An injectable hydrogel loaded with 4-octylitaconic acid, its preparation method and its application

Abstract

This invention discloses an injectable hydrogel loaded with 4-octyl itaconic acid, its preparation method, and its applications, belonging to the field of biomedical materials technology. The injectable hydrogel is formed by enzymatic cross-linking of a hyaluronic acid-tyramine complex to create a three-dimensional network, with 4-octyl itaconic acid encapsulated within this network structure. This invention allows for precise implantation of the hydrogel into the intervertebral disc lesion via minimally invasive injection, achieving local high-concentration drug accumulation. The three-dimensional network of the hydrogel serves as a drug reservoir, enabling continuous and controllable release of 4-OI, prolonging the drug's duration of action and achieving long-term therapeutic effects. Simultaneously, the loaded 4-OI specifically activates the Nrf2 / GPX4 antioxidant pathway in nucleus pulposus cells, mechanistically antagonizing ferroptosis and matrix degradation, thus achieving a dual function of mechanical support and biological therapy. It exhibits good biocompatibility and solves the problems of difficult targeted drug delivery, short-lived efficacy, and lack of disease-modifying effects in existing treatments for intervertebral disc degeneration.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to an injectable hydrogel loaded with 4-octylitaconic acid, its preparation method, and its application. Background Technology

[0002] Intervertebral disc degeneration (IVDD) is the main pathological basis for chronic low back pain, and its occurrence and development are closely related to the progressive loss of nucleus pulposus cells and the degradation of the extracellular matrix. Recent studies have revealed that oxidative stress-induced ferroptosis in nucleus pulposus cells is a key mechanism driving this pathological process. Therefore, targeting and activating the core intracellular antioxidant defense pathway—the nuclear factor E2-related factor 2 (Nrf2) / glutathione peroxidase 4 (GPX4) axis—has become a promising therapeutic strategy for intervening in IVDD. 4-Octylitaconic acid (4-OI), as a specific small molecule agonist of Nrf2, has been shown to effectively antagonize ferroptosis by activating this pathway at both cellular and animal levels.

[0003] However, efficiently delivering therapeutic molecules such as 4-OIs into avascular intervertebral disc tissue and achieving long-term residence and sustained release presents significant challenges. Systemic administration struggles to achieve effective therapeutic concentrations at the lesion site and is prone to causing systemic side effects. Local injection of free drugs suffers from rapid diffusion and clearance, as well as short duration of action, failing to meet the long-term management needs of chronic degenerative diseases.

[0004] Injectable hydrogels offer a potential solution to the aforementioned challenges. Among them, hyaluronic acid-tyramine (HA-Tyr) hydrogels, based on enzymatic crosslinking, are considered promising local delivery carriers due to their good biocompatibility, tunable gel kinetics, and structure similar to the natural extracellular matrix. However, current applications of HA-Tyr hydrogels primarily focus on their function as physical barriers or growth factor carriers.

[0005] A significant gap exists in current technological solutions: no treatment system exists that can intelligently integrate small molecule drugs (4-OIs) with a well-defined mechanism (activating Nrf2 / GPX4 to antagonize ferroptosis) with injectable hydrogel carriers to construct a synergistic treatment system that combines "mechanical support," "in-situ retention," "long-acting sustained release," and "mechanism-targeted therapy." Particularly concerning is the complex pathological microenvironment of IVDD; how to achieve continuous and efficient regulation of the core disease pathways through material design remains a critical issue that urgently needs to be addressed in this field. Summary of the Invention

[0006] The purpose of this invention is to provide an injectable hydrogel loaded with 4-octylitaconic acid, its preparation method, and its application, which can be injected in situ, continuously release drugs, and specifically activate cell protection pathways to delay intervertebral disc degeneration, thus solving the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] An injectable hydrogel loaded with 4-octylitaconic acid, using a hyaluronic acid-tyramine complex as the gel matrix, forms a three-dimensional network structure through enzymatic cross-linking, with 4-octylitaconic acid encapsulated inside the three-dimensional network structure.

[0009] According to another aspect of the present invention, a method for preparing an injectable hydrogel loaded with 4-octyl itaconic acid as described above is provided, comprising the following steps:

[0010] S1. Synthesis of hyaluronic acid-tyramine complex:

[0011] Hyaluronic acid and tyramine were coupled in MES buffer under the catalysis of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide. After the reaction, the mixture was purified by dialysis and lyophilized to obtain HA-Tyr solid.

[0012] S2. Preparation of precursor solution:

[0013] The obtained HA-Tyr solid was dissolved in PBS buffer to prepare a solution of a certain concentration, and DMSO solution containing 4-octylitaconic acid was added to it. After stirring, the solution was mixed evenly to obtain a drug-containing prodrug solution.

[0014] S3, In-situ gelation:

[0015] Horseradish peroxidase and hydrogen peroxide solution were added sequentially to the obtained drug precursor solution to initiate an enzymatic cross-linking reaction of tyramine groups, rapidly forming HA-Tyr hydrogel loaded with 4-octylitaconic acid within 1-2 seconds.

[0016] Preferably, in S1, the mass ratio of hyaluronic acid to tyramine in the hyaluronic acid-tyramine complex is (5-10):1; and the molecular weight of the hyaluronic acid is 100k-200kDa.

[0017] Preferably, in S1, the pH value of the MES buffer is 5.0-6.0, and the concentration is 0.1-0.2 mol / L; the molar ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 1:1, and the EDC:NHS:Tyr ratio is 2.5~3:2.5~3:1.

[0018] Preferably, in step S1, the coupling reaction temperature is 20-25℃ and the reaction time is 2-4h; dialysis purification uses a dialysis bag with a molecular weight cutoff of 3500Da, and dialysis with deionized water for 3-5 days. On the first day, the bag is changed 3 times in 8 hours or 4 times in 6 hours, and then changed once every 12 hours for a total of 3-5 days.

[0019] Preferably, in step S2, the obtained HA-Tyr solid is dissolved in PBS buffer to prepare a solution with a mass-volume concentration of 1% w / v.

[0020] Preferably, in S2, the final concentration of 4-octylitaconic acid is 50-200 μM.

[0021] Preferably, in step S2, the volume ratio of DMSO solution to PBS buffer is ≤1:20, the stirring speed is 200-300 r / min, and the stirring time is 10-20 min.

[0022] Preferably, in step S3, the final concentration of horseradish peroxidase is 10 U / mL, and the final concentration of hydrogen peroxide is 0.003% w / v.

[0023] According to another aspect of the invention, an application is provided of an injectable hydrogel loaded with 4-octylitaconic acid as described above, wherein the injectable hydrogel loaded with 4-octylitaconic acid is used in a medicament for the prevention, delay, or treatment of intervertebral disc degeneration.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] This invention precisely implants hydrogel into intervertebral disc lesions via minimally invasive injection, achieving local high-concentration drug accumulation. The three-dimensional network of the hydrogel serves as a drug reservoir, enabling continuous and controllable release of 4-OI, prolonging the drug's duration of action and achieving long-term therapeutic effects. Simultaneously, the hydrogel itself provides temporary mechanical support and simulates the highly hydrated microenvironment of the nucleus pulposus. The loaded 4-OI specifically activates the Nrf2 / GPX4 antioxidant pathway in nucleus pulposus cells, mechanistically antagonizing ferroptosis and matrix degradation, thus achieving a dual function of mechanical support and biological therapy. This hydrogel exhibits ultra-fast (second-level) gelation characteristics, making it suitable for clinical operation. It has a balanced water content >96%, good biocompatibility, and suitable mechanical strength and enzyme-responsive degradation characteristics. Furthermore, in vitro and in vivo experiments have confirmed that this hydrogel can effectively delay intervertebral disc degeneration in model animals, maintain disc height and proteoglycan content, and its effects are directly related to upregulating Nrf2 / GPX4 expression and inhibiting oxidative damage. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the rapid enzymatic gelation process of the hydrogel of the present invention;

[0027] Figure 2 This is a photograph of the macroscopic morphology of the hydrogel after equilibrium swelling according to the present invention.

[0028] Figure 3 This is a microscopic morphology diagram of the hydrogel of the present invention;

[0029] Figure 4 These are schematic diagrams of representative X-ray images of the four groups of rats at 4 and 8 weeks of age, as presented in this invention.

[0030] Figure 5 This is a bar chart showing the results of the DHI three-way ANOVA combined with Tukey post-hoc test in this invention.

[0031] Figure 6 This is a schematic diagram of the HE staining and SO-FG staining results of the present invention;

[0032] Figure 7 This is a schematic diagram of the immunohistochemical (IHC) results of different groups according to the present invention;

[0033] Figure 8 This diagram illustrates the preparation steps of the injectable hydrogel loaded with 4-octylitaconic acid according to the present invention. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] To address the challenges of targeted drug delivery, short-lived efficacy, and lack of disease-modifying effects in current treatments for intervertebral disc degeneration, see [reference needed]. Figure 1-8 This embodiment provides the following technical solution:

[0036] An injectable hydrogel loaded with 4-octylitaconic acid is formed by enzymatic cross-linking of a hyaluronic acid-tyramine (HA-Tyr) complex to form a three-dimensional network, and 4-octylitaconic acid (4-OI) is encapsulated in the network structure.

[0037] It should be noted that injectable hydrogels are biomaterials that exist in a liquid form in vitro and are implanted into the body via injection, subsequently transforming rapidly into a solid gel under physiological conditions. Their unique sol-gel phase transition characteristics make them promising for a wide range of applications in the biomedical field, such as drug delivery, tissue engineering, and wound healing. Based on their formation mechanisms, injectable hydrogels can be broadly classified into two categories: physically cross-linked and chemically cross-linked. Among these, enzymatic cross-linking, as a highly efficient chemical cross-linking method, has attracted considerable attention due to its mild reaction conditions, strong controllability, and excellent biocompatibility. The core principle of enzymatic cross-linking lies in inducing covalent bonding of active functional groups on polymer chains through the catalytic action of specific enzymes, thereby constructing a three-dimensional network structure. For example, horseradish peroxidase (HRP) can effectively catalyze the oxidative cross-linking reaction of tyramine groups in the presence of hydrogen peroxide (H2O2), forming a stable hydrogel structure. This cross-linking mechanism not only possesses rapid gelation capabilities but also allows for precise control of the gelation rate and mechanical properties by adjusting enzyme concentration and reactant ratios, providing an important theoretical basis for the design and optimization of injectable hydrogels.

[0038] Hyaluronic acid (HA) is a naturally occurring linear polysaccharide composed of alternating D-glucuronic acid and N-acetylglucosamine linked by β-1,4 glycosidic bonds. Its excellent biocompatibility, biodegradability, and low immunogenicity make it an ideal substrate for injectable hydrogels. In recent years, hydrogels based on hyaluronic acid and its derivatives have been extensively studied in tissue engineering. For example, chemically modifying hyaluronic acid by introducing functional groups (such as thiol groups and acrylates) can significantly improve its mechanical and functional properties. Studies have shown that modified hyaluronic acid derivatives can form stable hydrogel structures through various cross-linking mechanisms (such as thiol-ene click reactions and Michael addition reactions), while also endowing them with intelligent properties such as self-healing and thermoresponsiveness. Furthermore, hyaluronic acid-based hydrogels are particularly prominent in cartilage repair; their porous network structure provides a favorable growth microenvironment for cells and promotes the secretion and deposition of extracellular matrix. These research advances indicate that hyaluronic acid and its derivatives demonstrate enormous development potential in the field of injectable hydrogels.

[0039] Among them, 4-Octyl Itaconic Acid (4-OI) is an unsaturated fatty acid derivative with hydrophobic properties. Its molecular structure contains double bonds and carboxyl functional groups, which provides a unique chemical basis for its application in the field of biomaterials.

[0040] Example 1

[0041] A method for preparing an injectable hydrogel loaded with 4-octyl itaconic acid includes the following steps:

[0042] S1. Synthesis of HA-Tyr complex: Hyaluronic acid (HA) was dissolved in MES buffer at pH 5.5, tyramine (Tyr) was added, and the mixture was reacted at room temperature for 2-4 h under the catalysis of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The reaction solution was neutralized with NaOH, dialyzed, and lyophilized to obtain HA-Tyr white solid.

[0043] S2. Preparation of precursor solution: Dissolve HA-Tyr white solid in PBS buffer to prepare a 1% (w / v) solution. Prepare a stock solution of 4-OI with DMSO and add it to the HA-Tyr solution to make the final concentration of 4-OI 100 μM. Mix well to obtain the drug-containing precursor solution.

[0044] S3. Formation of hydrogel: Add horseradish peroxidase (HRP) (final concentration 10 U / mL) to the above drug precursor solution, mix well, and immediately add hydrogen peroxide (H2O2) solution (final concentration 0.003%). Gently stir for about 1-2 seconds. The solution quickly transforms into a non-flowing gel, thus obtaining the HA-Tyr hydrogel loaded with 4-OI. Figure 1 As shown, the gel is macroscopically transparent and elastic, and microscopically has a porous structure, such as... Figure 2-3 As shown.

[0045] It should be noted that the linear chain structure of hyaluronic acid contains abundant carboxyl groups, which can be chemically modified to introduce active groups and provide sites for subsequent cross-linking reactions. Tyramine (Tyr) is a compound containing phenolic hydroxyl groups, and the active groups in its molecular structure can undergo coupling reactions with HA to form stable covalent bonds. In addition, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), as commonly used coupling reagents, can effectively activate the carboxyl groups in the HA molecule and promote its condensation reaction with Tyr. EDC forms an O-acylisourea intermediate with the carboxyl group, which further reacts with NHS to generate NHS ester, thereby improving the reaction efficiency and enhancing the stability of the product. The selection of these raw materials and reagents is not only based on the matching of their chemical properties and reaction mechanisms, but also takes into account their safety and feasibility in biomedical applications.

[0046] The successful implementation of the coupling reaction depends on the precise control of reaction conditions, including the selection of the reaction solvent, temperature, time, and optimization of the raw material ratio. This invention chooses MES buffer as the reaction solvent mainly because it has a suitable pH range and good buffering capacity, effectively maintaining the stability of the reaction system. Experimental results show that the coupling efficiency of HA and Tyr is highest when the reaction temperature is controlled at room temperature (25°C). Excessively high temperatures may lead to side reactions, thereby reducing the purity of the product. Furthermore, the reaction time also has a significant impact on the degree of coupling. The experiment used 2-4 hours, within which HA and Tyr can react fully, while avoiding excessive cross-linking or incomplete reaction.

[0047] To obtain high-purity HA-Tyr complex, this invention employs a combination of dialysis purification and lyophilization for post-processing. First, the reaction solution is transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in deionized water for 3-5 days to remove unreacted Tyr, EDC, NHS, and other small molecule impurities. Subsequently, the dialyzed solution is lyophilized to obtain a white solid HA-Tyr complex. Gel formation experiments confirm that grafting has occurred and the complex can form a gel.

[0048] It should be noted that dissolving HA-Tyr solid in PBS buffer is a key step in preparing the precursor solution. The concentration selection takes into account the mechanical properties, drug loading capacity, and injectability of the hydrogel. In this invention, 1% (w / v) is selected as the dissolution concentration of HA-Tyr. This concentration can ensure the fluidity of the solution while giving the hydrogel suitable mechanical strength. During the experiment, it was found that the dissolution rate is affected by factors such as solution temperature, stirring rate, and dissolution time. In order to improve the dissolution efficiency, a magnetic stirrer was used to stir at 300 rpm for 20 min at room temperature to ensure that the HA-Tyr solid was completely dissolved.

[0049] The final concentration of 4-OI was determined to be 50-200 μM through preliminary experiments, with 100 μM being the optimal concentration. This concentration effectively exerts its biological activity while avoiding cytotoxicity or rapid drug release caused by excessively high concentrations. To achieve uniform distribution of 4-OI in the HA-Tyr solution, dimethyl sulfoxide (DMSO) was used as a co-solvent. 4-OI was pre-dissolved in DMSO and then added dropwise to the HA-Tyr solution. During the mixing process, a vortex shaker was used to shake at 1000 rpm for 5 minutes to ensure that the two components were fully and uniformly mixed.

[0050] It should be noted that horseradish peroxidase (HRP) and hydrogen peroxide (H2O2), as classic initiators for enzymatic cross-linking reactions, are widely used in the preparation of hydrogels due to their high efficiency and biocompatibility. HRP can catalyze the decomposition of H2O2 to generate highly reactive oxygen free radicals. These free radicals further attack the phenolic hydroxyl groups in Tyr molecules, initiating oxidative cross-linking reactions, thereby forming a three-dimensional network structure. The final concentration of horseradish peroxidase was chosen to be 10 U / mL, and the final concentration of hydrogen peroxide was chosen to be 0.003% w / v. This ratio can ensure rapid gelation while avoiding side reactions or increased hydrogel brittleness caused by excessively high initiator concentrations.

[0051] Specifically, using hyaluronic acid and tyramine as raw materials, a hyaluronic acid-tyramine covalent complex is synthesized through the coupling reaction of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide. Horseradish peroxidase and hydrogen peroxide are used as the cross-linking catalytic system to construct an enzymatically cross-linked three-dimensional network hydrogel. Simultaneously, the anti-inflammatory active substance 4-octylitaconic acid is encapsulated within the gel network, resulting in a rapidly moldable, biocompatible injectable drug-loaded hydrogel. The prepared HA-Tyr hydrogel can complete in-situ gelation within 1-2 seconds, exhibiting fast molding speed and good injection fluidity, making it suitable for minimally invasive injection drug delivery. The stable three-dimensional network structure of the gel enables long-term sustained release of 4-octylitaconic acid, effectively improving drug bioavailability and avoiding the defects of rapid metabolism and strong irritation of free drugs. The process is simple, the reaction conditions are mild, there are no toxic residual solvents, and the raw materials have good biocompatibility, demonstrating excellent prospects for industrial application.

[0052] Example 2

[0053] The process of treating intervertebral disc degeneration in rats with HA-Tyr hydrogel loaded with 4-OI is as follows:

[0054] 1) Model establishment and grouping: The degeneration model of the intervertebral disc in the coccyx (Co7 / 8, Co8 / 9) of SD rats was induced by acupuncture. The rats were randomly divided into four groups: sham operation + blank HA-Tyr hydrogel group, sham operation + 4-OI / HA-Tyr hydrogel group, model + blank HA-Tyr hydrogel group, and model + 4-OI / HA-Tyr hydrogel group.

[0055] 2) Treatment intervention: During the modeling surgery, the corresponding hydrogel (approximately 20 μL) prepared in Example 1 was injected into the target intervertebral disc nucleus pulposus using a syringe;

[0056] 3) Efficacy evaluation: X-ray examinations were performed at 4 and 8 weeks postoperatively to calculate and analyze the intervertebral disc height index (DHI). Intervertebral disc tissue was taken at 8 weeks postoperatively for histological (HE, Safranin O and Fast Green staining) and immunohistochemical (Nrf2, GPX4, 4-HNE, MMP13) analysis.

[0057] 4) Results: X-ray showed that disc height loss was significantly suppressed in the model + 4-OI / HA-Tyr group, and the disc height density (DHI) was significantly higher in the model + blank hydrogel group. Figure 4-5 As shown; histological examination revealed that the nucleus pulposus structure of the model +4-OI / HA-Tyr group was more intact, and the content of proteoglycans was higher, such as Figure 6 As shown; immunohistochemistry revealed that the expression of Nrf2 and GPX4 proteins was upregulated in the nucleus pulposus of this group, while the expression of oxidative damage marker 4-HNE and matrix-degrading enzyme MMP13 was downregulated, such as Figure 7 As shown in the figure; the results indicate that 4-OI / HA-Tyr hydrogel can effectively delay intervertebral disc degeneration in rats by activating the Nrf2 / GPX4 pathway.

[0058] Specifically, an enzymatically cross-linked HA-Tyr injectable hydrogel loaded with the Nrf2-specific agonist 4-OI was constructed, exhibiting synergistic therapeutic effects against a novel mechanism of intervertebral disc degeneration (ferroptosis). Through the combination of "injectable in-situ molding and hydrogel sustained release" technologies, the challenge of local drug delivery to the intervertebral disc was solved, achieving precise and long-term drug delivery. The application of combining materials science carrier technology with 4-OI, which has clear cellular and molecular pharmacological effects, in the preparation of anti-intervertebral disc degeneration drugs is novel.

[0059] In summary, this invention precisely implants hydrogel into intervertebral disc lesions via minimally invasive injection, achieving local high-concentration drug accumulation. The three-dimensional network of the hydrogel serves as a drug reservoir, enabling continuous and controllable release of 4-OI, prolonging the drug's duration of action and achieving long-term therapeutic effects. Simultaneously, the hydrogel itself provides temporary mechanical support and simulates the highly hydrated microenvironment of the nucleus pulposus. The loaded 4-OI specifically activates the Nrf2 / GPX4 antioxidant pathway in nucleus pulposus cells, mechanistically antagonizing ferroptosis and matrix degradation, thus achieving a dual function of mechanical support and biological therapy. This hydrogel exhibits ultra-fast (second-level) gelation characteristics, making it suitable for clinical operation. It has a balanced water content >96%, good biocompatibility, and suitable mechanical strength and enzyme-responsive degradation characteristics. Furthermore, in vitro and in vivo experiments have confirmed that this hydrogel can effectively delay intervertebral disc degeneration in model animals, maintain disc height and proteoglycan content, and its effects are directly related to upregulating Nrf2 / GPX4 expression and inhibiting oxidative damage.

[0060] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0061] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An injectable hydrogel loaded with 4-octyl itaconic acid, characterized in that, Using a hyaluronic acid-tyramine complex as a gel matrix, a three-dimensional network structure is formed by enzymatic cross-linking, and the three-dimensional network structure encapsulates 4-octylitaconic acid.

2. A process for the preparation of injectable hydrogel loaded with 4- octyl itaconic acid as claimed in claim 1, wherein, Includes the following steps: S1. Synthesis of hyaluronic acid-tyramine complex: Hyaluronic acid and tyramine were coupled in MES buffer under the catalysis of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide. After the reaction, the mixture was purified by dialysis and lyophilized to obtain HA-Tyr solid. S2. Preparation of precursor solution: The obtained HA-Tyr solid was dissolved in PBS buffer to prepare a solution of a certain concentration, and DMSO solution containing 4-octylitaconic acid was added to it. After stirring, the solution was mixed evenly to obtain a drug-containing prodrug solution. S3, In-situ gelation: Horseradish peroxidase and hydrogen peroxide solution were added sequentially to the obtained drug precursor solution to initiate an enzymatic cross-linking reaction of tyramine groups, rapidly forming HA-Tyr hydrogel loaded with 4-octylitaconic acid within 1-2 seconds.

3. The injectable hydrogel loaded with 4-octyl itaconic acid according to claim 2, characterized in that, In S1, the mass ratio of hyaluronic acid to tyramine in the hyaluronic acid-tyramine complex is (5-10):1; the molecular weight of the hyaluronic acid is 100k-200kDa.

4. The injectable hydrogel loaded with 4-octyl itaconic acid according to claim 3, characterized in that, In S1, the pH value of the MES buffer is 5.0-6.0, and the concentration is 0.1-0.2 mol / L; the molar ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 1:1, and the EDC:NHS:Tyr ratio is 2.5~3:2.5~3:

1.

5. The injectable hydrogel loaded with 4-octyl itaconic acid according to claim 4, characterized in that, In S1, the coupling reaction temperature is 20-25℃ and the reaction time is 2-4h; dialysis purification uses a dialysis bag with a molecular weight cutoff of 3500Da, and dialysis with deionized water for 3-5 days. On the first day, the bag is changed 3 times in 8 hours or 4 times in 6 hours, and then changed once every 12 hours for a total of 3-5 days. The coupling reaction temperature is monitored in real time, the real-time temperature deviation is calculated, and it is determined whether the coupling reaction temperature is abnormal. The real-time temperature deviation is obtained using the following formula: ； In the formulae: represents the real-time monitoring temperature at time t; represents the set temperature of the coupling reaction, which is the process allowable standard interval [20, 25°C]; Based on the determination of the coupling reaction temperature, a three-level temperature threshold determination and alarm system is implemented to achieve precise control of the coupling reaction temperature. The three-level temperature threshold determination and alarm modes are as follows: When the process is effective, When the process is effective, fine-tuning the coupling reaction temperature restores, When the process is ineffective.

6. The injectable hydrogel loaded with 4-octyl itaconic acid according to claim 5, characterized by, In step S2, the obtained HA-Tyr solid is dissolved in PBS buffer to prepare a solution with a mass-volume concentration of 1% w / v.

7. The injectable hydrogel loaded with 4-octyl itaconic acid according to claim 6, characterized in that, In S2, the final concentration of 4-octylitaconic acid is 50-200 μM.

8. The injectable hydrogel loaded with 4-octyl itaconic acid according to claim 7, characterized in that, In step S2, the volume ratio of DMSO solution to PBS buffer is ≤1:20, the stirring speed is 200-300 r / min, and the stirring time is 10-20 min.

9. An injectable hydrogel loaded with 4-octylitaconic acid according to claim 8, characterized in that, In S3, the final concentration of horseradish peroxidase is 10 U / mL, and the final concentration of hydrogen peroxide is 0.003% w / v.

10. An application of the injectable hydrogel loaded with 4-octyl itaconic acid as described in claim 1, characterized in that, The injectable hydrogel loaded with 4-octyl itaconic acid is used in drugs for the prevention, delay, or treatment of intervertebral disc degeneration.

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