Gallium metal organic framework material, preparation method and application thereof

Abstract

The application discloses a gallium metal organic framework material, which is prepared through the following steps: (1) dissolving 2-amino terephthalic acid and itaconic acid in an organic solvent to obtain solution A; (2) dissolving gallium nitrate hydrate in an organic solvent to obtain solution B containing gallium ions; and (3) mixing solution A and solution B, heating under the condition of 50-70 DEG C for 3-5 hours, centrifuging and collecting the precipitate, and purifying to obtain the gallium metal organic framework material. The gallium metal organic framework material is constructed by combining itaconic acid and 2-amino terephthalic acid as a double ligand and taking gallium ions as a metal node, so that the technical problems of short biological half-life and low cell uptake efficiency of free itaconic acid are solved, the itaconic acid and the gallium ions are coordinated to play an anti-inflammatory and antibacterial role, to remove planktonic bacteria, and to have strong antibiofilm activity, and the gallium metal organic framework material can be used for preparing a drug for treating pyogenic arthritis.

Description

Technical Field

[0001] This invention belongs to the field of organic framework materials technology, specifically relating to gallium metal organic framework materials, their preparation methods, and applications. Background Technology

[0002] Septic arthritis (SA) is a devastating orthopedic emergency characterized by rapid joint destruction, high recurrence rates, and poor functional prognosis. These pathological processes are primarily driven by persistent intracellular bacterial infection and unresolved infection-induced inflammation. With an aging population and increased invasive medical procedures and immunosuppressive therapy, synovial joints, especially the knee, are increasingly vulnerable to iatrogenic or exogenous bacterial invasion. Among these pathogens, the incidence of methicillin-resistant Staphylococcus aureus (MRSA) infection continues to rise. Bacterial invasion can induce a strong phagocytic response and activate innate immune defenses; when the bacterial load is too high, the pathogens are often not completely eliminated. Although vancomycin remains the first-line antibiotic for treating MRSA, its clinical efficacy is limited, and the recurrence rate is high. This challenge stems primarily from the persistence of bacteria within macrophages, followed by their re-release into the joint cavity. Therefore, thorough clearance of intra-articular bacteria, especially intracellular pathogens residing within macrophages, is crucial for the effective treatment of SA. Infection in synovial inflammatory syndrome (SA) can induce oxidative stress and inflammatory pyroptosis in macrophages, leading to synovial inflammation, cartilage damage, and joint dysfunction. Persistent inflammatory stimulation also promotes inflammatory senescence in macrophages; senescent cells amplify inflammatory signals by secreting senescence-associated secretory phenotypes (SASPs), ultimately driving chronic joint damage. However, effectively and precisely controlling infection-induced excessive joint inflammation remains an unmet need in the current clinical treatment of SA. Traditional antibiotic treatment often fails to eliminate bacteria residing within macrophages and also fails to alleviate subsequent inflammatory pyroptosis and inflammation-related cellular senescence, all of which contribute to chronic joint damage.

[0003] Iron is a crucial element essential for microbial survival and biofilm formation, participating in various enzymatic reactions and metabolic processes. Due to the similarity between gallium and iron in coordination properties and ionic radii, bacterial iron uptake systems struggle to effectively distinguish between the two, allowing gallium ions to competitively interfere with and disrupt bacterial iron metabolism, thus endowing gallium with significant antibacterial activity. Furthermore, gallium ions can exert anti-inflammatory effects by regulating the production of pro-inflammatory cytokines and nitric oxide in activated immune cells. The endogenous metabolite itaconic acid (ITA) shows potential in the treatment of inflammatory and infectious diseases because it can inhibit NLRP3 inflammasome-mediated inflammatory pyroptosis and suppress bacterial metabolism by competitively inhibiting isocitrate lyase (ICL). However, the free form of ITA has limitations such as rapid in vivo clearance, short biological half-life, and low cellular uptake efficiency, significantly limiting its in vivo therapeutic efficacy. Therefore, there is an urgent need to develop gallium metal-organic framework materials that simultaneously target multiple pathological mechanisms of acute sarcopenia (SA). Summary of the Invention

[0004] The purpose of this invention is to provide gallium metal-organic framework materials and their preparation methods to solve the technical problem that existing materials cannot simultaneously target multiple pathological mechanisms of pyogenic arthritis and have good therapeutic effects on pyogenic arthritis.

[0005] According to a first aspect of the present invention, a gallium metal-organic framework material is provided, which is prepared by the following steps:

[0006] (1) Dissolve 2-aminoterephthalic acid and itaconic acid in an organic solvent to obtain solution A;

[0007] (2) Dissolve gallium nitrate hydrate in an organic solvent to obtain a solution B containing gallium ions;

[0008] (3) Mix solution A and solution B, heat at 50-70℃ for 3-5 hours, centrifuge and collect the precipitate, purify, and obtain the final product.

[0009] This invention relates to a gallium metal-organic framework (MOF) material based on the antibacterial and immunomodulatory properties of gallium ions and itaconic acid. By coordinating gallium ions with ITA and ATA, using gallium ions as metal nodes, and itaconic acid and 2-aminoterephthalic acid (ATA) as ligands, a metabolite-engineered dual-ligand gallium MOF material is constructed. This MOF material can not only efficiently eliminate free bacteria and intracellular bacteria within macrophages, but also inhibit inflammatory signaling pathways, reduce M1 polarization, inflammatory pyroptosis, and the senescent phenotype of activated macrophages.

[0010] In some embodiments, the molar ratio of gallium ions, itaconic acid, and 2-aminoterephthalic acid is (1-1.2):(0.4-0.6):(0.4-0.6). Preferably, the molar ratio of gallium ions, itaconic acid, and 2-aminoterephthalic acid is 1:0.5:0.5.

[0011] In some embodiments, the organic solvent is selected from N,N-dimethylformamide (DMF). Choosing DMF as the organic solvent results in gallium metal-organic framework materials with more uniform structures.

[0012] In some embodiments, the gallium metal-organic framework material has a porous structure; the hydrated particle size of the gallium metal-organic framework material is 60-420 nm.

[0013] In some implementations, the average pore size of the gallium metal-organic framework material is 2.0-2.5 nm.

[0014] According to a second aspect of the present invention, a method for preparing a gallium metal-organic framework material is provided, comprising the following steps:

[0015] (1) Dissolve 2-aminoterephthalic acid and itaconic acid in an organic solvent to obtain solution A;

[0016] (2) Dissolve gallium nitrate hydrate (Ga(NO3)3·xH2O) in an organic solvent to obtain a solution B containing gallium ions;

[0017] (3) Mix solution A and solution B, heat at 50-70℃ for 3-5 hours, centrifuge and collect the precipitate, purify, and obtain the final product.

[0018] In some embodiments, step (3) involves heating at 60°C for 4 hours. The purpose of heating at this temperature is that ITA decomposes at 120°C, and heating at this temperature avoids the decomposition of itaconic acid due to excessive temperature, while also ensuring that both 2-aminoterephthalic acid and itaconic acid can react with Ga. 3+ reaction.

[0019] In some implementations, centrifugation is performed by centrifuging at 8000×g for 30 minutes.

[0020] In some embodiments, in step (3), the purification method is to wash the precipitate three times with DMF and water respectively, then dialyze it in ultrapure water for 96 hours, changing the water every 4 hours, and finally freeze-drying it at -60°C to obtain powder.

[0021] According to a third aspect of the present invention, the use of gallium metal-organic framework materials in the preparation of medicaments for treating pyogenic arthritis is provided.

[0022] This invention develops a gallium-based metal-organic framework material capable of simultaneously targeting multiple pathological mechanisms of acute joint inflammation (SA). In a mouse SA model, it effectively controls joint infection, alleviates synovial inflammation, and protects cartilage structure, providing a potentially transformative therapeutic strategy for this disabling disease. This gallium-based metal-organic framework material can simultaneously intervene in multiple pathogenic mechanisms of SA, offering a potentially transformative strategy for its treatment.

[0023] The beneficial effects of this invention are as follows:

[0024] (1) In this invention, itaconic acid and 2-aminoterephthalic acid are combined as dual ligands and gallium ions are used as metal nodes to jointly construct gallium metal organic framework materials. This not only solves the technical problems of short biological half-life and low cell uptake efficiency of free itaconic acid, but also the coordination of itaconic acid with gallium ions exerts anti-inflammatory and antibacterial effects, clears airborne bacteria, and has strong anti-biofilm activity.

[0025] (2) The gallium metal-organic framework material of the present invention can coordinate the intervention of multiple pathogenic processes of SA, including bacterial infection, inflammation, pyroptosis and inflammatory senescence. It can also be effectively taken up by macrophages and clear intracellular bacteria resistant to vancomycin. It can be used to prepare drugs for treating septic arthritis. Attached Figure Description

[0026] Figure 1 (A) shows the preparation process of GIM; Figure 1 (B) shows the preparation process of GIM; Figure 1 (C) represents the hydrated particle size of GM and GIM; Figure 1 (D) represents the surface zeta potential of GM and GIM; Figure 1 (E) shows the UV-Vis absorption spectra of ATA, ITA, GM, and GIM; Figure 1 (F) shows the TEM and elemental distribution of GM and GIM; Figure 1 (G) represents the full XPS spectrum of GIM; Figure 1 (H) is the Ga2p plot of GIM in XPS analysis; Figure 1 (I) is the Ga 3d plot of GIM in XPS analysis; Figure 1 (J) is the C1s plot of GIM in XPS analysis; Figure 1 (K) is the O 1s plot of GIM in XPS analysis; Figure 1 (L) represents Ga in GIM under different pH conditions. 3+ The release curve; Figure 1 (M) shows the release curves of ITA from GIM under different pH conditions;

[0027] Figure 2 (A) is the full XPS spectrum of GM; Figure 2(B) is the Ga 2p plot of GM in XPS analysis; Figure 2 (C) is the Ga 3d plot of GM in XPS analysis; Figure 2 (D) is the C1s plot of GM in XPS analysis; Figure 2 (E) is the O 1s plot of GM in XPS analysis; Figure 2 (F) is the TGA curve of GIM; Figure 2 (G) is the TGA curve of GM; Figure 2 (H) is the N2 adsorption-desorption isotherm of GIM; Figure 2 (I) shows the aperture distribution of the GIM;

[0028] Figure 3 (A) shows MRSA colonies on TSA plates in each group; Figure 3 (B) Quantitative analysis of CFUs from MASA colonies in each group;

[0029] Figure 4 (A) PI / SYTO staining flow cytometry analysis of MRSA in each group; Figure 4 (B) represents the PI for each group + Quantitative analysis of bacterial population proportions; Figure 4 (C) represents each group of SYTO + Quantitative analysis of bacterial population proportions;

[0030] Figure 5 (A) Crystal violet staining images of MRSA biofilms in each group during the biofilm formation experiment; Figure 5 (B) represents the biofilm inhibition rate of each group in the biofilm formation experiment; Figure 5 (C) Quantitative analysis of CFUs in biofilms of each group in the biofilm formation experiment;

[0031] Figure 6 (A) Crystal violet staining images of MRSA biofilms in each group during the biofilm disruption experiment; Figure 6 (B) represents the biofilm disruption rate in each group during the biofilm disruption experiment; Figure 6 (C) Quantitative analysis of CFUs in biofilms of each group in the biofilm disruption experiment;

[0032] Figure 7 (A) shows the colocalization of GM and GIM with lysosomes in RAW264.7 cells; Figure 7 (B) shows the co-localization of GM and GIM with MSRA in RAW264.7 cells; Figure 7 (C) shows the colocalization analysis of GM and lysosomes; Figure 7 (D) shows the colocalization analysis of GIM and lysosomes; Figure 7 (E) shows the co-location analysis of GM and MSRA; Figure 7(F) shows the co-location analysis of GIM and MSRA; Figure 7 (G) are representative images of MRSA colonies in RAW264.7 cells on TSA plates after different treatments; Figure 7 (H) represents the quantitative analysis of CFUs of MRSA in RAW264.7 cells;

[0033] Figure 8 (A) Flow cytometry analysis of RAW264.7 cells in each group; Figure 8 (B) represents Arg1 for each group + / iNOS + Quantitative analysis of cell subset ratios; Figure 8 (C) represents the IL-6 level in the cell supernatant of each group; Figure 8 (D) represents the TNF-α level in the cell supernatant of each group; Figure 8 (E) represents the IL-4 level in the cell supernatant of each group; Figure 8 (F) represents the IL-10 level in the cell supernatant of each group; Figure 8 (G) shows CLSM images of RAW264.7 cells stained with DCFH-DA in each group; Figure 8 (H) represents the quantitative analysis of the average fluorescence intensity of RAW264.7 cells in each group; Figure 8 (I) Representative bright-field images of β-galactosidase staining in cells of each group; Figure 8 (J) Western blot analysis of p16 and p21 expression in RAW264.7 cells of each group; Figure 8 (K) is a schematic diagram of the preparation of conditioned medium; Figure 8 (L) shows the results of toluidine blue and alicin blue staining of chondrocytes after treatment with conditioned medium in each group; Figure 8 (M) Western blot analysis of the expression of COL2A1, SOX9, MMP13 and ADAMTS-5 in chondrocytes after treatment with conditioned medium in each group; Figure 8 (N) are CLSM immunofluorescence images of chondrocytes after treatment with conditioned medium in each group;

[0034] Figure 9 (A) Cell viability of chondrocytes treated with different concentrations of GIM; Figure 9 (B) Cell viability of RAW264.7 cells treated with different concentrations of GIM; Figure 9 (C) Western blot analysis of iNOS and CD206 in RAW264.7 cells of each group; Figure 9 (D) Quantification of iNOS density in RAW264.7 cells of each group; Figure 9 (E) Quantification of CD206 density in RAW264.7 cells of each group; Figure 9(F) represents β-Gal after different treatments + Quantitative analysis of the proportion of RAW264.7 cells; Figure 9 (G) Western blot quantification of p16 density in RAW264.7 cells in each group; Figure 9 (H) Western blot quantification of p21 density in RAW264.7 cells of each group; Figure 9 (I) Western blot quantification of COL2A1 chondrocyte density in each group under conditioned medium. Figure 9 (J) Western blot quantification of SOX9 density in chondrocytes under conditioned medium in each group; Figure 9 (K) represents the Western blot density of MMP13 in chondrocytes under conditioned medium in each group. Figure 9 (L) Western blot quantification of ADAMTS-5 density in chondrocytes under conditioned medium in each group; Figure 9 (M) represents the average fluorescence intensity of ACAN in chondrocytes under the action of conditioned medium in each group; Figure 9 (N) represents the average fluorescence intensity of MMP3 in chondrocytes under conditioned medium in each group.

[0035] Figure 10 (A) Representative bright-field images of RAW264.7 cells in each group; Figure 10 (B) CLSM images of RAW264.7 cells labeled with NLRP3 and GSDMD in each group; Figure 10 (C) Quantitative analysis of the average fluorescence intensity of NLRP3 in RAW264.7 cells of each group; Figure 10 (D) Quantitative analysis of the average fluorescence intensity of GSDMD in RAW264.7 cells of each group; Figure 10 (E) Western blot analysis of NLRP3, GSDMD and caspase-1 expression in RAW264.7 cells of each group; Figure 10 (F) ELISA quantitative analysis of IL-1β levels in RAW264.7 cell supernatant of each group; Figure 10 (G) ELISA quantitative analysis of IL-18 levels in RAW264.7 cell supernatant of each group; Figure 10 (H) is Figure 10 ; Figure 10 (I) The relative intracellular ATP levels of RAW264.7 cells in each group; Figure 10 (J) Western blot analysis of p16 and p21 expression in RAW264.7 cells of each group; Figure 10 (K) shows the β-Gal staining pattern of RAW264.7 cells in each group; Figure 10 (L) is a schematic diagram of the preparation of conditioned medium for each group; Figure 10 (M) shows the toluene blue and acetate blue staining of chondrocytes; Figure 10 (N) is a CLSM immunofluorescence image of chondrocytes;

[0036] Figure 11 (A) Quantification of the relative expression density of NLRP3 in RAW264.7 cells of each group; Figure 11 (B) Quantification of the relative expression density of GSDMD in RAW264.7 cells of each group; Figure 11 (C) Quantification of the relative expression density of caspase-1 protein in RAW264.7 cells of each group; Figure 11 (D) represents the quantification of the percentage of RAW264.7 cells with β-galactosidase in each group; Figure 11 (E) Quantification of p16 protein density in RAW264.7 cells of each group; Figure 11 (F) Quantification of p21 protein density in RAW264.7 cells of each group; Figure 11 (G) represents the quantification of the average fluorescence intensity of ACAN in chondrocytes of each group; Figure 11 (H) represents the quantification of the average fluorescence intensity of MMP3 in chondrocytes of each group;

[0037] Figure 12 (A) is a schematic diagram of the establishment and treatment of a mouse model of suppurative arthritis; Figure 12 (B) shows representative images of the knee joints of mice in each group; Figure 12 (C) White blood cell counts in each group of mice; Figure 12 (D) represents the red blood cell count of mice in each group; Figure 12 (E) Count of lymphocytes in each group of mice; Figure 12 (F) represents the monocyte count of mice in each group; Figure 12 (G) represents the neutrophil count in each group of mice; Figure 12 (H) shows CLSM images of GFP-labeled MRSA in the synovium of mice in each group; Figure 12 (I) MRSA colony diagrams formed on TSA plates by each group of synovial flushing fluids; Figure 12 (J) Quantification of MRSA colony CFUs formed on TSA plates by each group of synovial flushing fluids; Figure 12 (K) is a diagram of MRSA colonies formed on TSA plates in the tissues surrounding the knee joint in each group; Figure 12 (L) Quantification of MRSA colony CFUs formed on TSA plates in the tissues around the knee joint in each group;

[0038] Figure 13 (A) H&E staining images of synovium in each group; Figure 13 (B) is a diagram of safranin O staining of each group of cartilage; Figure 13 (C) shows the TRAP staining pattern of subchondral bone in each group; Figure 13 (D) represents the inflammation score for each group; Figure 13 (E) OARSI scores for each group; Figure 13 (F) represents the cartilage degeneration score for each group; Figure 13 (G) TRAP groups + Quantitative analysis of cell proportions; Figure 13 (H) shows CLSM immunofluorescence staining images of iNOS cells in synovial macrophages from each group; Figure 13 (I) CLSM immunofluorescence staining images of Arg1 in synovial macrophages of each group; Figure 13 (J) shows CLSM immunofluorescence staining images of aggregated proteins in cartilage of each group; Figure 13 (K) Immunohistochemical staining images of MMP13 in cartilage of each group; Figure 13 (L) Quantification of the average fluorescence intensity of iNOS colocalized in F4 / 80 in synovial macrophages of each group; Figure 13 (M) represents the average fluorescence intensity of Arg1 colocalized in F4 / 80 synovial macrophages in each group; Figure 13 (N) represents the quantification of the average fluorescence intensity of ACAN in the cartilage of each group; Figure 13 (O) represents MMP13 in the cartilage of each group. + Quantitative analysis of cell proportions;

[0039] Figure 14 (A) is an immunofluorescence staining image of NLRP3 and GSDMD in synovial tissue using CLSM; Figure 14 (B) Quantitative analysis of the average fluorescence intensity of NLRP3 in synovial tissue; Figure 14 (C) Quantitative analysis of the average fluorescence intensity of GSDMD in synovial tissue; Figure 14 (D) is an immunohistochemical staining image of p-p65 in synovial tissue; Figure 14 (E) is an immunohistochemical staining image of phosphorylated p-p38 in synovial tissue; Figure 14 (F) shows the immunohistochemical staining of p16 in synovial tissue; Figure 14 (G) is an immunohistochemical staining image of p21 in synovial tissue; Figure 14 (H) represents the quantitative analysis of the average optical density of p-p65 in synovial tissue; Figure 14 (I) Quantitative analysis of the average optical density of p-p38 in synovial tissue; Figure 14 (J) is a quantitative analysis of the average optical density of p16 in synovial tissue; Figure 14 (K) represents the quantitative analysis of the average optical density of p21 in synovial tissue;

[0040] Figure 15 (A) H&E staining images of organ and tissue sections in each group; Figure 15(B) Immunofluorescence staining image of COL2A1 in a section of mouse knee joint cartilage tissue; Figure 15 (C) Quantitative analysis of COL2A1 fluorescence intensity in mouse knee joint cartilage tissue sections;

[0041] Figure 16 (A) is the GIM preparation process; Figure 16 (B) is the therapeutic target of GIM in septic arthritis; Figure 16 (C) illustrates the mechanism of action of GIM in suppurative arthritis. Detailed Implementation

[0042] The present invention will now be described in further detail with reference to the accompanying drawings, but the embodiments of the present invention are not limited thereto. The raw materials and reagents involved in the following embodiments are all commercially available.

[0043] In this invention, data are expressed as mean ± standard deviation. Statistical analysis was performed using an unpaired two-sided Student's t-test (ns, no statistically significant difference; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).

[0044] I. Materials and Reagents

[0045] All chemicals are of analytical grade and require no further purification before use.

[0046] 2-Aminoterephthalic acid, gallium(III) nitrate hydrate, acetic acid, and N,N-dimethylformamide (DMF) were purchased from Maclean's. Itaconic acid (ITA) was purchased from Ruihao. Unless otherwise specified, all other reagents were purchased from Sigma-Aldrich. All aqueous solutions were prepared using ultrapure water (18.2 MΩ·cm) purified using a Millipore purification system (Millipore, USA).

[0047] II. Instruments

[0048] High-resolution transmission electron microscopy (HRTEM) and energy dispersive spectroscopy (EDS) were performed using a JEM-2100F microscope with an accelerating voltage of 200 kV.

[0049] X-ray photoelectron spectroscopy (XPS) analysis was performed using an Escalab 250Xi system (Thermo Fisher Scientific).

[0050] The instrument used for ultraviolet-visible absorption spectroscopy analysis was a UV-2600i photometer (Shimadzu).

[0051] The nitrogen adsorption-desorption test was performed using a surface area and porosity analyzer (Micromeritics Tristar 3020, USA). The cumulative pore volume was calculated using the Barrett-Joyner-Halenda (BJH) model through the adsorption branch of the adsorption isotherm.

[0052] Example 1

[0053] This embodiment provides a method for preparing gallium metal-organic framework (GIM) materials, including the following steps:

[0054] 2-Aminoterephthalic acid (40 mg, 0.22 mmol) and itaconic acid (28.6 mg, 0.22 mmol) were dissolved in 5 mL of DMF and stirred at room temperature for 15 minutes to obtain solution A. Gallium nitrate hydrate (Ga(NO3)3·xH2O, 110 mg, 0.44 mmol) was dissolved in 10 mL of DMF and stirred for 15 minutes to obtain solution B. Solutions A and B were mixed and stirred for 15 minutes, then heated at 60 °C for 4 hours. After cooling, the mixture was centrifuged at 8000 × g for 20 minutes, the precipitate was collected, washed three times each with DMF and ultrapure water, then dialyzed against ultrapure water for 96 hours, changing the water every 4 hours. Finally, the mixture was lyophilized at -60 °C to obtain a powder, which was stored at room temperature.

[0055] Comparative Example 1

[0056] This comparative example provides another method for preparing gallium metal-organic framework (GM) materials, including the following steps:

[0057] 2-Aminoterephthalic acid (80 mg, 0.44 mmol) was dissolved in 5 mL of DMF containing 30% (v / v) acetic acid and stirred at room temperature for 15 minutes to obtain solution A. Gallium nitrate hydrate (Ga(NO3)3·xH2O, 110 mg, 0.44 mmol) was dissolved in 10 mL of DMF containing 30% acetic acid and stirred for 15 minutes to obtain solution B. Solutions A and B were mixed and stirred for 15 minutes, then transferred to a PTFE-lined stainless steel autoclave and reacted at 120 °C for 12 hours. After cooling to room temperature, the reaction mixture was centrifuged at 8000 × g for 30 minutes, and the precipitate was collected. The precipitate was washed three times each with DMF and ultrapure water, and then dialyzed against ultrapure water for 96 hours, changing the water every 4 hours. The final product was obtained by lyophilization at -60 °C and stored at room temperature.

[0058] The preparation process of GIM and GM in this invention is as follows: Figure 1 As shown in (A) and (B), GIM and GM are characterized below.

[0059] 1. Characterization

[0060] Dynamic light scattering was used to characterize the hydration size and zeta potential of GM and GIM. The results are as follows: Figure 1 (C) and Figure 1 As shown in (D), GIM and GM have similar hydration particle sizes, with GIM's hydration particle size distribution ranging from 60 to 420 nm (see Figure 1). Figure 1 (C)); however, the zeta potentials of the two show opposite trends, which may be attributed to the additional introduction of ITA ligands.

[0061] UV-Vis absorption spectra of GM and GIM (see) Figure 1 (E) shows that ATA and ITA have characteristic absorption peaks at 241 nm and 219 nm, respectively. Corresponding absorption characteristics were also observed in GIM, indicating that the two ligands maintain structural integrity within the framework and are co-distributed within the GIM framework. The absorption characteristics of GM also indicate that its composition is as expected. These results demonstrate the successful preparation of GIM as a material.

[0062] The morphology and elemental distribution of GM and GIM were analyzed using transmission electron microscopy (TEM) combined with energy-dispersive X-ray spectroscopy (EDS). The results are shown in Figure 1(F). As can be seen from the figure, Ga, C, and O elements are uniformly distributed in both GM and GIM.

[0063] The chemical bonding states of Ga, C, and O on the surfaces of GM and GIM were studied using X-ray photoelectron spectroscopy (XPS), and the results are as follows: Figure 1 As shown in (G)-(K), the XPS spectra of GIM show two distinct peaks for Ga 2p at 1114.85 eV and 1118.02 eV, while the Ga 3d peak is located at 20.64 eV; the binding energies for C 1s and O 1s are located at 288.6 eV and 531.77 eV, respectively. The observed positive shifts in the binding energies of Ga 2p and Ga 3d, accompanied by co-shifts in the O 1s and carboxylate C 1s signals, indicate a redistribution of electron density during coordination. This type of multi-element co-shift is a typical feature of Ga-O coordination bond formation in GIM. Similar XPS features have also been observed in GM (see [link to relevant data]). Figure 2 (A)-(E)) indicates that they have similar coordination environments.

[0064] Further investigation into the pH-responsive release behavior of ITA and Ga ions, and Ga in GIM 3+ The release kinetics were assessed by dialysis. GIM (1 mg mL) was added... -11 mL of solution was placed into a dialysis bag (MWCO 3500 Da) and immersed in PBS buffer at pH 6.5 or 7.4. Samples were taken at predetermined time points (0, 2, 4, 6, 8, 10, 12, 24, 36, 48, 72, and 96 hours), and Ga was determined using inductively coupled plasma mass spectrometry (ICP-MS, PerkinElmer NexION 300X, USA). 3+ concentration.

[0065] The release behavior of ITA was analyzed by UV-Vis spectroscopy. A standard curve (characteristic absorption peak at 219 nm) was established. GIM (1 mg) was dispersed in 5 mL of deionized water at pH 6.5 or 7.4 and gently stirred. The supernatant was collected at predetermined time points to measure the absorbance, thereby calculating the release concentration.

[0066] Ga in GIM under different pH conditions 3+ The release curve of ITA is as follows Figure 1 (L) and Figure 1 As shown in (M). The figure shows that under slightly acidic conditions (pH=6.5), the release rates of both ITA and Ga are faster than under physiological conditions (pH=7.4).

[0067] Thermogravimetric analysis results of GIM and GM ( Figure 2 (F)-(G)) indicates that the quality of GM and GIM begins to gradually decrease above 100°C.

[0068] Nitrogen adsorption-desorption isotherms and pore size distribution of GIM were tested, and the results are as follows: Figure 2 (H) and Figure 2 As shown in (I), GIM has a porous structure with an average pore size of approximately 2.39 nm.

[0069] 2. In vitro antibacterial and anti-infective effects of GIM

[0070] To evaluate the antibacterial activity of GIM against free and intracellular bacteria, methicillin-resistant Staphylococcus aureus (MRSA) was selected as the model pathogen.

[0071] 2.1 Culture of methicillin-resistant Staphylococcus aureus (MRSA)

[0072] GFP-labeled MRSA (GFP-MRSA) was provided by Liang Haifeng of Guangzhou Medical University. The strain was stored at -80℃ and, after resuscitation, inoculated onto tryptone soybean agar (TSA) plates and cultured at 37℃ for 24 hours. GFP fluorescence was observed using a stereomicroscope, and single colonies were selected for monoclonal amplification. After the strain reached the logarithmic growth phase, it was washed with PBS to obtain a bacterial suspension, and the concentration of the bacterial suspension was adjusted to a standard value (OD). 600=1.0), it should be noted that PBS was used to dilute to OD 600 =1.0 is used for experiments.

[0073] 2.2 Antibacterial test

[0074] Bacterial suspension OD 600 Adjust to 1, add 200 μL of suspension and corresponding treatment to each group: PBS group, vancomycin (Vanco) group (10 μg / mL). -1 Vancomycin), ITA group (5 mM ITA), GM group (based on Ga content, detected by ICP-MS; 50 μg / mL) -1 ), GIM group (calculated as Ga, 50 μg mL) -1 Incubate at 37°C on a shaker for 6 hours, then dilute and spread on TSA plates for colony counting.

[0075] Figure 3 (A) shows MRSA colonies on TSA plates for each group. Figure 3 (B) Quantitative analysis of MASA colonies (CFU) in each group showed that the antibacterial experiment results indicated that at an initial bacterial density of 10... 8 CFU·mL -1 Under these conditions, the ITA and GM groups only showed moderate sterilization effects. In contrast, the GIM group had a sterilization efficiency exceeding 99%, comparable to the Vanco group.

[0076] 2.3 Flow cytometry analysis

[0077] After 6 hours of antibacterial incubation, the MRSA in each group was fixed, permeabilized, and blocked, and then incubated with FITC-iNOS and APC-Arg1 antibodies for flow cytometry analysis.

[0078] PI / SYTO staining and flow cytometry analysis of MRSA in each group are shown below. Figure 4 (A); PIs in each group + Quantitative analysis of bacterial population proportions is available in [link to relevant documentation]. Figure 4 (B); SYTO groups + Quantitative analysis of bacterial population proportions is available in [link to relevant documentation]. Figure 4 (C). Flow cytometry analysis of bacterial viability showed that the SYTO cells in the GIM group... + The bacterial population was significantly lower than that of the GM and ITA groups, further demonstrating that the GIM group had superior sterilization efficiency.

[0079] 2.4 Experiment on biofilm formation and disruption

[0080] Given that biofilm formation plays a crucial role in bacterial resistance, the inhibitory effect of GIM on biofilm formation was further evaluated. 1% sucrose was added to TSB to promote stable biofilm formation.

[0081] Biofilm formation experiment: The concentration OD of the bacterial suspension was determined using PBS. 600 =1, and then inoculated into 48-well plates, 200 μL per well, adding the treatment (vancomycin, ITA, GM, and GIM added at the same amounts as in the antibacterial experiment), fixed after 24 hours, stained with crystal violet, and the OD was measured. 560 Parallel experiments were conducted to verify the results by ultrasonic dispersion and colony counting.

[0082] Biofilm formation experiment: The concentration OD of the bacterial suspension was determined using PBS. 600 =1, and then inoculated into 48-well plates, 200 μL per well, and cultured until the formation of a stable biofilm was observed. The appropriate treatment was added, and after 24 hours, the plate was fixed, stained with crystal violet, and the OD was measured. 560 .

[0083] The formulas for calculating biofilm inhibition rate and destruction rate are as follows:

[0084] Inhibition rate (%) = (OD) blank -OD experimental ) / (OD blank -OD PBS )×100%;

[0085] Damage rate (%) = (OD) blank -OD experimental ) / (OD blank -OD PBS )×100%.

[0086] The results showed that GIM significantly inhibited the formation of MRSA biofilms compared to other drugs (see...). Figure 5 (A)), its inhibition rate is as high as 99.18% (see Figure 5 (B) This result is also supported by quantitative analysis of bacterial counts within biofilms (see [link]). Figure 5 (C)). Furthermore, the disruptive effect of GIM on mature biofilms was assessed. The results showed that vancomycin was ineffective in degrading mature biofilms (see [link]). Figure 6 (A)), while GIM achieved a biofilm disruption rate of 92.84% (see [link]). Figure 6 (B)), and showed significant bactericidal activity against bacteria within biofilms (see [link]). Figure 6 (C)).

[0087] 2.5 Intracellular bacterial infection experiment

[0088] To establish an intracellular infection model, RAW264.7 macrophages were co-cultured with MRSA. Co-localization analysis was performed to verify whether GM and GIM can target intracellular MRSA.

[0089] (1) Cy5.5 tags GM and GIM

[0090] GM and GIM (10 mg) were separately dispersed in 10 mL of ultrapure water and stirred at room temperature for 15 minutes to obtain solution A. Cy5.5-NHS (1 mg) was dissolved in 100 μL of DMF to obtain solution B. Solution B was added dropwise to solution A under light-protected conditions and stirred overnight. The mixture was placed in a dialysis bag (MWCO 200 Da) and dialyzed against ultrapure water in the dark for 7 days, with frequent water changes. The product was collected by centrifugation, washed 3 times, and lyophilized for storage. Cy5.5-GM and Cy5.5-GIM were obtained.

[0091] (2) RAW264.7 cell culture: RAW264.7 cells were purchased from ATCC and cultured in high glucose DMEM and 10% FBS without penicillin or streptomycin. 60%-80% of cells were confluent and passaged.

[0092] (3) Establishment and validation of intracellular bacterial infection model

[0093] RAW264.7 cells were seeded in six-well plates, infected with GFP-MRSA at MOI=10, and extracellular bacteria were removed after 15-90 minutes. 10 μg / mL vancomycin was added, and the plates were incubated for 2 hours. Intracellular localization was observed using confocal microscopy after LysoTracker Red and Hoechst 33342 staining to verify the successful establishment of the intracellular bacterial infection model. The presence of MRSA within macrophages confirmed the successful establishment of the intracellular bacterial infection model.

[0094] (4) Intracellular antibacterial activity detection: After treatment, 1×10⁻⁶ cells were collected. 5 Cells were lysed to release intracellular bacteria, centrifuged and resuspended, and then plated on TSA plates for colony counting.

[0095] (5) Cy5.5-GM, Cy5.5-GIM co-localize with lysosomes and GFP-MRSA

[0096] After establishing the intracellular bacterial infection model, Cy5.5-GM or Cy5.5-GIM (Ga elemental mass concentration, 10 μg / mL) was added. -1 Images were visualized using confocal laser scanning microscopy (CLSM) and Hoechst 33342 staining, with colocalization analysis performed using ImageJ.

[0097] The results showed that GM and GIM were significantly co-localized with lysosomes and MRSA within macrophages, indicating that GM and GIM can be endocytosed and transported to the lysosomal compartment, where they interact with intracellular bacteria (see [link to study]). Figure 7 (A) and Figure 7 (B)).

[0098] Subsequently, the intracellular bactericidal activity of GIM was evaluated in MRSA-infected macrophages.

[0099] Colony counting experiments showed that GIM could clear almost all intracellular MRSA (>99%), while vancomycin showed almost no bactericidal effect, which may be related to its limited ability to enter host cells (see...). Figure 7 (G) and Figure 7 (H)).

[0100] In summary, the experimental results show that GIM has strong antibacterial activity against free bacteria, can effectively inhibit and destroy bacterial biofilms, and more importantly, can efficiently remove intracellular MRSA.

[0101] 3. In vitro anti-inflammatory effects of GIM

[0102] The above results confirm that GIM can effectively clear free and intracellular MRSA. However, reducing bacterial load is only the first step in treating suppurative arthritis (SA). Bacterial infection and macrophage-mediated synovitis induce oxidative stress, which is one of the important driving factors for joint tissue damage. Given that GIM... 3+ Given that ITA possesses potential anti-inflammatory activity, we hypothesized that GIM may exert an immunomodulatory role after being internalized by pro-inflammatory macrophages. To verify this hypothesis, we constructed a lipopolysaccharide (LPS)-induced inflammatory macrophage model using RAW264.7 cells and systematically investigated the immunomodulatory effects of GIM.

[0103] (1) Macrophage inflammation model: RAW264.7 cells were used at a dose of 1×10⁻⁶. 5 Cells were added to the well plate at a density of 100 cells / well and cultured for 6 hours, followed by the addition of 100 ng / mL LPS. -1 , to be trained for 24 hours.

[0104] (2) Cell viability assay: Different concentrations of GIM (0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μg / mL) were used. -1 Macrophages were used to validate the model and chondrocytes, which were cultured for 24 hours and then the absorbance was measured at 450 nm using the CCK-8 assay.

[0105] The results of the CCK-8 experiment show (see...) Figure 9 (A) Figure 9 (B) GIM exhibits low cytotoxicity against chondrocytes and macrophages. Based on this result, subsequent experiments selected 50 μg·mL⁻¹ Ga. -1 GM and GIM are used as working concentrations.

[0106] (3) Flow cytometry analysis: RAW264.7 cells were seeded into 48-well plates and then divided into groups. The Control group was treated with PBS, while the LPS, ITA, GM, and GIM groups were treated with 100 ng / mL PBS. -1 LPS and ITA groups were treated with 10 mM ITA, while GM and GIM groups were treated with GM and GIM (50 μg·mL⁻¹, calculated as Ga, respectively). -1 After culturing for 24 hours, the MRSA of each group was fixed, permeabilized, and blocked, and then incubated with FITC-iNOS and APC-Arg1 antibodies for flow cytometry analysis.

[0107] First, we evaluated whether GIM could regulate macrophage M1 / M2 polarization and inhibit the inflammatory response. Flow cytometry analysis showed that both GM and GIM could increase the M2 / M1 ratio, with GIM having a more significant effect (see...). Figure 8 (A) and Figure 8 (B)).

[0108] (4) ELISA detection of macrophage cytokine secretion: After treatment, the supernatant was collected, centrifuged, and IL-6, TNF-α, IL-4, IL-10, IL-1β, and IL-18 were measured.

[0109] ELISA analysis showed that GIM significantly reduced the levels of pro-inflammatory cytokines while enhancing the secretion of anti-inflammatory cytokines, with effects superior to GM and ITA (see [link to ELISA]). Figure 8 (C)–(F)).

[0110] (5) Western Blot: Macrophages were lysed, treated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to PVDF membrane, blocked with 5% BSA, incubated with primary and secondary antibodies, and chemiluminescent imaging was performed.

[0111] Figure 9 Western blot results (C)-(E) further validated that both GM and GIM can increase the LPS-induced M2 / M1 ratio in macrophages. Persistent inflammation can disrupt the antioxidant defense system, leading to excessive accumulation of reactive oxygen species (ROS) and triggering oxidative damage.

[0112] (6) Intracellular ROS detection: DCFH-DA fluorescent probe was used for staining for 30 minutes, Hoechst 33342 for 10 minutes, fluorescence intensity was observed under a microscope, and ImageJ was used for quantification.

[0113] The effects of GIM on macrophage oxidative stress were further investigated, and the staining results are as follows: Figure 8 (G) and Figure 8 As shown in (H), both ITA and GM can partially reduce LPS-induced ROS elevation, while GIM almost completely eliminates the ROS elevation. These results indicate that GIM can effectively inhibit macrophage inflammatory activation and oxidative stress.

[0114] (7) Assessment of cellular senescence phenotypes

[0115] Staining was performed using an aging-related β-gal assay kit, followed by microscopic observation and β-gal quantification using ImageJ. + Cell ratio.

[0116] Inflammation-driven oxidative stress is a key contributing factor to "inflammatory aging." In inflammatory aging (SA), tissue damage caused by inflammatory aging may become a significant obstacle to tissue repair and functional recovery after bacterial clearance. Given the significant anti-inflammatory activity of GIM, it is hypothesized that GIM may exert a joint-protective effect by alleviating inflammation-induced macrophage senescence. To verify this, cellular senescence phenotypes were evaluated in RAW264.7 cells. SA-β-gal staining results (see...) Figure 8 (I) and Figure 9 (F) shows that GIM can significantly reduce the senescence level of macrophages.

[0117] Western blot analysis of the aging marker proteins p16 and p21 using the aforementioned method further supports this conclusion (see [link to analysis]). Figure 8 (J) Figure 9 (G)-(H)). In summary, these results indicate that GIM can alleviate inflammation-induced macrophage senescence.

[0118] 3.8 Effects of GIM-treated macrophages on chondrocytes

[0119] Macrophage senescence and its associated senescence-associated secretory phenotype (SASP) can disrupt the anabolic-catabolic balance of chondrocytes, leading to cartilage degradation, which is one of the important pathological features of senescence-associated chondrocyte syndrome (SA). To evaluate the effects of GIM-treated macrophages on chondrocytes, this invention collected the conditioned medium from GIM-treated macrophages and used it to culture primary chondrocytes. The flowchart is shown below. Figure 8 (K).

[0120] Preparation of primary mouse chondrocytes: Cartilage from the femoral and tibial plateau joints of 6-7 day old C57BL / 6J suckling mice was isolated, digested with type II collagenase for 6 hours, centrifuged and resuspended in DMEM / F12 medium containing 10% FBS.

[0121] Macrophage conditioned medium: After RAW264.7 cells were treated with different drugs, the supernatant was collected and diluted 2-fold. The diluted supernatant was added to the above-mentioned DMEM / F12 medium containing 10% FBS and cultured for 48 hours for subsequent analysis. The supernatant of the control group was the normal culture medium of RAW264.7 cells.

[0122] Immunofluorescence staining of cells: After fixation and blocking, cells were incubated with primary antibody and secondary antibody for 2 hours, and then visualized using CLSM. Toluidine blue and aricin blue staining of chondrocytes: After treatment with conditioned medium, cells were fixed with 4% paraformaldehyde, stained with toluidine blue and aricin blue for 30 minutes, and then imaged and recorded.

[0123] Toluidine blue and alnic blue staining were used to assess glycosaminoglycan (GAG) secretion. The staining results are as follows: Figure 8 As shown in (L), LPS-activated macrophage conditioned medium significantly promoted the loss of GAGs in chondrocytes, while conditioned medium derived from GIM-treated macrophages effectively reversed this phenomenon. Western blot and immunofluorescence analyses indicated that GIM could alleviate chondrocyte metabolic imbalance induced by macrophage-derived inflammatory signals (see [L]). Figure 8 (M)-(N) and Figure 9 (I)-(N)).

[0124] In summary, these results indicate that GIM can not only inhibit the inflammatory response of pro-inflammatory macrophages and inflammation-induced cellular senescence, but also improve the metabolic homeostasis of chondrocytes by regulating and activating the secretory phenotype of macrophages.

[0125] 4. In vitro antipyretic effect of GIM

[0126] Bacterial infection can induce reactive oxygen species (ROS) generation and metabolic disorders, thereby promoting NLRP3 inflammasome activation, triggering Caspase-1-mediated IL-1β secretion and pyroptosis. Based on these findings, it is hypothesized that GIM may further alleviate inflammation and chondrocyte metabolic imbalance in septic arthritis (SA) by inhibiting macrophage pyroptosis. To verify this hypothesis, pyroptosis was induced in RAW264.7 cells using LPS and Nigericin (Nig).

[0127] The pyrolysis model: RAW264.7 with 1×10 5Cells were added to wells at a density of 100 ng / well and cultured for 6 hours. The control group received no treatment, while other groups received 100 ng / mL LPS. -1 After culturing for 23 hours, 10 µM Nigericin was added and treated for 1 hour. The ITA group received 10 mM ITA, while the GM and GIM groups received GM and GIM (calculated as Ga, 50 μg / mL), respectively. -1 (Culturing for 24 hours). Morphological observation.

[0128] LPS / Nig stimulation resulted in significant cytoplasmic vacuolation in macrophages, a typical characteristic of pyroptosis; however, GIM treatment almost completely eliminated this phenotype. Figure 10 (A)). Consistently, GIM significantly downregulated the expression of NLRP3, GSDMD, and Caspase-1 in pyroptotic macrophages (see [A]). Figure 10 (B)-(E), Figure 11 (A)-(C)), and significantly reduced the secretion of inflammasome-dependent IL-1 family cytokines IL-1β and IL-18 (see [link to relevant documentation]). Figure 10 (F)-(G)).

[0129] Inflammation-related oxidative stress can rapidly deplete intracellular NAD(H) and ATP, thus maintaining NAD... + Homeostasis and ATP levels are crucial for preventing energy depletion and subsequent cell damage. NAD + / NADH detection uses enhanced NAD + / NADH assay kit (WST-8). Intracellular ATP assay was performed using an enhanced ATP assay kit.

[0130] The results show that ( Figure 10 (H)-(I)), GIM significantly alleviated inflammation-induced macrophage NAD. + And ATP decreased. Next, the effect of GIM on macrophage senescence was examined, and it was found that GIM significantly inhibited the senescence phenotype induced by pyroptosis activation (see...). Figure 10 (J)-(K), Figure 11 (D)-(F)).

[0131] It is known that macrophage pyroptosis disrupts the anabolic-degradative balance of chondrocytes. To assess whether GIM protects chondrocytes by inhibiting macrophage pyroptosis, conditioned medium obtained after treating pyroptosis-induced macrophages was used for primary chondrocytes (see...). Figure 10 (L)), cultured for 48 h. Toluidine blue and Alcian blue staining ( Figure 10(M) showed that GIM-treated macrophages could rescue glycosaminoglycan loss in chondrocytes induced by conditioned medium in pyroptotic macrophages. Immunofluorescence staining ( Figure 10 (N) Figure 11 (G)-(H)) showed that GIM can alleviate the catabolism-synthesis imbalance in chondrocytes.

[0132] In summary, GIM not only inhibits LPS / Nig-induced macrophage pyroptosis and related senescence, but also improves chondrocyte metabolic disorders caused by pyroptotic macrophages.

[0133] 5. Antibacterial effect of GIM in SA

[0134] To evaluate the in vivo therapeutic effect of GIM, according to Figure 12 (A) Schematic diagram for in vivo animal experiments. With IACUC approval, a suppurative arthritis model was established using 10-week-old male C57BL / 6J mice. MRSA was injected into the knee joint of the mice (Normol group did not develop the model, 1×10⁻⁶). 6 CFUmL -1 After 3 days, the patients were randomly assigned to different groups: the Normal and PBS groups received 8 μL of PBS per injection, the Vanco group received 10 μg / mL vancomycin per injection, the ITA group received 10 mM ITA, and the GM and GIM groups received 50 μg / mL GM and GIM (calculated as Ga, respectively). -1 Except for the Normol group, the solvent for all other groups was PBS.

[0135] Each group received intra-articular injections of the aforementioned drugs every two days until the end of the experiment, for a total of approximately two weeks. SA model mice treated with PBS exhibited typical SA characteristics such as joint swelling, while the GIM treatment group significantly alleviated these symptoms (see...). Figure 12 (B)).

[0136] In vivo distribution of GFP-MRSA: The GFP-MRSA model was established. The method differed from that used to establish the septic arthritis model only in that GFP-MRSA was injected. Knee joints were harvested 14 days later, stained with DAPI, and the distribution was observed under a fluorescence microscope.

[0137] Furthermore, analysis of complete blood count (CBC) using cardiac blood samples, including white blood cell, red blood cell, lymphocyte, monocyte, and neutrophil percentages, revealed that GIM significantly restored SA-induced hematological abnormalities, exhibiting superior efficacy compared to vancomycin. This suggests that GIM can effectively clear infection and restore host immune function (see [link to relevant documentation]). Figure 12 (C)-(G)).

[0138] To visually observe the intra-articular bacterial load after treatment, a suppurative arthritis model was established using GFP-MASA. Fluorescence imaging showed that the GIM group almost completely eliminated MRSA-related fluorescence in the synovial tissue, while the Vanco group still had residual fluorescence, indicating that GIM had a superior bacterial clearance effect within the infected joint (see...). Figure 12 (H)). Bacterial cultures of knee joint lavage fluid and periarticular tissue homogenates showed no colony formation in the GIM group (see [link]). Figure 12 (I)-(L)), further confirming the potent in vivo bactericidal activity of GIM. Furthermore, H&E staining evaluation of major organs and tissues showed no significant pathological abnormalities, indicating that GIM has good systemic safety (see [link to study]). Figure 15 (A)).

[0139] 6. In vivo joint protection effect of GIM in SA

[0140] Given the potent antibacterial and anti-inflammatory activity of GIM, it was hypothesized that it could protect the joint structure of SA mice. To verify this hypothesis, knee joints of treated mice were collected for histological evaluation.

[0141] Quantitative analysis of bacteria in synovial fluid and periarticular tissues: Tissue homogenate and joint lavage fluid were diluted and plated for colony counting.

[0142] Histological evaluation: Knee joint immobilization and EDTA decalcification for 3 weeks; paraffin sections were stained with H&E, Safranin O, tartrate-resistant acid phosphatase (TRAP), and analyzed for synovitis (OARSI) score, inflammation score (International Osteoarthritis Research Society), and cartilage degeneration score; TRAP-positive cells were quantified using ImageJ. Synovial membrane was collected for H&E staining to observe pathological changes.

[0143] H&E staining results showed that GIM significantly reduced synovial thickness and alleviated synovial inflammation, which was more pronounced compared to the PBS and Vanco groups (see [link to H&E staining results]). Figure 13 (A) and Figure 13 (D)). Safranin O staining further showed that GIM significantly reduced cartilage degeneration (see [reference needed]). Figure 13 (A) Figure 13 (D) and Figure 13 (F)). Inflammatory stimuli can drive osteoclast activation. TRAP staining results in subchondral bone ( Figure 13 (C) and Figure 13 (G) showed that the GIM group had the lowest proportion of TRAP-positive osteoclasts, suggesting that GIM effectively inhibits osteoclast-mediated bone resorption and matrix degradation during SA. Immunofluorescence staining of synovial macrophages showed that GIM significantly reduced M1 macrophage infiltration while increasing the level of M2 macrophages (GIM). Figure 13 (H), Figure 13 (I) Figure 13 (L) and Figure 13 (M)). Furthermore, immunofluorescence and immunohistochemistry showed that GIM significantly upregulated cartilage synthesis markers and downregulated degradation markers, exhibiting effects superior to vancomycin (M). Figure 13 (J) Figure 13 (K) Figure 13 (N) Figure 13 (O) and Figure 15 (B)-(C)). In summary, the results indicate that GIM can significantly protect the synovium and cartilage structure of SA mice, and its therapeutic effect is superior to vancomycin.

[0144] To further verify the mechanism by which GIM alleviates synovial inflammation in vivo, immunofluorescence and immunohistochemistry were performed on synovial tissue based on transcriptomic and in vitro results to detect pyroptosis and key markers of the MAPK and NF-κB pathways.

[0145] Immunofluorescence staining of tissues: antigen retrieval, 5% BSA blocking, primary antibody incubation overnight, secondary antibody incubation for 2 hours, DAPI nuclear staining, and fluorescence microscopy imaging.

[0146] Immunohistochemistry: Antigen retrieval, blocking with 5% BSA and 3% H2O2 (volume fraction), incubation with primary antibody overnight, secondary antibody for 2 hours, DAB staining, hematoxylin counterstaining, and observation under an optical microscope. Wash with PBS 3 times for 5 minutes each time.

[0147] result( Figure 14 (A)-(C)) showed that the GIM group had the lowest expression of NLRP3 and GSDMD, indicating that GIM effectively inhibited synovial pyroptosis; at the same time, GIM significantly reduced the expression of NF-κB and MAPK pathway activation markers p-p65 and p-p38, suggesting that it may alleviate synovial inflammation by inhibiting these pathways (see [link to relevant documentation]). Figure 14 (D) Figure 14 (E) Figure 14 (H), Figure 14 (I)). Furthermore, GIM significantly reduced the expression of synovial cell senescence markers p16 and p21 ( Figure 14 (F) Figure 14 (G) Figure 14 (J) Figure 14 (K)). In summary, GIM may alleviate SA-induced synovial inflammation by inhibiting pyroptosis and NF-κB / MAPK pathway activation.

[0148] 7. Conclusion

[0149] Suppurative arthritis (SA) is a medical emergency caused by bacterial infection of the synovial joint, characterized by rapid joint destruction and severe functional impairment. In SA, bacteria can directly induce the release of proteolytic enzymes through their virulence factors, accelerating cartilage degradation. Simultaneously, bacterial infection of macrophages triggers oxidative stress and pyroptosis, promoting synovial inflammation and further exacerbating joint damage. Furthermore, infection-induced macrophage inflammation maintains a low-grade chronic inflammatory microenvironment, leading to persistent joint damage. Current treatment strategies primarily rely on high-dose antibiotics to control the initial infection. However, due to the lipid bimolecular barrier effect of the host cell membrane, many antibiotics struggle to effectively penetrate the cell, failing to completely eliminate intracellular bacteria and increasing the risk of persistent or recurrent infection. Moreover, traditional treatments often neglect the joint inflammation and subsequent pathological remodeling in SA.

[0150] Trivalent gallium (Ga) 3+ It can competitively interfere with bacterial iron metabolism through a unique iron mimicry mechanism, thereby exhibiting potent antibacterial activity. Furthermore, Ga... 3+ Itaconic acid (ITA) exerts its anti-inflammatory effect by inhibiting the release of pro-inflammatory factors from macrophages. Itaconic acid (ITA) is an endogenous immune metabolite that inhibits macrophage pyroptosis by regulating NLRP3 inflammasome activation and simultaneously exerts its antibacterial effect by competitively inhibiting bacterial isocitrate lyase (ICL) and interfering with bacterial energy metabolism. This invention utilizes Ga… 3+ A novel dual-ligand gallium metal-organic framework (GIM) was synthesized by combining with two organic ligands (ITA and ATA), forming a structure based on Ga-O coordination bonds. The preparation process is described in [link to preparation details]. Figure 16 (A)

[0151] Figure 16 (B) demonstrates the therapeutic targets of GIM in septic arthritis, including free bacterial clearance, intracellular bacterial clearance, inflammation suppression, pyroptosis inhibition, and senescence reduction. Figure 16 (C) demonstrates the mechanism of action of GIM in suppurative arthritis, including: (1) clearing free MRSA; and (2) being taken up by cells and clearing MRSA in cells. Specifically, experimental results show that GIM can effectively clear planktonic bacteria and has strong anti-biofilm activity. More importantly, GIM can be effectively taken up by macrophages and clear vancomycin-resistant intracellular bacteria. In addition to antibacterial activity, GIM can promote macrophage polarization from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype, thereby alleviating macrophage-mediated chondrocyte metabolic imbalance. Transcriptome analysis shows that GIM may exert its biological effects through NOD-like receptors, MAPK, and NF-κB signaling pathways. Subsequent experiments further demonstrated that GIM significantly inhibited macrophage pyroptosis associated with these pathways. In addition, GIM can significantly reduce inflammation and pyroptosis-induced macrophage senescence.

[0152] In a mouse SA model, GIM treatment effectively controlled intra-articular bacterial infection, inhibited pro-inflammatory polarization of synovial macrophages, and reduced pyroptosis and senescence of synovial cells. Therefore, GIM significantly alleviated synovial inflammation and cartilage degeneration, ultimately providing a powerful joint protection effect.

[0153] In summary, GIM can coordinate interventions in multiple pathogenic processes of SA, including bacterial infection, inflammation, pyroptosis, and inflammatory senescence, providing a promising and translational comprehensive treatment strategy for the effective treatment of SA.

[0154] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A gallium metal-organic framework material, characterized in that, It is prepared through the following steps: (1) Dissolve 2-aminoterephthalic acid and itaconic acid in an organic solvent to obtain solution A; (2) Dissolve gallium nitrate hydrate in an organic solvent to obtain a solution B containing gallium ions; (3) Mix solution A and solution B, heat at 50-70℃ for 3-5 hours, centrifuge and collect the precipitate, purify, and obtain the final product.

2. The gallium metal-organic framework material according to claim 1, characterized in that, The molar ratio of gallium ions, itaconic acid and 2-aminoterephthalic acid is (1-1.2):(0.4-0.6):(0.4-0.6).

3. The gallium metal-organic framework material according to claim 2, characterized in that, The molar ratio of gallium ions, itaconic acid, and 2-aminoterephthalic acid is 1:0.5:0.

5.

4. The gallium metal-organic framework material according to claim 1, characterized in that, The organic solvent is selected from N,N-dimethylformamide.

5. The gallium metal-organic framework material according to any one of claims 1-4, characterized in that, The pore size of gallium metal-organic framework materials is 2.0-2.5 nm.

6. A method for preparing the gallium metal-organic framework material according to any one of claims 1-5, comprising the following steps: (1) Dissolve 2-aminoterephthalic acid and itaconic acid in an organic solvent to obtain solution A; (2) Dissolve gallium nitrate hydrate in an organic solvent to obtain a solution B containing gallium ions; (3) Mix solution A and solution B, heat at 50-70℃ for 3-5 hours, centrifuge and collect the precipitate, purify, and obtain the final product.

7. The preparation method according to claim 6, characterized in that, In step (3), the mixture is heated at 60°C for 4 hours.

8. The preparation method according to claim 6, characterized in that, The centrifugation method is to centrifuge at 7000-9000×g for 15-30 minutes.

9. The preparation method according to claim 6, characterized in that, In step (3), the purification method is to wash the precipitate three times with DMF and water respectively, then dialyze it in ultrapure water for 96 hours, changing the water every 4 hours, and finally freeze-drying it at -60℃ to obtain powder.

10. The use of the gallium metal-organic framework material according to any one of claims 1-5 in the preparation of a medicament for treating pyogenic arthritis.

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