Intervertebral disc regeneration and repair hydrogel system with inflammation chemotactic factor removal effect as well as preparation method and application of intervertebral disc regeneration and repair hydrogel system

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an intervertebral disc regeneration and repair hydrogel system with an inflammation chemotactic factor removal effect and a preparation method and application thereof. The intervertebral disc regeneration and repair hydrogel system disclosed by the invention is prepared from glycosaminoglycan, EDC (Ethylene Dichloride), sulfo-NHS (N-Hydroxysuccinimide) and StarPEG (StarPEG). The hydrogel-lipidosome drug delivery system can capture inflammatory chemokines (such as MCP1 and IL8) and regulate the inflammatory environment, the hydrogel-lipidosome drug delivery system is prepared into a drug preparation to be injected into an excision cavity of an aging IVDD mouse model, it is found that a precursor solution is gelatinized to form hydrogel, alpha-Klotho circular RNA (circRNA) with a regeneration and repair function can be continuously released, and the effect of the alpha-Klotho circular RNA is achieved. Furthermore, in-vivo intervertebral disc cell ECM anabolism and intervertebral disc regeneration are promoted, so that IVDD is relieved, and the good practical application value is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an intervertebral disc regeneration and repair hydrogel system with inflammatory chemokine clearing effect, and a preparation method and application thereof. Background Art

[0002] Intervertebral Disc Degeneration (IVDD) is a chronic degenerative disease that leads to progressive destruction of the intervertebral disc and significantly affects the quality of life. Currently, there is no approved effective therapy to prevent the progression of IVDD, and clinical treatment mainly adopts symptomatic relief and ultimate spinal fusion.

[0003] The inflammatory microenvironment of the intervertebral disc plays a crucial role in the occurrence and progression of disc degeneration. Disc degeneration is often accompanied by the overexpression of inflammatory factors, such as monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α), which further aggravate the local inflammatory response by attracting the infiltration and activation of immune cells. The inflammatory microenvironment not only destroys the normal metabolic balance of disc cells, but also accelerates the decomposition of the extracellular matrix of the disc cells by promoting the expression of matrix metalloproteinases (MMPs) and the ADAMTS protein family. In addition, inflammation can also induce apoptosis and senescence of disc cells and reduce their ability to secrete key structural proteins (such as collagen II and proteoglycans), thereby weakening the structural integrity and function of the disc. More seriously, the chronic inflammatory state will form a continuous harmful cycle, leading to a significant decrease in the repair ability of the disc and further aggravating the process of disc degeneration. Therefore, regulating or improving the inflammatory microenvironment of the disc is considered to be an important strategy to delay disc degeneration and promote disc regeneration.

[0004] The intervertebral disc is characterized by its avascular nature, which makes systemic drug delivery to treat degeneration limited. In addition, the intervertebral disc is surrounded by ligaments and muscles, forming a shock-absorbing system that can withstand multi-directional forces. Clinically, surgical discectomy is the first choice for IVDD patients, but the residual cavity further increases the risk of disc degeneration. In addition, the overexpression of inflammatory chemokines (such as MCP1 and IL8) exacerbates chronic inflammation and significantly hinders disc regeneration. In recent years, injectable hydrogels have received widespread attention in the treatment of IVDD due to their minimally invasive properties, ability to adapt to the injured area, and the potential for controlled therapeutic loading and release.

[0005] Therefore, there is an urgent need for a hydrogel system that can regulate the inflammatory microenvironment of the degenerative intervertebral disc, promote the repair and regeneration of the intervertebral disc, and rebuild the normal structure and functional properties of the intervertebral disc. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a disc regeneration and repair hydrogel system with inflammatory chemokine clearing effect, and its preparation method and application. The disc regeneration and repair hydrogel system has the effect of clearing inflammatory chemokines, can capture inflammatory chemokines (such as MCP1 and IL8), and regulate the inflammatory microenvironment of the disc. It was prepared as a pharmaceutical preparation and injected into the resection cavity of the aged IVDD mouse model. It was found that the precursor solution was gelled to form a hydrogel, which can continuously release circular RNA (circRNA) with regeneration and repair function, thereby promoting the ECM anabolism of intervertebral disc cells and intervertebral disc regeneration in the body, thereby alleviating IVDD, and has good practical application value. Based on the above research, the present invention is completed.

[0007] Specifically, the present invention is implemented through the following technical solutions:

[0008] The first aspect of the present invention provides a hydrogel system for intervertebral disc regeneration and repair with the efficacy of clearing inflammatory chemokines. The hydrogel system for intervertebral disc regeneration and repair consists of glycosaminoglycan, EDC, sulfo-NHS and StarPEG.

[0009] The second aspect of the present invention provides a method for preparing the above-mentioned intervertebral disc regeneration and repair hydrogel system, the preparation method comprising:

[0010] S1. Dissolve glycosaminoglycan, EDC, sulfo-NHS and StarPEG separately in pre-cooled deionized water.

[0011] S2. Mix EDC and sulfo-NHS to form an EDC-sulfo-NHS mixture:

[0012] S3, mixing the glycosaminoglycan solution with the EDC-sulfo-NHS mixture to form a glycosaminoglycan-EDC-sulfo-NHS mixture;

[0013] S4. Add the StarPEG solution into the glycosaminoglycan-EDC-sulfo-NHS mixture to obtain.

[0014] The third aspect of the present invention provides the use of the above-mentioned intervertebral disc regeneration and repair hydrogel system in the preparation of intervertebral disc degeneration drugs.

[0015] A fourth aspect of the present invention provides a pharmaceutical preparation, wherein the pharmaceutical preparation comprises the above-mentioned intervertebral disc regeneration and repair hydrogel system and a pharmaceutical active ingredient.

[0016] Beneficial technical effects of one or more of the above technical solutions:

[0017] The hydrogel-liposome drug delivery system of the present invention can capture inflammatory chemokines (such as MCP1 and IL8) and regulate the inflammatory environment. It was prepared as a drug preparation and injected into the resection cavity of an aged IVDD mouse model. It was found that the precursor solution gelled to form a hydrogel, which can continuously release circular RNA (circRNA) with regeneration and repair functions, thereby promoting the in vivo reduction of the inflammatory microenvironment of the degenerative intervertebral disc, promoting the anabolism of the intervertebral disc cell ECM and the regeneration of the intervertebral disc tissue, thereby alleviating IVDD, and has good practical application value for the treatment of spinal degeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0019] Figure 1 : is a GAG hydrogel system in an embodiment of the present invention, wherein (a) is a TEM image of a GAG hydrogel; (b) is a scanning electron microscope (SEM) image of a GAG hydrogel; (c) is a change in the modulus of a GAG hydrogel;

[0020] Figure 2 Schematic diagram of live / dead cell staining randomly captured by the high-content screening system in an embodiment of the present invention, wherein (a) is a live / death staining image showing the result of 2-day co-culture of NPPCs with GAG gel; (b) is the MTT assay result showing the cell proliferation after 0, 2, 4, 6 and 8 days of co-culture of NPPCs with different hydrogels (GAG and PEG); (c) is the ELISA result showing the level of inflammatory factors in the culture medium after inflammatory macrophages were co-incubated with different hydrogels (GAG and PEG) for 1 day; (d) is the RT-PCR result showing the expression of ECM synthesis / decomposition related genes (ACAN, COL2, SOX9 and MMP2) in NPPCs after co-incubation with conditioned medium, and the conditioned medium was collected after 1 day of co-incubation with different hydrogels (GAG and PEG gel); (e) is the detection of inflammatory chemokine adsorption capacity;

[0021] Figure 3Schematic diagram of the experiment of the hydrogel system in slowing down the IVD degeneration process in an embodiment of the present invention, wherein (a) is a schematic diagram of the animal experiment, showing the in vivo delivery process of the NT-KLNP@GAG Gel system; (b) is a live imaging image of the mouse intervertebral disc; (c) is a live imaging image of the control group in vivo retention; (d) is a live imaging image of NT-KLNP retention in vivo; (e) is a live imaging image of NT-KLNP@GAG Gel retention in vivo; (f) is a HE, Safranin O and PicrosiriusRed staining image, showing the repair and regeneration effect of the NT-KLNP@GAG hydrogel delivery system injected into the degenerated intervertebral disc at 4 weeks and 8 weeks. DETAILED DESCRIPTION

[0022] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0023] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention.

[0024] As mentioned above, in the prior art, surgical discectomy is the preferred intervention for IVDD patients, but the residual cavity further increases the risk of disc degeneration. In order to further fill the cavity after clinical surgical discectomy, the present invention provides a chemokine-clearing hydrogel-liposome drug delivery system.

[0025] In view of this, in a specific embodiment of the present invention, a hydrogel system for intervertebral disc regeneration and repair with the effect of clearing inflammatory chemokines is provided, wherein the hydrogel system for intervertebral disc regeneration and repair is composed of glycosaminoglycan, EDC, sulfo-NHS and StarPEG.

[0026] In another specific embodiment of the present invention, in the above-mentioned intervertebral disc regeneration and repair hydrogel system, the glycosaminoglycan is a heparin molecule with a molecular weight of MW 14,000, and the molar ratio of the glycosaminoglycan to StarPEG is 0.1-0.6. Preferably, the molar ratio of the glycosaminoglycan to StarPEG is 0.17.

[0027] In another specific embodiment of the present invention, in the above-mentioned intervertebral disc regeneration and repair hydrogel system, the molar ratio of EDC to sulfo-NHS is (1-3):1, preferably, the molar ratio of EDC to sulfo-NHS is 2:1.

[0028] In another specific embodiment of the present invention, the molar ratio of glycosaminoglycan to EDC is 1:5.

[0029] In another specific embodiment of the present invention, a method for preparing the above-mentioned intervertebral disc regeneration and repair hydrogel system is provided, and the preparation method comprises:

[0030] S1. Dissolve glycosaminoglycan, EDC, sulfo-NHS and StarPEG separately in pre-cooled deionized water.

[0031] S2. Mix EDC and sulfo-NHS to form an EDC-sulfo-NHS mixture:

[0032] S3, mixing the glycosaminoglycan solution with the EDC-sulfo-NHS mixture to form a glycosaminoglycan-EDC-sulfo-NHS mixture;

[0033] S4. Add StarPEG to the glycosaminoglycan-EDC-sulfo-NHS mixture to obtain GAGGel.

[0034] Wherein, in the GAG ​​Gel, the concentration of glycosaminoglycan is 11.8±1.0μg / μL to 12.27±0.4μg / μL, and the concentration of StarPEG is 12.5±0.9% to 38.3±1.9%. Preferably, the concentration of glycosaminoglycan is 12.27±0.4μg / μL, and the concentration of StarPEG is 38.3±1.9%.

[0035] In the GAG ​​Gel, the concentration of EDC is 27.5-37.5 μM, and the concentration of sulfo-NHS is 13.75-18.75 μM. Preferably, the concentration of EDC is 30 μM, and the concentration of sulfo-NHS is 15 μM.

[0036] In another specific embodiment of the present invention, there is provided use of the above-mentioned intervertebral disc regeneration and repair hydrogel system in the preparation of intervertebral disc degeneration drugs.

[0037] In another specific embodiment of the present invention, a pharmaceutical preparation is provided, wherein the drug comprises the above-mentioned intervertebral disc regeneration and repair hydrogel system and a pharmaceutically active ingredient. Preferably, the pharmaceutically active ingredient is selected from any one of mRNA, circleRNA, and polypeptide.

[0038] In another specific embodiment of the present invention, the above-mentioned intervertebral disc regeneration and repair hydrogel system or drug preparation can be in the form of an injection; specifically, the above-mentioned intervertebral disc regeneration and repair hydrogel system or drug preparation can be injected into the puncture site of in vitro diagnosis to achieve a therapeutic effect. Experiments have shown that it has the ability to treat intervertebral disc degeneration and regulate the immune microenvironment. The drug dosage form is an injection.

[0039] In another specific embodiment of the present invention, a pharmaceutical preparation NT-KLNP@GAG Gel is provided, and the preparation method thereof comprises: mixing GAG Gel with NT-KLNP to obtain the pharmaceutical preparation.

[0040] In another specific embodiment of the present invention, the preparation method of the NT-KLNP comprises:

[0041] 1. Preparation of DSPE-PEG-Vas: Mix an anhydrous ethanol solution of DSPE-PEG-MAL and an anhydrous ethanol solution of vasculotide, and stir to prepare DSPE-PEG-Vas;

[0042] 2. Weigh DSPE-PEG-Vas, T12, PEG-DMG, DOPE and cholesterol and dissolve them in anhydrous ethanol to prepare a lipid stock solution with a concentration of 5 mg / mL. At the same time, accurately weigh T12 and dissolve it in anhydrous ethanol to prepare a stock solution with a concentration of 10 mg / mL; according to the molar mass of each lipid component converted into the added volume, the above lipid stock solutions of the prescribed volume are respectively drawn and fully mixed to obtain a lipid ethanol phase; use an enzyme-free sodium dihydrogen phosphate-citrate buffer with a pH of 5 to dilute α-Klotho-circRNA to a certain concentration to obtain an α-Klotho-circRNA aqueous phase;

[0043] 3. The α-Klotho-circRNA aqueous phase and the lipid ethanol phase were mixed in a mass ratio of 1:10 and reacted in a microfluidic system. After the reaction was completed, the mixture of the α-Klotho-circRNA aqueous phase and the lipid ethanol phase was dialyzed to remove ethanol. After ultrafiltration and concentration, circRNA-loaded liposomes were obtained, named Vas-Klotho@LNP, abbreviated as NT-KLNP.

[0044] Among them, the nucleotide sequence of linearized α-Klotho-circRNA is shown in SEQ ID NO.1, its NCBI reference sequence number is NM_013823.2, and the structural formula of T12 is shown in Formula I.

[0045] Formula I:

[0046] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0047] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0048] Description of experimental materials and reagents used in the examples

[0049] Example 1

[0050] Preparation of GAG hydrogel and its performance verification

[0051] StarPEG-GAG hydrogels were prepared as 300 μm thick planar layers and attached to glass substrates. Specifically, heparin (MW 14,000), EDC (Sigma-Aldrich), N-hydroxysulfosuccinimide (sulfo-NHS), and amino-terminally functionalized four-arm StarPEG (MW 10,000) were dissolved in deionized water on ice. A 5-fold molar excess of EDC was used according to the amount of heparin carboxylic acid groups. The heparin and EDC / sulfo-NHS (molar ratio of EDC to sulfo-NHS was 2:1) solution was placed on ice (about 2-4°C) for 15 minutes to activate the heparin carboxylic acid groups. Star-PEG was dissolved in MilliQ-water on ice and then added to the activated heparin mixture to obtain GAG hydrogels, wherein the molar ratio of heparin to StarPEG was set to 0.50, 0.25, and 0.17, respectively, to prepare three GAG ​​hydrogels with different moduli. In the three different GAG Gels, the concentrations of heparin were 11.8±1.0μg / μL, 12.67±3.3μg / μL, and 12.27±0.4μg / μL, respectively; the concentrations of StarPEG were 12.5±0.9%, 21.4±0.7%, and 38.3±1.9%, respectively; the concentrations of EDC were 27.5μM, 30μM, and 37.5μM, respectively; and the concentrations of sulfo-NHS were 13.75μM, 15μM, and 18.75μM, respectively.

[0052] Comparison of the storage modulus and water absorption related properties of the three different gels shows that the expansion and viscoelasticity experiments of the gels confirm that the storage modulus increases with the increase in the degree of crosslinking. Increasing the number of crosslinks will form a denser network, which is harder (higher storage modulus), and the water absorption rate will be limited (lower swelling) due to the large number of covalent bridges in the gel, resulting in a larger retraction force. Therefore, we selected a hydrogel with a molar ratio of heparin to StarPEG of 0.17 for subsequent experiments (the highest degree of crosslinking, more suitable for local injection into the intervertebral disc).

[0053] The microstructure of the hydrogel was observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Figure 1 a and 1b). In addition, the storage modulus (G′) value was always higher than the loss modulus (G″) value during the entire test, and the G′ value was approximately ten times the G″ value, indicating that the hydrogel was successfully formed ( Figure 1 c).

[0054] By using live / dead cell staining fields captured randomly by a high-content screening system, the experimental results showed that the hydrogel had minimal adverse effects on cell proliferation, such as Figure 2In addition, the cell viability was evaluated at different time points (0, 2, 4, 6, and 8 days) by the methylthiazolyl tetrazolium (MTT) method, and the results showed that the cell viability of NPPCs did not change significantly over time, as shown in Figure 2. Figure 2 As shown in b. To evaluate the ability of GAG hydrogel to bind chemokines in an inflammatory environment associated with IVDD, we conducted a co-culture experiment. First, the culture medium extracted from macrophages cultured in an inflammatory state for 3 days was collected as a conditioned medium, and the GAG ​​hydrogel system was co-incubated with the conditioned medium cultured with macrophages activated by inflammation. After the hydrogel was co-incubated with the macrophage conditioned medium for 24 hours, the concentration of unbound cytokines or chemokines was measured using an enzyme-linked immunosorbent assay (ELISA). The results showed that GAG Gel exhibited a relatively high ability to bind inflammatory chemokines ( Figure 2 c) In addition, NPPCs were co-cultured with the conditioned medium after hydrogel adsorption for 24 h, and the expression of ECM synthesis / decomposition-related genes was evaluated by RT-PCR after 3 days. Figure 2 The results of d showed that after GAG Gel adsorbed inflammatory factors, it could effectively promote the expression of ECM synthesis-related genes (ACAN, COL2, and SOX9), while down-regulating the expression of MMP2. These findings indicate that GAG adsorption of chemokines can create a favorable microenvironment for the differentiation of NPPCs and intervertebral disc repair.

[0055] In this patent, we systematically investigated the ability of hydrogels containing different sulfation patterns to bind the chemokines MCP1 and IL8. These chemokines are involved in attracting immune cells to inflammatory responses. The results showed that the chemokines were effectively adsorbed by all GAG-based hydrogels within 24 hours, regardless of their sulfation pattern. Removal of the 6-O-sulfate group significantly affected the interaction with MCP1, while the lack of the N-sulfate group had no significant effect on the overall binding. In contrast, the adsorption capacity of the hydrogels for IL8 was not affected by the specific sulfate position ( Figure 2 e).

[0056] Example 2

[0057] Exploring the effect of GAG Gel delivery system on IVD regeneration in vivo

[0058] Experimental methods

[0059] 1. Preparation of NT-KLNP Liposomes Containing circRNA

[0060] 1.1 Preparation of DSPE-PEG-Vas: An anhydrous ethanol solution of DSPE-PEG-MAL and an anhydrous ethanol solution of vasculotide were mixed at a molar ratio of 1:1 and stirred to prepare DSPE-PEG-Vas;

[0061] 1.2 DSPE-PEG-Vas, T12, PEG-DMG, DOPE and cholesterol (molar ratio of 2:40:3:10:45) were weighed and dissolved in anhydrous ethanol to prepare a lipid stock solution with a concentration of 5 mg / mL. At the same time, T12 was accurately weighed and dissolved in anhydrous ethanol to prepare a stock solution with a concentration of 10 mg / mL; according to the molar mass of each lipid component converted into the added volume, the above lipid stock solutions of the prescribed volume were respectively taken and fully mixed to obtain a lipid ethanol phase; α-Klotho-circRNA was diluted to 1 mg / mL using an enzyme-free sodium hydrogen phosphate-citrate buffer at pH 5 to obtain an α-Klotho-circRNA aqueous phase; wherein, the in vitro synthesis of α-Klotho-circRNA was completed by Gisai Biotechnology Co., Ltd., the nucleotide sequence of linearized α-Klotho-circRNA is shown in SEQ ID NO.1, and the structural formula of T12 is shown in Formula I.

[0062] Formula I:

[0063] 1.3 The α-Klotho-circRNA aqueous phase and the lipid ethanol phase were simultaneously injected into the microfluidic system (INano) at a mass ratio of 1:10 and a flow rate of 1:3. TM L, Micro&Nano (Shanghai) Biologics Co. Ltd.), using a microfluidic chip to quickly mix the two. Then, the mixture was dialyzed in RNase-free PBS (pH 7.4) to remove ethanol and concentrated by ultrafiltration to obtain LNP loaded with circRNA, named Vas-Klotho@LNP, abbreviated as NT-KLNP.

[0064] 2. Preparation of NT-KLNP@GAG Gel

[0065] Gel was prepared according to the preparation method of GAG Gel in Example 1, wherein heparin (molecular weight 14,000), EDC (Sigma-Aldrich), N-hydroxysulfosuccinimide (sulfo-NHS) and amino-terminally functionalized four-arm StarPEG (molecular weight 10,000) were dissolved in deionized water on ice, and EDC was used in a 5-fold molar excess according to the amount of heparin carboxylic acid groups. The heparin and EDC / sulfo-NHS (the molar ratio of EDC to sulfo-NHS was 2:1) solution was placed on ice (about 2-4°C) for 15 minutes to activate the heparin carboxylic acid groups to obtain a heparin-EDC-sulfo-NHS mixture, star-PEG was dissolved in MilliQ-water on ice, and then the NT-KLNP and star-PEG prepared above were added to the heparin-EDC-sulfo-NHS mixture to obtain NT-KLNP@GAG Gel. Among them, in NT-KLNP@GAG Gel, the molar ratio of heparin to StarPEG was 0.17, the concentration of NT-KLNP was 0.1ug / uL, the concentration of glycosaminoglycan was 12.27±0.4μg / μL, the concentration of StarPEG was 38.3±1.9%, the concentration of EDC was 30μM, and the concentration of sulfo-NHS was 15μM.

[0066] Exploring the effect of GAG Gel delivery system on IVD regeneration in vivo

[0067] The IVD model of aged mice was established by acupuncture, followed by minimally invasive injection. This study included: non-acupuncture control group, acupuncture injury group, and NT-KLNP@GAG Gel injection group. Tests were performed at different time points after treatment ( Figure 3 a). First, we evaluated the degradation of NT-KLNP and NT-KLNP@GAG Gel within the IVD (0, 3, 6, 9, 12, and 15 days). Briefly, mice were anesthetized by inhalation of anesthetics. Subsequently, a 29-gauge needle was carefully inserted into the center of the intervertebral disc (C7-8) under X-ray guidance after preoperative disinfection. Then, 5 μL of the mixture (NT-KLNP solution or NT-KLNP@GAG gel) was injected into the puncture site of the intervertebral disc using a syringe. Both NT-KLNP solution and NT-KLNP@GAG gel were labeled with rhodamine red, and their degradation was observed by IVIS spectral imaging system (PerkinElmer, USA) at 0, 3, 6, 9, 12, and 15 days, respectively.

[0068] The results are as follows Figure 3As shown in Figures b to e, the NT-KLNP solution degraded rapidly, with only 28.8% remaining on the 6th day; in contrast, due to the presence of the hydrogel, the degradation rate of NT-KLNP@GAG Gel was significantly slowed down, with approximately 54.9% remaining intact on the 6th day and 23.0% still retained on the 15th day.

[0069] To further evaluate the regeneration and repair of IVD, the disc tissue was stained with H&E, Safranin O, and Picrosirius Red. The results showed that at weeks 4 and 8, only the acupuncture group showed significant cell infiltration and severely damaged disc structure. At week 4, in addition to the control group, the NT-KLNP@GAG Gel group showed more complete nucleus pulposus tissue and a more orderly annulus fibrosus structure. By week 8, the histological score of the NT-KLNP@GAG Gel group was close to that of the control group ( Figure 3 f).

[0070] The above results indicate that the hydrogel-carrying delivery system exhibits excellent efficacy in slowing down IVD degeneration and has great potential to promote IVD regeneration.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A hydrogel system for intervertebral disc regeneration and repair with inflammatory chemokine removal effect, characterized in that: The intervertebral disc regeneration and repair hydrogel system consists of glycosaminoglycan, EDC, sulfo-NHS and StarPEG.

2. The intervertebral disc regeneration and repair hydrogel system according to claim 1, characterized in that: The glycosaminoglycan is a heparin molecule with a molecular weight of MW 14,000, and the molar ratio of the glycosaminoglycan to StarPEG is 0.1-0.

6.

3. The intervertebral disc regeneration and repair hydrogel system according to claim 1, characterized in that: The molar ratio of EDC to sulfo-NHS is (1-3):

1. Preferably, the molar ratio of EDC to sulfo-NHS is 2:

1.

4. The intervertebral disc regeneration and repair hydrogel system according to claim 1, characterized in that: The molar ratio of glycosaminoglycan to EDC was 1:

5.

5. The method for preparing the intervertebral disc regeneration and repair hydrogel system according to claim 1, characterized in that: The preparation method comprises: S1. Dissolve glycosaminoglycan, EDC, sulfo-NHS and StarPEG separately in pre-cooled deionized water. S2. Mix EDC and sulfo-NHS to form an EDC-sulfo-NHS mixture: S3, mixing the glycosaminoglycan solution with the EDC-sulfo-NHS mixture to form a glycosaminoglycan-EDC-sulfo-NHS mixture; S4. Add StarPEG to the glycosaminoglycan-EDC-sulfo-NHS mixture to obtain GAG Gel.

6. The preparation method according to claim 5, characterized in that: In GAG Gel, the concentration of EDC is 27.5-37.5 μM, and the concentration of sulfo-NHS is 13.75-18.75 μM. Preferably, the concentration of EDC is 30 μM, and the concentration of sulfo-NHS is 15 μM.

7. The preparation method according to claim 3, characterized in that: In the GAG ​​Gel, the concentration of glycosaminoglycan is 11.8±1.0μg / μL to 12.27±0.4μg / μL, and the concentration of StarPEG is 12.5±0.9% to 38.3±1.9%. Preferably, the concentration of glycosaminoglycan is 12.27±0.4μg / μL, and the concentration of StarPEG is 38.3±1.9%.

8. Use of the intervertebral disc regeneration and repair hydrogel system according to any one of claims 1 to 3 in the preparation of intervertebral disc degeneration drugs.

9. A pharmaceutical preparation, characterized in that The drug comprises the intervertebral disc regeneration and repair hydrogel system according to any one of claims 1 to 3 and a pharmaceutically active ingredient. Preferably, the pharmaceutically active ingredient is selected from any one of mRNA, circleRNA, and polypeptide.

10. The pharmaceutical preparation according to claim 9, characterized in that The drug dosage form is injection.

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