0.5-10 mg / mL.

4. The preparation method of CH@SHP099-NPs composite hydrogel according to claim 3, characterized in that: In step (2), the buffer solution is PBS buffer solution with a pH of 7.2-7.6; the reaction time in the dark is 10-14 hours.

5. The method for preparing the CH@SHP099-NPs composite hydrogel according to claim 4, characterized in that: In step (3), the mass ratio of PLGA-PEG-cLABL block copolymer to SHP099 is 10:1, and the concentration of SHP099 in the organic solvent is 0.5-2.0 mg / mL.

6. The method for preparing the CH@SHP099-NPs composite hydrogel according to claim 5, characterized in that: In step (3), the organic solvent is a mixture of dichloromethane and acetonitrile in a volume ratio of 1:

1.

7. The method for preparing the CH@SHP099-NPs composite hydrogel according to claim 6, characterized in that: In step (4), the final concentration of SHP099 NPs is 0.8-1.2 mg / mL; the final concentration of Genipin is 0.3-0.7 mg / mL.

8. The method for preparing CH@SHP099-NPs composite hydrogel according to claim 7, characterized in that: The heating reaction in step (4) is carried out at a temperature of 35-39°C for 4-8 hours.

9. CH@SHP099-NPs composite hydrogel prepared by the preparation method according to any one of claims 1-8.

10. The use of the CH@SHP099-NPs composite hydrogel of claim 9 in the preparation of a medicament for relieving or treating osteoarthritis.

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