Application of Mn3O4 nanoparticles in relieving intervertebral disc degeneration, biological material and preparation method of biological material

Abstract

The invention relates to the technical field of biological medicine. The invention aims to provide an application of Mn3O4 nanoparticles in relieving intervertebral disc degeneration. The manganous-manganic oxide nanoparticles Mn3O4NPs are added into an interventional drug for treating intervertebral disc degeneration as a nano-enzyme. According to the invention, Mn3O4 nanoparticles are used as a nano active catalytic enzyme, have multienzyme catalytic activity similar to superoxide dismutase, catalase and glutathione peroxidase, can effectively remove reactive oxygen species (ROS) and regulate ECM metabolism, significantly reduce cell apoptosis, delay cell senescence, inhibit inflammation and enhance autophagy, and can be used for preparing a medicine for treating hyperplasia. Therefore, annulus fibrosus AF repair in intervertebral disc degeneration treatment is promoted. The Mn3O4 nano particles have good biocompatibility, metabolites of the Mn3O4 nano particles can be used for other physiological processes, and no toxic or side effect is generated in in-vivo circulation. Meanwhile, the enzyme is stable in low-oxygen and low-pH adverse microenvironment of the intervertebral disc, and has in-vivo stability which is not possessed by other active catalytic enzymes.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of Mn3O4 nanoparticles in alleviating intervertebral disc degeneration. Background Art

[0002] Annulus fibrosus (AF) injury plays a key role in intervertebral disc degeneration (IVDD), and oxidative stress and imbalance of extracellular matrix (ECM) metabolism are considered to be the main pathogenic factors of AF injury. However, effective methods to repair damaged AF are still lacking.

[0003] Intervertebral disc (IVD) degeneration (IVDD) is a common degenerative spinal disease and one of the main causes of low back pain. With the aging of the population, the incidence of IVDD continues to rise. IVDD not only causes pain and a decline in quality of life in individuals, and even leads to disability, but also imposes a huge economic burden on the social medical system. However, since IVDD involves complex biological processes such as structural damage, release of inflammatory mediators, changes in enzyme activity, and cell death, the current treatment options for IVDD are very limited. Existing treatments mainly include conservative treatment and surgery, but the effects are limited, the recurrence rate is high, and there are many complications. Given the complex pathological mechanisms of IVDD, finding more effective treatment options is crucial to mitigate its effects.

[0004] The intervertebral disc is composed of the nucleus pulposus (NP), the annulus fibrosus (AF), and the cartilaginous endplates. These structures may change with aging and other factors. The pathogenesis of disc degeneration is complex and involves mechanical, biochemical, and genetic factors. Among them, the annulus fibrosus ruptures due to long-term mechanical load and plays a key role in disc degeneration. The annulus fibrosus is a tough fibrous tissue structure surrounding the nucleus pulposus, which is mainly used to protect the nucleus pulposus and maintain the structural integrity of the intervertebral disc. During the degeneration process, the annulus fibrosus may be torn or damaged due to aging, long-term mechanical pressure, or acute injury. This damage may cause the herniation or extrusion of the nucleus pulposus, compress the nerve roots, cause symptoms such as pain, numbness, and stiffness, and reduce the height and stability of the intervertebral disc. In addition, annulus fibrosus damage may trigger local inflammation and accelerate disc degeneration. Clinically, the rate of recurrent nucleus pulposus herniation after discectomy can be as high as 26% because the annulus fibrosus defect is not resolved. Therefore, repairing annulus fibrosus damage is crucial for the treatment of IVDD.

[0005] Oxidative stress is mainly manifested as an increase in the level 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 decrease in small molecule antioxidants such as vitamin C, vitamin E and glutathione. Oxidative stress is considered to be the main pathogenic factor of annular fibrosis injury. In IVDD, oxidative stress is closely related to cell death, cell 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 essential for slowing the progression of IVDD and have great potential. Although many natural substances with antioxidant properties (such as quercetin and mangiferin) show good therapeutic effects in vitro, their poor stability in the adverse microenvironment of low oxygen and low pH in the intervertebral disc greatly limits their application.

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

[0007] However, there is no relevant research on whether such an excellent nanoparticle as Mn3O4 NPs can really participate in the specific treatment of IVDD. At the same time, since the intervertebral disc is a relatively "vascular-free" structure, it is difficult for drugs to directly penetrate into the intervertebral disc through the blood circulation. In addition, repeated local injections may accelerate the progression of IVDD. Therefore, it is also very necessary to design a drug delivery system that can accurately deliver drugs to the lesion site and maintain an effective concentration. Summary of the invention

[0008] The purpose of the present invention is to provide an application method of Mn3O4 nanoparticles in alleviating intervertebral disc degeneration, to provide a new idea for IVDD treatment, to effectively alleviate intervertebral disc degeneration, and to enhance the treatment effect of IVDD.

[0009] To achieve the above-mentioned purpose of the invention, the technical solution adopted by the present invention is: application of Mn3O4 nanoparticles in alleviating intervertebral disc degeneration, adding manganese tetraoxide nanoparticles Mn3O4 NPs as nanozymes to interventional drugs for treating intervertebral disc degeneration.

[0010] Preferably, manganese tetraoxide nanoparticles Mn3O4 NPs are added to a hydrogel, and the hydrogel is used as a delivery medium to form an injectable.

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

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

[0013] Preferably, a biomaterial Mn3O4@ChS-HA for treating intervertebral disc degeneration includes manganese tetraoxide nanoparticles Mn3O4 NPs, and a hydrogel ChS-HA composed of dopamine-grafted oxidized 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] Preferably, the preparation method of the biomaterial Mn3O4@ChS-HA for treating intervertebral disc degeneration comprises the following steps:

[0015] S1. Raw material preparation:

[0016] Preparation of manganese tetraoxide nanoparticles Mn3O4 NPs:

[0017] Oleic acid was added to a 0.2wt / v% KMnO4 aqueous solution at a volume ratio of 2:100, and stirred at room temperature for 6 hours; then centrifuged at 8000rpm for 5 minutes, and the precipitate was washed with deionized water and ethanol in sequence; the precipitate was collected and dried at 80°C for 10 hours, the brown solid was ground and calcined in an oven at 200°C for 5 hours, and then ground again and passed through a 50-mesh sieve to obtain Mn3O4 NPs powder;

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

[0019] Oxidized chondroitin sulfate (OChS) was prepared using NaIO4 as an oxidant, and carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were further used to activate the carboxyl groups on the oxidized chondroitin sulfate (OChS). After adding dopamine, the mixture was stirred at room temperature in the dark for 24 hours to obtain dopamine-grafted oxidized chondroitin sulfate (dop-OChS).

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

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

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

[0023] Mn3O4 NPs were added at 2 mg / ml to a 5% wt / v dop-OChS solution and mixed evenly 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 Mn3O4 NPs.

[0024] The beneficial effects of the present invention are mainly embodied 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, can effectively remove reactive oxygen species (ROS) and regulate ECM metabolism, significantly reduce cell apoptosis, delay cell senescence, inhibit inflammation, enhance autophagy, and thus promote the repair of annulus fibrosus AF in the treatment of intervertebral disc degeneration.

[0026] 2. Mn3O4 nanoparticles have good biocompatibility, and their metabolites can be used in other physiological processes without toxic side effects in the body. At the same time, they are stable in the adverse microenvironment of low oxygen and low pH in the intervertebral disc, and have in vivo stability that other active catalytic enzymes do not have.

[0027] 3. It is preferred that the Mn3O4 nanoparticles are loaded on the hydrogel. The special hydrogel delivery medium simulates the natural environment of the intervertebral disc, due to its excellent tissue compatibility, ability to fill irregular wounds, and effect of reducing scars and pain. At the same time, it allows local, controllable and sustained release of Mn3O4 NPs. It not only has excellent injectability and biocompatibility, but also has good mechanical properties and adhesion. It can closely adhere to the defect of the annulus fibrosus and provide support for the intervertebral disc, and continuously play the effects of clearing ROS, 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 annular fiber damage, and slowing down the progression of IVDD, making it the basis for adjuvant treatment of IVDD through photothermal therapy (PTT). The photothermal therapy combined with MPTT intervention can further enhance its tissue penetration, achieve therapeutic efficacy, and improve the overall treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The basic physical properties of Mn3O4 NPs of the present invention are shown in the following statistical diagram:

[0030] Figure 2 Characterization statistics of the injectable ChS-HA hydrogel of the present invention:

[0031] Figure 3 The photothermal performance statistics of injectable Mn3O4@ChS-HA hydrogel under 1064nm laser irradiation;

[0032] Figure 4 Statistical diagram for evaluating the effect of Mn3O4 NPs on the viability of AFCs using live / dead cell staining assay;

[0033] Figure 5 Statistical graph of the ability of Mn3O4@ChS-HA to inhibit oxidative stress and cell apoptosis;

[0034] Figure 6 Statistical diagram of Mn3O4@ChS-HA promoting AFCs autophagy and inhibiting aging and inflammation;

[0035] Figure 7 Statistical diagram of the ability of Mn3O4@ChS-HA to regulate the balance of anabolic / catabolic metabolism;

[0036] Figure 8 Statistical diagram of the differences in biological behaviors caused by Mn3O4@ChS-HA treatment;

[0037] Fig. 9 This is a statistical diagram of the therapeutic effect of Mn3O4 NPs@ChS-HA in rats;

[0038] Fig.10 This is a statistical chart showing that Mn3O4@ChS-HA delays IVDD progression through the IL-17 signaling pathway;

[0039] Fig.11 A schematic diagram of the overall concept of the present invention;

[0040] Fig.12 Statistical diagram of the photothermal performance of Mn3O4@ChS-HA in vivo;

[0041] Fig.13 Representative statistical graphs of HE staining of rat heart, liver, spleen, lung and kidney. DETAILED DESCRIPTION

[0042] The present invention innovatively developed a 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 and solve 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 the present invention, an injectable photothermal hydrogel loaded with manganese tetraoxide Mn3O4 nanoparticles was developed, which consisted of dopamine-modified oxychondroitin sulfate (dop-OChS) and adipic acid dihydrazine-modified hyaluronic acid (ADH-HA), loaded with Mn3O4 nanoparticles (Mn3O4 NPs), called Mn3O4@ChS-HA. This hydrogel not only mimics the natural environment of the intervertebral disc, but also has excellent mechanical properties to support the intervertebral disc. Its strong adhesion enables it to firmly adhere to the AF defect site and achieve controlled and sustained release of Mn3O4NPs. In addition, compared with other metal nanoparticles, Mn3O4NPs 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 demonstrated that Mn3O4@ChS-HA hydrogel significantly reduced cell apoptosis, delayed cell senescence, inhibited inflammation, and enhanced autophagy, thereby promoting AF repair.

[0044] Furthermore, it improved disc height and maintained ECM integrity in an AF defect rat model. Mild photothermal therapy (MPTT) further enhanced these effects, promoted tissue repair and delayed IVDD progression. Overall, the MPTT-nanozyme-hydrogel system Mn3O4@ChS-HA exhibited multiple biological functions by regulating redox homeostasis and promoting tissue regeneration, providing a promising strategy for IVDD treatment. This invention provides new insights into the treatment of IVDD, especially in repairing AF damage and delaying disease progression, and has potential clinical application value.

[0045] Below, the present invention will be described in detail with reference to the embodiments:

[0046] Example 1

[0047] Preparation and characterization of Mn3O4 NPs

[0048] 10 mL of oleic acid was added to 500 mL of KMnO4 aqueous solution (0.2 wt / v%) and stirred at room temperature for 6 hours. Then centrifuged (8000 rpm, 5 min), and the precipitate was washed with DI water and ethanol in turn, and then the precipitate was collected and dried at 80°C for 10 hours. The brown solid was ground and calcined in an oven at 200°C for 5 hours, ground again and passed through a 50-mesh sieve to obtain Mn3O4 NPs powder.

[0049] The Mn3O4 NPs powder was ultrasonically dispersed in deionized water. The micromorphology of Mn3O4 NPs was observed at 200 kV using a transmission electron microscope (TEM, Tecnai G2-F30, FEI Company, the Netherlands). The hydrodynamic diameter at 25 ° C was measured using a dynamic light scattering instrument (DLS, Dandong Better Instrument Co., Ltd., BeNano180Zeta, China). The infrared spectrum of Mn3O4 NPs was measured on a Fourier transform infrared spectrometer (Equinox55, Bruker, Germany) using the KBr pellet method. The valence state of manganese in Mn3O4 NPs was analyzed using an X-ray photoelectron spectrometer (XPS, AXIS-ULTRA DLD-600W, Shimadzu Corporation, Japan). The ultraviolet-visible spectrophotometer (UV 3600, Shimadzu, Japan) was used to determine the Mn3O4 NPs aqueous dispersion (1.0 mg mL -1 ) and its crystal structure was determined by X-ray diffractometer (X'Pert PRO, PANalytical, The Netherlands).

[0050] In addition, the catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD) activities of Mn3O4 NPs were determined1. First, a certain concentration of Mn3O4NPs was co-incubated with H2O2, and the absorbance change of the system at 240nm was recorded over time to characterize its CAT activity. Secondly, the classic coupling assay was used to evaluate the GPx activity by measuring the absorbance change of NADPH at 340nm in the reaction. That is, the following substances were added to the 4mL reaction system in the following order: different concentrations of Mn3O4NPs (0-50ng L -1 ), reduced glutathione (GSH, 2 mM), NADPH (200 μM), GR (1.7 units), H2O2 (1 mmol L -1 ), the solvent is phosphate buffer at pH 7.4, and the absorbance of the system at 340 nm is measured at 25°C for 2 min. Reaction rate υ (mol L -1 min -1 ) was calculated by Origin software according to formula (1), where ε is the extinction coefficient of 6.22 mmol L-1 cm -1 , l is the light path (1 cm), dA is the absorbance change, and dt is the time interval (s).

[0051]

[0052] The superoxide scavenging ability of Mn3O4 NPs and its SOD-like activity were determined using a SOD assay kit according to the instructions provided by the manufacturer.

[0053] Detailed results combined Figure 1 As shown in: (a) TEM image; (b) particle size and its distribution determined by DLS; (c) FTIR spectrum; (d) XRD spectrum; (ef) XPS spectrum; (g) CAT-like activity; (h) GPx-like activity; (i) SOD-like activity;

[0054] Its TEM images show that the synthesized Mn3O4 NPs are nanoflower-like structures with a particle size of about 60 nm (e.g. Figure 1 a). DLS test results show that the average hydration kinetic diameter of Mn3O4 NPs is 157nm ( Figure 1 b) Zeta potential is -0.20mV. Its FTIR spectrum shows that the -1 The stretching vibration peaks of Mn-O and Mn-O-Mn bonds appear at Figure 1 c). 1529cm -1 The peak at can be attributed to the stretching vibration peak of COO- in oleic acid on the surface of Mn3O4 NPs.

[0055] XRD results ( Figure 1 d) shows that the synthesized Mn3O4 NPs have a good crystal structure, similar to the tetragonal crystal structure of hausmannite. In addition, XPS was used to analyze the valence state of manganese. Figure 1 As shown in Figure ef, the center of the Mn 2p3 / 2 peak is at 642.6 eV, and the center of the Mn2p1 / 2 peak is at 654.4 eV. These results indicate that there is Mn in Mn3O4 NPs. 2+ and Mn 3+ Mixed valence.

[0056] This study also determined the CAT- and GPx-like activities of Mn3O4 NPs as well as SOD. Figure 1g It can be seen that in the presence of Mn3O4 NPs, the absorbance of hydrogen peroxide as a substrate decreases significantly over time, indicating that Mn3O4 NPs have the activity of catalyzing the decomposition of hydrogen peroxide similar to catalase. In addition, Mn3O4 NPs also have good GPx activity ( Figure 1 h). It uses intracellular GSH as a reducing agent to catalyze hydrogen peroxide to produce water and oxidized glutathione, while glutathione reductase (GR) can reduce the formation of oxidized glutathione (GSSG) and reduce it to GSH and NADPH. When the concentration of Mn3O4NPs in the reaction system is changed, the reaction rate increases proportionally with the increase in the amount of Mn3O4NPs added. In addition to the activity of CAT and GPx, Mn3O4NPs also exhibit superoxide dismutase (SOD)-like activity by catalyzing the dismutation of superoxide into H2O2 and oxygen ( Figure 1 i). Therefore, Mn3O4 NPs have similar activities to CAT, GPx, and SOD, which can effectively decompose hydrogen peroxide and scavenge peroxide and superoxide free radicals, and are expected to maintain redox homeostasis in the body.

[0057] Example 2

[0058] Preparation and characterization of injectable hydrogel ChS-HA loaded with Mn3O4 NPs:

[0059] First, the precursors of injectable ChS-HA hydrogels, adipoyl diacetyl hydrazine-modified hyaluronic acid (ADH-HA) and dopamine-grafted oxidized chondroitin sulfate (dop-OChS), were prepared. HA was activated by EDC and HOBT, and then reacted with adipoyl diacetyl hydrazine at room temperature to obtain adipoyl diacetyl hydrazine-modified hyaluronic acid (ADH-HA). Oxidized chondroitin sulfate (OChS) was prepared using NaIO4 as an oxidant. The carboxyl groups on OChS were further activated by EDC and NHS, and dopamine was added and stirred at room temperature in the dark for 24 hours to obtain dopamine-grafted OChS (dop-OChS). Its chemical structure was characterized by attenuated total reflection Fourier transform infrared spectroscopy (Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy, ATR-FTIR, VERTEX 70, Bruker Spectrometer, Germany) and 1H NMR spectrometer (AVANCE III 400MHz, Bruker BioSpin, Switzerland).

[0060] Mn3O4 NPs (2 mg mL -1) and mixed evenly with 5% (wt / v) ADH-HA PBS solution at a volume ratio of 1:2 to obtain an injectable ChS-HA hydrogel loaded with Mn3O4 NPs (named Mn3O4@ChS-HA). In addition, an injectable ChS-HA hydrogel without Mn3O4 NPs was prepared and used as a control. The sol-gel transition time of the injectable ChS-HA hydrogel at 37°C was studied by the bottle inversion method. A rheometer (MCR302, Anton Paar, Australia) was further used 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) using a 50 mm diameter parallel plate fixture and a gap of 0.5 mm. The viscosity of Mn3O4@ChS-HA was measured as a function of shear rate (0.1-1000 s-1) at 25°C. The adhesion properties of the hydrogels were characterized by adhesion tests. The hydrogels were pressed onto different surface materials (such as plastics, paper, test tube caps, etc.), their adhesion effects on these materials were observed, and their performance was recorded by photographs. This method evaluates the adhesion ability of hydrogels on different materials and provides a preliminary basis for their application in biological environments.

[0061] Based on the physiological activities of chondroitin sulfate and hyaluronic acid, we prepared injectable ChS-HA hydrogels composed of dopamine-grafted oxidized chondroitin sulfate and ADH-modified hyaluronic acid to simulate the natural environment of the intervertebral disc.

[0062] Combination Figure 2 As shown, (a) 1H NMR spectra of ADH-HA and dop-OChS; (b) FTIR of ADH-HA; (c) FTIR of dop-OChS; (d) UV-Vis spectrum of dop-OChS; (e) bottle inversion method showing the sol-gel transition behavior of injectable hydrogel; (f) curve of modulus of hydrogel changing with shear strain; (g) change of modulus of hydrogel with shear frequency; (h) shear thinning characteristics of injectable hydrogel; (i) injectability of injectable hydrogel; (J) adhesion of injectable hydrogel.

[0063] The chemical structures of the prepared ADH-HA and dop-OChS were confirmed by 1H NMR spectra ( Figure 2 a) and FTIR spectra ( Figure 2 b) was confirmed. For ADH-HA, the peak around δ1.6-2.5 ppm corresponds to the methylene protons in ADH, while the peak at δ2.3-2.5 ppm indicates the presence of hydrazine groups ( Figure 2a). The backbone protons of hyaluronic acid appear around δ3.2-4.0ppm. For dop-OChS, the peak around δ6.5-7.5ppm corresponds to the protons on the aromatic ring of dopamine; the small peak at δ9.5-10.0ppm is the aldehyde proton in oxidized chondroitin sulfate. In addition, the peak around δ2.5-3.5ppm corresponds 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 appears at 1554cm-1 ( Figure 2 b), indicating that ADH was successfully modified. OChS showed an aldehyde absorption peak at 1725 cm-1, indicating that ChS had been oxidized to form OChS. Compared with OChS, a new peak corresponding to the C=C stretching vibration of the aromatic ring in dopamine appeared near 1500 cm-1 in the spectrum of dop-OChS, indicating that dopamine was grafted onto OChS to form dop-OChS ( Figure 2 c). The UV-vis spectrum of dop-OChS shows a maximum absorption peak at 280nm ( Figure 2 d), further indicating that dopamine was grafted onto the OChS backbone and was not oxidized.

[0064] The results of the bottle inversion method showed that the two precursors of the injectable ChS-HA hydrogel could form a stable gel after being mixed and placed at 37°C for 3 minutes ( Figure 2 e). Within the range of shear strain and shear frequency tested, the storage modulus (G') and loss modulus (G") of ChS-HA and Mn3O4@ChS-HA hydrogels maintained relatively stable values, and G' was always higher than G" ( Figure 2 f,g), indicating that even at higher strains and shear frequencies, the hydrogel can still maintain its elastic properties and is not prone to permanent deformation. These properties enable the ChS-HA hydrogel to withstand certain mechanical stresses in applications without significant deformation, maintaining its structural stability and functionality. In addition, the viscosity of the injectable ChS-HA hydrogel was studied as a function of shear rate. The results showed that its viscosity decreased with increasing shear rate, indicating that it has shear-thinning properties and flows easily when passing through a syringe (low viscosity at high shear rates), and once injected, it quickly recovers its viscosity to form a stable colloid (high viscosity at low shear rates) ( Figure 2 h), so that it can be injected into the degenerated intervertebral disc in a minimally invasive manner, reducing surgical trauma. The injectable hydrogel can be easily squeezed out of the syringe and form a hydrogel in situ ( Figure 2i), which further demonstrates its injectability and suitability for minimally invasive procedures. The adhesion properties of the hydrogel were characterized by an adhesion test. The hydrogel was pressed onto different surface materials (such as plastic, paper, test tube caps, etc.), its adhesion effect on these materials was observed, and its performance was recorded through photographs. This method evaluates the adhesion ability of the hydrogel on different materials and provides a preliminary basis for its application in biological environments. The picture shows ( Figure 2 j), the hydrogel can effectively adhere to different surfaces and show strong adhesion, indicating that it may have good stability in complex biological environments. In the repair process of intervertebral disc degeneration (IVDD), the hydrogel needs to continue to adhere to the surface of the intervertebral disc tissue under the action of dynamic mechanical force to provide a stable microenvironment to promote tissue regeneration. Through this adhesion test, the adaptability and potential application prospects of the hydrogel were preliminarily proved, providing a basic basis for its use in the treatment of IDD.

[0065] Example 3

[0066] Verification of photothermal performance of Mn3O4@ChS-HA hydrogel in near-infrared II region:

[0067] The preparations contained different concentrations (0, 0.5, 1, and 2 mg mL -1 ) of Mn3O4 NPs. 1 mL of each of the above composite systems was placed in a 4 mL cuvette and a near-infrared laser (MW-GX-1064 / 5000 mW, Changchun Laser Optoelectronics Technology Co., Ltd.) was used to inject the ChS-HA hydrogel in the NIR II region (1064 nm, 1.5 W cm -2 ) for 6 min. The distance between the laser and the sample was set to 10 cm. An infrared thermal imager (MobIR Air, AutoNavi) was used to monitor the temperature change of the system.

[0068] In addition, the concentration of Mn3O4@ChS-HA was determined to be 2 mg mL -1 The injectable Mn3O4@ChS-HA hydrogels were prepared at different laser power densities (0, 0.5, 1.0, 1.5 and 2 W cm -2 ) under 1064nm laser irradiation. Each experiment was carried out in parallel three times. The system was also tested at a laser density of 1.5W cm -2 The temperature changes during five laser on-off cycles under 400 W / cm2 were used to evaluate the photothermal stability. -2The system was irradiated with 1064nm laser for 15min and then cooled to room temperature naturally. The temperature change during the experiment was recorded with an infrared thermal imager and a temperature-time curve was drawn. The photothermal conversion efficiency of the system was calculated according to the photothermal efficiency formula.

[0069] The results are as follows Figure 3 As shown in Figure 2, (a) UV-Vis-NIR absorption spectrum of Mn3O4 NPs; (b) Effect of Mn3O4 NPs concentration in gel on heating (1.5 W cm -2 )(n=3); (c) Effect of laser power density on the photothermal properties of gel (the concentration of Mn3O4 NPs in the gel is 2 mg mL -1 )(n=3); (d) Changes in gel temperature during the laser irradiation on-off cycle experiment; (e) Temperature curve of the gel after NIR-II laser irradiation for 15 minutes and then naturally cooling to room temperature (Mn3O4 NPs concentration: 2 mg mL -1 ; Laser power density: 1.5W cm -2 ); (f) The linear relationship between time t and dimensionless constant -lnθ obtained from the cooling curve of (e);

[0070] First, the UV-Vis-NIR absorption spectrum of Mn3O4 NPs solid powder was tested. The results showed that it has good light absorption performance in both near-infrared region I and II ( Figure 3 a). Further study was conducted at a laser power density of 1.5 W cm -2 Temperature rise of injectable ChS-HA hydrogels containing different concentrations of Mn3O4 NPs under 1064nm laser irradiation. The results show that as the concentration of Mn3O4 NPs increases, the temperature rises faster and higher, indicating that the concentration of Mn3O4 NPs has a significant effect on the photothermal effect ( Figure 3 b) The concentration of Mn3O4 NPs is 2 mg mL -1 When the laser power density increases, the temperature of the hydrogel increases more significantly ( Figure 3 c). When the laser power density is 1.5 W cm -2 After irradiation for 6 minutes, the system temperature rose by 21.3℃.

[0071] In the laser irradiation on-off cycle experiment, the temperature change of the hydrogel showed good reversibility and stability, proving that it can still maintain excellent photothermal response performance in multiple cycles ( Figure 3 d). To calculate the photothermal conversion efficiency of Mn3O4@ChS-HA hydrogel under 1064 nm laser irradiation, the concentration of Mn3O4 NPs was 2 mg mL -1The injectable Mn3O4@ChS-HA hydrogel was irradiated by 1064 nm laser (1.5 W cm -2 )'s heating-cooling curve ( Figure 3 e). The linear relationship curve between time t and dimensionless constant -lnθ is obtained as follows: Figure 3 f. According to the disclosed formula and the A1064=0.425 of the system at this time, the photothermal conversion efficiency (η) of the injectable Mn3O4@ChS-HA hydrogel was calculated to be 46.3%. These results show that the injectable hydrogel has excellent NIR-II photothermal performance. Based on the above results, the concentration of Mn3O4 NPs in the injectable Mn3O4@ChS-HA hydrogel was fixed at 2 mg mL -1 , the power density of 1064nm laser irradiation is 1.5Wcm -2 .

[0072] The next step will be to further verify the adhesion and biocompatibility of the hydrogel in biological tissues through in vivo experiments to evaluate its effectiveness in practical applications.

[0073] Example 4

[0074] 4.1. Isolation and culture of rat AFCs:

[0075] AFCs were extracted from the annulus fibrosus tissue of the rat tail. First, the rats were euthanized under sterile conditions and the intervertebral discs were isolated from the coccygeal vertebrae. Next, the annulus fibrosus was separated from the nucleus pulposus. The annulus fibrosus tissue was cut into small pieces using clean scissors and placed in a 1.5 ml centrifuge tube. The tissue was transferred to a 15 ml centrifuge tube and digested with a 0.25% trypsin solution on a 37°C shaker for 30 minutes. The trypsin solution was removed and further digested with a type I collagenase digestion solution on a 37°C shaker for 2 hours. After digestion was completed, culture medium was added to terminate 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, and the cells were inoculated into a cell culture flask and cultured in a 37°C, 5% CO2 incubator.

[0076] 4.2 Interventional treatment of AFCs

[0077] First, 3D images of the samples containing different concentrations of Mn3O4 nanoparticles (0, 1, 2, 5, 8, 10, 20, and 30 μg mL -1) hydrogels, which were immersed in cell culture medium to obtain extracts, which were then used to treat AFCs to evaluate their cytotoxicity. To evaluate the effect of Mn3O4 nanoparticles on the survival of AFCs under oxidative stress, AFCs were pretreated with Mn3O4 nanoparticles for 24 hours and then treated with H2O2 for 6 hours.

[0078] 4.3. CCK-8 Cell Counting Kit Experiment

[0079] Cell viability was assessed using the CCK-8 kit assay. Briefly, AFCs were seeded in 96-well plates with 5,000 cells per well and cultured in a 37°C, 5% CO2 incubator for 24 h. After culture, AFCs were treated with different concentrations of the test compounds for 12 h. After treatment, CCK-8 working solution was added to each well, the plates were incubated at 37°C for 2 h, and the absorbance was measured at a wavelength of 450 nm using a microplate reader (Thermo Scientific, MA, USA).

[0080] 4.4 Calcein-AM / PI staining

[0081] After Calcein-AM / PI staining, the cells were incubated with a PBS solution containing Calcein-AM and propidium iodide at 37°C in the dark for 20 min, and then the excess dye was gently washed off with PBS. The cells were then observed under a fluorescence microscope (Olympus IX71; Olympus Inc., Tokyo, Japan) to distinguish between live cells (green fluorescence) and dead cells (red fluorescence).

[0082] 4.5 Flow cytometry analysis

[0083] Detection of AFCs cell death using PI staining: AFCs cell death was assessed by propidium iodide (PI) staining. PI selectively enters dead cells and is detected by flow cytometry (BD LSR II).

[0084] Annexin V-FITC / PI apoptosis detection kit was used to detect apoptosis of AFCs: Annexin V-FITC was used to label the phosphatidylserine on the outer membrane of apoptotic cells, and PI was used to distinguish necrotic cells. Fluorescence intensity was analyzed by flow cytometry (BD LSRII).

[0085] Detection of intracellular ROS in AFCs using DCFH-DA: AFCs were incubated with ROS-sensitive fluorescent dye DCFH-DA, and the fluorescence intensity was detected by flow cytometry or fluorescence microscopy (BD LSR II).

[0086] Detection of mitochondrial ROS using MitoSOX Red: AFCs were stained with MitoSOX Red, which selectively accumulates in mitochondria and fluoresces under superoxide oxidation and analyzed by flow cytometry (BD LSR II).

[0087] Mitochondrial membrane potential was detected using the JC-1 kit: AFCs were stained with JC-1 dye, which showed red fluorescence when the mitochondria were normal and green fluorescence when depolarized. The red-green fluorescence ratio was analyzed by flow cytometry (BD LSR II).

[0088] 4.6 SA-β-Gal staining

[0089] Detection of senescent cells: First, wash AFCs with PBS and fix them with β-Gal fixative at room temperature for 15 minutes. After three PBS washes, incubate AFCs in staining solution at 37°C overnight (without CO2). During this process, senescent cells will turn blue. Finally, wash the cells again and observe them under a microscope.

[0090] 4.7. Western blot analysis

[0091] After treatment, AFCs were lysed with a buffer containing protease and phosphatase inhibitors and protein quantification was performed. SDS loading buffer was added and boiled at 95°C to denature the proteins. Equal amounts of proteins were loaded onto SDS-PAGE gels and separated according to molecular weight. The proteins were transferred to PVDF membranes and blocked with 5% nonfat dry milk in TBST for 1 h. The membranes were incubated with primary antibodies overnight at 4°C and then with HRP-labeled secondary antibodies for 1 h at room temperature. After washing with TBST, protein bands were visualized by chemiluminescence using ECL substrate.

[0092] 4.8. Quantitative real-time PCR (qRT-PCR) analysis

[0093] After treatment, total RNA of AFCs was extracted using TRIzol reagent to ensure RNA concentration and purity, and RNA was converted into cDNA by measuring absorbance at 260 / 280 nm by NanoDrop2000. qRT-PCR reactions were set up and run using specific cycling conditions. Fluorescence data during amplification were collected, and Ct values ​​were recorded, and gene expression was analyzed by normalization to reference genes using the ΔΔCt method.

[0094] 4.9 RNA Sequencing Analysis

[0095] RNA-seq analysis was performed to explore potential regulatory mechanisms. After treatment, 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 length trimming, and alignment with the reference genome. Differentially expressed genes (DEGs) under different conditions were identified by DESeq2 (|log2FC|>1.5, P<0.05). Bioinformatics analyses, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, were subsequently performed to elucidate gene expression patterns and differences.

[0096] 4.10 Animal Experiments

[0097] The annulus fibrosus defect model is commonly used to study intervertebral disc degeneration and repair. In this model, intervertebral disc injury or degeneration is simulated by creating a defect in the outer annulus fibrosus of the intervertebral disc. Thirty male Sprague Dawley rats (weight 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. Subsequently, a longitudinal incision of about 2 cm was made along the midline behind the 6th / 7th caudal vertebra to expose the intervertebral disc. Then, an 18-gauge needle was used to puncture the intervertebral disc to generate a full-thickness annulus fibrosus defect with a diameter of 0.60 mm and a depth of 1.40 mm, minimizing damage to the nucleus pulposus. Only skin incision was performed in the sham operation group. About 50 μL of different hydrogels or PBS were immediately injected at the annulus fibrosus defect, which was then disinfected, sutured, and disinfected again. After surgery, NIR II (1064 nm, 1.5 W cm -2 ) Expose to light for 15 minutes for two weeks.

[0098] 4.11 Imaging examination

[0099] After treatment, the rats' caudal vertebrae were examined by X-ray and magnetic resonance imaging (MRI). The disc height index (DHI) was calculated by comparing the disc height with the vertebral body height using digital radiographic images. The water content of the corresponding disc was assessed using MRI T2-weighted images, and the degree of disc degeneration was assessed using the Pfirrmann grading system.

[0100] 4.12 Histology

[0101] For histology, after dewaxing and hydration, hematoxylin-eosin (H&E) staining and safranin O / fast green (SO / FG) staining were performed to evaluate the histopathological scores.

[0102] 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. 3% BSA was used to reduce nonspecific binding, and then the sections were incubated with specific primary antibodies at 4°C overnight. The antibodies used included: anti-type II collagen antibody (ab34712, 1:200, Abcam), anti-aggregin 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), and then HRP-labeled secondary antibodies were added and incubated at room temperature for 1 hour. After that, DAB was used for color development and hematoxylin was used for counterstaining of the nuclei. After dehydration and mounting, the presence and distribution of the target antigens were observed under an optical microscope.

[0103] 4.13. Detection of reactive oxygen species (ROS) in vivo

[0104] Dihydroethidium (DHE) staining was used to detect the level of reactive oxygen species (ROS) in vivo. Fresh, unfixed frozen tissue sections were prepared and placed on slides to dry. DHE working solution (2 μM in PBS) was added to the tissue sections and incubated in a dark humid chamber at 37°C for 30 minutes. After incubation, the sections were gently washed with PBS and sealed with an anti-fading mounting medium containing DAPI. The stained sections were observed using a fluorescence microscope.

[0105] 4.14 Immunofluorescence (IF) staining

[0106] Immunofluorescence (IF) staining is used to detect and locate specific proteins in tissues or cells. For cell samples, fixation was performed with 4% paraformaldehyde; for tissue samples, the process included dewaxing, hydration, and EDTA antigen retrieval in a water bath at 85°C for 25 minutes. This was followed by permeabilization with 0.5% Triton X-100 for 20 minutes. After blocking nonspecific binding with 3% BSA, the primary antibody was added and incubated overnight at 4°C. The antibodies used included: anti-Cleaved Caspase-3 antibody (9664S, 1:100, CellSignaling Technology), anti-TOM20 antibody (ab186735, 1:200, Abcam), 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-aggregin 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), and anti-p16 antibody (sc-1661, 1:50, SantaCruz). After rewarming and washing, goat anti-rabbit antibody labeled with 488 (ab150077, 1:200, Abcam), goat anti-rabbit antibody labeled with 594 (ab150080, 1:200, Abcam) or goat anti-mouse antibody (ab150116, 1:200, Abcam) was added and incubated in the dark for 1 hour. After washing, the slides were mounted with anti-fading medium containing DAPI and the samples were observed under a fluorescence microscope.

[0107] Example 5

[0108] Statistical analysis of animal experiment results:

[0109] 5.1. Live / dead cell staining assay was used to evaluate the effect of Mn3O4 NPs on the viability of AFCs.

[0110] like Figure 4As shown, (A) Effects of different concentrations (1-40 μg mL-1) of Mn3O4@ChS-HA treatment for 24 h on the viability of AFCs (n=6). (B) Effects of Mn3O4@ChS-HA on the viability of AFCs under oxidative stress conditions (n=6). (C) Representative images of Calcein-AM / PI staining of H2O2-treated AFCs after 24 h of pretreatment with Mn3O4@ChS-HA (scale bar = 200 μm). (D, E) Representative images and statistical analysis of PI staining flow cytometry of AFCs with or without 100 μmol L-1 H2O2 or 5 μg mL-1 Mn3O4 NPs treatment. (ns, no statistically significant difference; *p<0.05, ***p<0.001, ****p<0.0001).

[0111] The results showed that no significant AFCs death was observed at a concentration of 30 ng / mL ( Figure 4 ), indicating that Mn3O4 NPs have good biocompatibility. Next, the H2O2-induced oxidative stress model was used to further evaluate the effect of Mn3O4 NPs@ChS-HA on the viability of AFCs. CCK8 results showed that at a concentration of 5 ng / mL of Mn3O4 NPs, cell viability was significantly restored ( Figure 4 Live / dead cell staining and flow cytometry results showed that AFCs underwent massive cell death after exposure to H2O2, while Mn3O4NPs@ChS-HA intervention significantly improved cell survival rate ( Figure 4 ). Therefore, the prepared Mn3O4 NPs@ChS-HA in this study exhibited good biocompatibility and effectively rescued AFCs from oxidative stress-induced cell death.

[0112] 5.2 Performance of Mn3O4 NPs@ChS-HA on oxidative stress

[0113] First, we evaluated the ability of Mn3O4 NPs@ChS-HA to scavenge ROS.

[0114] like Figure 5 The following are statistical graphs of the ability of Mn3O4@ChS-HA to inhibit oxidative stress and cell apoptosis. (AG) Representative flow cytometry graphs and statistical analysis of ROS staining, Mito-SOX staining, JC-1 staining, and Annexin V-FITC / PI staining (100 μmol L -1 H2O2 or 5 μg mL -1(H, I) Representative Western blot images and statistical analysis of the expression levels of Bax, Bcl2, C-CASP3, and C-CASP9 in AFCs treated with or without Mn3O4NPs (100 μmol L -1 H2O2 or 5 μg mL -1 Under the conditions of AFCs treated or not treated with Mn3O4NPs, n=3). (J, K) Representative immunofluorescence staining images and statistical analysis of C-CASP3 (100 μmol L -1 H2O2 or 5 μg mL -1 Under the conditions of AFCs treated or not treated with Mn3O4NPs, n = 3, scale bar = 40 μm). (L) Representative immunofluorescence staining images and statistical analysis of dsDNA (100 μmol L -1 H2O2 or 5 μg mL - 1 AFCs treated with or without Mn3O4NPs, n=3, scale bar=20μm). (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0115] DCFH was used to detect the level of reactive oxygen species in cells. Flow cytometry results showed that Mn3O4NPs@ChS-HA significantly reduced the increase of intracellular ROS caused by H2O2 ( Figure 5 ). At the same time, we detected the levels of mitochondrial reactive oxygen species and mitochondrial membrane potential. The flow cytometry results showed that Mn3O4NPs@ChS-HA significantly reduced mitochondrial reactive oxygen species and improved mitochondrial membrane potential levels, maintaining the integrity of mitochondrial function ( Figure 5 ). Studies have shown that inhibiting oxidative stress can delay IVDD by reducing IVD cell apoptosis, so we evaluated the anti-apoptotic ability of Mn3O4 NPs@ChS-HA. Flow cytometry results showed that Mn3O4NPs@ChS-HA significantly alleviated AFCs apoptosis ( Figure 5 ). At the same time, we evaluated the expression of molecules related to the apoptotic pathway. Western blotting results showed that Mn3O4 NPs@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 ), and immunofluorescence further confirmed this result ( Figure 5 ).

[0116] ROS is one of the main inducers of DNA damage and can initiate a variety of cell death mechanisms. dsDNA, including mtDNA released into the cytoplasm and oxidized mtDNA, among which mitochondrial DNA (mtDNA) is susceptible to ROS. It has been reported that endogenous mitochondrial ROS (mtROS) can promote mtDNA leakage. We further detected the expression of dsDNA. Immunofluorescence results showed that dsDNA was observed to be translocated into the cytoplasm under oxidative stress conditions, and increased mitochondrial fission was observed, leading to mitochondrial dysfunction. Mn3O4 NPs@ChS-HA reduced dsDNA in the cytoplasm and inhibited mitochondrial fission, maintaining the integrity of mitochondrial function ( Figure 5 ). Therefore, Mn3O4 NPs@ChS-HA inhibits the increase of dsDNA and mitochondrial fission while scavenging intracellular ROS, maintaining the integrity of mitochondrial function and further exerting its anti-apoptotic effect.

[0117] 5.3, Mn3O4@ChS-HA promotes AFCs autophagy and inhibits aging and inflammation;

[0118] ROS is not only related to apoptosis, but also closely related to cell aging, inflammation and cell autophagy. Mitochondrial dysfunction plays a key role in the process of cell aging, and Mn3O4NPs@ChS-HA can maintain the integrity of mitochondrial function, so we evaluated the anti-aging ability of Mn3O4NPs@ChS-HA.

[0119] The results are as follows Figure 6 Figure 3 shows the statistical diagram of Mn3O4@ChS-HA promoting AFCs autophagy and inhibiting aging and inflammation. (A, B) Representative SA-β-Gal staining images and statistical analysis (100 μmol L -1 H2O2 or 5 μg mL -1 AFCs treated with or without Mn3O4NPs, n = 3). (C, D) Representative immunofluorescence staining images and statistical analysis of γ-H2AX (100 μmol L - 1 H2O2 or 5 μg mL -1 Under the conditions of AFCs treated or not treated with Mn3O4NPs, n=3, scale bar=20μm). (EG) Representative Western blotting images and statistical analysis of p16, p21, IL-1β, IL-6 and TNF-α (100μmol L -1 H2O2 or 5 μg mL -1(H) Statistical analysis of mRNA levels of IL-1β, IL-6, and TNF-α (100 μmol L -1 H2O2 or 5 μg mL -1 AFCs treated with or without Mn3O4NPs, n = 3). (I, J) Representative immunofluorescence staining images and statistical analysis of IL-6 (100 μmol L -1 H2O2 or 5 μg mL -1 Under the conditions of AFCs treated or not treated with Mn3O4NPs, n = 3, scale bar = 40 μm). (K, L) Representative immunofluorescence staining images and statistical analysis of LC3B (100 μmol L -1 H2O2 or 5 μg mL -1 Under the conditions of AFCs treated or not treated with Mn3O4NPs, n=3, scale bar=20μm). (M,N) Representative Western blotting images and statistical analysis of BECN1, ATG7, p62 and LC3B (100μmol L -1 H2O2 or 5 μg mL -1 AFCs treated with or without Mn3O4NPs, n=3). (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0120] SA--Gal staining results showed that Mn3O4 NPs@ChS-HA significantly reduced the SA--Gal positive ratio increased by H2O2 ( Figure 6 ). Meanwhile, immunofluorescence showed that Mn3O4 NPs@ChS-HA inhibited the expression of DNA damage marker - histone H2A family member X (-H2AX) ( Figure 6 Mn3O4 NPs@ChS-HA inhibited the mRNA expression and secretion of SASP molecules such as IL6, IL1 and tumor necrosis factor (TNF) ( Figure 6 ). Western blotting showed that Mn3O4 NPs@ChS-HA significantly reduced the increase of p21 and p16 induced by H2O2.

[0121] Autophagy can alleviate aging-inducing factors such as oxidative stress and DNA damage, and delay the aging process of cells. To evaluate whether the anti-aging effect of Mn3O4 NPs@ChS-HA is attributed to the increase in cellular autophagy, we performed autophagy-related analyses. First, we evaluated the expression of LC3B, a key marker protein in the autophagy process. Immunofluorescence staining showed that Mn3O4NPs@ChS-HA treatment significantly inhibited the decrease in LC3B expression caused by H2O2, and enhanced autophagy ( Figure 6 ). We then evaluated the expression of molecules related to the autophagy pathway. Western blotting results showed that Mn3O4 NPs@ChS-HA downregulated the expression of p62 and increased the expression of BECN1, ATG7, and LC3B ( Figure 6 ). Taken together, these results indicate that Mn3O4 NPs@ChS-HA can effectively alleviate oxidative stress-induced cellular senescence and inflammation by promoting cellular autophagy.

[0122] 5.4. The ability of Mn3O4@ChS-HA to regulate the anabolic / catabolic balance;

[0123] Extracellular matrix (ECM) plays a vital role in intervertebral disc degeneration (IVDD). Oxidative stress can lead to abnormal ECM metabolism in IVD and promote the progression of IVDD. To evaluate the ability of Mn3O4 NPs@ChS-HA to regulate anabolic / catabolic balance, we detected the expression of metabolism-related molecules such as Col1a1, Col2a1, Aggrecan, MMP13, and ASDMTS5.

[0124] The results are as follows Figure 7 Figure 3 shows the statistical diagram of the ability of Mn3O4@ChS-HA to regulate the balance of synthesis / catabolism. (A, B) Representative immunofluorescence staining and statistical analysis of Aggrecan, COL2A1, COL1A1, ADAMTS5 and MMP13 (100 μmol L -1 H2O2 or 5 μg mL -1 Under the conditions of AFCs treated or not treated with Mn3O4NPs, n=3, scale bar=40μm). (C) Statistical analysis of mRNA levels of Aggrecan, COL2A1, COL1A1, ADAMTS5 and MMP13 (100μmol L -1 H2O2 or 5 μg mL -1 Under the conditions of AFCs treated or not treated with Mn3O4NPs, n=3). (D, E) Representative Western blotting images and statistical analysis of Aggrecan, COL2A1, COL1A1, ADAMTS5 and MMP13 (100 μmol L -1 H2O2 or 5 μg mL -1 AFCs treated with or without Mn3O4NPs, n=3). (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0125] Immunofluorescence results showed that Mn3O4 NPs@ChS-HA significantly restored the H2O2-induced decrease in the synthesis of Aggrecan and COL2A1, while reducing the H2O2-induced increase in the levels of matrix-degrading enzymes matrix metallopeptidase 13 (MMP13), COL1A1, and a disintegrin and metalloproteinase with thrombospondin motif 5 (ADAMTS5) ( Figure 7 ). At the same time, immunoblotting analysis and qRT-PCR results also showed that oxidative stress led to a decrease in the expression of Col2a1 and Aggrecan and an increase in the expression of Col1a1, MMP13 and ASDMTS5, and Mn3O4 NPs@ChS-HA significantly reversed this result. In summary, these results indicate that Mn3O4 NPs@ChS-HA can effectively inhibit the increase in catabolism induced by oxidative stress and promote anabolism, thereby maintaining the integrity of the extracellular matrix.

[0126] 5.5. Differences in biological behaviors caused by Mn3O4 NPs@ChS-HA treatment;

[0127] Mn3O4 NPs@ChS-HA showed great therapeutic potential in H2O2-induced AFCs death, and to reveal its underlying mechanism, we performed RNA sequencing (RNA-seq) on the H2O2 group and the Mn3O4 NPs@ChS-HA+H2O2 group.

[0128] The results are as follows Figure 8 The following are statistical diagrams of the differences in biological behaviors 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+Mn3O4NPs group (n=3); (B) Volcano plot of DEGs; (C) Heat map of DEGs between the H2O2 group and the H2O2+Mn3O4NPs group; (D) GO enrichment analysis of identified DEGs; (E) KEGG enrichment analysis of DEGs; (F, G) Representative Western blotting images and statistical analysis of IL-17 (100 μmol L -1 H2O2 or 5 μg mL -1 Under the conditions of AFCs treated or not treated with Mn3O4NPs, n=3); (H) Statistical analysis of IL-17 mRNA expression level (100 μmol L -1 H2O2 or 5 μg mL -1 (I) Representative immunofluorescence staining and statistical analysis of IL-17 (100 μmol L -1 H2O2 or 5 μg mL -1AFCs treated with or without Mn3O4NPs, n=3, scale bar=40μm). (**p<0.01, ***p<0.001, ****p<0.0001).

[0129] The results of principal component analysis (PCoA) showed that there were significant differences between the H2O2 group and the Mn3O4 NPs@ChS-HA+H2O2 group ( Figure 8 ). According to the volcano Figure 8 As a result, there were 128 differentially expressed genes between the H2O2 group and the Mn3O4NPs@ChS-HA+H2O2 group, including 89 up-regulated genes and 39 down-regulated genes ( Figure 8 ) At the same time, heat Figure 8 Binary classification matrix data of upregulated genes (red) and downregulated genes (blue) are shown, indicating that the RNA profiles between the two groups were significantly different ( Figure 8 ).

[0130] Next, the structured bioinformatics annotation system GO (Gene Ontology) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to perform pathway enrichment analysis on DGEs and reveal the physiological and biochemical processes involved in DEGs. GO analysis was used to determine the 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 In addition, KEGG analysis showed that DEGs in AFCs were mainly enriched in “IL-17signaling pathwayand TNF signaling pathway” ( Figure 8 ). Among them, the IL-17 signaling pathway plays a vital role in the regulation of the immune system, especially in inflammatory response, tissue damage and repair, and cancer progression. qPCR, WB, and IF analysis results showed that Mn3O4 NPs@ChS-HA significantly inhibited the expression of IL-17RA, a key gene in the IL-17 signaling pathway. Therefore, the above results indicate that Mn3O4 NPs@ChS-HA mainly exerts its biological effects by regulating the IL-17 signaling pathway.

[0131] 5.6. The therapeutic effect of Mn3O4 NPs@ChS-HA in rats;

[0132] In order to study the in vivo photothermal performance of Mn3O4 NPs@ChS-HA, an infrared camera was used to detect thermal imaging of the hydrogel after implantation into the AF defect. Under 1064nm, 1.5W cm-2 NIR-II laser irradiation, the temperature of the rat coccygeal vertebra implanted with Mn3O4@ChS-HA increased significantly, reaching about 42°C within 6 minutes (Fig. Fig.12 Statistical diagram of the photothermal performance of Mn3O4@ChS-HA in vivo, (A) Photothermal images of ChS-HA and Mn3O4@ChS-HA under 1064nm near-infrared radiation under the coccyx of rats. (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 ability of NIR-II lasers and their low absorption properties, and verify the photothermal therapeutic effect achieved by Mn3O4@ChS-HA, establishing it as a promising physical therapy modality for IVDD treatment.

[0133] To evaluate the in vivo therapeutic effect of Mn3O4 NPs@ChS-HA, we established an annulus fibrosus defect model in the rat coccygeal intervertebral disc, and then injected different hydrogels into the defect, which could adhere well to the defect ( Fig. 9 ). X-ray examination, magnetic resonance imaging (MRI), H&E, SO / FG, and IHC were used to evaluate the repair of AF.

[0134] The results are as follows Fig. 9 The therapeutic effect of Mn3O4 NPs@ChS-HA in rats is shown in Figure 2. Specifically: (A) (B) Representative X-rays and T2 MRI images of rats in the sham or IVDD group; (B) Representative images of HE staining and SO / FG staining; (C) Statistical analysis of disc height index; (D) Statistical analysis of Pfirrmann grading; (E) Statistical analysis of relative signal intensity; (F) Statistical analysis of histological scores; (G, H) Immunohistochemical staining images (G) and statistical analysis (H) of Aggrecan, COL2A1, COL1A1, ADAMTS5 and MMP13 in disc tissue. (n = 6, scale bar = 500 μm, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0135] X-ray examination showed that Mn3O4 NPs@ChS-HA greatly rescued the reduction in disc height, as shown by the DHI value being closest to that of the SHAM group ( Fig. 9). The T2-weighted signal intensity on MRI can reflect the NP water content and is directly related to the degree of degeneration. Consistent with the X-ray examination results, compared with the other IVDD groups, the Mn3O4 NPs@ChS-HA group had the highest signal intensity and the highest NP water content, and the Pfirman grade was significantly improved ( Fig. 9 ). In addition, H&E and SO / FG staining results showed that the nucleus pulposus in the IVDD group was missing, the border was blurred, the contents were replaced by the annulus fibrosus, and the proteoglycans almost disappeared and were eventually replaced by collagen. In contrast, the Mn3O4NPs@ChS-HA group showed a significant recovery of the nucleus pulposus volume and vacuolar matrix, and showed a relatively rich proteoglycan content, while the histological score was significantly improved ( Fig. 9 ). Immunohistochemical staining showed that Mn3O4NPs@ChS-HA rescued the imbalance of ECM metabolism caused by acupuncture, as manifested by increased expression levels of matrix proteins Aggrecan and COL2A1, and decreased expression levels of COL1A1, MMP13, and ADAMTS5 ( Fig. 9 ). The above results all indicate that Mn3O4 NPs@ChS-HA can significantly delay the progression of IVDD.

[0136] Meanwhile, considering the risk of leakage after disc injection, we evaluated the toxicity of Mn3O4NPs@ChS-HA to major organs such as heart, liver, spleen, lung, and kidney. H&E staining showed that no obvious damage was observed to major organs such as heart, liver, spleen, lung, and kidney during Mn3O4NPs@ChS-HA treatment (e.g. Fig.13 , Representative images of HE staining of rat heart, liver, spleen, lung, and kidney, taken from tissue samples at 4 weeks, scale bar = 100 μm. ) Therefore, Mn3O4 NPs@ChS-HA exhibited good biocompatibility in vivo.

[0137] 5.7. Mn3O4@ChS-HA delays IVDD progression via IL-17 signaling pathway

[0138] Next, we explored the possible mechanism of Mn3O4 NPs@ChS-HA in delaying the progression of IVDD.First, we performed dihydroethidium staining on the freshly collected IVD tissues.

[0139] The results are as follows Fig.10The following is a statistical diagram of Mn3O4@ChS-HA delaying IVDD progression through the IL-17 signaling pathway. Specifically: (A) Representative images of ROS staining in IVD tissue; (BF) Representative immunofluorescence staining images of C-CASP3, H2AX, p16, p21, IL-6, LC3B, and IL-17 in IVD tissue; (G) Statistical analysis of ROS levels in IVD tissue; (HN) Statistical analysis of C-CASP3, H2AX, p16, p21, IL-6, LC3B, and IL-17 levels in IVD tissue. (n=6, scale bar=100μm, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0140] The results showed that acupuncture led to a significant increase in ROS levels in IVD tissues, while Mn3O4NPs@ChS-HA significantly reduced ROS levels in vivo. In addition, we performed immunofluorescence staining for apoptosis-related markers (C-CASP3), senescence-related markers (p21, p16, -H2AX), inflammation-related markers (IL-6), and autophagy-related markers (LC3B). The results showed that Mn3O4NPs@ChS-HA significantly reduced the levels of C-CASP3, p21, p16, -H2AX, and IL-6 in IVD tissues and increased the expression of LC3B. Finally, Mn3O4 NPs@ChS-HA treatment significantly inhibited the expression of IL-17RA, a key protein in the IL-17signaling pathway, as confirmed by immunofluorescence staining experiments. Taken together, these results indicate that Mn3O4NPs@ChS-HA significantly inhibits the IL-17signaling pathway, thereby exerting its multifunctional biological effects.

[0141] In summary, this study successfully developed a multifunctional MPTT-nanozyme-hydrogel system, Mn3O4@ChS-HA, which showed significant potential through multiple mechanisms and played an important role in alleviating intervertebral disc degeneration (IVDD). On the one hand, by simulating the natural environment within the disc, Mn3O4@ChS-HA not only showed excellent injectability and biocompatibility, but also had good mechanical properties and adhesion, enabling it to tightly adhere to the annulus fibrosus defect and provide support for the disc. On the other hand, Mn3O4 nanoparticles exhibited significant CAT, GPx, and SOD-like activities, which could effectively reduce the level of ROS in cells and mitochondria, thereby exerting anti-apoptotic effects, delaying aging, and promoting autophagy. In addition, after combining with MPTT, the system was able to further inhibit inflammation, promote the repair of annulus fibrosus damage, and delay the progression of IVDD.

[0142] Hydrogel-based drug delivery systems represent a promising therapeutic strategy for the treatment of IVDD. Hydrogels are widely used in a variety of fields including bone disease, trauma repair, and inflammatory diseases due to their three-dimensional network structure, excellent biocompatibility, mechanical properties, injectability, and drug loading capacity.

[0143] 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 can form a stable gel under physiological conditions, ensuring its long-term residence at the injury site, which is a key factor for local IVDD treatment. In addition, ChS-HA enhances tissue adhesion and provides structural support to the injury site, simulating the natural disc environment. Therefore, ChS-HA is an ideal drug delivery system that can ensure the sustained release of Mn3O4 nanoparticles at the injury site, reduce the need for repeated administration, and reduce the potential complications of systemic treatment. Notably, after loading with Mn3O4 nanoparticles, the system exhibited photothermal properties (≤42°C), further enhancing its therapeutic effect by promoting annular tissue repair and slowing the in vivo progression of IVDD. The combination of MPTT and Mn3O4 nanoparticles enhanced the multifunctional biological effects of the hydrogel, which is consistent with the results of previous studies showing that combined MPTT can effectively relieve inflammation, inhibit cell apoptosis, and promote ECM synthesis. There is growing evidence that controlled MPTT can synergize with nanoparticle-based therapies to improve tissue repair and slow disease progression. Our study showed that MPTT obtained under near-infrared light irradiation could inhibit the release of pro-inflammatory cytokines and enhance autophagy, further enhancing the anti-inflammatory and anti-aging effects of Mn3O4@ChS-HA.

[0144] Oxidative stress is a key driver of IVDD progression. By inhibiting oxidative stress, IVDD progression can be slowed by inhibiting apoptosis, delaying cellular senescence, inhibiting inflammation, and regulating ECM metabolism. In recent years, metal nanomaterials with enzyme-like activities have received extensive attention. Monodispersed nanoflower-like Mn3O4 nanoparticles possess intrinsic SOD, CAT, and GPx-like activities, showing significant antioxidant properties. These enzyme-like activities significantly reduced ROS levels in AFCs, which are known to cause annulus fibrosus 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 indicate that Mn3O4@ChS-HA is able to maintain cellular homeostasis under oxidative stress, highlighting the role of oxidative stress in promoting apoptosis, inflammation, and senescence in IVDD.

[0145] On the other hand, oxidative stress can lead to imbalanced ECM metabolism in the intervertebral disc, which is another key factor in the progression of IVDD. In IVDD, due to the reduction in cell number and dysfunction, the catabolic process of ECM increases, while the anabolic process decreases, leading to imbalanced ECM metabolism. In this study, Mn3O4@ChS-HA effectively regulated ECM metabolism and maintained the integrity of ECM by downregulating the expression of MMP13 and ADAMTS5, while upregulating the expression of key components of the intervertebral disc matrix COL2A1 and agrin. At the same time, in vivo experiments confirmed that Mn3O4@ChS-HA successfully alleviated the reduction of 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. In addition, these therapeutic effects were further enhanced after MPTT. The above results indicate that Mn3O4@ChS-HA has important 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 disc degeneration.

[0146] In conclusion, the MPTT-nanozyme-hydrogel system (Mn3O4@ChS-HA) is a promising biomaterial capable of treating IVDD by addressing IVDD-associated oxidative stress and ECM imbalance. Its multifunctional properties combined with the innovative combination of photothermal therapy provide a powerful platform for future therapeutic applications in degenerative disc disease. In addition, the system's nontoxicity in major organs suggests that Mn3O4@ChS-HA has good biocompatibility, which is a key factor for future clinical translation.

[0147] The MPTT-nanozyme-hydrogel system (Mn3O4@ChS-HA) represents a promising multifunctional therapeutic platform capable of treating IVDD by resolving oxidative stress, regulating ECM metabolism, and promoting annulus fibrosus tissue repair. Its combination with mild photothermal therapy further enhances its therapeutic capacity.

Claims

1. The application of Mn3O4 nanoparticles in alleviating intervertebral disc degeneration is characterized by: Manganese tetraoxide nanoparticles Mn3O4 NPs are added as nanozymes to interventional drugs for the treatment of intervertebral disc degeneration.

2. The use of Mn3O4 nanoparticles according to claim 1 in alleviating intervertebral disc degeneration, characterized in that: Manganese oxide nanoparticles (Mn3O4 NPs) were added into the hydrogel, and the hydrogel was used as a delivery medium to form an injectable.

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

4. The use of Mn3O4 nanoparticles in alleviating intervertebral disc degeneration according to claim 3, characterized in that: The injection achieves therapeutic effect enhancement by cooperating with mild photothermal therapy MPTT.

5. The biomaterial Mn3O4@ChS-HA for treating intervertebral disc degeneration according to claim 1, characterized in that: It includes manganese tetraoxide nanoparticles Mn3O4 NPs, and a hydrogel ChS-HA composed of dopamine-grafted oxidized chondroitin sulfate dop-OChS and ADH-modified hyaluronic acid ADH-HA; Together they constitute the biomaterial Mn3O4@ChS-HA with 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: The following steps are involved: S1. Raw material preparation: Preparation of manganese tetraoxide nanoparticles Mn3O4 NPs: Oleic acid was added to a 0.2wt / v% KMnO4 aqueous solution at a volume ratio of 2:100, and stirred at room temperature for 6 hours; then centrifuged at 8000rpm for 5 minutes, and the precipitate was washed with deionized water and ethanol in sequence; the precipitate was collected and dried at 80°C for 10 hours, the brown solid was ground and calcined in an oven at 200°C for 5 hours, and then ground again and passed through a 50-mesh sieve to obtain Mn3O4 NPs powder; Preparation of dopamine-grafted oxidized chondroitin sulfate dop-OChS: Oxidized chondroitin sulfate (OChS) was prepared using NaIO4 as an oxidant, and carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were further used to activate the carboxyl groups on the oxidized chondroitin sulfate (OChS). After adding dopamine, the mixture was stirred at room temperature in the dark for 24 hours to obtain dopamine-grafted oxidized chondroitin sulfate (dop-OChS). Preparation of ADH-modified hyaluronic acid ADH-HA: HA was activated by carbodiimide EDC and 1-hydroxybenzotriazole HOBT, and then reacted with adipoyl diacetyl hydrazide ADH at room temperature to obtain adipoyl diacetyl hydrazide-modified hyaluronic acid (ADH-HA); Preparation of S2 and Mn3O4@ChS-HA: Mn3O4 NPs were added at 2 mg / ml to a 5% wt / v dop-OChS solution and mixed evenly 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 Mn3O4 NPs.

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