Application of diatomic iron-iron site nano-enzyme in preparation of targeted osteoarthritis treatment medicine by relieving oxidative stress and cartilage degeneration

Abstract

The invention relates to a diatomic iron-iron site nano-enzyme constructed by relieving oxidative stress and cartilage degeneration and used for targeted osteoarthritis treatment. According to the invention, a nitrogen-doped porous carbon anchored diatomic iron nano enzyme catalyst (Fe2-NCs) with Fe-Fe dimer coordination is developed by using a'subject-object 'strategy. The Fe2-NCs protect cartilage cells from oxidative stress induced apoptosis by modulating ROS and active nitrogen species (RNS) and promoting O2 release. In addition, Fe2-NCs restores mitochondrial function by inhibiting NOX4 expression, improving ATP production, and normalizing COXIV levels. In an in-vivo OA model, the Fe2-NCs can reduce the expression of a pro-inflammatory medium COX-2 through an NF-kappa B signal channel, inhibit the up-regulation of MMP-13 and delay the degradation of type II collagen. The invention provides a new theoretical framework and methodological approach for the treatment of osteoarthritis, and has important clinical and scientific significance.

Description

Technical Field

[0001] This invention relates to the technical field of nanozyme targeted therapy for arthritis, and in particular to the application of a diatomic iron-iron site nanozyme in the preparation of a drug for targeted treatment of osteoarthritis by reducing oxidative stress and cartilage degeneration. Background Technology

[0002] Osteoarthritis (OA) is a common degenerative joint disease. Increasing evidence highlights the crucial role of reactive oxygen species (ROS) in the pathophysiology of OA. Excessive ROS accumulation directly damages key cellular structures, disrupts redox homeostasis within mitochondria, and accelerates the degradation of the extracellular matrix (ECM). Furthermore, ROS-mediated oxidative stress induces the release of pro-inflammatory cytokines, further exacerbating chondrocyte apoptosis. Antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), have recently attracted widespread attention due to their catalytic responses in cells to remove ROS, thereby regulating redox status and protecting cells from oxidative damage. However, the low stability of these natural enzymes under pathological conditions limits their practical application. Therefore, there is an urgent need to improve the stability of these antioxidant enzymes to fully realize their therapeutic potential in regenerative medicine and other clinical applications. This study highlights the superior catalytic performance of single-atom NCs, which utilize the single metal atom and the strong interaction between the metal center and the support, endowing them with significant catalytic activity and selectivity for ROS removal. Although single-atom NCs are highly capable of mimicking the functions of natural oxidases, they face significant challenges in replicating the complex multinuclear structures found in natural enzymes. This hinders the efficient adsorption and desorption of reaction intermediates during catalysis, ultimately reducing the efficiency of ROS catalysis.

[0003] To overcome this limitation, atomically dispersed diatomic nanozymes with two adjacent metal atomic sites have emerged as a promising solution, closely resembling the multimetallic active sites commonly found in natural oxidases. Therefore, designing and constructing desired diatomic nanozymes with precise diatomic structures and understanding the underlying mechanisms of catalytic ROS removal are essential for next-generation biomimetic enzymes. Furthermore, natural antioxidant defense systems consist of complex networks of multiple antioxidant enzymes working synergistically to protect cells from oxidative stress. Notably, reported nanozymes mimicking these systems often encounter inherent limitations when mimicking multiple antioxidant activities. Specifically, these limitations are detailed below: Heteronuclear atoms readily generate harmful free radicals (·OH) in living organisms, and their uneven charge distribution leads to low catalytic efficiency.

[0004] Uncontrollable interatomic spacing – Precise synthesis of Fe2N6 configuration (iron atom spacing 2.18 ± 0.05 Å) overcomes the limitations of traditional diatomic synthesis methods, which struggle to control interatomic spacing, leading to uneven atomic dispersion. This results in diatomic atoms easily agglomerating (too small spacing leads to inactivation) or dispersing too far (too large spacing leads to loss of synergistic effect). By constructing homonuclear iron diatomic atoms (Fe-Fe) with a pyridine nitrogen-rich surface optimized electronic structure and a uniform coordination environment (Fe2N6), the energy barrier for OO bond dissociation is lowered, synergistically promoting the maximization of catalytic effect.

[0005] Single enzyme activity – under physiological pH, it can mimic SOD-CAT-OXD-GSH in various ways, regulate oxygen adsorption configuration, accelerate OO bond breaking, eliminate ROS, and efficiently decompose H2O2 to produce O2 (activity is improved compared to single-atom enzymes), without producing byproducts such as ·OH, thus significantly improving safety.

[0006] Biological inactivation—Based on its high specific surface area and pyridine-rich surface design, Fe2-NCs ensure long-term enzyme activity stability for 30 days, maintaining a catalytic activity retention rate of >90%. Fe2-NCs can effectively reduce ROS and protect chondrocytes from oxidative stress-induced apoptosis. By inhibiting NOX4 expression, restoring ATP levels, and normalizing COX IV expression, Fe2-NCs can enhance mitochondrial function. Furthermore, Fe2-NCs significantly downregulate the pro-inflammatory mediator COX-2, inhibit MMP-13-mediated cartilage degradation, and slow down type II collagen (COL2) breakdown by inhibiting the NF-κB signaling pathway.

[0007] Therefore, developing a biatomic NCs platform that can accurately construct homonuclear metal sites and simulate the activities of various enzymes will better simulate complex intracellular antioxidant defense systems and provide new ideas for OA treatment. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides the application of diatomic iron-iron site nanozymes in the preparation of targeted therapeutic drugs for osteoarthritis by mitigating oxidative stress and cartilage degeneration. This invention utilizes a "host-guest" strategy to develop a nitrogen-doped porous carbon-anchored diatomic iron nanozyme catalyst (Fe2-NCs) with Fe-Fe dimer coordination. In osteoarthritis, the synovium of the joint lining increases vascular permeability under inflammation. Simultaneously, the joint cavity lacks an effective lymphatic drainage system. This invention leverages the nanozyme's nanoscale size (typically in the 1-150 nm range), enabling it to more easily penetrate the increased permeability of synovial vascular endothelial cells under inflammatory conditions and infiltrate into the joint cavity from the bloodstream. Once inside the joint cavity, due to impaired lymphatic return, the nanozyme remains relatively long-term at the site of inflammation (near the synovium, synovial fluid, and cartilage surface), thus passively accumulating in the lesion area.

[0009] The purpose of this invention is to provide a diatomic iron-iron site nanozyme for the preparation of a targeted treatment for osteoarthritis by reducing oxidative stress and cartilage degeneration. The diatomic iron-iron site nanozyme is an Fe2 diatomic site dimer embedded in nitrogen-modified carbon defect sites and loaded on the surface of a three-dimensional porous carbon framework.

[0010] In some embodiments of the present invention, the iron-iron site nanozyme has a bridging bond with a distance of 2.18 ± 0.05 Å between iron atoms.

[0011] In some embodiments of the present invention, the activity concentration of the diatomic iron-iron site nanozyme is 5~50 μg / ml.

[0012] In some embodiments of the present invention, the size of the diatomic iron-iron site nanozyme is 1~150 nm.

[0013] In some embodiments of the present invention, the diatomic iron-iron site nanozyme is prepared by the following method: Zinc and iron sources were dissolved in a ternary mixed solvent to obtain solution A; 2-methylimidazole was dissolved in a binary mixed solvent to obtain solution B. Solution A was added to solution B to obtain a mixture. The mixture was kept at 25±2℃ for 12~48 hours to promote the formation of encapsulated iron species of ZIF-8 to obtain a colloidal product. The product was washed and dried to obtain the precursor material. The precursor material was pulverized and heated to carbonize, thus obtaining the diatomic iron-iron site nanozyme (diatomic iron nanozyme catalyst Fe2-NCs).

[0014] In some embodiments of the present invention, the zinc source is selected from zinc nitrate and / or zinc acetate; the iron source is selected from ferric acetate and / or ferrocene tetracarbonyl dimer.

[0015] In some embodiments of the present invention, the molar ratio of the zinc source to the iron source is (3~8):(0.015~0.06).

[0016] In some embodiments of the present invention, the ternary mixed solvent comprises N,N-dimethylformamide, methanol, and ethylene glycol; the volume ratio of N,N-dimethylformamide, methanol, and ethylene glycol is (1~3):1:1.

[0017] In some embodiments of the present invention, the binary mixed solvent comprises N,N-dimethylformamide and methanol; the volume ratio of N,N-dimethylformamide and methanol is 3:(1~2).

[0018] In some embodiments of the present invention, the conditions for heating and carbonization are as follows: under an inert atmosphere, the heating rate is 4~6℃ / min, the temperature is 900~950℃, and the time is 3~4 hours.

[0019] The inert atmosphere contains inactive gases including nitrogen and / or argon.

[0020] The technical solution of the present invention has the following advantages compared with the prior art: The Fe2-NCs prepared in this invention exhibit antioxidant enzyme-like activities, including SOD, CAT, OXD, and GSH-Px, effectively mimicking the natural antioxidant system. Compared with single-atom iron nanozyme catalysts (Fe1-NCs), Fe2-NCs show superior catalytic performance, supported by density functional theory (DFT) calculations. This indicates that the synergistic effect of the Fe-Fe dimer structure enhances the oxygen adsorption configuration, lengthens the O2 / O2 bond distance, and lowers the energy barrier of the critical transition state, thereby accelerating the breaking of the O2 / O2 chain and improving catalytic activity. In vitro experiments show that Fe2-NCs protect chondrocytes from oxidative stress-induced apoptosis by regulating ROS and reactive nitrogen species (RNS) and promoting O2 release. Furthermore, Fe2-NCs restore mitochondrial function by inhibiting NOX4 expression, improving ATP production, and normalizing COXIV levels. In an in vivo OA model, Fe2-NCs may reduce the expression of the pro-inflammatory mediator COX-2 through the NF-κB signaling pathway, inhibit the upregulation of MMP-13, and delay the degradation of type II collagen. This invention provides a new theoretical framework and methodological approach for the treatment of osteoarthritis, and has important clinical and scientific significance. Attached Figure Description

[0021] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein... Figure 1 The diagram shows the structural characterization results of the Fe2-NCs nanozyme obtained in this invention; (a) schematic diagram of Fe2-NCs synthesis, (b, c) TEM images of Fe2-NCs, (d) STEM image, (e) HRTEM image, (f) HAADF-STEM image, and (gj) corresponding EDX elemental mapping images of AC-HAADF-STEM and Fe2-NCs.

[0022] Figure 2 These are the X-ray diffraction (XRD) pattern and regional electron diffraction pattern of this invention.

[0023] Figure 3 This is the multimodal spectral characteristic of the iron coordination environment of the present invention.

[0024] Figure 4 This invention presents a three-dimensional charge density difference mapping diagram and an electron localization function (ELF) analysis diagram.

[0025] Figure 5 This invention relates to a multi-enzyme catalytic cascade of Fe2-NCs nanozymes.

[0026] Figure 6 These are the experimental results of the Fe2-NCs stability test of this invention.

[0027] Figure 7 This is the atomic-scale oxygen interaction mechanism in the Fe1 / Fe2-NCs catalytic system of this invention.

[0028] Figure 8 This invention describes the in vitro biocompatibility and antioxidant activity of the nanozyme synthesized in ATDC5 cells.

[0029] Figure 9 These are the results of the hemolytic characteristics experiment of rat erythrocytes and the cell uptake characteristics experiment of ATDC5 cells of the present invention.

[0030] Figure 10-11 This is a laser confocal microscope image showing the cell uptake results of the present invention.

[0031] Figure 12 This is a diagram showing the cytotoxicity test results of this invention.

[0032] Figure 13 This is a graph showing the relationship between Fe2-NCs dosage and cell density.

[0033] Figure 14 This invention relates to RNA sequencing analysis of ATDC5 cells treated with H2O2-induced iron nanozymes.

[0034] Figure 15 The nanozyme synthesized in this invention can effectively inhibit inflammation of chondrocytes through COX-2 and reduce mitochondrial damage to chondrocytes caused by oxidative stress through NOX4.

[0035] Figure 16 This is an in vivo therapeutic evaluation of the Fe2-NCs of this invention in ACLT-induced OA.

[0036] Figure 17 This is a diagram showing the results of histological analysis of key organs using H&E staining, as described in this invention.

[0037] Figure 18 This is a schematic diagram of the synthesis and mechanism of Fe2-NCs of the present invention, which acts as SOD, CAT and GSH-Px to scavenge ROS and GSH in sequence to alleviate OA. Detailed Implementation

[0038] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0039] The reagents and kits used in this invention are all commonly used in the field and can be purchased from the market. The use of the kits is a conventional method in the field.

[0040] Example I. Preparation and Structural Characterization of Fe2-NCs 1. Preparation steps: By... 1.19 g (4 mmol) and Fe2(CO)9 (11 mg, 0.03 mmol) were dissolved in a ternary mixed solvent system containing 36 mL of N,N-dimethylformamide, 12 mL of methanol, and 12 mL of ethylene glycol to obtain a homogeneous metal precursor solution (solution A). The solution was sonicated at 25 °C (40 kHz, 150 W) to ensure complete dissolution. 2-Methylimidazole (1.314 g, 16 mmol) was dissolved in a binary solvent mixture of 12 mL of N,N-dimethylformamide and 8 mL of methanol under magnetic stirring (500 rpm) until optical clarity was achieved, yielding solution B. The synthesis involved gradually introducing solution A into solution B under vigorous mechanical stirring (800 rpm). The resulting mixture was kept under ambient conditions (25 ± 2 °C) for 24 hours to promote the formation of encapsulated iron species from ZIF-8. Colloidal products were separated by high-speed centrifugation (10000×g, 5 min), followed by three consecutive washing cycles with anhydrous methanol to remove residual reactants. Final purification was achieved by vacuum drying (60℃, 0.1 MPa) for 12 hours to obtain the Fe2(CO)9@ZIF-8 precursor material. The Fe2(CO)9@ZIF-8 precursor material was mechanically pulverized and uniformly distributed in a quartz combustion vessel, then placed in the isothermal zone of a horizontal tube furnace (GSL-1700X, Hefei Kejing). A controlled carbonization process was performed under continuous argon flow (99.99% purity, 100 sccm), with the following thermal distribution: linear heating to a target temperature of 950℃ at a rate of 5℃ / min, isothermal holding for 3 hours, followed by passive cooling to ambient conditions (25±2℃). The resulting pyrolytic composite material, designated Fe2-NCs, was ready for use without post-treatment.

[0041] Preparation method of single-atom iron nanozymes Fe1-NCs: Its preparation method is similar to that of Fe2-NCs, the difference being that the iron source is changed to (Fe(acac)2). Pure NCs support: a support without metal single or two atoms.

[0042] 2. Structural characterization: To accurately design atomically dispersed Fe2-NCs, a "host-guest" strategy was employed, and the characterization results are as follows: Figure 1 As shown in Figure a, firstly, non-carbonyl diferric iron (Fe2(CO)9) with inherent Fe-Fe metallic bonds (bond length: 2.52 Å) was incorporated into a zeolite imidazole salt framework-8 (ZIF-8) via solution-phase impregnation. Molecular-level encapsulation utilized the tetrahedral cavities (11.6 Å) of ZIF-8 interconnected through narrow pores (3.4 Å) to achieve size-specific encapsulation of the Fe2(CO)9 guest molecules (molecular diameter approximately 9.5 Å), thus forming Fe2(CO2)9@ZIF-8. This ensured atomic dispersion of the Fe-Fe dimers while maintaining the rhombic dodecahedral morphology of ZIF-8. Secondly, a subsequent pyrolysis process led to Zn evaporation and Fe-Fe deposition on the ligand nodes of the ZIF-8 framework to obtain Fe2-NCs. The morphology and structural characterization of the Fe2-NCs were systematically performed using advanced electron microscopy techniques. Figure 1 The TEM image in b shows that the synthesized material maintains a distinct polyhedral structure with rhombic dodecahedral symmetry (average particle size: 110 nm) and shows no signs of morphological degradation. Meanwhile... Figure 1 The results also confirm that the integrity of the rhombohedral framework was maintained throughout the synthesis process. Figure 1 The middle section revealed a complex three-dimensional mesoporous network within the nanocatalyst. This hierarchical porous structure increases... It significantly improves the accessibility of active sites and partially exposes them, while optimizing mass transfer kinetics during the reactive oxygen species (ROS) catalytic cycle. Figure 1 The presence of 'e' indicates the absence of metallic iron aggregates or crystalline nanoparticles, confirming the atomic-level dispersion of iron species. Figure 1 The figure reveals a uniform distribution of diatomic iron anchored on a nitrogen-doped carbon matrix.

[0043] also, Figure 2 The corresponding power X-ray diffraction (XRD) pattern showed that, apart from the presence of large grains of iron-containing species, no other features related to Fe were observed. x The peaks corresponding to O or metallic Fe, and the electron diffraction pattern of the selected region also shows the spectrum of the amorphous phase. Figure 1 The g-value indicates that C, N, and Fe elements are uniformly dispersed throughout the structure of Fe2-NCs. The Fe content in Fe2-NCs and Fe1-NCs, measured by inductively coupled plasma atomic emission spectrometry (ICP-AES), was 1.42 wt% and 1.37 wt%, respectively. This invention reveals that Fe2-NCs exhibit excellent adsorption properties, with a Brunauer-Emmett-Teller (BET) surface area of ​​808.04 m².2 g -1 It significantly exceeds Fe1-NCs (640.21 m). 2 g -1 ) and NCs (407.89 m 2 g -1 This enhanced surface accessibility is crucial for optimizing catalytic performance, stemming from the formation of nitrogen vacancies and the anisotropic contraction of the carbon skeleton during pyrolysis. Raman spectroscopy analysis revealed a characteristic D band (1355 cm⁻¹). -1 ) and G-band (1572 cm) -1 Intensity. The Ig of Fe2-NCs compared to Fe1-NCs (1.128) and NCs (1.090) D / I G The increased ratio (1.263) indicates that the synergistic coupling between adjacent Fe-Fe sites promotes defect engineering within the graphite matrix, which helps to enhance π-electron delocalization and further maintain sufficient graphitization to achieve efficient charge transfer kinetics.

[0044] The electronic configuration and atomic coordination of Fe species in Fe2-NCs were systematically elucidated through synchrotron-based X-ray absorption spectroscopy (XAS) and X-ray photoelectron spectroscopy (XPS) analysis. Figure 3 Figure a shows that the Fe K edge absorption thresholds of Fe2-NCs and Fe1-NCs are located between Fe foil and Fe2O3, and the oxidation state of the intermediate Fe species (average value approximately +2) was confirmed by linear combination fitting. Figure 3 b indicates that Fe2-NCs are relative to Fe1-NCs at 2p 3 / 2 The binding energy shows a negative shift of 0.3 eV (710.8 vs 711.1 eV), indicating a change from isolated... In contrast, enhanced electron delocalization within the Fe-Fe dimer is beneficial for the transformation of ROS species. Extended X-ray absorption fine structure (EXAFS) analysis revealed different coordination information. Figure 3 (c). The main Fourier transform peak at 1.48 Å corresponds to... First-shell coordination (CN = 4.1 ± 0.2 for Fe1-NCs; CN = 5.3 ± 0.9 for Fe2-NCs). Notably, Fe2-NCs exhibit a secondary scattering path at 2.49 Å (R-space), a characteristic of Fe-Fe metallic bonding, confirming the formation of atomic-scale Fe-Fe dimers. Quantitative EXAFS fitting further validated the coexistence of Fe-N4 (77%) and Fe-Fe (23%) coordination environments in Fe2-NCs. Figure 3(d). DFT modeling determined the thermodynamically stable configuration of the Fe-Fe pair embedded in the graphite matrix (formation energy: -3.2 eV / atom), where adjacent Fe atoms adopt a bridging geometry with an interatomic distance of 2.18 Å. This unique diatomic structure facilitates cooperative electron transfer, as demonstrated by Bader charge analysis, which optimizes the reactive oxygen species (ROS) conversion kinetics through enhanced d-orbital hybridization. Figure 3 (d).

[0045] To elucidate the atomic coordination structure of iron sites in Fe2-NCs, synchrotron-based soft X-ray absorption near-edge structure (XANES) and XPS were systematically employed. CK-edge XANES spectra (…) Figure 3 E(e) shows three distinct resonances at 286.9 eV (π*C=C), 289.9 eV (σ*CNM), and 294.2 eV (σ*CC), corresponding to transitions from C1s to antibonding orbitals. Supplementary XPS analysis deconvolved the C1s peak at 283.5 eV (graphite sp2 C=C), 284.8 eV (CN coordination), and 287.1 eV (C-C σ bond), respectively. NK-side XANES revealed four characteristic excitations: pyridine N (peak 4, 398.5 eV), Fe-N coordination (peak 5, 400.1 eV), pyrrole N (peak 6, 402.8 eV), and graphite N (peak 7, 405.3 eV). Figure 3 (f). High-resolution N 1s XPS quantified the nitrogen speciation, with binding energies of 399.2 eV (Fe Nx), 400.4 eV (pyrrole N), 401.3 eV (pyridine N), and 403.0 eV (graphite N), confirming the Fe-N binding during pyrolysis. x The motif was successfully preserved. Wavelet transform EXAFS analysis resolved two prominent scattering paths in Fe2-NCs: a major Fe-N coordination shell at 1.5 Å and a minor Fe-Fe metallic bond at 2.3 Å, clearly confirming atomic Fe-Fe dimerization. Figure 3 (g).

[0046] The evolution of electronic configuration within Fe2-NCs was rigorously studied using DFT simulations. For example... Figure 3 Projected density of states (PDOS) analysis of Fe2-NCs shows that, compared to Fe1-NCs, Fe2-NCs exhibit enhanced dp orbital hybridization between adjacent Fe sites, manifested as a significant downward shift of the d-band center. Three-dimensional charge density difference mapping ( Figure 4Images a and b) show a significant electron redistribution around the Fe-Fe dimer, characterized by electron accumulation regions along the Fe-Fe interatomic axes and depletion regions at the Fe-N coordination sites. This asymmetric charge distribution establishes an optimized electronic environment for ROS activation, quantified by enhanced adsorption energies. Electron localization function (ELF) analysis ( Figure 4 (c) and (d) reveal that the delocalization index in Fe2-NCs is higher than that in Fe1-NCs, indicating enhanced metallic properties between iron atoms. The maximum interatomic ELF along the Fe-Fe direction indicates partial covalent bonding while maintaining ionic Fe-N interactions.

[0047] II. Enzyme Activity Evaluation and Theoretical Analysis 1. Superoxide dismutase (SOD) activity detection: Based on the McCord-Friedovich principle, WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylphenyl)-2H-tetrazole sodium salt) was used instead of the traditional nitroblue tetrazolium as the colorimetric probe. The nanozyme activity was evaluated using an optimized enzymatic method. Scavenging capacity. The catalytic system consisted of xanthine oxidase (XOD, 0.025 U / mL) as a superoxide generator, 300 μM xanthine substrate, 0.1 mM EDTA (ethylenediaminetetraacetic acid) for metal ion chelation, and 100 μM WST-8 dissolved in 50 mM PBS (pH 7.0±0.1). Reaction kinetics were tracked at 450 nm using a UV-Vis spectrophotometer (Shimadzu UV-2600i) with 1 nm spectral resolution. Half-maximum inhibition concentration (IC50) 50 The concentration of nanozyme required to inhibit the WST-8 oxidation rate by 50% is defined as a quantitative indicator of SOD mimicry performance. A control experiment verified the specificity of the assay by parallel measurement of natural bovine erythrocyte SOD (Sigma-Aldrich S9697, 3000 U / mg).

[0048] 1.1 Results: Under physiological conditions, superoxide dismutase (SOD), as an essential antioxidant enzyme, catalyzes the dismutation of superoxide radicals (O2·-) into hydrogen peroxide (H2O2) and oxygen (O2), preventing further damage from secondary free radicals. This process is accompanied by the dismutation of (ONOO) - The oxygen is produced and then converted into non-toxic H2O and O2 by catalase (CAT). Figure 5 Figure a shows the enzyme-like properties of Fe2-NCs. First, the SOD activity of the prepared nanozyme was evaluated using the WST-8 assay system, which reacts with the O2·- ions of xanthine and with xanthine oxidase (XOD) to form a water-soluble methylxanthate dye with an absorption wavelength of 450 nm. Figure 5 (b) Fe2-NCs (inhibition rate: 94.30%) significantly inhibited the formation of formamide compared to Fe1-NCs (50.50%) and NCs (3.20%), indicating that Fe2-NCs effectively scavenge superoxide anions. Figure 5 As shown in Figure c, the inhibition rate of methylphenidate formation exhibits dose-dependent behavior. The IC50 of Fe2-NCs... 50 The value was determined to be Significantly lower than Fe1-NCs (>100) This highlights the superior SOD-like activity of Fe2-NCs compared to Fe1-NCs. Furthermore, the response signal of Fe2-NCs at 560 nm was lower than that of the control group (Ctrl) and Fe1NCs, indicating that Fe2-NCs possess enhanced SOD-like activity and a greater ability to scavenge O2·-. Fe2-NCs exhibit SOD activity similar to that of natural SOD. (Sigma, CAS: 9054-89-1), while Fe1-NCs and NCs exhibited... and The above results indicate that Fe2-NCs are promising candidates for SOD simulation applications, and their performance is gradually enhanced after incorporation into the Fe-Fe structure.

[0049] 2. Catalase-like Activity (CAT) Detection: The CAT-like catalytic performance of the nanozyme was quantitatively evaluated using two complementary methods. First, the release of molecular oxygen was tracked in real time using a precision dissolved oxygen detection system (JPB-607A, Shanghai Instrument & Electronics Scientific Instruments). The reaction was carried out under isothermal control (25.0±0.5°C) in 0.1 M phosphate buffer (pH 7.4±0.1). The nanozyme suspension (1 mg / mL) was introduced into the reaction chamber, followed by the addition of H2O2 substrate (final concentration 50 mM). Oxygen saturation levels were recorded at 15-second intervals over 10 minutes. Subsequently, based on the enzyme H2O2 decomposition kinetics, the activity was detected using a commercial catalase activity analysis system (Sigma-Aldrich MAK436) and a catalase detection kit. Finally, under a nitrogen atmosphere, electron spin resonance spectroscopy (Bruker EMXplus, X-band) was used to monitor the activity of the nanozyme containing 100 μM DMPO spin traps and 1 mM Fe2O3. 2+ Hydroxyl radical (·OH) kinetics in a Fenton-like system with 10 mM H2O2. Characteristic quartet signals (1:2:2:1 intensity ratio) were recorded at g=2.006 and a modulation amplitude of 1 G.

[0050]

[0051] 2.2 Results: The obtained Fe2-NCs also exhibited catalase-like (CAT) properties. After uniformly mixing H2O2 with the prepared samples, the absorbance of H2O2 at 240 nm was measured over time to quantitatively compare the decomposition ability of the prepared samples on H2O2. After 120 minutes of reaction, the absorbance of the reaction solution of Fe2-NCs, Fe1-NCs, and NCs at 240 nm decreased from 18.05 to 1.98 and 8.15, respectively, indicating that Fe2-NCs had a stronger kinetic rate for decomposing H2O2. Figure 5 (d). Figure 5 The data in section e show that the decomposition of H2O2 on Fe2-NCs and Fe1-NCs follows Michaelis-Menten kinetics, and the K+ of H2O2 on Fe2-NCs is significantly different. M The value is about half that of Fe1-NC, and far lower than the K of the natural CAT enzyme. M The value (CAS: 9001-05-2, Aladdin) indicates that Fe2-NCs have a more significant adsorption effect on H2O2 (see Table 1). The V0 value of Fe2-NCs... max The value was 1.74 times higher than Fe1-NCs and 2.64 times higher than the natural CAT enzyme, indicating that Fe2-NCs have a higher binding affinity for H2O2 and can more effectively convert H2O2 to O2. Introducing Fe2-NCs into H2O2 solution led to a significant increase in bubble formation and a significant increase in the oxygen concentration in the solution over time. Furthermore, the calculated CAT-like activity of Fe2-NCs was 85.31 U / µg, which is 1.54 times that of Fe1-NCs (55.43 U / µg). Figure 5 As shown in Figure f, the O2 generation on Fe2-NCs was evaluated using a dissolved oxygen analyzer. Fe2-NCs could generate approximately 15.52 µmol of O2 from 100 mM H2O2 within 400 seconds, which is three times higher than the 5.08 µmol generated by Fe1-NCs. In summary, compared with Fe1-NCs, Fe2-NCs can effectively inhibit the formation of hydroxyl radicals (·OH) in the Fenton reaction, indicating that Fe2-NCs have higher CAT-like activity and better H2O2 scavenging ability.

[0052] 3. Oxidase-like activity (OXD) detection: Oxidase detection kits were used. Finally, time-dependent spectrophotometric monitoring was performed at 652 nm using a multimode microplate reader (SpectraMax i3x, Molecular Devices). Catalytic kinetics were analyzed by fitting kinetic curves using SoftMax Pro 7.0 software, and the initial reaction rate was calculated based on the linear phase of the absorbance-time plot to quantify OXD-like activity.

[0053]

[0054] 3.1 Results: After the reaction, the absorbance of ox-TMB at 652 nm for Fe2-NCs reached 2.25, while that for Fe1-NCs and NCs reached 1.58 and 0.13, respectively. After 60 minutes of reaction, the absorbance of the blue product catalyzed by Fe2-NCs to produce TMB was 2.28, which was 1.82 times that of Fe1-NCs (1.25, ...). Figure 5 (g). Calculations showed that the OXD activity of Fe2-NCs was 93.9 U / µg, which is 1.43 times that of Fe1-NCs (65.6 U / µg). This invention demonstrates that Fe2-NCs exhibit stronger catalytic activity than Fe1-NCs by observing the increase in characteristic absorbance of TMB oxidation over time. The OXD-like activity of Fe2-NCs follows typical Michaelis-Menten kinetics (…). Figure 5 (h). K m and V max The values ​​further indicate that the OXD activity of Fe2-NCs is higher than that of Fe1-NCs (Table 2). The OXD reaction rate on Fe2-NCs is 4.07 µmol / L. The activity of Fe2-NCs is 3.18 times that of Fe1-NCs (1.28 µmol / ). These results indicate that Fe2-NCs have higher OXD-like activity and superior ability to catalyze the oxidation of substrates to H2O or H2O2 compared to Fe1-NCs.

[0055] 4. Glutathione peroxidase (GSH-Px) activity assay: The simulated catalytic efficiency of GPx was evaluated using an enzyme-coupled method with glutathione reductase. The assay was performed using a glutathione peroxidase kit, and kinetic analysis was conducted using a UV-Vis spectrophotometer with continuous spectrophotometric measurements (λ=340 nm). The extinction coefficient (ε) was... This is used to calculate enzyme activity based on NADPH oxidation kinetics.

[0056] 4.1 Results: (e.g.) Figure 5 As shown in Figure i, the GSH-Px activity of Fe2-NCs is far higher and The reaction rate was 90.38 mM·min for Fe2-NCs. -1 Compared to Fe1-NCs (58.34 mM·min) -1 , Figure 5 The concentration of GSH in the middle (j) was 1.55 times higher, indicating that it has higher GSH-Px-like activity and superior ability to catalyze the oxidation of GSH to non-toxic glutathione disulfide (GSSG).

[0057] 5. Stability test of Fe2-NCs Long-term stability is another performance indicator for evaluating nanozymes. For example... Figure 6 As shown, Fe2-NCs maintained their initial activity for 30 days in SOD, CAT, and GSH-Px-like activity tests, far exceeding that of natural enzymes. Furthermore, the activity of Fe2-NCs reached its optimal performance within a pH range of 5 to 9. These results indicate that Fe2-NCs exhibit SOD, CAT, and GSH-Px-like activities comparable to natural enzymes, with superior stability.

[0058] III. Mechanism and Theoretical Simulation of Fe2-NCs To further elucidate the relationship between the atomic-scale configuration of the designed nanozyme and its superior enzyme mimicry properties, DFT simulations were performed to calculate the reaction free energy of H2O generated on Fe2-NCs and Fe1-NCs. As a Brønsted base, it typically exists in biological fluids as a hydrogen peroxide radical (HO2·). Theoretical calculations indicate that O2 adsorbed at the Fe-Fe sites of Fe2-NCs can accelerate the breaking of the OO bond. This is because more electrons can be transferred to the empty orbitals of O2, resulting in better activation. Figure 7 The adsorption model of O2 molecules on the synthesized nanozymes is shown in Figure 1. The adsorption energies of O2 on Fe2-NCs and Fe1-NCs are -1.12 eV and -0.82 eV, respectively. Under these conditions, the Fe-O bond energies of Fe2-NCs and Fe1-NCs are 1.83 Å and 1.97 Å, respectively, indicating that the adsorption process at the atomic sites is most favorable for Fe-Fe dimers.

[0059] The O2 OO bonds adsorbed on Fe1-NCs are approximately 1.29 Å long, attributed to superoxide, while O2 on Fe2-NCs tends to form a redox-like structure, in which the O2 OO bonds are significantly longer, at 1.34 Å. This indicates that the redox-like structure of Fe2-NCs can extend the O2 OO bond distance and accelerate its breakage. Generally, the SOD-like activity of nanozymes involves four basic steps. Transition state simulation (… Figure 7 China and Figure 7 (f) indicates that, compared to Fe1-NCs, the synergistic effect in Fe2-NCs lowers the reaction energy barrier for intermediate release of O2 or H2O. This reduction in energy barrier, coupled with the increased reaction rate at each step, was identified as the main factor enhancing reactivity. Simultaneously, PDOS results show a significant overlap between the 3d orbitals of Fe and the 2p orbitals of O in the Fe-Fe dimer, explaining the strong activation ability of O2 (f). Figure 7 Zhongg and Figure 7 (h).

[0060] Based on the above simulation results, the reaction pathways for O2 activation and conversion on Fe2-NCs and Fe1-NCs are as follows: Figure 7 As shown in i and j in 7. For Fe2-NCs, the adsorbed O2 preferentially dissociates rather than forms As an intermediate. Therefore, the oxygen reduction reaction on Fe2-NCs follows the oxygen dissociation mechanism (ODM), in which the intermediate sequentially transforms from... Evolved into It eventually evolved into This pathway is more favorable under mild reaction conditions because it facilitates a smoother electron transfer process and meets the molecular mechanism requirements for highly efficient catalysis. Conversely, the oxygen reduction pathway on Fe1-NCs was simulated using an adsorption evolution mechanism (AEM), in which the intermediate undergoes structural rearrangement and relaxation upon O2 adsorption, ultimately evolving into… and Therefore, due to the low oxidation state of Fe sites and strong adsorption capacity for O2 in Fe2-NCs, the reaction mechanism of Fe-Fe sites changes from AEM to ODM, which accelerates the overall reaction rate, makes the process more favorable for oxygen reduction, and improves the catalytic activity of SOD and CAT.

[0061] IV. The in vitro efficacy of Fe2-NCs in improving oxidative stress and alleviating mitochondrial dysfunction Experimental steps: CCK-8: Detects cell viability; DCFH-DA: Detects reactive oxygen species; Annexin V-FITC / PI: Detects apoptosis; JC-1: Detects mitochondrial potential. All assays were performed using standard kits, representing routine detection methods in this field.

[0062] 5.1 Results: To further verify the ability of Fe2-NCs to scavenge ROS and inhibit apoptosis, in vitro cell experiments were conducted. After ATDC5 cells were incubated with the synthesized nanozyme at gradually increasing concentrations for 24 hours, a significant decrease in cell viability was observed. The degree of cytotoxicity was concentration-dependent with the nanozyme treatment. The group co-incubated with Fe2-NCs showed higher cell viability, maintaining 81.5% cell viability at a high concentration of 200 µg / mL, which was higher than the groups co-incubated with NCs (59.6%) and Fe1-NCs (69.9%), indicating that Fe2-NCs have superior biocompatibility (…). Figure 8 (a) Figure 9 Hemolytic properties of rat erythrocytes showed that Fe2-NCs did not exhibit significant hemolysis. To evaluate the cellular uptake characteristics of ATDC5 cells, RhB-PEG@Fe2-NCs (…) were synthesized. Figure 9Laser confocal microscopy images showed a dose- and time-dependent increase in cellular uptake. Figure 10 and Figure 11 These findings collectively demonstrate that the Fe2-NCs nanozymes prepared in this invention possess excellent biocompatibility and are promising candidates for further application in biological systems. Then, an in vitro model of oxidative damage was established using H2O2 to explore the potential therapeutic applications of Fe2-NCs in the context of oxidative stress-related diseases, particularly their ability to alleviate cell damage and improve cell survival. Fe2-NCs exhibited the best ability to attenuate H2O2-induced oxidative damage, significantly reducing intracellular ROS levels and thereby improving cell survival. Figure 8 (b) This highlights its efficacy as a regulator of cellular elasticity in resisting oxidative damage.

[0063] Figure 8 The results show that the activity of SOD, CAT and GSH-Px in chondrocytes subjected to H2O2-induced oxidative stress on Fe2-NCs can be effectively restored, which can provide strong cytoprotective effects, alleviate H2O2-induced oxidative damage, support the maintenance of cellular redox homeostasis, and ultimately enhance cell viability and function. Figure 8 The image shows the intracellular ROS scavenging ability of the synthesized nanozymes. The fluorescence of Fe2-NCs is negligible due to its lower ROS levels compared to Fe1-NCs and NCs, indicating that Fe2-NCs possess excellent ROS scavenging efficacy. The results show that high H2O2 content significantly increases intracellular malondialdehyde (MDA) levels. The antioxidant properties of Fe2-NCs are as follows... Figure 8 As shown in Figure e, it significantly reduced the formation of MDA and alleviated cell damage caused by oxidative stress.

[0064] It is well known that excessive ROS not only leads to inflammation but also induces chondrocyte apoptosis and mitochondrial dysfunction in osteoarthritis (OA), accelerating disease progression. For example... Figure 8 As shown in figure f, the protective effect of the synthesized nanozymes on mitochondrial health was monitored. Compared with Fe1-NCs and NCs, the Fe2-NCs group exhibited trace amounts of green fluorescence and significant red fluorescence signals, very similar to the baseline, thus further enhancing its strong ability to protect mitochondrial function from H2O2-induced cellular damage. Higher H2O2 content will limit cellular bioenergetics and impair mitochondrial function by reducing adenosine 5'-triphosphate (ATP) concentration. Figure 5 g showed that Fe2-NCs could significantly restore ATP levels, highlighting their enhanced protective potential. To assess the effect of oxidative stress on chondrocyte apoptosis, flow cytometry was used to analyze H2O2-induced apoptotic events in ATDC5 cells. Figure 8The apoptosis rate of Fe2-NCs was significantly reduced to approximately 10.48%, far lower than that of Fe1-NCs (15.69%) and those without nanozymes (22.45%), indicating that Fe2-NCs effectively protected chondrocytes from oxidative stress-induced apoptosis, thereby significantly reducing the incidence of cell death. Fe2-NCs induced minimal cell death in cells containing H2O2. Figure 12 Compared with the control group, it did not show significant toxicity and maintained normal cell morphology. With increasing Fe2-NCs dosage, cell density gradually increased ( Figure 13 The results indicate that the synthesized Fe2-NCs effectively neutralized excess ROS, thereby alleviating oxidative stress and significantly reducing apoptosis and cell death. These results highlight that Fe2-NCs not only significantly alleviated oxidative stress-induced cell damage but also promoted the recovery of mitochondrial function by enhancing cellular energy metabolism. Therefore, Fe2-NCs play a crucial role in protecting chondrocytes from apoptosis. Further analysis showed that Fe2-NCs exhibited a more pronounced role in maintaining mitochondrial integrity and cellular energy homeostasis, highlighting their great potential as a therapeutic agent for restoring mitochondrial function under oxidative stress conditions.

[0065] V. Molecular Mechanism of Fe2-NCs in Treating OA Transcriptomic analysis was performed using next-generation sequencing services provided by Aksomics Corporation (Shanghai, China). Chondrocyte specimens were isolated from mouse models using a TRIzol-based RNA extraction protocol (Invitrogen), including chloroform phase separation followed by isopropanol precipitation. The experimental groups consisted of three distinct sets: 1. Unstimulated chondrocytes (NSCs) – physiological baseline control; 2. Oxidative stress model (H2O2) – 200 μM H2O2 stimulation; 3. Therapeutic intervention (H2O2 + Fe2NCs) – H2O2 + 50 μg / mL iron-based nanozymes. RNA integrity was validated using a Bioanalyzer 2100 (RIN ≥ 8.0) before enrichment of A-tailed RNA with oligomeric (dT) magnetic beads. Strand-specific libraries were prepared using the KAPA Stranded mRNASeq Kit (KAPA Biosystems) according to Illumina-compatible protocols. High-throughput sequencing was performed on the NovaSeq 6000 platform (Illumina) in 150 bp paired-end mode, with three biological replicates per group to ensure statistical robustness. Bioinformatics involved included: raw read alignment with the mm10 reference genome using HISAT2 (v2.2.1); transcriptional quantification using feature counting (v2.0.1); and differential expression analysis via the limma-voom pipeline (R v4.2.1). Cross-cohort comparison strategies: OA pathogenesis: NSC vs H2O2 (FDR < 0.05, log2FC ≥ |0.3|); therapeutic mechanisms: H2O2 vs H2O2 + Fe2NCs (FDR < 0.05, log2FC ≥ |0.3|). Functional annotation utilized: GO terminology enrichment analysis (cluster Profiler v4.4.4); KEGG pathway annotation (KOBAS v3.0); and heatmap visualization (complex heatmap v2.12.1).

[0066] Female C57BL / 6J mice (n=32, 10 weeks old, weighing 18-20g) were obtained from Beijing VitaRiver Laboratory Animal Technology Co., Ltd. (SPF grade, certification number SCXK-2022-0012). The mice were acclimatized to the environment (temperature 22-24°C, humidity 55±5%, light duration 12:12 hours) in IVC isolation cages within an AAALAC-certified animal facility, with free access to autoclaved food and acidified water. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Fudan University Pudong Medical Center (IACUC-20230920-02) in accordance with the ARRIVE 2.0 guidelines. Figure 8As shown in Figure a, osteoarthritis modeling was achieved through unilateral right anterior cruciate ligament transection (ACLT) surgery under 2% isoflurane anesthesia. Postoperative analgesia was maintained for 72 hours with buprenorphine SR (1.0 mg / kg). After a 7-day recovery period, animals were randomly assigned to four experimental groups: 1) sham surgery group, 2) ACLT group, 3) Fe1-NCs+ACLT group, and 4) Fe2-NCs+ALCT group. Final surgery was performed 8 weeks later according to a carbon dioxide asphyxiation protocol. Synovial joints were harvested for subsequent micro-CT and histomorphometric analysis.

[0067] To systematically evaluate the biosafety and potential toxicity of nanozymes, healthy male Sprague-Dawley (SD) rats were used as experimental subjects. Prior to the experiment, the rats underwent an acclimatization feeding period to ensure optimal physiological conditions. Throughout the experiment, different concentrations of nanozymes were injected via lateral ventricle to elucidate their effects on the central nervous system. Currently, an equal volume of 0.9% sodium chloride solution was injected to establish a control group. Treatment time and dosage were strictly controlled in all groups to maintain the reliability of the experimental results. After administration, the rats were euthanized according to ethical guidelines and subsequently dissected. Key organs harvested included the brain, heart, liver, spleen, lungs, and kidneys, selected based on their critical roles in the organism and their representative responses to exogenous substances. The excised organs underwent standard histological processing, including paraffin embedding, sectioning, and hematoxylin-eosin (HE) staining. Morphological changes induced by nanozymes in various organs were analyzed by careful examination of HE-stained tissue sections, and a comprehensive assessment of their biosafety and toxicity characteristics was conducted.

[0068] To gain a deeper understanding of the molecular mechanisms underlying the therapeutic effects of Fe2-NCs in osteoarthritis (OA), RNA transcriptome analysis was performed on three experimental groups: normal chondrocytes (NCs group), H2O2-induced chondrocytes (H2O2 group), and H2O2-induced cells treated with Fe2-NCs (H2O2+Fe2-NCs group). Clean readings were mapped to a reference genome for analysis of gene expression levels in protein-coding genes. Figure 14Figure a shows the cluster analysis of differentially expressed genes (DEGs). Statistical analysis of DEGs revealed significant differences between the H2O2 and NC groups, identifying a total of 205 DEGs (p<0.05), of which 154 were upregulated and 51 were downregulated, indicating that H2O2 damages and degenerates chondrocytes by regulating the expression of a series of genes. Compared with the H2O2 group, the H2O2+Fe2-NCs group showed only 113 DEGs (p<0.05), with 36 genes upregulated and 77 genes downregulated in the H2O2 group. This suggests that Fe2-NCs intervention significantly remodeled the gene expression profile altered by oxidative stress and may alleviate the pathological changes in chondrocytes by inhibiting or repairing molecular pathways related to cell damage.

[0069] Further cross-analysis of DEGs revealed 41 cross-DEGs, which are potential therapeutic targets for Fe2-NCs in OA treatment. Figure 14 (b and c). A specific gene, cyclooxygenase-2 (PTGS2, also known as COX2), was significantly upregulated in the H2O2 group and significantly downregulated in the H2O2+Fe2-NCs group, suggesting that Fe2-NCs exert an anti-inflammatory effect by inhibiting COX2 expression, potentially alleviating joint degeneration. Subsequently, pathway analysis was performed on DEGs in the H2O2+Fe2-NCs group using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Figure 14 The enrichment scores were plotted along the horizontal axis (d). Enrichment analysis showed that the NF-κB signaling pathway had the highest enrichment score. Considering the crucial role of NF-κB activation in the progression of cartilage damage and the pathogenesis of arthritis, it can be concluded that Fe2-NCs can exert anti-inflammatory and chondrogenic effects in OA by regulating the NF-κB signaling pathway. Gene Ontology (GO) analysis and cell function analysis (…) Figure 14 The study (e.g.) suggests that Fe2-NCs will exert therapeutic effects in OA through multiple mechanisms, including modulating inflammatory signaling pathways, alleviating oxidative stress, and restoring cellular homeostasis.

[0070] The pathogenesis of osteoarthritis (OA) is closely related to the inflammatory response, and the upregulation of inflammatory mediators is a key indicator of disease progression. Transcriptome analysis further confirmed these observations, allowing for the investigation of the specific mechanisms involved in the inflammatory response. Figure 15Tables a and b show the expression levels of phosphorylated nuclear factor κb (p-NF-κb) and COX-2 quantified by polymerase chain reaction (qPCR). Notably, the qPCR results are highly consistent with transcriptomic findings, highlighting the key role of the synthesized nanozyme in significantly reducing COX-2 expression and inhibiting NF-κB pathway activation. To further elucidate the role of ROS in OA, the expression of NADPH oxidase 4 (NOX4) was specifically investigated. qPCR analysis results showed that Fe2-NCs effectively attenuated NOX4 expression, reduced ROS production, and mitigated mitochondrial damage. Figure 15 (c) Western blot analysis and immunofluorescence further validated the above results. Figure 15 (df). Fe2-NCs nanozymes can effectively reverse the rapid expression of p-NF-κB and COX-2 induced by H2O2. By alleviating ROS accumulation and inhibiting the activation of inflammatory signaling pathways, the synthesized Fe2-NCs nanozymes show promising therapeutic potential in combating inflammation and protecting cartilage from degeneration.

[0071] VI. Inhibiting the COX-2 / NF-κB signaling pathway and alleviating NOX4-activated Fe2-NCs-induced mitochondrial dysfunction During the initiation and progression of osteoarthritis (OA), excessive degradation of type II collagen (COL2), a decisive marker of matrix metalloproteinase-13 (MMP-13) cartilage degeneration, impairs ECM remodeling and regeneration, further exacerbating cartilage degradation and accelerating OA progression. Figure 15 As shown in Figure d, compared with the control group, H2O2 stimulation in chondrocytes significantly increased MMP-13 expression, thereby promoting COL2 degradation. Fe2-NCs are a novel class of antioxidants that can effectively inhibit H2O2-induced upregulation of MMP-13 in chondrocytes, thereby alleviating COL2 degradation and slowing the progression of OA. Therefore, the significantly reduced expression levels of MMP-13 and other inflammation-related proteins provide a promising therapeutic approach for OA intervention.

[0072] Typically, abnormally elevated ROS levels lead to mitochondrial dysfunction, subsequently triggering chondrocyte apoptosis, amplifying the inflammatory cascade, and accelerating ECM degradation of cartilage. This study investigated mitochondrial integrity using cytochrome c oxidase subunit IV (COX IV) as a mitochondrial marker. Figure 15(i.e., in H2O2-induced oxidative stress chondrocytes, NOX4 expression was significantly upregulated while COXIV levels were significantly decreased, indicating mitochondrial damage during oxidative stress. Interestingly, a significant decrease in NOX4 expression and a significant increase in COXIV levels were observed in the Fe2-NCs group, suggesting that Fe2-NCs provide effective protection against ROS-induced mitochondrial damage, indicating that their mitochondrial protective effect is attributed to the comprehensive restoration of mitochondrial function.) Figure 15 As shown in Figure j, under H2O2 environment, Fe2-NCs nanozymes can effectively exert SOD-like and CAT-like effects, reduce oxidative stress-induced mitochondrial damage by normalizing ATP and downregulating NOX4, protect mitochondrial function, and avoid energy deficiency.

[0073] VII. In vivo therapeutic effects of Fe2-NCs on OA Detailed imaging of the knee joint of each mouse was performed using the high-resolution Skyscan 1276 miniature CT scanner (Brück, Germany), obtaining high-precision imaging data of its internal structures. The scanning process ensured comprehensive coverage of all joint components, resulting in high-resolution three-dimensional images that laid the foundation for subsequent analysis. After scanning, the original images were reconstructed using the 3D reconstruction software NRecon (version 1.7.4.2, Bück, Germany) to delineate regions of interest (ROIs). This step not only improved the accuracy of data processing but also laid a solid foundation for further research. To analyze the selected ROIs, CT Analyzer (version 1.20.3.0, Bück, Germany) software was used to maintain consistent evaluation parameters. This software calculated several key parameters, including total tissue volume (TV), bone volume (BV), subchondral bone plate thickness, and osteophyte size. These parameters are crucial for elucidating the progression of osteoarthritis and its associated pathological changes. To ensure the objectivity and accuracy of the analysis results, two professional radiologists independently assessed the severity of osteoarthritis and the maturity of osteophytes using a blinded approach during the imaging evaluation. The double-blind assessment method significantly reduces the potential subjective bias of assessors, thereby improving the reliability and validity of the survey results.

[0074] In this study, mouse knee joint tissues were collected for detailed histological evaluation and analysis. Knee joint tissues were first fixed in 4% paraformaldehyde at room temperature for one week, a process performed without light exposure to prevent any potential light-induced effects on the tissues. After fixation, tissue samples were transferred to a 10% EDTA solution for decalcification, which may continue for up to one month to effectively remove bone mineral content while maintaining the integrity of cellular structures. After decalcification, tissue samples were embedded in paraffin for subsequent sectioning. The thickness of the embedded tissue sections was set to 7 µm, suitable for further microscopic observation and analysis. After sectioning, the samples were stained with hematoxylin and eosin (H&E) to observe fine morphological structures, including cell nuclei, cytoplasm, and overall tissue morphological characteristics. Furthermore, Safran Red O / Fast Green staining was used to assess the distribution of glycosaminoglycans (GAGs) in the knee joint tissues, which is crucial for understanding the health status of articular cartilage. By combining these two staining techniques, a more comprehensive assessment of cartilage morphological changes and functional status was achieved. To quantify the degree of cartilage degeneration, three-dimensional computed tomography (3D-CT) imaging was combined with histological staining results, and the Osteoarthritis Research Society International (OARSI) scoring system was used to assess cartilage degeneration. The OARSI scoring system is a widely recognized standard that effectively assesses cartilage degeneration in osteoarthritis, taking into account multiple aspects such as cartilage thickness, structural integrity, and cell viability.

[0075] To further explore the therapeutic potential of synthesized nanozymes for osteoarthritis (OA), an OA mouse model (ACLT) was established by anterior cruciate ligament transection. Figure 16 (a) After successful model induction, five experimental groups were formed: Sham, ACLT, ACLT+NCs, ACLT+Fe1-NCs, and ACLT+Fe2-NCs. In this study, all samples were collected 8 weeks post-surgery, and a three-dimensional model of the mouse knee joint was generated using micro-CT scanning and reconstruction techniques, which facilitated a comprehensive and intuitive assessment of the knee joint condition. Figure 16 (b) Quantitative analysis of micro-CT images showed that ACLT surgery led to significant changes in key skeletal features. Specifically, the Fe2-NCs group (bone volume fraction: 45.63, osteophyte size: 1.25, osteophyte maturity: 1.38, subchondral bone plate thickness: 0.0539) showed significant treatment improvements in bone volume fraction and osteophyte size, while cartilage degeneration was also significantly reduced. Figure 16The results (cf) indicate that Fe2-NCs successfully and effectively intervened in OA. In contrast, the reduction in knee OA symptoms in the Fe1-NCs (43.80, 1.38, 1.63, 0.0635) and NCs (32.78, 2.88, 2.75, 0.096) groups was negligible. These results collectively highlight the beneficial therapeutic role of synthetic Fe2-NCs nanozymes in treating ACLT-induced OA. Figure 8 The results showed that the Fe2-NCs+ACLT score of the International Osteoarthritis Research Society (OARSI) was approximately 4.13, which was lower than that of other control groups, indicating that the synthesized Fe2-NCs nanozyme effectively reduced cartilage damage after ACLT-induced injury and promoted recovery.

[0076] Eight weeks after ACLT surgery, the protective effect of Fe2-NCs nanozymes on cartilage was evaluated through comprehensive histological analysis. Figure 16 H&E and Safranin O / Fast Green histological staining showed severe degeneration of knee cartilage in mice receiving ACLT, exhibiting characteristics of osteoarthritis (OA). Specifically, irregular surface wear and erosive cracks were evident, accompanied by severe damage to the cartilage matrix and cellular structure, further indicating that the natural repair mechanisms of cartilage were severely impaired. These changes ultimately led to significant thinning of the cartilage layer, a significant decrease in chondrocyte density, and a significant narrowing of the joint space. In contrast, the Fe2-NCs group showed a significant reduction in ACLT-induced cartilage damage. The overall cartilage layer in these groups was significantly thickened, and the structural integrity of the cartilage tissue was significantly restored. Cell arrangement appeared to be more orderly, and the chondrocyte count was significantly higher than in the ACLT group. In addition, the joint space was significantly wider, supporting the conclusion that the synthesized nanozyme effectively alleviated cartilage damage and provided protective benefits to the joint structure. The effects on arthritis-related inflammatory markers and gene expression profiles were investigated by immunofluorescence analysis. Figure 16 (j). The results showed that, compared with the knee joints of mice that underwent ACLT surgery alone, treatment with synthetic nanozymes significantly reduced the expression of key markers associated with the pathogenesis of OA and cartilage degeneration, including COX-2, p-NF-κB, and MMP13. In the knee joints of treated mice, the expression of type II collagen was significantly upregulated. These results indicate that synthetic nanozymes alleviate ACLT-induced OA by inhibiting inflammation and promoting cartilage repair. It has been reported that NF-κB is activated under oxidative stress, activating downstream pro-inflammatory protein kinase COX-2 and the oxidative stress-related protein NOX4 via phosphorylation. Subsequently, p-NF-κB induces inflammation in a transcriptionally and non-transcriptionally dependent manner. Figure 16 (k). Therefore, it is reasonable to speculate that Fe2-NCs alleviate oxidative stress-induced mitochondrial damage and achieve OA repair by interfering with the NF-κB signaling pathway.

[0077] VIII. In vivo biosafety and toxicity of Fe2-NCs Biosafety and toxicity are key considerations for the potential clinical translation of novel nanomaterials. Therefore, the biosafety of the synthesized nanozymes was rigorously evaluated in vivo. Eight weeks post-ACLT surgery, comprehensive anatomical and histopathological evaluations of organs from all groups of mice were performed. To rule out any potential organ toxicity caused by the nanozymes, histological analysis of key organs (heart, liver, spleen, lung, and kidney) was performed using H&E staining. Figure 17 The results showed no evidence of cell damage, necrosis, or inflammatory cell infiltration in organs and tissues exposed to nanozymes, confirming their safety. All these findings lay a solid foundation for subsequent clinical studies.

Claims

1. The application of a diatomic iron-iron site nanozyme in the preparation of a targeted therapeutic drug for osteoarthritis by alleviating oxidative stress and cartilage degeneration, wherein the diatomic iron-iron site nanozyme is an Fe2 diatomic site dimer embedded in nitrogen-modified carbon defect sites and loaded on the surface of a three-dimensional porous carbon framework.

2. The application according to claim 1, characterized in that, The iron-iron site nanozyme has a bridging bond with a distance of 2.18 ± 0.05 Å between iron atoms.

3. The application according to claim 1, characterized in that, The active concentration of the diatomic iron-iron site nanozyme is 5-50 μg / ml.

4. The application according to claim 1, characterized in that, The size of the diatomic iron-iron site nanozyme is 1-150 nm.

5. The application according to claim 1, characterized in that, The diatomic iron-iron site nanozyme was prepared by the following method: Zinc and iron sources were dissolved in a ternary mixed solvent to obtain solution A; 2-methylimidazole was dissolved in a binary mixed solvent to obtain solution B. Solution A was added to solution B to obtain a mixture. The mixture was kept at 25±2℃ for 12-48 hours to promote the formation of encapsulated iron species of ZIF-8 to obtain a colloidal product. The product was washed and dried to obtain the precursor material. The precursor material was pulverized and heated to carbonize, yielding the diatomic iron-iron site nanozyme.

6. The application according to claim 5, characterized in that, The zinc source is selected from zinc nitrate and / or zinc acetate; the iron source is selected from ferric acetate and / or tetracarbonyl ferrocene dimer.

7. The application according to claim 5, characterized in that, The molar ratio of the zinc source to the iron source is (3~8):(0.015~0.06).

8. The application according to claim 5, characterized in that, The ternary mixed solvent comprises N,N-dimethylformamide, methanol, and ethylene glycol; the volume ratio of N,N-dimethylformamide, methanol, and ethylene glycol is (1~3):1:

1.

9. The application according to claim 5, characterized in that, The binary mixed solvent comprises N,N-dimethylformamide and methanol; the volume ratio of N,N-dimethylformamide to methanol is 3:(1~2).

10. The application according to claim 5, characterized in that, Conditions for carbonization by heating: under an inert atmosphere, the heating rate is 4~6℃ / min, the temperature is 900~950℃, and the time is 3~4 hours.

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