Chitosan-based gradient hydrogel as well as preparation method and application thereof

Abstract

The invention relates to the technical field of hydrogel and preparation thereof, and discloses chitosan-based gradient hydrogel as well as a preparation method and application of the chitosan-based gradient hydrogel. Thiolated chitosan and curcumin-loaded multi-vesicle liposome are dissolved in an alpha-ketoglutaric acid aqueous solution; and dissolving the N-hydroxysuccinimide acrylate and the hydroxyapatite in another deionized water, mixing the two solutions, and adjusting the pH value of the system to be neutral to obtain the bone layer gel. According to the method for preparing the gel, hydroxyapatite is replaced by cartogenin to obtain the cartilage layer gel. And placing the bone layer gel on the bottom layer and the cartilage layer gel on the top layer, and integrally preparing to obtain the chitosan-based gradient hydrogel. The chitosan-based osteochondral gradient hydrogel constructed by the invention can better simulate an osteochondral gradient structure and an osteochondral interface thereof, the cartilage layer gel promotes cartilage differentiation, the bone layer gel promotes osteogenic differentiation, and slow release of curcumin regulates inflammation microenvironment and maintains 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 subchondral bone, so the combination of hydrogel with 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 subchondral bone in biphasic structures.

[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 underlying diseases 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, 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 kartogenin (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, kartogenin 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 prepares the chitosan-based gradient hydrogel by an integrated method, 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) 300 mg of curcumin-loaded thiolated chitosan (CSSH) and multi-walled 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, and in solution A, the content of curcumin was 30 μM; 50 mg of N-hydroxysuccinimide acrylate (ACC-NHS) and 100 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 curcumin-loaded thiolated chitosan (CSSH) and multi-walled 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, and in solution C, the content of curcumin was 30 μM; 50 mg of N-hydroxysuccinimide 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, and the concentration of KGN in the mixed solution was 20 μM, and then 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 2 of the chitosan-based gradient hydrogel was obtained by an integrated preparation method.

[0044] Example Four.

[0045] 1) 400 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 150 mg of hydroxyapatite (HAP) were dissolved in 2.5 mL of deionized water to obtain solution B; 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; 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 30 μM; the pH value of the mixed solution two was adjusted to neutral with 0.5 M NaOH to obtain the bottom cartilage layer gel; and thus sample 3 of the chitosan-based gradient hydrogel was obtained by the integrated preparation method.

[0046] Performance test.

[0047] 1) Adhesion performance test.

[0048] The sample 1 of the chitosan-based gradient hydrogel prepared in Example 1 was adhered to a plastic plate, and the bone layer gel was in direct contact with the plastic plate, as shown in Figure 2 It can be seen from Figure 2 that the gel has good adhesion performance and adheres to the plastic plate, in which the interface between the bone layer gel and the cartilage layer gel of the chitosan-based gradient hydrogel is indicated by an arrow.

[0049] 2) Morphology observation.

[0050] The SEM internal morphology of the chitosan-based gradient hydrogel is shown in Figure 3 : The prepared chitosan-based gradient hydrogel was rapidly frozen with liquid nitrogen, then freeze-dried with a vacuum freeze dryer to obtain a sponge-like dry state hydrogel sample, and finally the sample was brittle fractured with liquid nitrogen, gold sprayed and placed in a scanning electron microscope to observe its internal morphology.

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

[0052] 3) Test of the chitosan-based gradient hydrogel in directional osteogenic differentiation and chondrogenic differentiation.

[0053] The bone layer gel and the cartilage layer gel were respectively used to induce the osteogenic differentiation and chondrogenic differentiation of the mesenchymal stem cells, and the results are shown in Figure 4 Figure 5 It can be seen from Figure 4 and Figure 5 that the bone layer gel can promote the expression level of the osteogenic-related genes (ALP, Runx2, OCN, Coll) of the stem cells, and the cartilage layer gel can promote the expression level of the chondrogenic-related genes (SOX9, ACAN, Col 2) of the stem cells. The results show that the bone-cartilage gel can directionally promote the osteogenic differentiation and chondrogenic differentiation.

[0054] 4) Animal experiment of the chitosan-based gradient hydrogel in integrated repair of bone-cartilage defects.

[0055] A rat bone-cartilage defect model was constructed, and a cylindrical bone-cartilage defect (diameter 2.5 mm, depth 3 mm) was manufactured by drilling to expose the knee joint. The experiment was divided into a control group (blank control) and a chitosan-based gradient hydrogel group, the repair site of the defect was observed and photographed by gross observation and micro-CT scanning, and the repair condition was analyzed, and the results are shown in Figure 6 .

[0056] Figure 6 In the above, (A) is the repair condition of the control group, it can be seen from the gross observation that the repaired defect site is obvious, and there is an obvious defect edge between the surrounding tissues, and it can be seen from the CT image that the bone defect is not well remodeled. (B) is the chitosan-based gradient hydrogel group, the gross observation can see that the defect site has a certain regeneration tissue filling, the edge between the defect surface and the surrounding tissues is not very obvious, and the regeneration tissue is better fused with the surrounding tissues, and the CT image can see that the bone defect is remodeled. It can be seen from Figure 6 that the chitosan-based gradient hydrogel can effectively promote the integrated repair of the bone-cartilage defect.

[0057] It should be noted that any combination of the technical features of the above embodiments can be made, and in order to make the description simple, all possible combinations of the technical features in the above embodiments are not described, however, as long as the combination of the technical features does not exist contradictory, it should be considered as the scope of the present disclosure.

[0058] ​The above embodiments only express several implementation ways of the present application, and the description is relatively specific and detailed, but it should not be understood as a limitation to the scope of the patent right of the present application. It should be noted that, for ordinary skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, which are all within the protection scope of the present application. The parts not described in the specific embodiments are all prior art or common knowledge.

[0059] It should be further noted that, in the present application, the term "prepared from" is synonymous with "comprising". The terms "comprising", "including", "having" and "with" or any other variation thereof, used in the present application, are intended to cover non-exclusive inclusion. For example, a composition, step, method, article or device comprising the listed elements does not necessarily limit to only those elements, but can include other elements not explicitly listed or inherent to such composition, step, method, article or device.

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.

10. The application of the chitosan-based gradient hydrogel as described in claim 9 in biomedical materials.

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