Chitosan-based gradient hydrogel, and preparation method and application thereof

Abstract

The present application relates to the technical field of hydrogel and its preparation, and discloses a chitosan-based gradient hydrogel, a preparation method and application thereof, wherein thiolated chitosan and curcumin-loaded multivesicular liposomes are dissolved in an alpha-ketoglutaric acid aqueous solution, N-hydroxysuccinimidyl acrylate and hydroxyapatite are dissolved in another deionized water, and the two solutions are mixed, and the pH value of the system is adjusted to neutral to obtain a bone layer gel. According to the above gel preparation method, the hydroxyapatite is replaced by cartilage root to obtain a cartilage layer gel. The bone layer gel is placed at the bottom layer, and the cartilage layer gel is placed at the top layer, and a chitosan-based gradient hydrogel is prepared integrally. The chitosan-based bone-cartilage gradient hydrogel constructed by the present application can better simulate the bone-cartilage gradient structure and the bone-cartilage interface, the cartilage layer gel promotes chondrogenesis, the bone layer gel promotes osteogenesis, and the slow release of curcumin adjusts the inflammatory microenvironment to maintain the phenotype.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of hydrogels and their preparation, in particular to a chitosan-based gradient hydrogel and a preparation method and application thereof. BACKGROUND

[0002] Chitosan is a natural polysaccharide, which has the advantages of good biocompatibility, degradability, antibacterial property, cationic property (electrostatic interaction with anionic glycosaminoglycans (GAG) in the extracellular matrix (ECM)), low cost, etc. However, chitosan also has the disadvantages of poor water solubility under physiological conditions, poor gel mechanical properties, low cell matrix interaction, etc. Thiolated chitosan (CSSH) can improve the water solubility and gelation of chitosan, and increase the gel strength, but the gel lacks adhesion and the ability to promote bone-cartilage regeneration, making it difficult to form a firm bond with the subchondral bone, so the combination of hydrogel with the subchondral bone, directional osteogenesis and chondrogenic differentiation is a problem to be solved.

[0003] Hydroxyapatite (HAP) is the main component of natural bone tissue, which has good biocompatibility, safety, non-toxicity and bone conduction. Studies have shown that short, nanorod-shaped hydroxyapatite (HAP) has osteogenic ability without the assistance of any growth factors, so short, nanorod-shaped hydroxyapatite (HAP) is suitable for use as a bone induction layer for repairing the subchondral bone in the biphasic structure.

[0004] Kato Genin (KGN) is a synthetic small molecule commonly used in the study of osteoarthritis (OA), and studies have shown that Kato Genin (KGN) can promote the differentiation of bone marrow mesenchymal stem cells (BMSCs) into chondrocytes, but Kato Genin (KGN) is not soluble in water and is easily released quickly in vivo, resulting in low drug utilization. In addition, bone-cartilage defects in clinical practice are often accompanied by the underlying disease of osteoarthritis, and curcumin (Cur) is a polyphenol with anti-inflammatory effects. Studies have found that curcumin can inhibit inflammation, inhibit chondrocyte apoptosis and promote cartilage repair, which is beneficial to maintaining the phenotype of cartilage, but curcumin is not soluble in water, which restricts its application due to poor water solubility and low bioavailability.

[0005] Some existing scaffolds and gels for simulating bone-cartilage repair can be found in Chinese patent application publications CN119950811A, CN119564937A, CN117467163A, CN119818730A, etc.

[0006] In addition, since cartilage destruction is always accompanied by subchondral bone damage, which affects the metabolism of cartilage and further accelerates its deterioration. Therefore, when designing treatment strategies, the cartilage and subchondral bone are gradually regarded as a whole structure. A Chinese invention patent application with publication number CN118490884A discloses a double-layer hydrogel for articular cartilage repair, which comprises an upper layer hydrogel and a lower layer hydrogel, wherein the upper layer hydrogel contains oxidized sodium alginate, catechol chitosan and puerarin; the lower layer hydrogel contains oxidized sodium alginate, catechol chitosan, curcumin and zinc oxide nanoparticles. The invention uses catechol-modified chitosan as a crosslinking agent, and catechol chitosan has the characteristics of biodegradability, low cytotoxicity, etc.; at the same time, the zinc oxide nanoparticles loaded with curcumin can participate in the formation of hydrogel under the crosslinking of catechol chitosan, which meets the preparation conditions of double-drug hydrogel, and the product has good mechanical properties and biocompatibility, and can be better applied to in vivo articular cartilage repair. However, the above invention still has deficiencies in simulating the bone-cartilage gradient structure, the bone-cartilage interface, and promoting the differentiation of bone marrow mesenchymal stem cells into chondroblasts and osteoblasts. SUMMARY

[0007] The purpose of the present application is to provide a chitosan-based gradient hydrogel and a preparation method thereof, which can better simulate the bone-cartilage gradient structure and its bone-cartilage interface, maintain the cartilage phenotype, and promote the differentiation of bone marrow mesenchymal stem cells into chondroblasts and osteoblasts, to solve one or more technical problems existing in the prior art, at least provide a beneficial choice or create conditions.

[0008] To achieve the above purpose, the technical scheme adopted by the present application is as follows.

[0009] The preparation method of the chitosan-based gradient hydrogel comprises the following steps: 1) preparing curcumin (Cur) loaded multivesicular liposomes (CMLVs), hydrogenated soybean phosphatidylcholine (HSPC) and curcumin are added to warm water and ultrasonic treatment to prepare curcumin (Cur) loaded multivesicular liposomes (CMLVs); 2) preparing an upper gel, thiolated chitosan (CSSH) and curcumin (Cur) loaded multivesicular liposomes (CMLVs) are dissolved in an alpha-ketoglutaric acid (KA) aqueous solution, N-hydroxysuccinimidyl acrylate (ACC-NHS) and hydroxyapatite (HAP) are dissolved in another deionized water, and the two solutions are mixed to obtain a mixed solution one, and the pH value of the mixed solution one is adjusted to neutral to obtain the upper gel; 3) preparing a lower gel, thiolated chitosan (CSSH) and curcumin (Cur) loaded multivesicular liposomes (CMLVs) are dissolved in an alpha-ketoglutaric acid (KA) aqueous solution, N-hydroxysuccinimidyl acrylate (ACC-NHS) and KGN are dissolved in another deionized water, and the two solutions are mixed to obtain a mixed solution two, and the pH value of the mixed solution two is adjusted to neutral to obtain the lower gel.

[0010] In the present application, the prepared upper gel is a bone layer gel, and the prepared lower gel is a cartilage layer gel, the bone layer gel is a dense porous structure, the cartilage layer gel is a porous structure with relatively larger holes, and a biomimetic bone-cartilage interface is formed between the two.

[0011] During preparation, one of the cartilage layer gel and the bone layer gel is directly prepared on the surface of the other and integrated into a gel.

[0012] In the preparation process of the chitosan-based gradient hydrogel, the thiol groups of thiolated chitosan and the double bonds of N-hydroxysuccinimidyl acrylate (ACC-NHS) undergo Michael addition reaction, the thiol groups react with each other to form disulfide bonds, and then crosslinking to form a gel. Among them, alpha-ketoglutaric acid (KA) can give the gel physical adhesion, and the hydroxysuccinimidyl group of N-hydroxysuccinimidyl acrylate (ACC-NHS) can crosslink with the amino groups of human tissues, giving it chemical crosslinking adhesion, thereby further improving the adhesion of the gel and solving the problem of poor adhesion of thiolated chitosan (CSSH) gel.

[0013] Among them, KGN is first dissolved in DMSO to prepare a mother liquor with a concentration of 5mM, and then added to deionized water to prepare a corresponding concentration.

[0014] Preferably, in the mixed solution one and the mixed solution two, the concentration of the thiolated chitosan (CSSH) is 40mg / mL-80mg / mL, and further preferably 40mg / mL-60mg / mL.

[0015] Preferably, the concentration of the alpha-ketoglutaric acid in the mixed solution one and the mixed solution two is 1 mg / mL-10 mg / mL, further preferably 1 mg / mL.

[0016] Preferably, the concentration of the hydrogenated soy phosphatidylcholine (HSPC) in the mixed solution one and the mixed solution two is 25-45 mM, further preferably 45 mM, and the concentration of the curcumin is 10 μM-30 μM, further preferably 30 μM.

[0017] Preferably, in the preparation step of the curcumin-loaded (Cur) multi-vesicular liposome (CMLVs), the temperature of the warm water is 37℃-60℃, further preferably 60℃, and the ultrasonic treatment time is 15-30 minutes, preferably 15 minutes.

[0018] Preferably, the concentration of the N-hydroxysuccinimidyl acrylate (ACC-NHS) in the mixed solution one is 10 mg / mL-20 mg / mL, further preferably 10 mg / mL, and the concentration of the hydroxyapatite (HAP) is 10 mg / mL-90 mg / mL, further preferably 10 mg / mL-30 mg / mL.

[0019] Preferably, the concentration of the N-hydroxysuccinimidyl acrylate (ACC-NHS) in the mixed solution two is 10 mg / mL-20 mg / mL, further preferably 10 mg / mL, and the concentration of the Kato Genin (KGN) is 10 μM-30 μM, further preferably 30 μM.

[0020] The application provides a preparation method of a chitosan-based gradient hydrogel, which uses thiolated chitosan as a base material, and uses an integrated gelation method to construct a chitosan-based bone-cartilage gradient hydrogel and simulate the bone-cartilage gradient structure and the bone-cartilage interface. The chitosan-based gradient hydrogel can be adhered to a joint bone-cartilage defect site, the cartilage layer gel can load Kato Genin (KGN) and curcumin (Cur) to promote chondrogenesis, the bone layer gel can be combined with hydroxyapatite (HAP) and curcumin (Cur) to promote osteogenesis, and the slow release of curcumin (Cur) can adjust the inflammatory microenvironment to maintain the phenotype, so as to integrally repair the bone-cartilage defect site.

[0021] In addition, when the chitosan-based gradient hydrogel is prepared by using the preparation method of the application, the content of thiolated chitosan, hydroxyapatite, alpha-ketoglutaric acid, Kato Genin, curcumin, etc. can be adjusted to control the gelation time, mechanical strength, internal morphology, adhesion performance, swelling performance, degradation performance, and bone-cartilage differentiation promotion performance of the hydrogel, so as to obtain a chitosan-based gradient hydrogel with controllable performance.

[0022] The application provides a preparation method of a chitosan-based gradient hydrogel.

[0023] The application has at least the following advantages and beneficial effects.

[0024] Firstly, the raw material chitosan of the application is derived from shrimp and crab shells and is a natural recyclable resource, and alpha-ketoglutaric acid is a key intermediate metabolite in the tricarboxylic acid cycle and has functions of antioxidation and energy metabolism. In addition to loading and slow-releasing drugs, the multivesicular liposome can reduce the friction of the slip surface through the phosphatidylcholine head group of hydrated phospholipid lipids, can simulate self-renewal and a thin friction-lubricated lipid molecular thickness boundary layer, and can play a role in lubricating joints. The bone layer gel loaded with curcumin (Cur) and hydroxyapatite (HAP) can regulate osteogenic differentiation, and the cartilage layer gel loaded with curcumin (Cur) and kato genin (KGN) can regulate chondrogenic differentiation. In addition, the bone layer gel and the cartilage layer gel can be used to prepare chitosan-based osteochondral gradient gel through an integrated construction method to simulate the osteochondral gradient structure and the osteochondral interface.

[0025] Secondly, the reaction system condition of the chitosan-based gradient hydrogel prepared by the application is mild, good in operability and controllable in performance.

[0026] Thirdly, the application can regulate the gelation time, mechanical strength, internal morphology, adhesion performance, swelling performance, degradation performance and osteochondral differentiation performance of the hydrogel by adjusting the content of thiolated chitosan, hydroxyapatite, alpha-ketoglutaric acid, kato genin and curcumin, so that the chitosan-based gradient hydrogel with controllable performance is obtained.

[0027] Fourthly, the chitosan-based gradient hydrogel prepared by the application does not need to add initiators and crosslinking agents, has a fast gelation speed, good adhesion performance, mechanical performance, cell compatibility and biological activity, has a bionic osteochondral interface, the bone layer gel can be directional osteogenic differentiation, the cartilage layer gel can be directional chondrogenic differentiation, the shape is adjustable, and the performance is controllable. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The preparation flow chart of the chitosan-based gradient hydrogel in the present application is shown.

[0029] Figure 2 The physical sample chart of the chitosan-based gradient hydrogel prepared in the present application is shown.

[0030] Figure 3 The SEM internal morphology chart of the chitosan-based gradient hydrogel prepared in the present application is shown.

[0031] Figure 4 The expression chart of the osteogenic differentiation genes of the chitosan-based gradient hydrogel prepared in the present application is shown.

[0032] Figure 5 The expression chart of the chondrogenic differentiation genes of the chitosan-based gradient hydrogel prepared in the present application is shown.

[0033] Figure 6 The comparison chart of the chitosan-based gradient hydrogel prepared in the present application for promoting bone-cartilage integration repair is shown. Figure 6 In the chart, A is a blank control group, and B is an experimental group. DETAILED DESCRIPTION

[0034] The specific embodiments of the present application are further described below to make the technical solutions of the present application and the beneficial effects thereof more clear and explicit. The following description of the embodiments is exemplary and is intended to explain the present application, and cannot be understood as a limitation of the present application.

[0035] Additional aspects and advantages of the present application will become apparent in the description that follows, or will be appreciated by practice of the present application.

[0036] REFERENCE Figure 1As shown, the application provides a chitosan-based gradient hydrogel, and the basic concept is that the curcumin (Cur) loaded multivesicular liposomes (CMLVs) and hydroxyapatite (HAP) are placed in the bottom bone layer gel, the curcumin (Cur) loaded multivesicular liposomes (CMLVs) and Kato Genin (KGN) are placed in the top cartilage layer gel, and the chitosan-based gradient hydrogel is integrally prepared. In the process of integrally preparing the chitosan-based gradient hydrogel, the hydrogel main body is crosslinked by thiolated chitosan (CSSH), N-hydroxysuccinimidyl acrylate (ACC-NHS) and alpha-ketoglutaric acid (KA); the thiol group of thiolated chitosan and the double bond of N-hydroxysuccinimidyl acrylate (ACC-NHS) undergo Michael addition reaction, the thiol group and the thiol group react to form disulfide bond, and then crosslinking forms the chitosan-based gradient hydrogel; wherein the alpha-ketoglutaric acid (KA) gives the gel physical adhesion, and the hydroxysuccinimidyl group of N-hydroxysuccinimidyl acrylate (ACC-NHS) can be crosslinked with the amino group of the tissue to give it chemical crosslinking adhesion, thereby further improving the adhesion of the gel, and the gradient gel can mimic the bone-cartilage interface, the bone layer gel can be oriented to bone differentiation, and the cartilage layer gel can be oriented to cartilage differentiation, thereby solving the problems of poor integration of the gel and subchondral bone, difficult oriented osteogenesis and chondrogenic differentiation.

[0037] The application integrally prepares the chitosan-based gradient hydrogel, and the prepared chitosan-based gradient hydrogel has good adhesion performance, biological activity and the like, and can provide a new research direction, research foundation and idea for the preparation and application of biological medical materials in the field of tissue engineering and regenerative medicine.

[0038] Example one.

[0039] Hydrogenated soybean phosphatidylcholine (HSPC) and curcumin are ultrasonically treated in water at 60℃ for 15 minutes to prepare curcumin (Cur) loaded multivesicular liposomes (CMLVs). The dispersion concentration of hydrogenated soybean phosphatidylcholine (HSPC) in the subsequent mixed solution is 45mM, and the dispersion concentration of curcumin in the subsequent mixed solution is 30μM.

[0040] Example two.

[0041] 1) 200 mg of thiolated chitosan (CSSH) and curcumin-loaded (Cur) multivesicular liposomes (CMLVs) were dissolved in 2.5 mL of an aqueous solution of alpha-ketoglutaric acid (KA) with a concentration of 2 mg / mL to obtain solution A, in which the content of curcumin was 30 μM; 50 mg of N-hydroxysuccinimidyl acrylate (ACC-NHS) and 50 mg of hydroxyapatite (HAP) were dissolved in 2.5 mL of deionized water to obtain solution B, and solution A and solution B were mixed in a volume ratio of 1:1 to obtain mixed solution one, and then the pH value of the mixed solution one was adjusted to neutral with 0.5 M NaOH to obtain the bottom bone layer gel. 2) On the upper surface of the bone layer gel, 200 mg of thiolated chitosan (CSSH) and curcumin-loaded (Cur) multivesicular liposomes (CMLVs) were dissolved in 2.5 mL of an aqueous solution of alpha-ketoglutaric acid (KA) with a concentration of 2 mg / mL to obtain solution C, in which the content of curcumin was 30 μM; 50 mg of N-hydroxysuccinimidyl acrylate (ACC-NHS) and KGN were dissolved in 2.5 mL of deionized water to obtain solution D, and solution C and solution D were mixed in a volume ratio of 1:1 to obtain mixed solution two, in which the concentration of KGN was 10 μM, and the pH value of the mixed solution two was adjusted to neutral with 0.5 M NaOH to obtain the bottom cartilage layer gel; thereby the sample 1 of chitosan-based gradient hydrogel was obtained by an integrated preparation method.

[0042] Example Three.

[0043] 1) Dissolve 300 mg of thiolated chitosan (CSSH) and curcumin-loaded multivesicular liposomes (CMLVs) in 2.5 mL of 2 mg / mL α-ketoglutarate (KA) aqueous solution to obtain solution A, in which the curcumin content is 30 μM; dissolve 50 mg of N-hydroxysuccinimide acrylate (ACC-NHS) and 100 mg of hydroxyapatite (HAP) in 2.5 mL of deionized water to obtain solution B. Mix solution A and solution B at a volume ratio of 1:1 to obtain mixed solution one. Then adjust the pH of mixed solution one to neutral with 0.5 M NaOH to obtain the underlying bone layer gel. 2) On the upper surface of the bone layer gel, 200 mg of thiolated chitosan (CSSH) loaded with curcumin (Cur) multivesicular liposomes (CMLVs) were dissolved in 2.5 mL of α-ketoglutarate (KA) aqueous solution with a concentration of 2 mg / mL to obtain solution C. The curcumin content in solution C was 30 μM. 50 mg of N-hydroxysuccinimide acrylate (ACC-NHS) and captopril (KGN) were dissolved in 2.5 mL of deionized water to obtain solution D. Solution C and solution D were mixed at a volume ratio of 1:1 to obtain mixed solution two. The concentration of captopril (KGN) in the mixed solution was 20 μM. The pH of mixed solution two was then adjusted to neutral with 0.5 M NaOH to obtain the bottom cartilage layer gel. Thus, sample 2 of chitosan-based gradient hydrogel was obtained through an integrated preparation method.

[0044] Example 4.

[0045] 1) Dissolve 400 mg of thiolated chitosan (CSSH) and curcumin-loaded multivesicular liposomes (CMLVs) in 2.5 mL of 2 mg / mL α-ketoglutarate (KA) aqueous solution to obtain solution A, in which the curcumin content is 30 μM; dissolve 50 mg of N-hydroxysuccinimide acrylate (ACC-NHS) and 150 mg of hydroxyapatite (HAP) in 2.5 mL of deionized water to obtain solution B. Mix solution A and solution B at a volume ratio of 1:1 to obtain mixed solution one. Then adjust the pH of mixed solution one to neutral with 0.5 M NaOH to obtain the underlying bone layer gel. 2) On the upper surface of the bone layer gel, 200 mg of thiolated chitosan (CSSH) and curcumin-loaded multivesicular liposomes (CMLVs) were dissolved in 2.5 mL of an aqueous solution of α-ketoglutarate (KA) at a concentration of 2 mg / mL to obtain solution C. Solution C contained 30 μM curcumin. 2.5 mL of curcumin-loaded multivesicular liposomes (CMLVs) containing 30 μM curcumin and 1 mg / mL α-ketoglutarate (KA) aqueous solution were also dissolved. Solution D was prepared by dissolving 50 mg of N-hydroxysuccinimide acrylate (ACC-NHS) and captopril (KGN) in 2.5 mL of deionized water. Solution C and solution D were then mixed at a volume ratio of 1:1 to obtain mixed solution II, in which the concentration of captopril (KGN) was 30 μM. The pH of mixed solution II was then adjusted to neutral with 0.5 M NaOH to obtain the underlying cartilage layer gel. Thus, sample 3 of chitosan-based gradient hydrogel was obtained through an integrated preparation method.

[0046] Performance testing.

[0047] 1) Adhesion performance test.

[0048] Sample 1 of the chitosan-based gradient hydrogel prepared in Example 1 was adhered to a plastic plate, with the bone layer gel in direct contact with the plastic plate, such as... Figure 2 As shown. From Figure 2 As can be seen, the gel has excellent adhesive properties and adheres to the plastic plate. The arrow points to the interface between the bone layer gel and the cartilage layer gel of the chitosan-based gradient hydrogel.

[0049] 2) Morphological observation.

[0050] SEM internal morphology of chitosan-based gradient hydrogel as follows Figure 3 As shown: The prepared chitosan-based gradient hydrogel was rapidly frozen with liquid nitrogen, and then freeze-dried using a vacuum freeze dryer to obtain a sponge-like dry hydrogel sample. Finally, the sample was subjected to brittle fracture with liquid nitrogen, sputtered with gold, and placed under a scanning electron microscope to observe its internal morphology.

[0051] from Figure 3As can be seen, the chitosan-based gradient hydrogel has a biomimetic osteocartilage interface (the dotted interface indicated by the arrow), the bone layer gel has a dense porous structure, and the cartilage layer gel has a porous structure with relatively large pores.

[0052] 3) Testing of directed osteogenic and chondrogenic differentiation in chitosan-based gradient hydrogels.

[0053] Bone layer gel and cartilage layer gel were used to induce osteogenic and chondrogenic differentiation of bone marrow mesenchymal stem cells, respectively, and the results were as follows: Figure 4 , Figure 5 As shown. From Figure 4 and Figure 5 It can be observed that bone layer gel can promote the expression levels of osteogenic-related genes (ALP, Runx2, OCN, Col 1) in stem cells, while cartilage layer gel can promote the expression levels of chondrogenic-related genes (SOX9, ACAN, Col 2) in stem cells. The results indicate that osteochondral gel can directionally promote both osteogenic and chondrogenic differentiation.

[0054] 4) Animal experiments on the integrated repair of bone-cartilage defects using chitosan-based gradient hydrogels.

[0055] A rat model of bone-cartilage defect was constructed. The knee joint was surgically exposed, and a cylindrical bone-cartilage defect (2.5 mm in diameter and 3 mm in depth) was created using a drill. The experiment was divided into a control group (blank control) and a chitosan-based gradient hydrogel group. The repaired areas were observed macroscopically and photographed, and micro-CT scans were performed. The repair status was analyzed, and the results are as follows: Figure 6 As shown.

[0056] Figure 6 In the comparison, (A) shows the repair status of the control group. Gross observation reveals a clearly defined defect with distinct edges between the repaired area and surrounding tissue. CT scans show significant bone loss, indicating poor bone remodeling. (B) shows the chitosan-based gradient hydrogel group. Gross observation shows some filling of the defect area with regenerated tissue. The edges between the defect surface and surrounding tissue are not very distinct, and the regenerated tissue integrates better with the surrounding tissue. CT scans show bone remodeling. Figure 6 It can be seen that chitosan-based gradient hydrogels can effectively promote the integrated repair of bone-cartilage defects.

[0057] It should be noted that the technical features of the above embodiments can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0058] The above embodiments merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Parts not described in the specific embodiments are all prior art or common knowledge.

[0059] It should also be noted that, in this invention, the term "prepared from" is synonymous with "comprising". The terms "comprising", "including", "having", "containing", or any other variations thereof, as used in this invention, are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus comprising the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such a composition, step, method, article, or apparatus.

Claims

1. A method for preparing a chitosan-based gradient hydrogel, characterized in that, The preparation steps are as follows: 1) Preparation of curcumin-loaded multivesicular liposomes (CMLVs): Hydrogenated soybean phosphatidylcholine (HSPC) and curcumin were added to warm water and ultrasonically treated to prepare curcumin-loaded multivesicular liposomes (CMLVs). 2) To prepare the upper gel, thiolated chitosan (CSSH) and curcumin-loaded multivesicular liposomes (CMLVs) were dissolved in an aqueous solution of α-ketoglutarate (KA) to obtain solution A. N-hydroxysuccinimide acrylate (ACC-NHS) and hydroxyapatite (HAP) were dissolved in deionized water to obtain solution B. Solution A and solution B were mixed to obtain mixed solution one. The pH of mixed solution one was adjusted to neutral to obtain the upper gel. 3) To prepare the lower gel, thiolated chitosan (CSSH) and curcumin-loaded multivesicular liposomes (CMLVs) were dissolved in an aqueous solution of α-ketoglutarate (KA) to obtain solution C. N-hydroxysuccinimide acrylate (ACC-NHS) and captopril (KGN) were dissolved in deionized water to obtain solution D. The above solutions C and D were mixed to obtain mixed solution two. The pH value of mixed solution two was adjusted to neutral to obtain the lower gel. One of the upper and lower gels is prepared directly on the other surface, forming an integral gel.

2. The method for preparing a chitosan-based gradient hydrogel according to claim 1, characterized in that, In the preparation steps of the curcumin-loaded multivesicular liposomes (CMLVs), the warm water temperature is 37℃-60℃, and the ultrasonic treatment time is 15-30 minutes.

3. The method for preparing a chitosan-based gradient hydrogel according to claim 1, characterized in that, The mixing ratio of solution A and solution B is 1:1, and the mixing ratio of solution C and solution D is also 1:1, by volume.

4. The method for preparing a chitosan-based gradient hydrogel according to claim 1 or 3, characterized in that, In both the first and second mixed solutions, the concentration of hydrogenated soybean phosphatidylcholine (HSPC) is 25-45 mM, and the concentration of curcumin is 10 μM-30 μM.

5. A method for preparing a chitosan-based gradient hydrogel according to claim 1 or 3, characterized in that, In both the first and second mixed solutions, the concentration of the thiolated chitosan (CSSH) is 40 mg / mL to 80 mg / mL.

6. A method for preparing a chitosan-based gradient hydrogel according to claim 1 or 3, characterized in that, In both the first and second mixed solutions, the concentration of α-ketoglutarate is 1 mg / mL to 10 mg / mL.

7. The method for preparing a chitosan-based gradient hydrogel according to claim 1, characterized in that, In the first mixed solution, the concentration of N-hydroxysuccinimide acrylate (ACC-NHS) is 10 mg / mL to 20 mg / mL, and the concentration of hydroxyapatite (HAP) is 10 mg / mL to 90 mg / mL.

8. The method for preparing a chitosan-based gradient hydrogel according to claim 1, characterized in that, In the second mixed solution, the concentration of N-hydroxysuccinimide acrylate (ACC-NHS) is 10 mg / mL-20 mg / mL, and the concentration of captopril (KGN) is 10 μM-30 μM.

9. The chitosan-based gradient hydrogel prepared by the method for preparing a chitosan-based gradient hydrogel according to any one of claims 1-8.

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