Metal-glucocorticoid nanoparticle and preparation method and application thereof

Abstract

The present application relates to a kind of metal-glucocorticoid nanoparticles and its preparation method and application, belong to the biomedicine technical field.The preparation method includes: betamethasone sodium phosphate solution is mixed with sodium hyaluronate solution, then zinc ion source solution is added, and metal-glucocorticoid nanoparticles are obtained by reaction.Zn 2+ It can be the best performance enhancing ion of BetP, can reduce the binding free energy of BetP-glucocorticoid receptor, thereby significantly improving the binding stability of both. Realize the pH-responsive drug release and CD44 targeted delivery. It can effectively inhibit systemic inflammatory response, and significantly relieve secondary osteoporosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a metal-glucocorticoid nanoparticle, its preparation method, and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by persistent synovial inflammation and progressive joint destruction. The harm of RA is not limited to local joint inflammation; if the disease is poorly controlled, the persistent inflammatory microenvironment will lead to irreversible cartilage damage, bone erosion, and joint deformities, ultimately causing permanent functional impairment.

[0004] Glucocorticoids (GCs), such as betamethasone, are important drugs in the clinical treatment of rheumatoid arthritis (RA). The anti-inflammatory effect of GCs essentially depends on the efficient binding of the drug to the glucocorticoid receptor (GR) and the subsequent nuclear translocation process. Long-term use of GCs can induce glucocorticoid-induced osteoporosis, with approximately 50% of long-term users experiencing varying degrees of bone loss. Therefore, GCs face a clinical contradiction in RA treatment: while effectively suppressing the inflammatory response, they also accelerate systemic bone loss by inhibiting osteoblast activity and promoting osteoclast formation.

[0005] How to improve the anti-inflammatory efficacy of GCs while maintaining bone homeostasis remains an important scientific problem that needs to be solved in the field of RA treatment. Summary of the Invention

[0006] In view of the current state of technology, the purpose of this invention is to provide a metal-glucocorticoid nanoparticle, its preparation method and application, and to construct hyaluronic acid (HA) modified Zn 2+ Coordinated betamethasone phosphate (BetP) self-assembled nanoparticles (HA-ZnB NPs) achieve targeted delivery to the CD44 receptor on the surface of inflammatory macrophages. Compared with free BetP, HA-ZnB NPs exhibit superior joint-targeting ability, anti-inflammatory effect, chondroprotective effect, and ability to prevent systemic bone loss.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, a method for preparing metal-glucocorticoid nanoparticles includes the steps of: mixing a betamethasone sodium phosphate (BetP) solution with a sodium hyaluronate solution, then adding a zinc ion source solution, and reacting to obtain metal-glucocorticoid nanoparticles (HA-ZnB NPs).

[0008] Secondly, the metal-glucocorticoid nanoparticles prepared by the above-mentioned method are metal-glucocorticoid nanoparticles.

[0009] Thirdly, the above-mentioned metal-glucocorticoid nanoparticles are used in at least one of the following (a1) to (a2); (a1) To prepare products that inhibit rheumatoid arthritis; (a2) Prepare products that inhibit bone loss.

[0010] The beneficial effects of this invention are as follows: This invention discovers, based on MD simulation screening, that Zn 2+ As a potential potentiator of BetP, it can reduce the binding free energy of BetP-glucocorticoid receptors, thereby significantly improving the binding stability of the two. Based on this, hyaluronic acid-modified Zn-BetP self-assembled nanoparticles (HA-ZnB NPs) were constructed, achieving pH-responsive drug release and CD44-targeted delivery. In vitro experiments showed that HA-ZnB NPs could promote macrophage polarization towards the M2 type, inhibit fibroblast-like synovial cell invasion, and alleviate the adverse effects of BetP on osteogenic differentiation. In a mouse model of collagen-induced arthritis, HA-ZnB NPs preferentially accumulated in inflamed joints, effectively inhibiting systemic inflammatory responses, reducing the arthritis score from 7.5 to 2.0 (compared to 3.8 in the BetP group), and significantly alleviating secondary osteoporosis. Attached Figure Description

[0011] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0012] Figure 1Figures show the results of BetP molecular docking and molecular dynamics simulations. A is a schematic diagram of the GR structure. B is the three-dimensional molecular structure of BetP. C is the overall molecular docking conformation of BetP in GR-LBD. D is a magnified view of the specific molecular interactions and coordination environment between BetP and key amino acid residues in GR. E is the RMSD variation curve of the GR-BetP complex under different divalent metal ion conditions. F is the Rg analysis of the six simulation systems, and G is the RMSF analysis of the six simulation systems. H is the change in the number of intermolecular hydrogen bonds over time during molecular dynamics simulations. I is a time-series conformational snapshot of the BetP-Ca, BetP-Cu, BetP-Mg, and BetP-Sr systems during molecular dynamics simulations from 0 to 100 ns. J is the residue energy decomposition analysis of the BetP, BetP-Ca, BetP-Cu, and BetP-Sr systems.

[0013] Figure 2 This is a schematic diagram illustrating the bone-protective effect of HA-ZnB NPs in treating CIA mice in Example 1.

[0014] Figure 3 These are molecular dynamics simulations and physicochemical characterizations of HA-ZnB NPs from Example 1. B represents a snapshot of conformational changes during the 100 ns molecular dynamics simulation (0–100 ns) of the BetP and BetP-Zn systems. C represents the van der Waals interaction energies (ΔVDWAALS) and total binding free energy (ΔTOTAL) of the BetP system and different metal ion systems within the last 10 ns of the simulation trajectory. D represents the amino acid residue energy decomposition analysis of the BetP-Zn and BetP-Mg systems. E represents TEM images of ZB nanoparticles prepared with different BetP / Zn molar ratios of (2:1), (1:1), and (1:2), scale bar: 500 nm. F represents the Zn / P molar ratio in ZB nanoparticles with different formulations. G represents the BetP / Zn... 2+ Schematic diagram of coordination-formed nanoparticles. H is a high-magnification TEM image of HA-ZnB NPs. I is the EDS elemental distribution map of HA-ZnB NPs. Scale bars for H and I: left image 500 nm, right image 100 nm. J is the in vitro cumulative release curve of BetP from HA-ZnB NPs under different pH conditions (pH 5.5, 6.5, and 7.4).

[0015] Figure 4This is a characterization of the physicochemical properties and biological activity of HA-ZnB NPs. A shows representative TEM images of ZB prepared at different BetP:Zn molar ratios (4:1 and 1:4). B shows the total yield under different ZB molar ratios. C shows the hydrated particle size distribution of HA-ZnB NPs determined by DLS. D shows the Fourier transform infrared spectra of HA-ZnB, ZB, BetP, and HA. E shows the X-ray diffraction patterns of HA-ZnB, ZB, and BetP. F shows the full X-ray photoelectron spectroscopy (XPS) spectra of HA-ZnB, ZB, and BetP. G shows the high-resolution XPS spectrum of Zn 2p in HA-ZnB NPs. H shows the high-resolution XPS spectrum of P 2p in HA-ZnB NPs. I–K show the cell viability of RAW264.7 cells, FLSs, and MC3T3-E1 cells after treatment with different concentrations of HA-ZnB for 24 h, respectively. L is a representative image of Live / Dead staining in RAW264.7 cells, FLSs, and MC3T3-E1 cells. Scale bar: 100 μm.

[0016] Figure 5 This diagram illustrates the cellular targeted uptake and anti-inflammatory mechanism of HA-ZnB NPs. A shows the intracellular localization and co-localization of CD44 and HA-ZnB NPs as detected by immunofluorescence (IF), scale bar: 20 μm. B shows the relative mRNA expression levels of pro-inflammatory factors and anti-inflammatory / tissue repair-related genes in macrophages under different treatment conditions. C and D are immunofluorescence images of CD86 and CD206 expression in macrophages, scale bar: 20 μm. E and F are representative results of flow cytometry detection of CD86 and CD206 expression levels. G and H show the expression and quantitative analysis of iNOS, Arg-1, and IL-6 proteins detected by Western blot. I and J are GO functional enrichment analysis and KEGG pathway enrichment analysis of differentially expressed genes (DEGs), respectively. K shows the expression of proteins related to the NF-κB signaling pathway detected by Western blot. L shows the localization of GR and the cell nucleus as detected by immunofluorescence, scale bar: 10 μm. M and N represent fluorescence intensity line scanning analysis and co-localization quantitative analysis of GR nuclear translocation. O and P represent Western blot detection and quantitative analysis of GR protein expression after separation of nuclear and cytoplasmic proteins, respectively.

[0017] Figure 6This diagram illustrates the anti-inflammatory mechanism of HA-ZnB NPs. A shows representative images of CD44 immunofluorescence (IF) staining in RAW264.7 macrophages with and without LPS stimulation (scale bar: 20 μm). B shows the quantitative analysis of CD44 expression MFI. C and D are flow cytometry histograms of CD44 expression and their MFI quantitative analyses, respectively. E is a flow cytometry scatter plot showing the correlation between CD44 expression and HA-ZnB NP uptake in control and LPS-activated macrophages. F shows the quantitative analysis of HA-ZnB NP uptake in control and LPS-activated macrophages. G is a Pearson correlation heatmap between macrophages in the LPS group and the LPS+HA-ZnB group. H is a volcano plot of differentially expressed genes (DEGs) between the LPS group and the LPS+HA-ZnB group, where downregulated and upregulated genes are represented in orange and blue, respectively. I–K represent the results of gene set enrichment analysis for the NF-κB signaling pathway, TNF signaling pathway, and cellular response to interferon-β, respectively. L represents the Western blot analysis of proteins related to the NF-κB signaling pathway.

[0018] Figure 7 This image shows the results of an analysis of the invasive phenotype of FLSs mediated by HA-ZnB NPs by inhibiting the MAPK signaling pathway. A and B are schematic diagrams of the Transwell co-culture system of RAW264.7 macrophages and FLSs, respectively. C is a representative image showing the migration ability of FLSs through crystal violet staining, scale bar: 100 μm. D is a quantitative analysis of the number of migrating FLSs. E is a representative image of the FLSs scratch assay (0, 24, and 48 h after treatment), scale bar: 200 μm. F shows the relative mRNA expression levels of Mmp1, Mmp3, and Mmp13 genes in FLSs. G is a representative image of MMP-13 immunofluorescence staining, scale bar: 50 μm. H is a quantitative analysis of the mean fluorescence intensity (MFI) of MMP-13. I is a representative image of DKK-1 immunofluorescence staining. J is a quantitative analysis of the mean fluorescence intensity (MFI) of DKK-1. K is a representative image of phosphorylated p38 (p-p38) immunofluorescence staining. L represents the quantitative analysis of p-p38 mean fluorescence intensity (MFI). M and N represent the expression and quantitative analysis of MAPK signaling pathway-related proteins p-p38 / p38, p-JNK / JNK, and p-ERK / ERK, respectively, detected by Western blot. O is a schematic diagram illustrating the mechanism by which HA-ZnB NPs inhibit inflammatory activation and aggressive phenotype of FLSs.

[0019] Figure 8This diagram illustrates how HA-ZnB NPs rescue osteogenic inhibition induced by glucocorticoids (GCs) and restore bone remodeling balance. A represents ALP staining images of MC3T3-E1 cells treated with Ctrl, BetP, or HA-ZnB, scale bar: 100 μm. B represents Alizarin Red staining images of MC3T3-E1 cells under different treatments, showing the formation of mineralized nodules, scale bar: 100 μm. C represents the relative mRNA expression levels of osteogenic-related genes (Alp, Runx2, Ocn, Opn, Osterix, and Bmp2) in MC3T3-E1 cells. D represents ALP immunofluorescence staining images of MC3T3-E1 cells under different treatments, scale bar: 50 μm. E represents OCN immunofluorescence staining images of MC3T3-E1 cells under different treatments, scale bar: 50 μm. F represents the quantitative analysis of mean fluorescence intensity (MFI) of ALP and OCN. G represents the Western blot analysis of osteogenic-related proteins. H represents the quantitative analysis of the relative expression levels of related proteins after normalization with β-actin as an internal control. I shows representative TRAP staining images under different treatment conditions, indicating osteoclast differentiation; scale bar: 100 μm. J shows representative images of phalloidin fluorescence staining in osteoclasts, showing F-actin (red) and cell nuclei (blue); scale bar: 100 μm.

[0020] Figure 9 These are images of the HA-ZnB NPs hemolysis experiment, CIA mouse treatment, and routine blood analysis. A shows the results of the HA-ZnB NPs hemolysis experiment. B shows representative in vivo fluorescence imaging images of major organs in CIA mice at 0, 1, 6, and 12 hours after systemic drug administration. C shows the record of weight changes in CIA mice. D shows representative photographs of the mouse hind paws at different time points. E shows representative Micro-CT images of the mouse hind paws. F shows representative images of IL-1β and IL-6 immunohistochemical (IHC) staining. G shows the quantitative analysis of the number of IL-1β and IL-6 positive cells. H shows the serum IL-6 concentration in CIA mice. I–N represent the routine peripheral blood parameters of CIA mice, including white blood cell count (WBC), lymphocyte count (Lymph), red blood cell count (RBC), granulocyte count (Gran), granulocyte percentage (Gran%), and platelet count (PLT).

[0021] Figure 10This diagram illustrates the in vivo therapeutic effects of HA-ZnB NPs in CIA mice. A is a schematic diagram of the DBA / 1J mouse CIA model establishment and treatment intervention process. B shows representative in vivo fluorescence imaging images of CIA mice at 0, 1, 6, and 12 h after systemic drug administration. C shows the clinical arthritis scores of each group of mice. D shows representative photographs of the hind feet of mice in the control group, CIA group, free BetP group, and HA-ZnB group at the end of treatment. E shows representative three-dimensional (3D) reconstruction images of the mouse hind feet. F shows representative histological images of the hind foot joint tissues in the control group, CIA group, free BetP group, and HA-ZnB group, including H&E staining, Masson staining, Safranin O-Fast Green staining, and Toluidine Blue staining (scale bar, 100 μm). G shows representative images of IL-1β and IL-6 immunofluorescence (IF) staining (scale bar, 100 μm). H represents the quantitative analysis of mean fluorescence intensity (MFI) of IL-1β and IL-6. I represents representative images of immunohistochemical (IHC) staining for TNF-α and MMP-13. J represents the quantitative analysis of the number of TNF-α and MMP-13 positive cells.

[0022] Figure 11 This is a schematic diagram illustrating how HA-ZnB NPs promote bone formation and alleviate secondary osteoporosis in CIA mice. A shows representative Micro-CT 3D reconstructed images of the distal femur in the control, CIA, free BetP, and HA-ZnB groups, scale bar: 1 mm (overall view) / 500 μm (enlarged view). B–E represent quantitative analyses of Micro-CT morphological parameters of the distal femur cancellous bone. F shows representative H&E stained images of distal femur tissue sections, scale bar: 1 mm (overall view) / 100 μm (enlarged view). G shows representative images of CK, ALP, and TRAP immunohistochemical (IHC) staining, scale bar: 100 μm. H shows quantitative analysis of the number of CK, TRAP, and ALP positive cells (per field of view). I shows representative images of RUNX2 and OCN immunofluorescence (IF) staining in bone tissue, scale bar: 100 μm. J shows quantitative analysis of the mean fluorescence intensity (MFI) of RUNX2 and OCN.

[0023] Figure 12 These are representative images of H&E staining of tissue sections from the heart, liver, spleen, lungs, and kidneys. Detailed Implementation

[0024] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0025] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0026] Unless otherwise specified, the experimental methods described in the following examples were performed under standard conditions. All raw materials and reagents used in the following examples are commercially available, unless otherwise indicated.

[0027] All data were statistically analyzed using GraphPad Prism 9.0 software, and results are expressed as mean ± standard deviation. In vitro experiments were performed at least three independent replicates; in vivo experiments included six mice per group (n = 6). Student's t-test was used for comparisons between two groups; one-way ANOVA was used for comparisons of three or more groups. A p-value < 0.05 was considered statistically significant.

[0028] This study was approved by the Medical Ethics Committee of the School of Stomatology, Shandong University.

[0029] Example 1, Zn 2+ Screening of the best coordinating ions for BetP-GR and construction of HA-ZnB NPs.

[0030] I. Screening of optimal coordinating ions.

[0031] Divalent metal ions have attracted widespread attention due to their dual functions of regulating protein-ligand interactions and promoting bone metabolism. Mg 2+ Ca 2+ 、Sr 2+ Cu 2+ and Zn 2+ Divalent cations can promote osteoblast proliferation and inhibit osteoclast bone resorption through multiple signaling pathways, thereby promoting bone formation. However, systematic research is still lacking on strategies that utilize biologically functional metal ions to synergistically optimize GCs in the treatment of RA, thereby enhancing anti-inflammatory activity and simultaneously restoring bone homeostasis.

[0032] GR is a member of the nuclear receptor superfamily and a key ligand-activated transcription factor mediating the anti-inflammatory effects of glucocorticoids, playing an important role in the regulation of immune responses. GR is mainly composed of a C-terminal ligand-binding domain (LBD), which is a sandwich structure consisting of 11 α-helices. The ligand-binding pocket is located in the lower part of the LBD. Figure 1 (A) After BetP binds to GR, it can induce a conformational change in the receptor, promote GR nuclear translocation, and further regulate the transcription of downstream genes. Figure 1 (BD in the text). To investigate the effect of different divalent metal ions on the BetP-GR interaction, the inventors selected Mg... 2+ Ca 2+ 、Sr 2+ Cu 2+ and Zn 2+A coordination system was constructed, and 100 ns all-atom molecular dynamics simulations were performed. This included obtaining the crystal structure of the human glucocorticoid receptor ligand-binding domain (PDB ID: 4P6X) from the RCSB Protein Data Bank (PDB) database. Molecular docking analysis was performed using AutoDock Vina software, and the receptor protein and ligand structures were preprocessed using AutoDock Tools software. The grid spacing was set to 0.375 Å. The Vina search algorithm was used to generate ligand-binding conformations, which were then sorted according to predicted binding affinity. The conformation with the optimal binding energy was selected as the initial structure for subsequent molecular dynamics simulations. The inventors used the open-source software GROMACS 2021.5 for molecular dynamics simulations. All simulation systems were set to a temperature of 300 K and a pressure of 1 bar, with periodic boundary conditions. The system was placed at the center of the simulation chamber, and the minimum distance between the system edge and the simulation chamber boundary was set to 1.0 nm. The receptor protein topology file was generated using the pdb2gmx tool, and the protein molecule was described using the AMBER ff14SB force field. The ligand topology file, compatible with GROMACS, was generated using AmberTools, and the ligand atomic parameters were processed using the GAFF force field. The TIP3P water model was used as the solvent model. After system construction, the steepest descent method was first used to minimize the energy of all atoms. Subsequently, under position constraints, 1000 ps constant particle number-constant volume-isothermal equilibrium and 1000 ps constant particle number-isothermal pressure-isothermal equilibrium were performed sequentially. After equilibrium was achieved, a formal MD simulation was performed for 100 ns with an integration step size of 2 fs. During the simulation, the Linear ConstraintSolver algorithm was used to constrain covalent bond lengths, and the Particle Mesh Ewald method was used to calculate long-range electrostatic interactions. The results show that all simulated systems can quickly reach equilibrium and maintain a stable conformation throughout the simulation. RMSD, Rg, RMSF, and hydrogen bond analyses all indicate that each system has good simulation stability. Figure 1 (EH in the middle).

[0033] Further extraction of conformations at different time points in the 100 ns simulated trajectory was used to analyze the dynamic behavior of the ligand within the binding pocket. Figure 3 B and Figure 1 (I) The results showed that in the BetP-only system, the ligands underwent significant translation and rotation within the binding cavity; Ca 2+ Cu 2+ Mg 2+ and Sr 2+The coordination system also exhibits significant conformational fluctuations, especially at the phosphate terminus of BetP. In contrast, BetP in the BetP-Zn system maintained a stable binding conformation throughout the simulation, with almost no significant positional or conformational changes, indicating that Zn²⁺… + It can significantly enhance the stability of the binding between BetP and GR.

[0034] After the simulation, the radius of gyration (Rg), root mean square deviation (RMSD), and root mean square fluctuation (RMSF) of the system were calculated using the built-in gmx module of GROMACS. The binding free energy of the system was calculated using the molecular mechanics / generalized Born surface area method. The calculated binding free energy further confirmed the above results. The total binding free energy (ΔTOTAL) of the BetP-Zn system is... 20.49 kcal / mol, significantly lower than the system with BetP acting alone ( 14.79 kcal / mol) and other metal ion coordination systems (such as Figure 3 (as shown in C and Table 1), illustrating Zn² + It can significantly enhance the binding affinity between BetP and GR. The improvement in binding free energy mainly stems from the enhanced van der Waals interaction (ΔVDWAALS = ...). 28.35 kcal / mol). Further residue energy decomposition analysis revealed that, compared to other metal ions, Zn... 2+ The system contains a greater number of amino acid residues that participate in favorable binding, and their energy contribution is higher. Figure 3 D and Figure 1 J in (of the Mg). Although MET-560 in Mg 2+ and Sr 2+ It also participates in ligand stabilization in the system, but its binding contribution is still significantly weaker than that of Zn. 2+ The above results indicate that Zn 2+ It is the best coordinating ion to enhance the binding stability of BetP-GR.

[0035] Table 1. Statistical table of the effects of different metal ions on the binding energy of BetP-GR

[0036] II. Construction of HA-ZnB NPs.

[0037] Solutions of betamethasone sodium phosphate (BetP, Aladdin Reagent, China) with concentrations of 0.025, 0.05, and 0.1 M were prepared, as were solutions of zinc nitrate hexahydrate (Zn(NO3)2•6H2O, Aladdin Reagent, China) with concentrations of 0.025, 0.05, and 0.1 M. Equal volumes of the two solutions at different concentrations were mixed to obtain BetP and Zn... 2+ Systems with molar ratios of 4:1, 2:1, 1:1, 1:2, and 1:4 were stirred at room temperature for 5 minutes to obtain Zn-containing systems. 2+ Liquid containing coordinated BetP nanoparticles (ZB) was prepared by centrifugation at 12000 r / min for 10 min, collecting the precipitate, and washing it three times with deionized water. The ZB was then stored at 4 °C for later use. TEM results showed that ZB obtained with different ratios exhibited irregular amorphous aggregates rather than uniformly dispersed nanoparticles, and displayed an interconnected network structure. Figure 3 E and Figure 4 (A) Where, when BetP and Zn 2+ The material yield reaches its maximum when the molar ratio is 1:1. Figure 4 (B in the text). Further analysis using ICP-MS revealed that the Zn and P content in samples with different ratios was approximately 1. Figure 3 The F in the figure indicates that the coordination products have a consistent composition regardless of the feed ratio. Therefore, subsequent experiments all used BetP and Zn. 2+ The formulation with a molar ratio of 1:1 was used. The above results suggest that Zn 2+ Coordination with phosphate groups is the main driving force for ZB self-assembly, while hydrophobic interactions between steroidal skeletons may further stabilize its supramolecular structure. Figure 3 (G in the middle).

[0038] Based on the self-assembly capability, sodium hyaluronate (HA) was introduced to modify Zn to construct HA-ZnBNPs. The construction method included: mixing 1 mL of 0.1 M BetP solution with 1 mL of sodium hyaluronate solution (40 mg / mL, Yuanye Biotechnology, China), stirring at room temperature for 5 min, then adding 1 mL of 0.1 M Zn(NO3)2•6H2O solution to the system, stirring for 5 min, to obtain HA-modified Zn. 2+Liquid containing coordinated BetP nanoparticles (HA-ZnB NPs) was prepared by centrifugation at 12000 r / min for 10 min, followed by collection of the precipitate. The precipitate was then washed three times with deionized water to obtain HA-ZnB NPs, which were stored at 4 ℃ for later use. TEM results showed that after HA modification, the originally randomly aggregated ZB was successfully transformed into uniformly dispersed nanoparticles with regular morphology. Figure 3 The H in the HA-ZnB NPs was further confirmed by the elemental surface scanning results. The C, O, N, and Zn elements were uniformly distributed within the nanoparticles. Figure 3 The DLS analysis results showed that the average hydrated particle size of HA-ZnB NPs was approximately 100 nm, and the particle size distribution was relatively uniform. Figure 4 (C in the text). Subsequently, FT-IR, XRD, and XPS were used to further characterize the composition and structure of HA-ZnB NPs. FT-IR results showed that the characteristic absorption peak of the phosphate group in BetP increased from 985 cm⁻¹. -1 and 1099 cm -1 Move to 999 cm respectively -1 and 1118 cm -1 The prompt is Zn 2+ Successful coordination with phosphate groups was achieved, while the characteristic absorption peak of HA was still retained in HA-ZnB NPs. Figure 4 (D in the text). XRD results show that the original BetP crystal diffraction peaks are similar to those of Zn. 2+ The complete disappearance after coordination indicates that the material has transformed into an amorphous structure. Figure 4 The E in the middle). XPS analysis further detected the characteristic peak of Zn element in HA-ZnB NPs ( Figure 4 The composition of HA-ZnB NPs was semi-quantitatively calculated based on XPS elemental composition analysis. The results show that HA-ZnB NPs consist of approximately 72 wt% BetP, 11 wt% Zn, and 17 wt% HA, as shown in Table 2.

[0039] Table 2 Elemental Content Statistics of HA-ZnB NPs

[0040] The in vitro release behavior of BetP from HA-ZnBNPs was evaluated using sodium acetate buffer (Shanghai Yuanye Biotechnology, China) at different pH values ​​(5.5, 6.5, and 7.4). 10 mg of HA-ZnBNPs were dispersed in 10 mL of the corresponding buffer and cultured with shaking at 37 ℃ and 100 r / min. At preset time points, 1 mL of the release solution was collected, and an equal volume of fresh buffer was added to maintain a constant total volume. The absorbance of the release solution at 240 nm was measured using a UV-Vis spectrophotometer (UV-2600i, SHIMADZU, Japan), and the cumulative release rate of BetP was calculated based on a standard curve. The results showed that the nanoparticles exhibited a significant pH-responsive release characteristic. Figure 3 (J in the text). Under physiological conditions (pH 7.4), BetP was released cumulatively by approximately 41.0% within 72 h; however, in an acidic environment, drug release was significantly accelerated, with a cumulative release of approximately 67.1% within 24 h at pH 5.5, indicating that HA-ZnB NPs can achieve rapid drug release in an inflammatory acidic microenvironment.

[0041] The cytocompatibility of HA-ZnB NPs was evaluated using a cell counting kit-8 (CCK-8; GlpBio, USA) and Live / Dead staining. The method involved seeding RAW264.7 cells, FLSs, and MC3T3-E1 cells into 96-well plates and adding culture medium containing different concentrations of HA-ZnB NPs (12.5, 25, 50, 100, and 200 μg / mL). Ordinary culture medium was used as a control. After 24 h of culture, the original culture medium was discarded, and 10 μL of CCK-8 working solution and 90 μL of culture medium were added to each well. The plates were incubated at 37 ℃ in the dark for 2 h. The absorbance (optical density, OD) at 450 nm was measured using a microplate reader (SPECTROstar Nano, Germany), and cell viability was calculated. In the Live / Dead staining experiment, RAW264.7 cells, FLSs, and MC3T3-E1 cells were seeded in 24-well plates and treated with different concentrations of HA-ZnB NPs for 24 h. Staining was then performed using a Live / Dead Cell Viability / Death Staining Kit (Solarbio, China). After incubation in the dark for 20 min, the cells were washed three times with PBS, and cell viability was observed using a fluorescence microscope (IX73, Olympus, Japan). The results showed that after co-culturing with RAW264.7 macrophages, FLSs, and MC3T3-E1 cells for 24 h, when the concentration of HA-ZnB NPs was below 50 μg / mL, no significant cytotoxicity was observed in any of the three cell types. Figure 4(IL in the HA-ZnB NPs). Combined with XPS compositional analysis, it was found that Zn accounts for approximately 11 wt% of the total mass of HA-ZnB NPs. Therefore, the Zn² released by 12.5 μg / mL HA-ZnB NPs is... + The concentration was consistent with the effective concentration range reported in the literature for anti-inflammatory activity. Based on this, 12.5 μg / mL was selected as the working concentration of HA-ZnB NPs for all subsequent in vitro experiments.

[0042] Example 2: CD44-targeted uptake and anti-inflammatory effects of HA-ZnB NPs.

[0043] I. Cellular uptake experiment.

[0044] RAW264.7 cells were fed at a rate of 1×10⁻⁶. 6 Cells were seeded at a density of [number] cells / dish in laser confocal culture dishes (Biosharp, China) and cultured for 24 h with or without lipopolysaccharide (LPS; 100 ng / mL, Solarbio, China). The medium was then replaced with medium containing 12.5 μg / mL Rhodamine B (Solarbio, China)-labeled HA-ZnB NPs, and incubated for another 2 h. After treatment, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and then blocked with 5% bovine serum albumin (BSA; Yuanye Biotechnology, China) for 1 h. CD44 primary antibody (ab189524, Abcam, UK) was added, and the cells were incubated overnight at 4 °C. The next day, CoraLite488-labeled goat anti-rabbit IgG (H+L) fluorescent secondary antibody (SA00013-2, Proteintech, USA) was added, and the cells were incubated at room temperature in the dark for 1 h. Fluorescence signals of HA-ZnB NPs and CD44 were quantitatively detected using flow cytometry (BD Biosciences, USA), and data analysis was performed using FlowJo software (FlowJo LLC, USA). Before confocal microscopy, cell nuclei were counterstained with DAPI (Abcam, UK), and fluorescence images were subsequently acquired using a laser confocal microscope (FV3000, Olympus, Japan). Semi-quantitative analysis of fluorescence intensity was performed using ImageJ 1.44 software (NIH, USA). Results showed that LPS stimulation significantly upregulated CD44 expression on the surface of RAW264.7 cells. Figure 6The HA-mediated uptake of HA-ZnB NPs by RAW264.7 cells under inflammatory conditions was significantly enhanced compared to normal macrophages, as both immunofluorescence (IF) and flow cytometry results confirmed that RAW264.7 cells under inflammatory conditions exhibited significantly enhanced uptake of HA-ZnB NPs. Figure 5 A in Figure 6 (E and F in the text). After achieving efficient cellular uptake, HA-ZnBNPs exhibited superior anti-inflammatory activity compared to free BetP. RT-qPCR results showed that HA-ZnB NPs could more significantly downregulate the expression of pro-inflammatory factors IL-1β, IL-6, and Tnf-α, while further promoting the expression of anti-inflammatory related genes Arg-1, IL-10, and Tgf-β. Figure 5 The results of immunofluorescence and flow cytometry showed that HA-ZnB NPs significantly reduced the expression of CD86, a marker of M1 macrophages, and increased the expression of CD206, a marker of M2 macrophages, compared with free BetP. Figure 5 (CF in the middle).

[0045] II. Analysis of anti-inflammatory effects.

[0046] Total cellular protein was extracted using RIPA lysis buffer (Solarbio, China) containing protease and phosphatase inhibitors. Nucleoplasmic protein separation experiments were performed using a nucleoplasmic and cytoplasmic protein extraction kit (Beyotime, China) according to the manufacturer's instructions. Protein concentration was determined using a BCA protein assay kit (Beyotime, China). An equal volume of protein (30 μg) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, ACE, China) and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The PVDF membrane was blocked with TBST buffer containing 5% skim milk powder at room temperature for 1 h, followed by incubation with primary antibody at 4 °C overnight. The primary antibodies used by the inventors included: iNOS (22226-1-AP, Proteintech, USA), IL-6 (DF6087, Affinity, China), Arg-1 (16001-1-AP, Proteintech, USA), p-p65 (AF3387, Affinity, China), p65 (BF8005, Affinity, China), p-IκBα (AF2002, Affinity, China), IκBα (AF5002, Affinity, China), and glucocorticoid receptor (GR; AF5004, Affinity, China). The following day, after washing the membrane with TBST, the corresponding horseradish peroxidase (HRP)-labeled secondary antibody (Solarbio, China) was added and incubated at room temperature for 1 hour. Color development was performed using an enhanced chemiluminescence reagent, and the gray values ​​of the protein bands were quantitatively analyzed using ImageJ software. In the nuclear protein isolation experiment, Lamin B1 (AF5161, Affinity, China) and β-actin (66009-1-Ig, Proteintech, USA) were used as internal controls for nuclear and cytoplasmic proteins, respectively; β-actin was used as the internal control protein in all other experiments. Western blot results further confirmed that HA-ZnB NPs can more effectively downregulate iNOS and IL-6 protein expression, while upregulating Arg-1 protein levels. Figure 5 The presence of G and H in the data indicates that HA-ZnB NPs can more effectively promote macrophage polarization towards the M2 type.

[0047] Transcriptome sequencing analysis was performed on each group of cells. The methods included: extracting total RNA from different cell types using TRIzol reagent (AG, China) according to the reagent instructions. Specifically, RAW264.7 cells, FLSs, and MC3T3-E1 cells were seeded in 6-well plates and cultured overnight. RAW264.7 cells were stimulated with LPS for 24 h and treated with or without BetP and HA-ZnB NPs; the RAW264.7-FLSs co-culture system was stimulated with LPS and treated with or without HA-ZnB NPs; MC3T3-E1 cells were treated with either BetP or HA-ZnB NPs in osteogenic induction medium for 14 days. After treatment, total RNA was extracted from each group of cells, and the RNA concentration and purity were determined spectrophotometrically. 1 μg of total RNA was used to synthesize cDNA using a reverse transcription kit. Subsequently, PCR amplification was performed using SYBR Green Master Mix (AG, China) on a real-time quantitative PCR instrument. The amplification program was as follows: pre-denaturation at 95 °C for 30 s; followed by 40 cycles, each cycle consisting of 5 s denaturation at 95 °C and 30 s annealing / extension at 60 °C. Gapdh was used as the internal control gene, and 2^ The relative expression levels of the target genes were calculated using the ΔΔCt method. The detection indicators included: inflammation and anti-inflammatory related genes Il-1β, Il-6, Tnf-α, Arg-1, Il-10, and Tgf-β in RAW264.7 cells; matrix metalloproteinase related genes Mmp1, Mmp3, and Mmp13 in FLSs; and osteogenic related genes Alp, Runx2, Ocn, Opn, Osterix, and Bmp2 in MC3T3-E1 cells. All experiments were independently repeated three times. The primer sequences for the target gene and the internal reference gene Gapdh used by the inventors are shown in Table 3.

[0048] Table 3 Primer sequences of the target gene and internal reference gene Gapdh used.

[0049] Principal component analysis and differential expression analysis showed that the LPS group and the LPS+HA-ZnB group had significantly different gene expression profiles. Figure 6 G and H in the sample. GO enrichment analysis, KEGG pathway analysis, and GSEA analysis all indicated that HA-ZnB NPs significantly inhibited inflammation-related signaling pathways, especially the NF-κB and TNF signaling pathways. Figure 5 The I and J in Figure 6 The Western blot results further validated the above analysis results, showing that HA-ZnB NPs could more significantly inhibit the phosphorylation levels of p65 and IκBα compared with free BetP. Figure 5 K in Figure 6 The presence of L in the formula indicates that it can more effectively inhibit the activation of the NF-κB signaling pathway. Given that GR is the main target for BetP's pharmacological effects, the inventors further evaluated the effect of HA-ZnB NPs on GR activation. Immunofluorescence and co-localization analyses showed that, compared with free BetP, HA-ZnB NPs treatment significantly enhanced GR accumulation in the cell nucleus (…). Figure 5 LN in (the context of LN).

[0050] Immunofluorescence (IF) staining was used to evaluate the nuclear translocation of GR in RAW264.7 cells. RAW264.7 cells were seeded in laser confocal microscopy dishes and treated with either BetP or HA-ZnB NPs for 2 h. After treatment, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% bovine serum albumin. GR primary antibody was then added and incubated overnight at 4 °C. The next day, the cells were incubated with the corresponding fluorescently labeled secondary antibody, and the nuclei were counterstained with DAPI. Fluorescence images were acquired using laser confocal microscopy. The fluorescence intensity distribution of line scans in designated areas was analyzed using ImageJ software to evaluate the spatial colocalization of GR and nuclear signals. Simultaneously, the JACoP plugin in ImageJ was used to analyze the overlap between GR and DAPI fluorescence signals and calculate the colocalization percentage to quantitatively evaluate the nuclear translocation level of GR.

[0051] Nucleoplasmic protein isolation experiments further confirmed that HA-ZnB NPs could significantly increase the expression of GR protein in the cell nucleus, while reducing the retention level of GR in the cytoplasm. Figure 5 In summary, HA-ZnB NPs can promote GR nuclear translocation, enhance GR signaling pathway activity, and further inhibit NF-κB inflammatory signaling, thereby promoting macrophage polarization towards the anti-inflammatory M2 type. Figure 3 (A in the middle).

[0052] Example 3: Phenotypic analysis of HA-ZnB NPs inhibiting the invasiveness of FLSs.

[0053] FLSs are key effector cells in the progression of RA from acute inflammation to chronic joint destruction, exhibiting abnormal proliferation, migration, and invasion characteristics similar to tumor cells. Previous studies have shown that the large amounts of pro-inflammatory cytokines secreted by inflammatory macrophages can continuously activate the mitogen-activated protein kinase (MAPK) signaling pathway in FLSs, thereby promoting their abnormal proliferation, migration, and matrix degradation. To investigate whether HA-ZnB NPs, after inhibiting the inflammatory response of macrophages, can further regulate the pathological activation of FLSs, the inventors established a Transwell co-culture system of RAW264.7 cells and FLSs, using LPS-stimulated RAW264.7 cells to continuously provide inflammatory stimulation signals (…). Figure 7 The methods (A and B in the text) included: RAW264.7 cells were seeded in the upper chamber of a Transwell chamber, and FLSs were seeded in the lower chamber. The two cell types communicated through soluble factors without direct cell contact. RAW264.7 cells were stimulated with LPS and treated with or without HA-ZnB NPs, and the migration ability of FLSs was then evaluated. In the Transwell migration assay, FLSs were seeded in the upper chamber of the Transwell chamber, and the lower chamber was added with the appropriate conditioned medium, and cultured for 24 h. Cells that migrated to the lower surface of the filter membrane were fixed with 4% paraformaldehyde and stained with crystal violet (Solarbio, China), followed by microscopic observation and quantitative analysis. In addition, the migration ability of FLSs was further evaluated using a scratch assay. FLSs were seeded in 6-well plates and cultured to approximately 90% confluence. A straight scratch was made in the center of the cell monolayer using a sterile pipette tip. After washing with PBS to remove exfoliated cells, the cells were cultured in serum-free medium containing different treatment factors. Images were taken at 0, 24, and 48 h using an inverted microscope to evaluate cell migration and scratch healing.

[0054] The results showed that FLSs exhibited significantly enhanced migration and invasion capabilities in the inflammatory microenvironment; while HA-ZnBNPs treatment significantly inhibited both migration and invasion capabilities of FLSs. Figure 7 Consistent with the phenotypic changes described above, RT-qPCR and Western blot results showed that HA-ZnB NPs significantly reduced the expression levels of matrix metalloproteinase (MMP)-related genes and proteins. Figure 7 In addition, under inflammatory stimulation, the expression of the Wnt signaling pathway antagonist Dickkopf-1 (DKK-1) was significantly increased, while its expression was significantly decreased after treatment with HA-ZnB NPs. Figure 7(I and J in the text). Since DKK-1 is an important promoter of bone erosion in RA, the above results suggest that HA-ZnBNPs can effectively reduce FLSs-mediated cartilage destruction and bone resorption potential. Considering that the MAPK signaling pathway is an important mechanism for regulating the pathological activation of FLSs, the inventors further examined the activation of the p38, JNK, and ERK signaling pathways. Western blot analysis (primary antibodies used included: p-p38 (AF4001, Affinity, China), p38 (BF8015, Affinity, China), p-JNK (AF3318, Affinity, China), JNK (AF6318, Affinity, China), p-ERK1 / 2 (AF1015, Affinity, China), ERK1 / 2 (BF8004, Affinity, China)) and immunofluorescence results showed that inflammatory stimulation significantly promoted the phosphorylation of p38, JNK, and ERK proteins, while treatment with HA-ZnB NPs significantly inhibited the phosphorylation levels of these proteins. Figure 7 (KN in the text). In summary, HA-ZnB NPs block the abnormally activated "macrophage-FLSs" interaction axis in RA by inhibiting the inflammatory response mediated by inflammatory macrophages, and effectively inhibit the invasiveness and matrix degradation phenotype of FLSs by inhibiting the MAPK signaling pathway, thereby playing a role in protecting cartilage and reducing joint damage.

[0055] Example 4: Analysis of the effect of HA-ZnB NPs in alleviating BetP-induced osteogenic differentiation inhibition.

[0056] To evaluate the regulatory effect of HA-ZnB NPs on osteogenic differentiation under BetP-induced osteogenic inhibition, the following methods were used: MC3T3-E1 cells were placed in osteogenic induction medium containing sodium β-glycerophosphate and ascorbic acid and subjected to different treatments. After 7 days of induction culture, ALP activity was detected using an ALP staining kit; after 21 days of induction culture, the formation of mineralized nodules was detected using Alizarin Red S staining. For immunofluorescence detection, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% BSA. ALP or osteocalcin (OCN) primary antibody was added, and the cells were incubated overnight at 4 °C; subsequently, the corresponding fluorescently labeled secondary antibody was added for incubation. Phalloidin was used to label F-actin, and DAPI was used to counterstain the cell nuclei. Fluorescence images were acquired using laser confocal microscopy, and the fluorescence intensity was semi-quantitatively analyzed using ImageJ software. In the osteoclast differentiation experiment, conditioned medium from MC3T3-E1 cells in different treatment groups was collected and added to a RAW264.7 cell culture system containing receptor activator of nuclear factor κB ligand (RANKL; 92649ES10, Yeasen, China) to induce osteoclast differentiation. After 7 days of induction, osteoclast formation was evaluated using a tartrate-resistant acid phosphatase (TRAP) staining kit. The results showed that BetP treatment significantly reduced alkaline phosphatase (ALP) activity and mineralized nodule formation in MC3T3-E1 cells, suggesting that BetP significantly inhibited osteogenic differentiation. Figure 8 (A and B in the original text). In contrast, although HA-ZnB NPs still contain BetP, treatment with them significantly restored the osteogenic differentiation capacity of cells. RT-qPCR results further showed that HA-ZnB NPs significantly upregulated the expression levels of osteogenic-related genes such as Alp, Runx2, Ocn, Opn, Osterix, and Bmp2. Figure 8 The results of immunofluorescence and Western blot (using primary antibodies including Wnt-3A (DF6113, Affinity, China) and β-catenin (AF6266, Affinity, China)) further confirmed that, compared with the BetP group, HA-ZnB NPs significantly increased the expression levels of ALP and OCN proteins. Figure 8The results showed that HA-ZnB NPs could effectively reverse BetP-induced osteogenic dysfunction. To further elucidate its osteogenic mechanism, the inventors examined changes in the classic Wnt / β-catenin signaling pathway. Wnt / β-catenin signaling is an important pathway regulating osteoblast differentiation and bone homeostasis. The results showed that BetP treatment significantly inhibited the expression of Wnt-3a and β-catenin proteins, while HA-ZnB NPs could significantly restore the expression levels of both (F). Figure 8 (G, H in the text), hinting at Zn 2+ It can effectively counteract the osteogenic inhibition induced by GCs by reactivating the Wnt / β-catenin signaling pathway. In addition to promoting osteoogenesis, the inventors further evaluated the effect of HA-ZnB NPs on osteoclast differentiation. An osteoclast model was established by inducing RAW264.7 cell differentiation using RANKL. TRAP staining results showed that, compared with the control group, the number and volume of multinucleated TRAP-positive osteoclasts were significantly increased after BetP treatment, while HA-ZnB NPs treatment significantly inhibited osteoclast formation. Figure 8 The F-actin ring staining results further confirmed that the F-actin ring structure characteristic of mature osteoclasts in the HA-ZnBNPs group was significantly reduced (I). Figure 8 (J in the text). The above results indicate that HA-ZnBNPs can not only promote osteogenic differentiation but also inhibit osteoclast formation, thereby helping to maintain the balance of bone remodeling.

[0057] Example 5: Analysis of the therapeutic effect of HA-ZnB NPs in CIA mice.

[0058] Before conducting formal in vivo experiments, a hemolysis test was first used to evaluate the blood compatibility of HA-ZnB NPs. The results showed that at all tested concentrations, the hemolysis rate of HA-ZnB NPs was less than 5%, indicating that it hardly caused hemolysis of erythrocytes, demonstrating good blood compatibility. Figure 9 (A in the middle).

[0059] Subsequently, the in vivo therapeutic effect of HA-ZnB NPs was evaluated in a CIA mouse model. Figure 10The first part (A) includes: establishing a collagen-induced arthritis (CIA) model using 8-10 week old male DBA / 1J mice. Twenty-four DBA / 1J mice were randomly divided into four groups of six each. One group served as the control group, while the other three groups underwent standard modeling procedures. Type II chicken collagen (20012, Chondrex, USA) was mixed with complete Freund's adjuvant (7009, Chondrex, USA), and then subcutaneously injected into the tail root of the mice to induce the CIA model. Once the mice developed clinical symptoms of arthritis, they were treated via tail vein injection with saline, free BetP (0.5 mg / kg), or HA-ZnB NPs (0.5 mg / kg), respectively. Rhodamine B-labeled HA-ZnB NPs were used to evaluate their in vivo targeting ability and biodistribution. Fluorescence imaging analysis was performed at different time points after drug administration using a small animal in vivo fluorescence imaging system (IVIS Spectrum, PerkinElmer, USA). During treatment, disease progression and the degree of local joint inflammation were monitored based on the clinical arthritis score. Each hind limb was scored from 0 to 4: 0 points, normal joint; 1 point, mild erythema and / or swelling; 2 points, moderate erythema and swelling; 3 points, severe swelling; 4 points, joint ankylosis and inability to bear weight. The scores of all four limbs were summed to obtain the total arthritis score for each mouse, with a maximum of 16 points. After the experiment, high-resolution micro-CT scans were performed on the hind paws and distal femurs of the mice to evaluate joint and bone tissue damage. After fixation, decalcification, and paraffin embedding, joint tissues were stained with hematoxylin-eosin (H&E), Masson's trichrome staining, Safranin O-Fast Green staining, and Toluidine Blue staining to evaluate synovial inflammation, cartilage damage, and collagen fiber changes. Immunofluorescence (IF) and immunohistochemistry (IHC) were used to detect the expression of IL-1β, IL-6, TNF-α, and MMP-13. Distal femoral tissues were stained with H&E, and IHC was used to detect CK and ALP levels, as well as tartrate-resistant acid phosphatase (TRAP) staining and IF was used to detect RUNX2 and OCN levels to evaluate bone formation and resorption. Immunofluorescence images were analyzed semi-quantitatively using ImageJ 1.44 software.

[0060] In vivo fluorescence imaging results showed that after tail vein injection, HA-ZnB NPs were continuously enriched in inflamed joints, demonstrating good inflammatory targeted delivery capabilities. Figure 10 B in Figure 9 (B in the formula). Consistent with its excellent targeting performance, HA-ZnB NPs significantly improved arthritis symptoms in CIA mice. The arthritis score decreased from 7.5 points in the model group to 2.0 points, while the free BetP group only decreased to 3.8 points. Figure 10 (C in the text). Furthermore, HA-ZnB NPs can significantly alleviate weight loss and hindfoot swelling during disease progression (C in the text). Figure 10 D in Figure 9 (C and D in the text). Micro-CT results showed that the CIA model group exhibited significant bone erosion and joint structure destruction, while HA-ZnB NPs treatment significantly protected the joint bone structure and reduced the degree of bone erosion, demonstrating superior therapeutic efficacy compared to the free BetP group. Figure 10 E in Figure 9 E in the text). Histological analysis further confirmed that HA-ZnB NPs can significantly alleviate pathological changes such as synovial hyperplasia, inflammatory cell infiltration, and cartilage degradation. Figure 10 The F in the image was used. Further immunofluorescence and immunohistochemistry were used to detect the level of local joint inflammation. The results showed that, compared with free BetP, HA-ZnBNPs significantly reduced the expression levels of IL-1β, IL-6, TNF-α, and MMP-13. Figure 10 GJ in the middle, Figure 9 The presence of F and G in the formula indicates that it has a stronger ability to inhibit local joint inflammation and matrix degradation.

[0061] Simultaneously, serum inflammatory factors and routine blood indicators in mice were analyzed. The CIA model group showed significantly elevated serum IL-6 levels, accompanied by neutrophilia and platelet elevation. Treatment with HA-ZnB NPs significantly improved all of these inflammatory indicators, demonstrating superior therapeutic efficacy compared to free BetP (…). Figure 9 (HN in the text). In summary, HA-ZnB NPs can effectively inhibit local and systemic inflammatory responses by enhancing targeted enrichment in inflamed joints, and significantly improve arthritis symptoms and joint tissue damage in CIA mice.

[0062] In the CIA model, persistently elevated levels of systemic inflammatory factors such as IL-6 and IL-1β severely disrupt bone metabolism balance, inducing secondary osteoporosis. This pathological process manifests as uncoupled bone remodeling, characterized by abnormally enhanced osteoclast activity and significantly suppressed osteoblast differentiation and bone formation. Although BetP has good anti-inflammatory effects, long-term use not only fails to effectively protect bone mass but also, due to high systemic exposure, can directly induce osteoblast apoptosis and inhibit their differentiation, thereby further exacerbating bone loss. Therefore, the inventors further evaluated the protective effect of HA-ZnB NPs against systemic bone loss in CIA mice. Micro-CT three-dimensional reconstruction results showed that the distal femur in the CIA model group exhibited typical osteoporotic features, including significantly thinned and fractured trabeculae and widened trabecular gaps. Figure 11 (A in the text). Quantitative analysis further confirmed that, compared with the normal group, the CIA model group had significantly lower BV / TV, Tb.N, and Tb.Th, while Tb.Sp was significantly higher (A in the text). Figure 11 The bone loss was further aggravated after treatment with free BetP, and the destruction of trabecular microstructure was more obvious. In contrast, HA-ZnB NPs significantly improved trabecular structure, increased BV / TV, Tb.N and Tb.Th, and significantly reduced Tb.Sp, showing excellent osteoprotective effects. H&E staining results further showed that the trabecular bone in the distal femur of CIA mice was significantly reduced and disordered, while the above pathological changes were significantly improved after treatment with HA-ZnB NPs; significant trabecular bone loss was still observed in the BetP group. Figure 11 F in the text). Further immunohistochemistry was used to evaluate bone remodeling. Results showed that the number of CK-positive and TRAP-positive osteoclasts was significantly increased in the CIA model group, while the activity of ALP-positive osteoblasts was significantly decreased (F in the text). Figure 11 The presence of G and H in the protein suggests increased bone resorption and suppressed bone formation. BetP treatment did not significantly improve these changes, while HA-ZnB NPs significantly reduced osteoclast numbers and restored osteoblast activity. Immunofluorescence assays also showed that RUNX2 and OCN expression was significantly reduced in the CIA model group, remained at low levels in the BetP group, while HA-ZnB NPs treatment significantly restored the expression of both osteoblast proteins. Figure 11 (I and J in the text).

[0063] In addition, the in vivo safety of HA-ZnB NPs was evaluated by H&E staining of major organs (heart, liver, spleen, lungs, and kidneys). The results showed that no significant tissue structural abnormalities or pathological damage were observed in any of the experimental groups. Figure 12As shown in the figure, HA-ZnB NPs have good in vivo biocompatibility. In summary, HA-ZnB NPs can significantly improve systemic bone loss in CIA mice, restore bone remodeling balance, and effectively alleviate secondary osteoporosis caused by rheumatoid arthritis.

[0064] The above embodiments construct a novel nanomedicine system based on a metal coordination enhancement strategy, achieving simultaneous improvement in the anti-inflammatory efficacy and osteoprotective effect of glucocorticoids. Existing glucocorticoid nanodelivery systems mainly reduce systemic toxicity by improving drug distribution in vivo and targeted enrichment in inflamed tissues; essentially, these are still pharmacokinetic optimization strategies and do not alter the intrinsic interaction between GCs and receptors. However, the inventors have demonstrated for the first time that Zn... 2+ Not only does it serve as a building block for nanostructures, but it can also act as a "pharmacological enhancer," directly strengthening the binding affinity between BetP and GR at the molecular level. Molecular dynamics simulations show that Zn... 2+ Coordination significantly enhanced the van der Waals interaction between BetP and the GR ligand binding pocket, reducing the binding free energy to the lowest level among all divalent metal ions, thereby stabilizing the BetP binding conformation and improving the GR binding affinity. This suggests that rational metal ion coordination may be a universal strategy for optimizing small molecule drug-receptor interactions. Based on the above theoretical foundation, the inventors further constructed a Zn... 2+ The -BetP coordination nanoplatform, combined with the introduction of hyaluronic acid, enhances the system's dispersion stability and CD44-mediated active targeting capability. The resulting HA-ZnB NPs exhibit excellent acid-responsive release characteristics, remaining stable under physiological conditions while rapidly releasing under acidic conditions in the inflammatory microenvironment, enabling precise drug delivery to arthritis lesions.

[0065] Cellular experiments showed that HA-ZnB NPs can efficiently target inflammatory macrophages through HA-CD44 interaction, increasing BetP and Zn levels. 2+ The enhanced intracellular delivery efficiency of HA-ZnB NPs further amplified glucocorticoid signaling, significantly inhibited NF-κB pathway activation, and promoted macrophage polarization from M1 to M2. Simultaneously, HA-ZnB NPs further blocked the aberrantly activated macrophage-FLS inflammatory axis in RA, effectively reducing FLS migration and invasion capacity and MMPs and DKK-1 expression by inhibiting the MAPK signaling pathway, thereby alleviating cartilage destruction and bone erosion. Notably, HA-ZnB NPs also remodeled the biological response of bone tissue during glucocorticoid treatment. Unlike the osteogenic inhibition caused by free BetP, HA-ZnB NPs, through Zn... 2+It reactivates the Wnt / β-catenin signaling pathway, restores osteoblast differentiation capacity, and simultaneously inhibits osteoclast formation, thereby re-establishing the balance of bone remodeling.

[0066] CIA animal experiments further validated the above mechanism. HA-ZnB NPs not only significantly reduced arthritis scores, improved joint inflammation and cartilage damage, but also significantly inhibited systemic inflammatory responses and effectively alleviated secondary osteoporosis. Simultaneously, due to their excellent inflammatory targeting ability, HA-ZnB NPs can reduce the exposure level of glucocorticoids in normal tissues, thereby reducing side effects such as neutrophilia caused by free BetP, and exhibiting good in vivo biocompatibility.

[0067] In summary, the above embodiments present, for the first time, a molecular dynamics-guided metal coordination nanomedicine design strategy. Using Zn... 2+ By enhancing the synergistic effects of BetP-GR binding, HA-mediated inflammation targeting, and acid-responsive release, HA-ZnBNPs achieve simultaneous improvement in inflammation suppression and bone homeostasis maintenance in rheumatoid arthritis, providing a promising new treatment strategy for rheumatoid arthritis and other inflammatory bone destructive diseases.

[0068] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing metal-glucocorticoid nanoparticles, characterized in that, Betamethasone sodium phosphate solution was mixed with sodium hyaluronate solution, and then zinc ion source solution was added to react and obtain metal-glucocorticoid nanoparticles.

2. The method for preparing metal-glucocorticoid nanoparticles as described in claim 1, characterized in that, The ratio of the betamethasone sodium phosphate, the sodium hyaluronate, and the zinc ions is (0.025~0.1) mol: (35~45) mg: (0.025~0.1) mol.

3. The method for preparing metal-glucocorticoid nanoparticles as described in claim 1, characterized in that, The concentration of the betamethasone sodium phosphate solution is 0.025~0.1M, the concentration of the sodium hyaluronate solution is 35~45 mg / mL, the concentration of zinc ions in the zinc ion source solution is 0.025~0.1M, and the betamethasone sodium phosphate solution, the sodium hyaluronate solution, and the zinc ion source solution are reacted in equal volume ratios.

4. The method for preparing metal-glucocorticoid nanoparticles as described in claim 1, characterized in that, The zinc ion source solution is an aqueous solution of Zn(NO3)2•6H2O.

5. The method for preparing metal-glucocorticoid nanoparticles as described in claim 1, characterized in that, The mixing time is 4-6 minutes, and the reaction time is 4-6 minutes.

6. The method for preparing metal-glucocorticoid nanoparticles as described in claim 1, characterized in that, The mixing and reaction were carried out at room temperature.

7. The method for preparing metal-glucocorticoid nanoparticles as described in claim 1, characterized in that, The reaction is a self-assembly reaction in which zinc metal coordinates with phosphate groups.

8. The method for preparing metal-glucocorticoid nanoparticles as described in claim 1, characterized in that, The process also includes the following steps: centrifuging the reaction system at 11000~13000 r / min for 8~12 min after the reaction is completed, collecting the precipitate, and washing it several times with deionized water to obtain the metal-glucocorticoid nanoparticles, which are then stored for later use.

9. Metal-glucocorticoid nanoparticles prepared by a method according to any one of claims 1-8.

10. An application of the metal-glucocorticoid nanoparticles as described in claim 9, characterized in that, The application includes at least one of the following (a1) to (a2); (a1) To prepare products that inhibit rheumatoid arthritis; (a2) Prepare products that inhibit bone loss.

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