Use of mn 3o 4 nanoparticles in alleviating intervertebral disc degeneration, biomaterial, and preparation method therefor

Abstract

The present invention relates to the technical field of biomedicine. The present invention aims to provide a use of Mn3O4 nanoparticles in alleviating intervertebral disc degeneration, trimanganese tetroxide nanoparticles Mn3O4 NPs are added as nanozymes to interventional drugs for treating intervertebral disc degeneration. The present invention uses Mn3O4 nanoparticles as nano-active catalytic enzymes, which have multi-enzyme catalytic activities similar to superoxide dismutase, catalase, and glutathione peroxidase, and can effectively scavenge reactive oxygen species (ROS) and control ECM metabolism, significantly reducing cell apoptosis, delaying cell senescence, inhibiting inflammation, and enhancing autophagy, thereby promoting annulus fibrosus AF repair in the treatment of intervertebral disc degeneration. Mn3O4 nanoparticles have good biocompatibility, and metabolites thereof can be used in other physiological processes, producing no toxic side effects in the in vivo circulation. Furthermore said nanoparticles perform stably in adverse microenvironments of low oxygen and low pH in intervertebral discs, and have in vivo stability that other active catalytic enzymes do not possess.

Description

Applications of Mn3O4 nanoparticles in alleviating intervertebral disc degeneration, biomaterials and their preparation methods Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of Mn3O4 nanoparticles in alleviating intervertebral disc degeneration. Background Technology

[0002] Annulus fibrosus (AF) injury plays a crucial role in intervertebral disc degeneration (IVDD), and oxidative stress and extracellular matrix (ECM) metabolic imbalance are considered major pathogenic factors for AF injury. However, effective methods for repairing damaged AF are still lacking.

[0003] Intervertebral disc (IVD) degeneration (IVDD) is a common degenerative spinal disease and a leading cause of lower back pain. With an aging population, the incidence of IVDD continues to rise. IVDD not only causes pain and a decline in quality of life, potentially leading to disability, but also places a significant economic burden on the social healthcare system. However, due to the complex biological processes involved in IVDD, including structural damage, release of inflammatory mediators, changes in enzyme activity, and cell death, current treatment options are very limited. Existing treatments mainly include conservative therapy and surgery, but their effectiveness is limited, with high recurrence rates and numerous complications. Given the complex pathological mechanisms of IVDD, finding more effective treatment options is crucial to mitigating its impact.

[0004] The intervertebral disc consists of the nucleus pulposus (NP), annulus fibrosus (AF), and cartilaginous endplates. These structures can change with age and other factors. The pathogenesis of intervertebral disc degeneration is complex, involving mechanical, biochemical, and genetic factors. Among these, the annulus fibrosus, which ruptures due to prolonged mechanical stress, plays a crucial role in intervertebral disc degeneration. The annulus fibrosus is a tough fibrous tissue structure surrounding the nucleus pulposus, primarily responsible for protecting the nucleus pulposus and maintaining the structural integrity of the intervertebral disc. During degeneration, the annulus fibrosus may tear or be damaged due to aging, prolonged mechanical pressure, or acute injury. This damage can lead to protrusion or extrusion of the nucleus pulposus, compressing nerve roots and causing symptoms such as pain, numbness, and stiffness, while also reducing the height and stability of the intervertebral disc. Furthermore, annulus fibrosus damage can trigger local inflammation, accelerating disc degeneration. Clinically, the recurrent nucleus pulposus protrusion rate after discectomy can be as high as 26% due to unresolved annulus fibrosus defects. Therefore, repairing annulus fibrosus damage is crucial for the treatment of intravertebral discectomy (IVDD).

[0005] Oxidative stress is primarily manifested as elevated levels of intracellular reactive oxygen species (ROS) and a relative decrease in antioxidants such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), accompanied by a reduction in small-molecule antioxidants (such as vitamin C, vitamin E, and glutathione). Oxidative stress is considered a major pathogenic factor in annulus fibrosus injury. In intraepithelial neoplasia (IVDD), oxidative stress is closely associated with cell death, cellular senescence, autophagy, extracellular matrix (ECM) remodeling, and inflammation. This highlights the important role of oxidative stress in the progression of IVDD. Therefore, antioxidants targeting oxidative stress are crucial for slowing the progression of IVDD and hold great potential. Although many natural substances with antioxidant properties (such as quercetin and mangiferin) have shown good therapeutic effects in vitro, their poor stability in the adverse microenvironment of the intervertebral disc (hypoxia, low pH) greatly limits their application.

[0006] Mn3O4 nanoparticles (Mn3O4NPs) with a monodisperse nanoflower-like structure possess the activity of three major antioxidant enzymes: SOD, CAT, and GPx. Furthermore, manganese (Mn), as an essential trace element for the human body, exhibits good biocompatibility, and its metabolites can be used in other physiological processes. Mn3O4 NPs not only possess multi-enzyme activity but also exhibit excellent photothermal properties, making them a promising candidate material for adjuvant treatment of IVDD via photothermal therapy (PTT). Near-infrared light is commonly used in PTT due to its strong tissue penetration. Although the high temperature of PTT (>50°C) may pose a potential risk of damage to surrounding healthy tissue, mild photothermal therapy (MPTT) (40-43°C) can promote tissue repair while exerting anti-inflammatory effects. Therefore, with its multi-enzyme activity and excellent photothermal properties, Mn3O4 NPs may be a promising candidate material for the treatment of IVDD.

[0007] However, whether such excellent nanoparticles as Mn3O4 NPs can truly participate in the specific therapeutic applications of IVDD remains unresolved. Furthermore, because the intervertebral disc is a relatively avascular structure, drugs cannot easily penetrate directly into it via blood circulation. In addition, repeated local injections may accelerate the progression of IVDD. Therefore, designing a drug delivery system capable of precisely delivering drugs to the lesion site and maintaining an effective concentration is also essential. Summary of the Invention

[0008] The purpose of this invention is to provide a method for applying Mn3O4 nanoparticles in alleviating intervertebral disc degeneration, offering a novel approach to IVDD treatment, effectively alleviating intervertebral disc degeneration, and improving the therapeutic effect of IVDD.

[0009] To achieve the above-mentioned objectives, the technical solution adopted in this invention is: the application of Mn3O4 nanoparticles in alleviating intervertebral disc degeneration, by adding manganese tetroxide nanoparticles (Mn3O4NPs) as nanoenzymes to interventional drugs for treating intervertebral disc degeneration.

[0010] Preferably, manganese tetroxide nanoparticles (Mn3O4NPs) are added to the hydrogel, and the hydrogel is used as a delivery medium to form an injection.

[0011] Preferably, the hydrogel is hydrogel ChS-HA, which is composed of dopamine-grafted chondroitin sulfate dop-OChS and ADH-modified hyaluronic acid ADH-HA.

[0012] Preferably, the injection achieves therapeutic benefit by combining with mild photothermal therapy (MPTT).

[0013] Preferably, a biomaterial Mn3O4@ChS-HA for treating intervertebral disc degeneration includes manganese tetroxide nanoparticles Mn3O4NPs and a hydrogel ChS-HA composed of dopamine-grafted chondroitin sulfate dop-OChS and ADH-modified hyaluronic acid ADH-HA; together they constitute the biomaterial Mn3O4@ChS-HA with an MPTT-nanozyme-hydrogel delivery system.

[0014] A preferred method for preparing Mn3O4@ChS-HA, a biomaterial for treating intervertebral disc degeneration, includes the following steps:

[0015] S1. Raw material preparation:

[0016] Preparation of manganese tetroxide nanoparticles (Mn3O4NPs):

[0017] Oleic acid was added to a 0.2 wt / v% KMnO4 aqueous solution at a volume ratio of 2:100 and stirred at room temperature for 6 hours. Then, the mixture was centrifuged at 8000 rpm for 5 min, and the precipitate was washed with deionized water and ethanol in sequence. The precipitate was then collected and dried at 80°C for 10 hours. The resulting brown solid was ground and calcined in an oven at 200°C for 5 hours. After grinding again, the solid was passed through a 50-mesh sieve to obtain Mn3O4NPs powder.

[0018] Preparation of dopamine-grafted chondroitin sulfate (dop-OChS):

[0019] Oxidized chondroitin sulfate OChS was prepared using NaIO4 as an oxidant. The carboxyl group on oxidized chondroitin sulfate OChS was further activated by carbodiimide EDC and N-hydroxysuccinimide NHS. After adding dopamine, the mixture was stirred at room temperature in the dark for 24 h to obtain dopamine-grafted oxidized chondroitin sulfate dop-OChS.

[0020] Preparation of ADH-modified hyaluronic acid (ADH-HA):

[0021] HA was activated with carbodiimide EDC and 1-hydroxybenzotriazole HOBT, and then reacted with adipicodiacetylhydrazine (ADH) at room temperature to obtain adipicodiacetylhydrazine-modified hyaluronic acid (ADH-HA).

[0022] Preparation of S2, Mn3O4@ChS-HA:

[0023] Mn3O4NPs were added to a 5% wt / v dop-OChS solution at a concentration of 2 mg / ml and mixed with a 5% wt / v ADH-HA PBS solution at a volume ratio of 1:2 to obtain an injectable hydrogel Mn3O4@ChS-HA loaded with Mn3O4NPs.

[0024] The beneficial effects of this invention are mainly reflected in:

[0025] 1. It uses Mn3O4 nanoparticles as nano-active catalytic enzymes, which have multi-enzyme catalytic activities similar to superoxide dismutase, catalase and glutathione peroxidase. It can effectively remove reactive oxygen species (ROS) and regulate ECM metabolism, significantly reduce cell apoptosis, delay cell senescence, inhibit inflammation, and enhance autophagy, thereby promoting the repair of annulus fibrosus (AF) in the treatment of intervertebral disc degeneration.

[0026] 2. Mn3O4 nanoparticles exhibit excellent biocompatibility, and their metabolites can be used in other physiological processes without producing toxic side effects during in vivo circulation. Furthermore, they demonstrate stability in the adverse microenvironment of the intervertebral disc, characterized by low oxygen and low pH, exhibiting in vivo stability not found in other active catalytic enzymes.

[0027] 3. Preferably, Mn3O4 nanoparticles are loaded onto a hydrogel. This special hydrogel delivery medium mimics the natural environment of the intervertebral disc, offering excellent tissue compatibility, the ability to fill irregular wounds, and reduction of scarring and pain. Simultaneously, it allows for the localized, controlled, and sustained release of Mn3O4 NPs. It not only possesses excellent injectability and biocompatibility but also good mechanical properties and adhesion, enabling it to adhere tightly to annulus fibrosus defects and provide support for the intervertebral disc, continuously exerting effects such as ROS scavenging, anti-apoptosis, anti-aging, and promoting autophagy.

[0028] 4. Mn3O4 nanoparticles also exhibit excellent photothermal properties. Combined with MPTT, they can fully exert the effects of Mn3O4@ChS-HA in inhibiting inflammation, promoting the repair of annulus fibrosus damage, and slowing the progression of IVDD, making them a basis for adjuvant treatment of IVDD using photothermal therapy (PTT). By combining MPTT with interventional photothermal therapy, its tissue penetration can be further enhanced, achieving a therapeutic benefit and improving the overall treatment outcome. Attached Figure Description

[0029] Figure 1 is a statistical chart showing the basic physical properties of the Mn3O4NPs of this invention:

[0030] Figure 2 is a statistical characterization chart of the injectable ChS-HA hydrogel of the present invention:

[0031] Figure 3 shows the photothermal performance of injectable Mn3O4@ChS-HA hydrogel under 1064 nm laser irradiation.

[0032] Figure 4 is a statistical graph showing the effect of Mn3O4NPs on AFCs activity as assessed by live / dead cell staining experiments.

[0033] Figure 5 is a statistical graph showing the ability of Mn3O4@ChS-HA to inhibit oxidative stress and apoptosis;

[0034] Figure 6 is a statistical graph showing how Mn3O4@ChS-HA promotes autophagy in AFCs and inhibits aging and inflammation.

[0035] Figure 7 is a statistical chart of the ability of Mn3O4@ChS-HA to regulate the balance of synthesis / catabolism;

[0036] Figure 8 is a statistical chart of the differences in biological behavior caused by Mn3O4@ChS-HA treatment;

[0037] Figure 9 shows the statistical graph of the therapeutic effect of Mn3O4 NPs@ChS-HA in rats;

[0038] Figure 10 is a statistical chart showing how Mn₃O₄@ChS-HA delays IVDD process through the IL-17 signaling pathway.

[0039] Figure 11 is a schematic diagram of the overall concept of the present invention;

[0040] Figure 12 shows the statistical diagram of the photothermal properties of Mn3O4@ChS-HA in vivo;

[0041] Figure 13 shows a representative statistical chart of HE staining of rat heart, liver, spleen, lung and kidney. Detailed Implementation

[0042] This invention innovatively develops an MPTT-nanozyme-hydrogel system Mn3O4@ChS-HA for the treatment of IVDD. This system can not only support the intervertebral disc, but also continuously remove ROS, solving the redox imbalance problem in IVDD. In addition, its combination with MPTT can further enhance its therapeutic effect, showing significant clinical application potential.

[0043] In this invention, an injectable photothermal hydrogel loaded with manganese tetroxide (Mn₃O₄) nanoparticles was developed. Composed of dopamine-modified chondroitin sulfate (dop-OChS) and adipic acid dihydrazine-modified hyaluronic acid (ADH-HA), it carries Mn₃O₄ nanoparticles (Mn₃O₄ NPs), referred to as Mn₃O₄@ChS-HA. This hydrogel not only mimics the natural environment of the intervertebral disc but also possesses excellent mechanical properties, supporting the disc. Its strong adhesion allows it to firmly attach to atrial fibrillation (AF) defects, enabling controlled and continuous release of Mn₃O₄ NPs. Furthermore, compared to other metal nanoparticles, Mn₃O₄ NPs exhibit catalytic properties similar to superoxide dismutase, catalase, and glutathione peroxidase, effectively scavenging reactive oxygen species (ROS) and regulating ECM metabolism. Both in vitro and in vivo experiments showed that Mn₃O₄@ChS-HA hydrogel significantly reduced apoptosis, delayed cell senescence, inhibited inflammation, and enhanced autophagy, thereby promoting AF repair.

[0044] Furthermore, in a rat model of atrial fibrillation (AF) defects, it improved intervertebral disc height and maintained ECM integrity. Mild photothermal therapy (MPTT) further enhanced these effects, promoting tissue repair and delaying IVDD progression. Overall, the MPTT-nanozyme-hydrogel system Mn₃O₄@ChS-HA exhibits multiple biological functions by modulating redox homeostasis and promoting tissue regeneration, providing a promising strategy for IVDD treatment. This invention offers new insights into the treatment of IVDD, particularly in repairing AF damage and delaying disease progression, and has potential clinical applications.

[0045] The present invention will now be described in detail with reference to embodiments: Example 1

[0046] Preparation and characterization of Mn3O4NPs

[0047] 10 mL of oleic acid was added to 500 mL of KMnO4 aqueous solution (0.2 wt / v%), and the mixture was stirred at room temperature for 6 hours. The mixture was then centrifuged (8000 rpm, 5 min), and the precipitate was washed successively with DI water and ethanol. The precipitate was then collected and dried at 80 °C for 10 hours. The resulting brown solid was ground, calcined in an oven at 200 °C for 5 hours, ground again, and passed through a 50-mesh sieve to obtain Mn3O4 NPs powder.

[0048] Mn3O4NPs powder was ultrasonically dispersed in deionized water. The microstructure of Mn3O4NPs was observed at 200 kV using a transmission electron microscope (TEM, Tecnai G2-F30, FEI, Netherlands). The hydrodynamic diameter at 25 °C was measured using a dynamic light scattering spectrometer (DLS, Dandong Better Instruments Co., Ltd., BeNano180Zeta, China). The infrared spectrum of Mn3O4NPs was measured using a Fourier transform infrared spectrometer (Equinox 55, Bruker, Germany) using the KBr pellet method. The valence state of manganese in Mn3O4NPs was analyzed using an X-ray photoelectron spectroscopy (XPS, AXIS-ULTRA DLD-600W, Shimadzu Corporation, Japan). The aqueous dispersion (1.0 mg / mL) of Mn3O4NPs was determined using a UV-Vis spectrophotometer (UV 3600, Shimadzu, Japan). -1 The ultraviolet-visible-near-infrared absorption curves of the sample were obtained. Its crystal structure was determined using an X-ray diffractometer (X′Pert PRO, Panaco GmbH, Netherlands).

[0049] In addition, the activities of catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD) in Mn3O4NPs were measured. First, a certain concentration of Mn3O4NPs was co-incubated with H2O2, and the change in absorbance at 240 nm over time was recorded to characterize CAT activity. Second, the classical coupling assay was used to evaluate GPx activity by measuring the change in NADPH absorbance at 340 nm during the reaction. Specifically, the following substances were added to a 4 mL reaction system in the following order: different concentrations of Mn3O4NPs (0–50 ng / L). -1 Reduced glutathione (GSH, 2 mM), NADPH (200 μM), GR (1.7 units), H2O2 (1 mmol L) -1 The solvent was a phosphate buffer solution with pH 7.4. The absorbance of the system at 340 nm was measured after 2 min of reaction at 25°C. The reaction rate υ (mol / L) was also measured. -1 min -1The extinction coefficient ε was calculated using Origin software according to formula (1), and ε is 6.22 mmol / L. -1 cm -1 l is the optical path length (1 cm), dA is the change in absorbance, and dt is the time interval (s).

[0050] (1)

[0051] The ability of Mn3O4NPs to scavenge superoxide and its SOD-like activity were determined using an SOD assay kit, according to the manufacturer's instructions.

[0052] Detailed results are shown in Figure 1, including (a) TEM image; (b) particle size and distribution determined by DLS; (c) FTIR spectrum; (d) XRD pattern; (ef) XPS plot; (g) CAT-like activity; (h) GPx-like activity; and (i) SOD-like activity.

[0053] TEM images show that the synthesized Mn3O4NPs are nanoflower-like structures with a particle size of approximately 60 nm (Figure 1a). DLS measurements show that the average hydration kinetic diameter of the Mn3O4NPs is 157 nm (Figure 1b). The zeta potential is -0.20 mV. FTIR spectra show peaks at 515 and 611 cm⁻¹. -1 Stretching vibration peaks of Mn-O and Mn-O-Mn bonds appeared at 1529 cm⁻¹ (Fig. 1c). -1 The peak at that location can be attributed to the stretching vibration peak of COO- in oleic acid on the surface of Mn3O4NPs.

[0054] XRD results (Figure 1d) show that the synthesized Mn3O4NPs have a good crystal structure, similar to the tetragonal crystal structure of manganese oxide. Furthermore, XPS analysis was used to determine the valence state of manganese. As shown in Figures 1e-f, the Mn 2p3 / 2 peak is centered at 642.6 eV, and the Mn 2p1 / 2 peak is centered at 654.4 eV. These results indicate the presence of Mn in the Mn3O4NPs. 2+ and Mn 3+ Mixed valence state.

[0055] This study also determined the CAT-like, GPx-like, and SOD-like activities of Mn3O4NPs. As shown in Figure 1g, the absorbance of hydrogen peroxide as a substrate decreased significantly over time in the presence of Mn3O4NPs, indicating that Mn3O4NPs possess catalase-like activity in catalyzing the decomposition of hydrogen peroxide. Furthermore, Mn3O4NPs also exhibited good GPx activity (Figure 1h). It utilizes intracellular GSH as a reducing agent to catalyze the production of water and oxidized glutathione from hydrogen peroxide, while glutathione reductase (GR) further reduces the formation of oxidized glutathione (GSSG) and reduces it to GSH and NADPH. When the concentration of Mn3O4NPs in the reaction system was changed, the reaction rate increased proportionally with the increase in the amount of Mn3O4NPs added. In addition to the activities of CAT and GPx, Mn3O4NPs also exhibit superoxide dismutase (SOD)-like activities by catalyzing the dismutation of superoxide into H2O2 and oxygen (Figure 1i). Therefore, Mn3O4NPs possess activities similar to CAT, GPx, and SOD, effectively decomposing hydrogen peroxide and scavenging peroxide and superoxide free radicals, and are expected to maintain redox homeostasis in vivo. Example 2

[0056] Preparation and characterization of injectable hydrogel ChS-HA loaded with Mn3O4NPs:

[0057] First, adipicohydrazide-modified hyaluronic acid (ADH-HA) and dopamine-grafted chondroitin sulfate (dop-OChS) were prepared as precursors for injectable ChS-HA hydrogels. HA was activated with EDC and HOBT, and then reacted with adipicohydrazide at room temperature to obtain adipicohydrazide-modified hyaluronic acid (ADH-HA). Chondroitin sulfate (OChS) was then prepared using NaIO4 as an oxidant. The carboxyl groups on OChS were further activated with EDC and NHS, and after adding dopamine, the mixture was stirred at room temperature in the dark for 24 h to obtain dopamine-grafted OChS (dop-OChS). Its chemical structure was characterized by attenuated total reflection-fourier transform infrared spectroscopy (ATR-FTIR, VERTEX 70, Bruker Spectroscopy GmbH, Germany) and 1H NMR spectroscopy (AVANCE III 400 MHz, Bruker BioSpin, Switzerland).

[0058] Add Mn3O4NPs (2 mg / mL) to a 5% (wt / v) dop-OChS solution. -1The soluble ChS-HA hydrogel loaded with Mn3O4 NPs was mixed with 5% (wt / v) ADH-HA PBS solution at a volume ratio of 1:2 to obtain an injectable ChS-HA hydrogel (named Mn3O4@ChS-HA) loaded with Mn3O4 NPs. An injectable ChS-HA hydrogel without Mn3O4 NPs was also prepared as a control. The sol-gel transition time of the injectable ChS-HA hydrogel at 37 °C was studied using the bottle inversion method. Further analysis was conducted using a rheometer (MCR302, Anton Paar, Australia) with a 50 mm diameter parallel plate clamp and a 0.5 mm gap to measure the storage modulus (G′) and loss modulus (G″) of the two injectable hydrogels as a function of shear strain and shear frequency (0.1 Hz–10 Hz). The viscosity of Mn3O4@ChS-HA was also measured at 25 °C as a function of shear rate (0.1–1000 s⁻¹). Adhesion tests were used to characterize the adhesive properties of the hydrogels. The hydrogels were pressed onto different surface materials (such as plastics, paper, and test tube caps), and their adhesion was observed and recorded photographically. This method evaluates the adhesion ability of hydrogels on different materials, providing preliminary evidence for their application in biological environments.

[0059] Based on the physiological activities of chondroitin sulfate and hyaluronic acid, we prepared an injectable ChS-HA hydrogel composed of dopamine-grafted oxidized chondroitin sulfate and ADH-modified hyaluronic acid to mimic the natural environment of the intervertebral disc.

[0060] Figure 2 shows: (a) 1H NMR spectra of ADH-HA and dop-OChS; (b) FTIR spectrum of ADH-HA; (c) FTIR spectrum of dop-OChS; (d) UV-Vis spectrum of dop-OChS; (e) sol-gel transition behavior of injectable hydrogels as shown by the bottle inversion method; (f) modulus of hydrogel as a function of shear strain; (g) modulus of hydrogel as a function of shear frequency; (h) shear thinning properties of injectable hydrogels; (i) injectability of injectable hydrogels; and (j) adhesiveness of injectable hydrogels.

[0061] The chemical structures of the prepared ADH-HA and dop-OChS were confirmed by 1H NMR (Fig. 2a) and FTIR (Fig. 2b). For ADH-HA, the peaks near δ 1.6–2.5 ppm correspond to the methylene protons in ADH, while the peaks near δ 2.3–2.5 ppm indicate the presence of hydrazine groups (Fig. 2a). The backbone protons of hyaluronic acid appear near δ 3.2–4.0 ppm. For dop-OChS, the peaks near δ 6.5–7.5 ppm correspond to the protons on the aromatic ring of dopamine; the small peaks near δ 9.5–10.0 ppm are the aldehyde protons in chondroitin sulfate oxidized. In addition, the peaks near δ 2.5–3.5 ppm correspond to the methyl and methylene protons in the polysaccharide structure. In the FTIR spectrum of ADH-HA, the characteristic peak of the amide II band in ADH appeared at 1554 cm⁻¹ (Fig. 2b), indicating successful modification of ADH. OChS exhibits an aldehyde absorption peak at 1725 cm⁻¹, indicating that ChS has been oxidized to form OChS. Compared to OChS, the dop-OChS spectrum shows a new peak near 1500 cm⁻¹ corresponding to the C=C stretching vibration of the aromatic ring in dopamine, indicating that dopamine is grafted onto OChS to form dop-OChS (Figure 2c). The UV-vis spectrum of dop-OChS shows a maximum absorption peak at 280 nm (Figure 2d), further demonstrating that dopamine is grafted onto the OChS framework and has not been oxidized.

[0062] The bottle inversion method experiment showed that a stable gel could be formed after mixing the two precursors of injectable ChS-HA hydrogel and standing at 37℃ for 3 min (Figure 2e). Within the tested range of shear strain and shear frequency variations, the storage modulus (G') and loss modulus (G'') of both ChS-HA and Mn3O4@ChS-HA hydrogels remained relatively stable, with G' always higher than G'' (Figure 2f, g). This indicates that even under high strain and shear frequency, the hydrogel can maintain its elastic properties and is not prone to permanent deformation. These properties enable ChS-HA hydrogels to withstand certain mechanical stresses in applications without significant deformation, maintaining their structural stability and functionality. Furthermore, the viscosity of the injectable ChS-HA hydrogel as a function of shear rate was investigated. The results showed that its viscosity decreased with increasing shear rate, indicating shear-thinning properties. This allows it to flow easily through the syringe (low viscosity at high shear rates), while rapidly recovering its viscosity after injection to form a stable colloid (high viscosity at low shear rates) (Figure 2h). This allows it to be injected into degenerated intervertebral discs via minimally invasive procedures, reducing surgical trauma. The injectable hydrogel can be easily extruded from the syringe and forms a hydrogel in situ (Figure 2i), further demonstrating its injectability and suitability for minimally invasive procedures. The adhesive properties of the hydrogel were characterized using an adhesion test. The hydrogel was pressed onto different surface materials (such as plastics, paper, and test tube caps), and its adhesion effect on these materials was observed and recorded using photographs. This method evaluated the hydrogel's adhesion ability on different materials, providing preliminary evidence for its application in biological environments. The images (Figure 2j) show that the hydrogel can effectively adhere to different surfaces, exhibiting strong adhesion, indicating that it may have good stability in complex biological environments. During the repair of intervertebral disc degeneration (IVDD), hydrogels need to continuously adhere to the surface of the intervertebral disc tissue under dynamic mechanical forces to provide a stable microenvironment to promote tissue regeneration. This adhesion test preliminarily demonstrates the adaptability and potential application prospects of hydrogels, providing a basis for their use in IDD treatment. Example 3

[0063] Verification of the near-infrared II region photothermal properties of Mn3O4@ChS-HA hydrogel:

[0064] The solutions contained different concentrations (0, 0.5, 1, and 2 mg / mL). -1Injectable ChS-HA hydrogels of Mn3O4NPs were prepared. 1 mL of each of the above composite systems was placed in a 4 mL cuvette, and a near-infrared laser (MW-GX-1064 / 5000mW, Changchun Laser Optoelectronics Technology Co., Ltd.) was used in the NIR II region (1064 nm, 1.5 W cm⁻¹). -2 The sample was irradiated for 6 minutes. The distance between the laser and the sample was set to 10 cm. The temperature change of the system was monitored using an infrared thermal imager (MobIR Air).

[0065] In addition, the concentration of Mn3O4@ChS-HA was determined to be 2 mg / mL. -1 Injectable Mn3O4@ChS-HA hydrogels were tested at different laser power densities (0, 0.5, 1.0, 1.5 and 2 W cm⁻¹). -2 The temperature rise of the system under 1064 nm laser irradiation was measured. Each experiment was performed in triplicate. The temperature rise of the system under laser density of 1.5 W / cm² was also measured. -2 The photothermal stability of the system was evaluated by measuring temperature changes during five laser on-off cycles. Additionally, the system was tested with a power density of 1.5 W / cm². -2 The system was irradiated with a 1064 nm laser for 15 minutes, then allowed to cool naturally to room temperature. Temperature changes during the experiment were recorded using an infrared thermal imager, and a temperature-time curve was plotted. The photothermal conversion efficiency of the system was calculated using the photothermal efficiency formula.

[0066] The results are shown in Figure 3, where (a) is the UV-Vis-NIR absorption spectrum of Mn3O4NPs; and (b) is the effect of Mn3O4NPs concentration in the gel on the temperature rise (1.5 W cm⁻¹). -2 (n=3); (c) Effect of laser power density on the photothermal properties of the gel (the concentration of Mn3O4NPs in the gel is 2 mg / mL) -1 (n=3); (d) Changes in gel temperature during laser irradiation on-off cycle experiments; (e) Temperature curves of gel after NIR-II laser irradiation for 15 minutes followed by natural cooling to room temperature (Mn3O4NPs concentration: 2 mg / mL) -1 Laser power density: 1.5 W / cm² -2 (f) The linear relationship between time t and the dimensionless constant -lnθ obtained from the cooling curve in (e);

[0067] First, the UV-Vis-NIR absorption spectrum of Mn3O4NPs solid powder was tested. The results showed that it has good light absorption performance in both the I and II regions of the near-infrared spectrum (Figure 3a). Further investigation was conducted at a laser power density of 1.5 W / cm². -2The temperature rise of injectable ChS-HA hydrogels containing different concentrations of Mn3O4NPs under 1064 nm laser irradiation was investigated. The results showed that the temperature rose faster and higher with increasing Mn3O4NPs concentration, indicating that the concentration of Mn3O4NPs has a significant impact on the photothermal effect (Figure 3b). The concentration of Mn3O4NPs was 2 mg / mL. -1 At this time, the temperature rise of the hydrogel is more significant with the increase of laser power density (Figure 3c). When the laser power density is 1.5 W / cm², the temperature rise is more pronounced. -2 After 6 minutes of irradiation, the system temperature rose by 21.3℃.

[0068] In the laser irradiation on-off cycle experiment, the temperature change of the hydrogel exhibited good reversibility and stability, proving that it can still maintain excellent photothermal response performance after multiple cycles (Figure 3d). To calculate the photothermal conversion efficiency of Mn3O4@ChS-HA hydrogel under 1064 nm laser irradiation, the concentration of Mn3O4NPs at 2 mg / mL was first determined. -1 Injectable Mn3O4@ChS-HA hydrogel under 1064 nm laser irradiation (1.5 W cm⁻¹) -2 The heating-cooling curve of the system is shown in Figure 3e. The linear relationship between time t and the dimensionless constant -lnθ is shown in Figure 3f. Based on the disclosed formula and the system's A1064 = 0.425 at this point, the injectable Mn3O... 4 The photothermal conversion efficiency (η) of the @ChS-HA hydrogel was 46.3%. These results indicate that the injectable hydrogel possesses excellent NIR-II photothermal properties. Based on these results, subsequent experiments fixed the concentration of Mn3O4 NPs in the injectable Mn3O4@ChS-HA hydrogel at 2 mg / mL. -1 The power density of 1064 nm laser irradiation is 1.5 W / cm². -2 .

[0069] The next step will be to further verify the adhesion and biocompatibility of the hydrogel in biological tissues through in vivo experiments, in order to evaluate its effectiveness in practical applications. Example 4

[0070] 4.1 Isolation and culture of rat AFCs:

[0071] AFCs were extracted from the annulus fibrosus tissue of rat tails. First, rats were aseptically euthanized, and the intervertebral discs were isolated from the tail vertebrae. Next, the annulus fibrosus was separated from the nucleus pulposus. Using clean scissors, the annulus fibrosus tissue was cut into small pieces and placed in 1.5 ml centrifuge tubes. The tissue was transferred to 15 ml centrifuge tubes and digested with 0.25% trypsin solution on a shaker at 37°C for 30 minutes. The trypsin solution was removed, and the tissue was further digested with type I collagenase solution on a shaker at 37°C for 2 hours. After digestion, culture medium was added to terminate the digestion. The mixture was filtered and the cell pellet was collected by centrifugation (1500 rpm, 5 minutes). The cell pellet was resuspended in DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. The cells were seeded into cell culture flasks and cultured in a 37°C, 5% CO₂ incubator.

[0072] 4.2 Intervention and Treatment of AFCs

[0073] First, Mn3O4 nanoparticles with different concentrations (0, 1, 2, 5, 8, 10, 20 and 30 µg mL) were prepared. -1 Hydrogels were prepared by immersing them in cell culture medium to obtain extracts, which were then used to treat AFCs to assess their cytotoxicity. To evaluate the effect of Mn3O4 nanoparticles on AFC survival under oxidative stress, AFCs were pretreated with Mn3O4 nanoparticles for 24 hours, followed by treatment with H₂O₂ for 6 hours.

[0074] 4.3 CCK-8 Cell Counting Kit Experiment

[0075] Cell viability was assessed using a CCK-8 assay kit. In short, AFCs were seeded in 96-well plates at 5000 cells per well and cultured adherently at 37°C in a 5% CO₂ incubator for 24 hours. After culture, AFCs were treated with different concentrations of the test compound for 12 hours. Following treatment, CCK-8 working solution was added to each well, and the plates were incubated at 37°C for 2 hours. Absorbance was measured at 450 nm using a microplate reader (Thermo Scientific, MA, USA).

[0076] 4.4 Calcein-AM / PI staining

[0077] After Calcein-AM / PI staining, the cells were incubated with PBS solution containing Calcein-AM and propidium iodide at 37°C in the dark for 20 minutes. Excess dye was gently washed away with PBS, and then observed under a fluorescence microscope (Olympus IX71; Olympus Inc., Tokyo, Japan) to distinguish between live cells (green fluorescence) and dead cells (red fluorescence).

[0078] 4.5 Flow cytometry analysis

[0079] Detection of AFC cell death using PI staining: AFC cell death was assessed by propidium iodide (PI) staining. PI selectively enters dead cells and is detected by flow cytometry (BD LSR II).

[0080] Apoptosis of AFCs was detected using the Annexin V-FITC / PI apoptosis detection kit: phosphatidylserine residues on the outer membrane of apoptotic cells were exposed by Annexin V-FITC labeling, and necrotic cells were distinguished by PI. Fluorescence intensity was analyzed by flow cytometry (BD LSR II).

[0081] Detection of intracellular ROS in AFCs using DCFH-DA: AFCs were incubated with the ROS-sensitive fluorescent dye DCFH-DA, and the fluorescence intensity was detected by flow cytometry or fluorescence microscopy (BD LSR II).

[0082] MitoSOX Red was used to detect mitochondrial ROS: AFCs were stained with MitoSOX Red, selectively accumulated in mitochondria, and fluoresced under superoxide oxidation. The results were analyzed by flow cytometry (BD LSR II).

[0083] Mitochondrial membrane potential was detected using the JC-1 kit: AFCs were stained with JC-1 dye, showing red fluorescence when mitochondria were normal and green fluorescence when depolarized. The red-green fluorescence ratio was analyzed by flow cytometry (BD LSR II).

[0084] 4.6 SA-β-Gal staining

[0085] Detection of senescent cells: First, wash AFCs with PBS and fix them with β-Gal fixative at room temperature for 15 minutes. After three washes with PBS, incubate the AFCs in staining solution overnight at 37°C (CO₂-free). During this process, senescent cells will turn blue. Finally, wash the cells again and observe them under a microscope.

[0086] 4.7 Western blot analysis

[0087] After treatment, AFCs were lysed with a buffer containing protease and phosphatase inhibitors, and protein quantification was performed. SDS loading buffer was added, and the protein was denatured by boiling at 95°C. Equal volumes of protein were loaded onto SDS-PAGE gels and separated according to molecular weight. The protein was transferred to a PVDF membrane and blocked with 5% skim milk in TBST for 1 hour. The membrane was incubated overnight with primary antibody at 4°C, and then incubated for 1 hour at room temperature with HRP-labeled secondary antibody. After washing with TBST, protein bands were visualized by chemiluminescence using ECL substrate.

[0088] 4.8 Quantitative Real-Time PCR (qRT-PCR) Analysis

[0089] After processing, total RNA was extracted from AFCs using TRIzol reagent to ensure RNA concentration and purity. Absorbance at 260 / 280 nm was measured using a NanoDrop 2000 to convert the RNA into cDNA. qRT-PCR reactions were set up and run under specific cycling conditions. Fluorescence data during amplification were collected, and Ct values ​​were recorded. Gene expression was analyzed by normalizing to a reference gene using the ΔΔCt method.

[0090] 4.9 RNA Sequencing Analysis

[0091] RNA-seq analysis was performed to explore potential regulatory mechanisms. After processing, high-quality RNA was extracted from AFCs, and RNA-Seq libraries were prepared by mRNA isolation or rRNA removal, fragmentation, and cDNA synthesis. Sequencing was performed on a high-throughput platform (Guangzhou Gene Island Biotechnology Co., Ltd.). Data preprocessing included quality control, read trimming, and alignment with a reference genome. Differentially expressed genes (DEGs) under different conditions were identified using DESeq2 (|log2FC| > 1.5, P < 0.05). Subsequent bioinformatics analyses, including Gene Ontology (GO) and Kyoto Genome Encyclopedia (KEGG) analyses, were performed to elucidate gene expression patterns and differential expression.

[0092] 4.10 Animal Experiments

[0093] A common model for studying intervertebral disc degeneration and repair is the annulus fibrosus defect. In this model, a defect is created in the outer annulus fibrosus of the intervertebral disc to simulate disc injury or degeneration. Thirty male Sprague Dawley rats (weighing 200-250 g) were divided into five groups according to different surgical procedures: sham operation group, IVDD group, IVDD+ChS-HA group, IVDD+Mn3O4@ChS-HA group, and IVDD+Mn3O4@ChS-HA+MPTT group. A longitudinal incision of approximately 2 cm was then made along the midline posterior to the 6th / 7th cervical vertebra to expose the intervertebral disc. The disc was then punctured with an 18-gauge needle to create a full-thickness defect of the annulus fibrosus with a diameter of 0.60 mm and a depth of 1.40 mm, minimizing damage to the nucleus pulposus. In the sham operation group, only a skin incision was made. Approximately 50 µL of different hydrogels or PBS were immediately injected into the annulus fibrosus defect, followed by disinfection, suturing, and further disinfection. Postoperatively, the annulus fibrosus defect was treated twice daily with NIR II (1064 nm, 1.5 W cm⁻¹) therapy. -2 Expose to light for 15 minutes for two weeks.

[0094] 4.11 Imaging examination

[0095] After treatment, X-ray and magnetic resonance imaging (MRI) were performed on the rats' caudal vertebrae. The intervertebral disc height index (DHI) was calculated by comparing the intervertebral disc height with the vertebral body height using digital radiographic images. The water content of the corresponding intervertebral discs was assessed using T2-weighted MRI images, and the degree of intervertebral disc degeneration was assessed using the Pfirrmann grading system.

[0096] 4.12. Histology

[0097] Histologically, after dewaxing and hydration, hematoxylin-eosin (H&E) staining and safranin O / rapid green (SO / FG) staining were performed to assess the histopathological score.

[0098] For immunohistochemical staining, tissue samples were first fixed, paraffin-embedded, and sectioned. The sections were then dewaxed and hydrated, and antigen retrieval was performed to expose the antigens. Non-specific binding was reduced using 3% BSA, and the sections were then incubated overnight at 4°C with specific primary antibodies. The antibodies used included: anti-type II collagen antibody (ab34712, 1:200, Abcam), anti-aggregating protein antibody (ab315486, 1:200, Abcam), anti-COL1A1 antibody (14695-1-AP, 1:200, Proteintech), anti-ADAMTS5 antibody (ab41037, 1:200, Abcam), and anti-MMP13 antibody (18165-1-AP, 1:200, Proteintech). HRP-labeled secondary antibody was then added and incubated at room temperature for 1 hour. Afterwards, DAB staining was performed, and hematoxylin contrast staining was used for nuclei. The sections were then dehydrated and mounted, and the presence and distribution of the target antigens were observed under a light microscope.

[0099] 4.13. Detection of reactive oxygen species (ROS) in vivo

[0100] In vivo reactive oxygen species (ROS) levels were detected using dihydroethidium (DHE) staining. Fresh, unfixed frozen tissue sections were prepared and air-dried on glass slides. DHE working solution (2 µM PBS) was added to the tissue sections, and they were incubated in a dark, humidified incubator at 37°C for 30 minutes. After incubation, the sections were gently washed with PBS and mounted using a DAPI-containing anti-fading mounting medium. The stained sections were observed using a fluorescence microscope.

[0101] 4.14 Immunofluorescence (IF) staining

[0102] Immunofluorescence (IF) staining is used to detect and locate specific proteins in tissues or cells. For cell samples, fixation with 4% paraformaldehyde was performed; for tissue samples, the process included dewaxing, hydration, and retrieval with EDTA antigen at 85°C for 25 minutes. This was followed by infiltration with 0.5% Triton X-100 for 20 minutes. After blocking nonspecific binding with 3% BSA, primary antibody was added and incubated overnight at 4°C. Antibodies used included: anti-Cleaved Caspase-3 antibody (9664S, 1:100, Cell Signaling Technology), anti-TOM20 antibody (ab186735, 1:200, Abcam), and anti-double-stranded DNA antibody (49156, 1:200, Antibody). System, anti-γH2AX antibody (AB81299, 1:200, Abcam), anti-IL6 antibody (DF6087, 1:200, Affinity), anti-LC3B antibody (ab192890, 1:200, Abcam), anti-type II collagen antibody (ab34712, 1:200, Abcam), anti-aggregating protein antibody (ab315486, 1:200, Abcam), anti-COL1A1 antibody (14695-1-AP, 1:200, Proteintech), anti-ADAMTS5 antibody (ab41037, 1:200, Abcam), anti-MMP13 antibody (ab39012, 1:200, Abcam), anti-p21 antibody (sc-6246, 1:50, Santa Cruz), anti-p16 antibody (sc-1661, 1:50, Santa Cruz). After rewarming and washing, add 488-labeled goat anti-rabbit antibody (ab150077, 1:200, Abcam), 594-labeled goat anti-rabbit antibody (ab150080, 1:200, Abcam), or goat anti-mouse antibody (ab150116, 1:200, Abcam), and incubate in the dark for 1 hour. After washing, mount with DAPI-containing anti-fading medium and observe the samples under a fluorescence microscope. Example 5 Statistical analysis of animal experiment results:

[0103] 5.1 The effect of Mn3O4NPs on the activity of AFCs was evaluated using a live / dead cell staining assay.

[0104] Figure 4 shows the following: (A) Effect of different concentrations (1-40 μg mL⁻¹) of Mn₃O₄@ChS-HA on AFC activity after 24 hours (n = 6). (B) Effect of Mn₃O₄@ChS-HA on AFC activity under oxidative stress (n = 6). (C) Representative Calcein-AM / PI staining of AFCs treated with H₂O₂ after 24 hours of Mn₃O₄@ChS-HA pretreatment (scale bar = 200 μm). (D, E) Representative flow cytometry images and statistical analysis of AFCs with and without 100 μmol L⁻¹ H₂O₂ or 5 μg mL⁻¹ Mn₃O₄ NPs. (ns, no statistically significant difference; *p < 0.05, ***p < 0.001, ****p < 0.0001).

[0105] The results showed that no significant AFC death was observed at a concentration of 30 ng / mL (Figure 4), indicating that Mn3O4NPs have good biocompatibility. Next, the effect of Mn3O4NPs@ChS-HA on AFC viability was further evaluated using a H₂O₂-induced oxidative stress model. CCK8 results showed that cell viability was significantly restored at a Mn3O4NPs concentration of 5 ng / mL (Figure 4). Live / dead cell staining and flow cytometry results indicated that AFCs underwent significant death after exposure to H₂O₂, and Mn3O4NPs@ChS-HA intervention significantly improved cell survival (Figure 4). Therefore, the Mn3O4NPs@ChS-HA prepared in this study exhibited good biocompatibility and effectively rescued AFCs from oxidative stress-induced cell death.

[0106] 5.2 Performance of Mn3O4NPs@ChS-HA under oxidative stress

[0107] First, we evaluated the ability of Mn3O4NPs@ChS-HA to scavenge ROS.

[0108] Figure 5 shows a statistical graph illustrating the ability of Mn3O4@ChS-HA to inhibit oxidative stress and apoptosis. (AG) Representative flow cytometry plots and statistical analysis of ROS staining, Mito-SOX staining, JC-1 staining, and Annexin V-FITC / PI staining (n = 3, under conditions of 100 μmol L⁻¹ H₂O₂ or 5 μg mL⁻¹ Mn₃O₄ NPs treatment or no AFCs treatment). (H, I) Representative Western blot plots and statistical analysis of Bax, Bcl2, C-CASP3, and C-CASP9 expression levels (n = 3, under conditions of 100 μmol L⁻¹ H₂O₂ or 5 μg mL⁻¹ Mn₃O₄ NPs treatment or no AFCs treatment). (J, K) Representative immunofluorescence staining images and statistical analysis of C-CASP3 (under conditions of AFCs treated with 100 μmol L⁻¹ H₂O₂ or 5 μg mL⁻¹ Mn₃O₄ NPs or without AFCs treatment, n = 3, scale bar = 40 μm). (L) Representative immunofluorescence staining images and statistical analysis of dsDNA (under conditions of AFCs treated with 100 μmol L⁻¹ H₂O₂ or 5 μg mL⁻¹ Mn₃O₄ NPs or without AFCs treatment, n = 3, scale bar = 20 μm). (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0109] Intracellular reactive oxygen species (ROS) levels were detected using DCFH. Flow cytometry results showed that Mn3O4NPs@ChS-HA significantly reduced H2O2-induced increases in intracellular ROS (Figure 5). Simultaneously, mitochondrial ROS and mitochondrial membrane potential levels were measured. Flow cytometry results showed that Mn3O4NPs@ChS-HA significantly reduced mitochondrial ROS and improved mitochondrial membrane potential, maintaining the integrity of mitochondrial function (Figure 5). Studies have shown that inhibiting oxidative stress can delay IVDD by reducing IVD cell apoptosis; therefore, we evaluated the anti-apoptotic ability of Mn3O4NPs@ChS-HA. Flow cytometry results showed that Mn3O4NPs@ChS-HA significantly alleviated AFC apoptosis (Figure 5). We also assessed the expression of molecules related to the apoptosis pathway. Western blot results showed that Mn3O4NPs@ChS-HA downregulated the expression of pro-apoptotic proteins cleaved caspase 3, cleaved caspase 9 and Bax, and increased the expression of anti-apoptotic protein Bcl-2 (Figure 5). Immunofluorescence further confirmed this result (Figure 5).

[0110] ROS are one of the major inducers of DNA damage and can initiate various cell death mechanisms. dsDNA, including mtDNA released into the cytoplasm and oxidized mtDNA, is particularly susceptible to ROS. Endogenous mitochondrial ROS (mtROS) have been reported to promote mtDNA leakage. We further examined dsDNA expression. Immunofluorescence results showed that under oxidative stress, dsDNA translocation into the cytoplasm was observed, along with increased mitochondrial fission, leading to mitochondrial dysfunction. Mn3O4NPs@ChS-HA reduced dsDNA in the cytoplasm and inhibited mitochondrial fission, maintaining the integrity of mitochondrial function (Figure 5). Therefore, Mn3O4NPs@ChS-HA inhibits the increase of dsDNA and mitochondrial fission while scavenging intracellular ROS, maintaining the integrity of mitochondrial function and thus exerting its anti-apoptotic effect.

[0111] 5.3 Mn₃O₄@ChS-HA promotes autophagy of AFCs and inhibits aging and inflammation;

[0112] ROS is not only associated with apoptosis, but also closely related to cellular senescence, inflammation, and autophagy. Mitochondrial dysfunction plays a crucial role in cellular senescence, and Mn3O4 NPs@ChS-HA can maintain the integrity of mitochondrial function. Therefore, we evaluated the anti-aging ability of Mn3O4 NPs@ChS-HA.

[0113] The results are shown in Figure 6, specifically a statistical graph of how Mn₃O₄@ChS-HA promotes autophagy in AFCs and inhibits senescence and inflammation. (A, B) Representative SA-β-Gal staining images and statistical analysis (under conditions of AFCs treated with 100 μmol L⁻¹ H₂O₂ or 5 μg mL⁻¹ Mn₃O₄ NPs or without AFCs treatment, n = 3). (C, D) Representative γ-H₂AX immunofluorescence staining images and statistical analysis (under conditions of AFCs treated with 100 μmol L⁻¹ H₂O₂ or 5 μg mL⁻¹ Mn₃O₄ NPs or without AFCs treatment, n = 3, scale bar = 20 μm). (EG) Representative Western blotting images and statistical analysis of p16, p21, IL-1β, IL-6, and TNF-α (n = 3, with or without AFCs treated with 100 μmol L⁻¹ H₂O₂ or 5 μg mL⁻¹ Mn₃O₄ NPs). (H) Statistical analysis of mRNA levels of IL-1β, IL-6, and TNF-α (n = 3, with or without AFCs treated with 100 μmol L⁻¹ H₂O₂ or 5 μg mL⁻¹ Mn₃O₄ NPs). (I, J) Representative immunofluorescence staining images and statistical analysis of IL-6 (n = 3, scale bar = 40 μm, with or without AFCs treated with 100 μmol L⁻¹ H₂O₂ or 5 μg mL⁻¹ Mn₃O₄ NPs). (K, L) Representative immunofluorescence staining images and statistical analysis of LC3B (under conditions of AFCs treated with 100 μmol L⁻¹ H₂O₂ or 5 μg mL⁻¹ Mn₃O₄ NPs or without AFCs treatment, n = 3, scale bar = 20 μm). (M, N) Representative Western blotting images and statistical analysis of BECN1, ATG7, p62 and LC3B (under conditions of AFCs treated with 100 μmol L⁻¹ H₂O₂ or 5 μg mL⁻¹ Mn₃O₄ NPs or without AFCs treatment, n = 3). (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0114] SA-Gal staining results showed that Mn3O4NPs@ChS-HA significantly reduced the proportion of SA-Gal positivity increased by H2O2 (Figure 6). Simultaneously, immunofluorescence showed that Mn3O4NPs@ChS-HA inhibited the expression of the DNA damage marker, histone H2A family member X (-H2AX) (Figure 6). Mn3O4NPs@ChS-HA inhibited the increased mRNA expression and secretion of SASP molecules such as IL6, IL1, and tumor necrosis factor (TNF) (Figure 6). Western blot analysis showed that Mn3O4 NPs@ChS-HA significantly reduced the H2O2-induced increase in p21 and p16.

[0115] Autophagy can alleviate aging-inducing factors such as oxidative stress and DNA damage, thus delaying the cellular aging process. To assess whether the anti-aging effect of Mn3O4NPs@ChS-HA is attributed to increased autophagy, we performed autophagy-related analyses. First, we evaluated the expression of LC3B, a key marker protein in autophagy. Immunofluorescence staining showed that Mn3O4NPs@ChS-HA treatment significantly inhibited the H2O2-induced decrease in LC3B expression and enhanced autophagy (Figure 6). Next, we assessed the expression of molecules related to the autophagy pathway. Western blot results showed that Mn3O4NPs@ChS-HA downregulated p62 expression and increased the expression of BECNIN1, ATG7, and LC3B (Figure 6). In conclusion, these results indicate that Mn3O4NPs@ChS-HA can effectively alleviate oxidative stress-induced cellular senescence and inflammation by promoting autophagy.

[0116] 5.4 The ability of Mn₃O₄@ChS-HA to regulate the balance of synthesis / catabolism;

[0117] The extracellular matrix (ECM) plays a crucial role in intervertebral disc degeneration (IVDD). Oxidative stress can lead to abnormal extracellular matrix metabolism in IVD, promoting its progression. To assess the ability of Mn3O4NPs@ChS-HA to regulate the anabolic / catabolic balance, we examined the expression of metabolism-related molecules such as Col1a1, Col2a1, Aggrecan, MMP13, and ASDMTS5.

[0118] The results are shown in Figure 7, specifically a statistical graph of the ability of Mn₃O₄@ChS-HA to regulate the balance of synthesis and catabolic metabolism. (A, B) Representative immunofluorescence staining images and statistical analysis of Aggrecan, COL2A1, COL1A1, ADAMTS5, and MMP13 (under conditions of AFCs treated with or without 100 μmol L⁻¹ H₂O₂ or 5 μg mL⁻¹ Mn₃O₄ NPs, n = 3, scale bar = 40 μm). (C) Statistical analysis of mRNA levels of Aggrecan, COL2A1, COL1A1, ADAMTS5, and MMP13 (under conditions of AFCs treated with or without 100 μmol L⁻¹ H₂O₂ or 5 μg mL⁻¹ Mn₃O₄ NPs, n = 3). (D, E) Representative Western blotting plots and statistical analysis of Aggrecan, COL2A1, COL1A1, ADAMTS5, and MMP13 (n = 3, with or without AFCs treated with 100 μmol L⁻¹ H₂O₂ or 5 μg mL⁻¹ Mn₃O₄ NPs). (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0119] Immunofluorescence results showed that Mn3O4NPs@ChS-HA significantly restored the H2O2-induced reduction in Aggrecan and COL2A1 synthesis, while simultaneously decreasing the H2O2-induced increases in matrix-degrading enzymes matrix metallopeptidase 13 (MMP13), COL1A1, and integrin and metalloproteinase 5 (ADAMTS5) with platelet-reactive protein motif 5 (Figure 7). Furthermore, Western blot analysis and qRT-PCR results also showed that oxidative stress led to decreased expression of Col2a1 and Aggrecan, while increasing expression of Col1a1, MMP13, and ADAMTS5; Mn3O4NPs@ChS-HA significantly reversed this outcome. In conclusion, these results indicate that Mn3O4NPs@ChS-HA can effectively inhibit oxidative stress-induced catabolism and promote anabolism, thereby maintaining the integrity of the extracellular matrix.

[0120] 5.5 Differences in biological behavior induced by Mn3O4 NPs@ChS-HA treatment;

[0121] Mn3O4 NPs@ChS-HA has shown great therapeutic potential in H2O2-induced AFCs death. To elucidate its underlying mechanisms, we performed RNA sequencing (RNA-seq) on both the H2O2 group and the Mn3O4 NPs@ChS-HA+H2O2 group.

[0122] The results are shown in Figure 8, which is a statistical graph of the differences in biological behavior caused by Mn3O4 NPs@ChS-HA treatment. Specifically: (A) Principal component analysis (PCoA) of differentially expressed genes (DEGs) between the H2O2 group and the H2O2+Mn₃O₄ NPs group (n = 3); (B) Volcano plot of DEGs; (C) Heatmap of DEGs between the H2O2 group and the H2O2+Mn₃O₄ NPs group; (D) GO enrichment analysis of identified DEGs; (E) KEGG enrichment analysis of DEGs; (F, G) Representative Western blotting plots and statistical analysis of IL-17 (under conditions of 100 μmol L⁻¹ H2O2 or 5 μg mL⁻¹ Mn₃O₄ NPs treatment or no AFCs treatment, n = 3); (H) Statistical analysis of IL-17 mRNA expression levels (100 μmol L⁻¹ H2O2 or 5 μg mL⁻¹ Mn₃O₄ (I) Representative immunofluorescence staining patterns and statistical analysis of IL-17 (100 μmol L⁻¹ H₂O₂ or 5 μg mL⁻¹ Mn₃O₄ NPs treated or untreated AFCs, n = 3, scale bar = 40 µm). (** p < 0.01, *** p < 0.001, **** p < 0.0001).

[0123] Principal component analysis (PCoA) results showed significant differences between the H2O2 group and the Mn3O4 NPs@ChS-HA+H2O2 group (Figure 8). According to the volcano plot in Figure 8, there were 128 differentially expressed genes between the H2O2 group and the Mn3O4 NPs@ChS-HA+H2O2 group, including 89 upregulated genes and 39 downregulated genes (Figure 8). Simultaneously, heatmap 8 shows the binary classification matrix data of upregulated genes (red) and downregulated genes (blue), indicating significant differences in RNA profiles between the two groups (Figure 8).

[0124] Next, pathway enrichment analysis of DGEs was performed using the structured bioinformatics annotation system GO (Gene Ontology) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) to reveal the physiological and biochemical processes involved in DEGs. GO analysis identified relevant biological pathways, cellular components, and molecular functions. The results showed that most DEGs were mainly enriched in "cellular process, metabolic process, biological process, and response to stimulus" (Figure 8). Furthermore, KEGG analysis showed that DEGs in AFCs were mainly enriched in "IL-17 signaling pathway and TNF signaling pathway" (Figure 8). The IL-17 signaling pathway plays a crucial role in the regulation of the immune system, especially in inflammatory responses, tissue damage and repair, and cancer progression. qPCR, WB, and IF analyses showed that Mn3O4 NPs@ChS-HA significantly inhibited the expression of the key gene IL-17RA in the IL-17 signaling pathway. Therefore, these results indicate that Mn3O4 NPs@ChS-HA mainly exerts its biological function by regulating the IL-17 signaling pathway.

[0125] 5.6. Therapeutic effects of Mn3O4 NPs@ChS-HA in rats;

[0126] To investigate the in vivo photothermal properties of Mn3O4 NPs@ChS-HA, thermal imaging of the hydrogel implanted at the AF defect site was performed using an infrared camera. Under irradiation with a 1064 nm, 1.5 W cm⁻² NIR-II laser, the temperature of the rat caudal vertebrae implanted with Mn3O4@ChS-HA significantly increased, reaching approximately 42°C within 6 minutes (Figure 12 shows the in vivo photothermal properties of Mn3O4@ChS-HA; (A) Photothermal images of ChS-HA and Mn3O4@ChS-HA under 1064 nm near-infrared radiation at the rat caudal vertebrae; (B) Temperature changes of ChS-HA and Mn3O4@ChS-HA). In contrast, no significant temperature increase was observed in the ChS-HA treatment group. These results highlight the effective tissue penetration capability and low absorption characteristics of the NIR-II laser, and validate the photothermal therapeutic effect achieved by Mn3O4@ChS-HA, establishing it as a promising physical therapy modality for IVDD treatment.

[0127] To evaluate the in vivo therapeutic effect of Mn3O4 NPs@ChS-HA, we established a rat caudal intervertebral disc annulus fibrosus defect model, and then injected different hydrogels into the defect site, which adhered well to the defect site (Figure 9). X-ray examination, magnetic resonance imaging (MRI), H&E, SO / FG, and IHC were used to evaluate the repair of AF.

[0128] The results are shown in Figure 9, illustrating the therapeutic effect of Mn3O4 NPs@ChS-HA in rats. Specifically: (A) Representative X-ray and T2 MRI images of rats in the sham-operated or IVDD group; (B) Representative images of HE staining and SO / FG staining; (C) Statistical analysis of intervertebral disc height index; (D) Statistical analysis of Pfirrmann grading; (E) Statistical analysis of relative signal intensity; (F) Statistical analysis of histological score; (G, H) Immunohistochemical staining images (G) and statistical analysis (H) of Aggrecan, COL2A1, COL1A1, ADAMTS5, and MMP13 in intervertebral disc tissue. (n = 6, scale bar = 500 µm, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0129] X-ray examination showed that Mn3O4 NPs@ChS-HA significantly salvaged the reduced intervertebral disc height, with the DHI value being closest to that of the SHAM group (Figure 9). T2-weighted signal intensity on MRI reflects NP water content and is directly related to the degree of degeneration. Consistent with the X-ray results, compared to other IVDD groups, the Mn3O4 NPs@ChS-HA group had the highest signal intensity and NP water content, along with a significant improvement in the Pfirman grade (Figure 9). Furthermore, H&E and SO / FG staining results showed that the IVDD group exhibited nucleus pulposus loss, blurred boundaries, replacement of contents by the annulus fibrosus, and near-complete disappearance of proteoglycans, eventually being replaced by collagen. In contrast, the Mn3O4 NPs@ChS-HA group showed significant recovery of nucleus pulposus volume and vacuolar matrix, along with relatively abundant proteoglycan content and a significantly improved histological score (Figure 9). Immunohistochemical staining showed that Mn3O4 NPs@ChS-HA rescued the ECM metabolic imbalance induced by acupuncture, manifested by increased expression levels of matrix proteins Aggrecan and COL2A1, and decreased expression levels of COL1A1, MMP13, and ADAMTS5 (Figure 9). These results indicate that Mn3O4 NPs@ChS-HA can significantly delay the progression of IVDD.

[0130] Meanwhile, considering the risk of leakage after intervertebral disc injection, we evaluated the toxicity of Mn3O4 NPs@ChS-HA to major organs such as the heart, liver, spleen, lungs, and kidneys. H&E staining showed that no significant damage was caused to major organs such as the heart, liver, spleen, lungs, and kidneys during Mn3O4 NPs@ChS-HA treatment (Figure 13, representative HE staining images of rat heart, liver, spleen, lungs, and kidneys, taken from tissue samples at 4 weeks, scale bar = 100 μm). Therefore, Mn3O4 NPs@ChS-HA exhibits good biocompatibility in vivo.

[0131] 5.7 Mn₃O₄@ChS-HA delays IVDD process through the IL-17 signaling pathway

[0132] Next, we explored the possible mechanism by which Mn3O4 NPs@ChS-HA delays IVDD progression. First, we stained freshly collected IVD tissues with ethidium dihydrosulfonate.

[0133] The results are shown in Figure 10, which is a statistical graph of how Mn₃O₄@ChS-HA delays IVDD progression through the IL-17 signaling pathway. Specifically: (A) Representative images of ROS staining in IVD tissues; (BF) Representative immunofluorescence staining images of C-CASP3, H2AX, p16, p21, IL-6, LC3B, and IL-17 in IVD tissues; (G) Statistical analysis of ROS levels in IVD tissues; (HN) Statistical analysis of C-CASP3, H2AX, p16, p21, IL-6, LC3B, and IL-17 levels in IVD tissues. (n = 6, scale bar = 100 μm, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0134] The results showed that acupuncture significantly increased ROS levels in IVD tissues, while Mn3O4 NPs@ChS-HA significantly reduced in vivo ROS levels. Furthermore, we performed immunofluorescence staining for apoptosis-related markers (C-CASP3), aging-related markers (p21, p16, -H2AX), inflammation-related markers (IL-6), and autophagy-related markers (LC3B). The results showed that Mn3O4 NPs@ChS-HA significantly reduced the levels of C-CASP3, p21, p16, -H2AX, and IL-6 in IVD tissues, while increasing LC3B expression. Finally, Mn3O4 NPs@ChS-HA treatment significantly inhibited the expression of IL-17RA, a key protein in the IL-17 signaling pathway, which was confirmed by immunofluorescence staining. In conclusion, these results indicate that Mn3O4 NPs@ChS-HA significantly inhibits the IL-17 signaling pathway, thereby exerting its multifunctional biological role.

[0135] In summary, this study successfully developed a multifunctional MPTT-nanozyme-hydrogel system—Mn3O4@ChS-HA—which demonstrates significant potential through multiple mechanisms and plays an important role in alleviating intervertebral disc degeneration (IVDD). On one hand, by mimicking the natural environment within the intervertebral disc, Mn3O4@ChS-HA exhibits excellent injectability and biocompatibility, as well as good mechanical properties and adhesion, enabling it to adhere tightly to the annulus fibrosus defect site and provide support for the intervertebral disc. On the other hand, Mn3O4 nanoparticles exhibit significant CAT, GPx, and SOD-like activities, effectively reducing intracellular and mitochondrial ROS levels, thereby exerting anti-apoptotic effects, delaying aging, and promoting autophagy. Furthermore, upon binding with MPTT, this system can further inhibit inflammation, promote the repair of annulus fibrosus damage, and delay the progression of IVDD.

[0136] Hydrogel-based drug delivery systems represent a promising therapeutic strategy for treating IVDD. Due to their three-dimensional network structure, excellent biocompatibility, mechanical properties, injectability, and drug loading capacity, hydrogels are widely used in various fields, including bone diseases, wound repair, and inflammatory diseases.

[0137] Based on the physiological activities of chondroitin sulfate and hyaluronic acid, we prepared an injectable thermosensitive ChS-HA hydrogel composed of dop-OChS and ADH-HA. ChS-HA forms a stable gel under physiological conditions, ensuring long-term retention at the injury site, a key factor in local IVDD treatment. Furthermore, ChS-HA enhances tissue adhesion and provides structural support to the injury site, mimicking the natural intervertebral disc environment. Therefore, ChS-HA is an ideal drug delivery system, ensuring sustained release of Mn3O4 nanoparticles at the injury site, reducing the need for repeated dosing, and lowering the potential complications of systemic therapy. Notably, the system exhibits photothermal properties (≤ 42℃) after loading Mn3O4 nanoparticles, further enhancing its therapeutic effect by promoting annulus fibrosus tissue repair and slowing the in vivo progression of IVDD. The combination of MPTT and Mn3O4 nanoparticles enhances the multifunctional biological effects of the hydrogel, consistent with previous studies showing that the combination of MPTT can effectively alleviate inflammation, inhibit apoptosis, and promote ECM synthesis. Mounting evidence suggests that controlled MPTT can synergistically enhance tissue repair and slow disease progression in conjunction with nanoparticle-based therapies. Our study demonstrates that MPTT induced by near-infrared light irradiation can inhibit the release of pro-inflammatory cytokines and enhance autophagy, thereby further amplifying the anti-inflammatory and anti-aging effects of Mn3O4@ChS-HA.

[0138] Oxidative stress is a key driver of IVDD progression. Inhibiting oxidative stress can slow IVDD progression by suppressing apoptosis, delaying cellular senescence, inhibiting inflammation, and regulating ECM metabolism. In recent years, metal nanomaterials with enzyme-like activities have attracted widespread attention. Monodisperse, flower-like Mn3O4 nanoparticles possess intrinsic SOD, CAT, and GPx-like activities, exhibiting significant antioxidant properties. These enzyme-like activities significantly reduce ROS levels in AFCs, which are known to cause ring of fibrous damage. By effectively scavenging ROS, Mn3O4@ChS-HA improved mitochondrial function and inhibited the translocation of double-stranded DNA to the cytoplasm, further reducing apoptosis, senescence, and inflammation in AFCs, while promoting autophagy, thereby minimizing cell and tissue damage and delaying IVDD progression. These results demonstrate that Mn3O4@ChS-HA can maintain cellular homeostasis under oxidative stress, highlighting the role of oxidative stress in promoting apoptosis, inflammation, and senescence in IVDD.

[0139] On the other hand, oxidative stress can lead to metabolic imbalance of the intervertebral disc emulsion (ECM), which is another key factor in the progression of intraepithelial disc disease (IVDD). In IVDD, due to reduced cell number and abnormal function, the catabolic processes of the ECM increase while the anabolistic processes decrease, resulting in ECM metabolic imbalance. In this study, Mn3O4@ChS-HA effectively regulated ECM metabolism and maintained ECM integrity by downregulating the expression of MMP13 and ADAMTS5, while upregulating the expression of key components of the intervertebral disc matrix, COL2A1 and aggregates. Simultaneously, in vivo experiments confirmed that Mn3O4@ChS-HA successfully alleviated the reduction in intervertebral disc height and maintained the water content of the nucleus pulposus, which is crucial for maintaining intervertebral disc function and delaying the progression of IVDD. Furthermore, these therapeutic effects were further enhanced after MPTT. These results indicate that Mn3O4@ChS-HA has significant potential in treating IVDD and slowing its progression by alleviating oxidative stress and maintaining ECM homeostasis. Furthermore, the combination with mild photothermal therapy further amplifies these therapeutic benefits, making it a promising treatment strategy for intervertebral disc degeneration.

[0140] In summary, the MPTT-nanozyme-hydrogel system (Mn3O4@ChS-HA) is a promising biomaterial capable of treating IVDD by addressing oxidative stress and ECM imbalance associated with IVDD. Its multifunctional properties, combined with the innovative photothermal therapy, provide a powerful platform for future therapeutic applications in degenerative disc diseases. Furthermore, the system's non-toxicity in major organs demonstrates the excellent biocompatibility of Mn3O4@ChS-HA, a key factor for future clinical translation.

[0141] The MPTT-nanozyme-hydrogel system (Mn3O4@ChS-HA) represents a promising multifunctional therapeutic platform capable of treating IVDD by addressing oxidative stress, modulating ECM metabolism, and promoting annulus fibrosus tissue repair. Its therapeutic capabilities are further enhanced by combination with mild photothermal therapy.

Claims

1. The application of Mn3O4 nanoparticles in alleviating intervertebral disc degeneration, characterized by: Manganese tetroxide nanoparticles (Mn3O4NPs) were added as nanozymes to interventional drugs for the treatment of intervertebral disc degeneration.

2. The application of Mn3O4 nanoparticles according to claim 1 in alleviating intervertebral disc degeneration, characterized in that: Manganese tetroxide nanoparticles (Mn3O4NPs) were added to a hydrogel, and the hydrogel was used as a delivery medium to form an injection.

3. The application of Mn3O4 nanoparticles according to claim 2 in alleviating intervertebral disc degeneration, characterized in that: The hydrogel is hydrogel ChS-HA, which is composed of dopamine-grafted chondroitin sulfate dop-OChS and ADH-modified hyaluronic acid ADH-HA.

4. The application of Mn3O4 nanoparticles according to claim 3 in alleviating intervertebral disc degeneration, characterized in that: The injectable drug achieves therapeutic benefit when combined with the mild photothermal therapy (MPTT).

5. The biomaterial Mn3O4@ChS-HA for treating intervertebral disc degeneration according to claim 1, characterized in that: Including manganese tetroxide nanoparticles Mn3O4NPs, and hydrogel ChS-HA composed of dopamine-grafted chondroitin sulfate dop-OChS and ADH-modified hyaluronic acid ADH-HA. Together, they constitute the biomaterial Mn3O4@ChS-HA with an MPTT-nanozyme-hydrogel delivery system.

6. The method for preparing the biomaterial Mn3O4@ChS-HA for treating intervertebral disc degeneration according to claim 5, characterized in that, Includes the following steps: S1. Raw material preparation: Preparation of manganese tetroxide nanoparticles (Mn3O4NPs): Oleic acid was added to a 0.2 wt / v% KMnO4 aqueous solution at a volume ratio of 2:100 and stirred at room temperature for 6 hours. Then, the mixture was centrifuged at 8000 rpm for 5 min, and the precipitate was washed with deionized water and ethanol in sequence. The precipitate was then collected and dried at 80°C for 10 hours. The resulting brown solid was ground and calcined in an oven at 200°C for 5 hours. After grinding again, the solid was passed through a 50-mesh sieve to obtain Mn3O4NPs powder. Preparation of dopamine-grafted chondroitin sulfate (dop-OChS): Oxidized chondroitin sulfate OChS was prepared using NaIO4 as an oxidant. The carboxyl group on oxidized chondroitin sulfate OChS was further activated by carbodiimide EDC and N-hydroxysuccinimide NHS. After adding dopamine, the mixture was stirred at room temperature in the dark for 24 h to obtain dopamine-grafted oxidized chondroitin sulfate dop-OChS. Preparation of ADH-modified hyaluronic acid (ADH-HA): HA was activated with carbodiimide EDC and 1-hydroxybenzotriazole HOBT, and then reacted with adipicodiacetylhydrazine (ADH) at room temperature to obtain adipicodiacetylhydrazine-modified hyaluronic acid (ADH-HA). Preparation of S2, Mn3O4@ChS-HA: Mn3O4NPs were added to a 5% wt / v dop-OChS solution at a concentration of 2 mg / ml and mixed with a 5% wt / v ADH-HA PBS solution at a volume ratio of 1:2 to obtain an injectable hydrogel Mn3O4@ChS-HA loaded with Mn3O4NPs.

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