Cartilage-imitating multifunctional drug release hydrogel material and preparation method thereof

Abstract

The invention belongs to the technical field of hydrogel preparation, and relates to a cartilage-imitating multifunctional drug release hydrogel material and a preparation method thereof. According to the invention, biocompatible high polymer materials such as polyvinyl alcohol, chitosan, grape seed protein, acyl chloride, sulphobetaine methacrylate and lecithin are used as raw materials, a cartilage structure is simulated to prepare the multilayer hydrogel, gelatin and plant polyphenol are mixed as an adhesive to improve the binding capacity with subosseous bones, and a drug is loaded to improve the biological activity. The preparation method of the hydrogel material comprises the five steps of preparation of high-strength anti-swelling precursor hydrogel, introduction of double bonds through surface modification, introduction of a poly SBMA molecular brush through interfacial polymerization, drug loading and preparation of an adhesion layer. The hydrogel has excellent anti-swelling ability, lubricating property, anti-pollution ability, free radical scavenging ability, self-adhesion characteristic, drug controlled release ability and biocompatibility, and has important significance for widening the application of the hydrogel in the field of artificial cartilage.

Description

Technical Field

[0001] The present invention belongs to the technical field of hydrogel preparation, and particularly relates to a cartilage-mimicking multifunctional drug-releasing hydrogel material and a preparation method thereof. Background Art

[0002] Articular cartilage, as a crucial "cushion" and "lubricant" between joints, continuously bears unimaginable huge pressure and frequent friction during daily activities such as walking, running, and jumping. Unfortunately, the self-repair ability of articular cartilage is extremely limited. Once damaged due to factors such as aging, overuse, and trauma, it is often difficult to heal on its own, thus triggering a series of problems such as joint pain, swelling, and limited mobility, seriously affecting the quality of life of patients. Existing cartilage replacement materials mainly consist of rigid materials such as titanium alloy, ultra-high molecular polymer, and ceramic materials. Although these materials have excellent mechanical properties, they have the following deficiencies: During use, due to the lack of good lubrication at the sliding interface and stress concentration problems, serious surface wear will occur, thus affecting the lifespan of the joint; the lack of a porous structure and water-containing environment of cartilage hinders the transmission of nutrients and metabolic wastes; they are powerless against possible bacterial infections and oxidation during the surgical process. In contrast, hydrogels, with a structure and water content similar to cartilage, are regarded as the most ideal cartilage replacement materials. An ideal cartilage replacement hydrogel should meet the following criteria: (1) high water content (>60%); (2) having a compressive strength close to that of natural cartilage (10 - 60 MPa) in a physiological environment; (3) a friction coefficient close to that of natural cartilage (0.03 - 0.001); (4) having anti-fouling ability; (5) having self-adhesive properties; (6) having biological activities such as antibacterial and free radical scavenging; (7) having biocompatibility (cell viability >95%).

[0003] Although some achievements have been made in hydrogel cartilage replacement materials and some individual properties are close to those of natural cartilage, there is still a large gap in comprehensive properties compared with natural cartilage. For example, Chinese Patent CN202311751245.3 discloses a preparation method and application of a high-strength, tough and wear-resistant hydrogel, and a high-strength, tough and wear-resistant polyvinyl alcohol / chitosan hydrogel is prepared. Its compressive strength is 6.25 MPa and the friction coefficient is 0.045. However, there is still a gap in bearing pressure and lubrication ability compared with natural cartilage, and it does not have the abilities of adhesion and antibacterial. CN202211686242.1 discloses a superhard, high-strength and tough hydrogel and its preparation method. Its compressive strength is 232.45 MPa, the friction coefficient is 0.18, and the water content is only 27%. There is still a gap in its lubrication ability and water content compared with natural cartilage, and it does not have the abilities of anti-pollution, self-adhesion and drug controlled release. CN202310072637.0 discloses a hydrogel composite material and its preparation method and application. Its compressive strength is 52 kPa and the adhesion strength is 21.9 kPa. However, this hydrogel does not have the abilities of anti-pollution, high lubrication and drug controlled release. CN201710756510.5 discloses a bioactive bionic cartilage material. Its compressive strength is 3.5 MPa and the friction coefficient is 0.05. It has biocompatibility. However, there is still a gap in bearing pressure and lubrication ability compared with natural cartilage, and it does not have the abilities of anti-pollution, self-adhesion and drug controlled release.

[0004] Therefore, developing a new type of bionic cartilage multifunctional hydrogel material that can meet the above seven requirements simultaneously is an urgent technical problem to be solved in this field. Summary of the Invention

[0005] To overcome the deficiencies of the prior art, the primary objective of the present invention is to provide a bionic cartilage multifunctional drug release hydrogel material. The present invention uses biocompatible polymer materials such as polyvinyl alcohol, chitosan, grape seed protein, acyl chloride, sulfobetaine methacrylate (SBMA), and lecithin as raw materials to mimic the cartilage structure to prepare a multi-layer hydrogel, uses gelatin and plant polyphenols mixed as adhesives to improve the binding ability with subchondral bone, and loads drugs to improve biological activity. The hydrogel of the present invention has high water content, high strength, excellent anti-swelling ability, lubrication performance, anti-pollution ability, free radical scavenging ability, self-adhesion characteristics, drug controlled release ability and biocompatibility.

[0006] Another objective of the present invention is to provide a preparation method of a bionic cartilage multifunctional drug release hydrogel material, including five steps: preparation of a high-strength anti-swelling precursor hydrogel, surface modification to introduce double bonds, interfacial polymerization to introduce poly-SBMA molecular brushes, drug loading, and preparation of an adhesion layer.

[0007] To achieve the above object, the present invention is realized through the following technical solutions: A preparation method of a cartilage-mimicking multifunctional drug-releasing hydrogel material, characterized in that the following steps are specifically carried out.

[0008] S01. Preparation of a high-strength anti-swelling precursor hydrogel 1) Heat 10-20 parts of polyvinyl alcohol, 1-5 parts of chitosan and 180-190 parts of deionized water at 90 °C for 6 hours to obtain a uniform solution, then inject the obtained solution into a mold and let it stand at room temperature to remove air bubbles; place it in a -20 °C freezer for 8-12 h, transfer it to room temperature and let it stand for 4-6 h, and perform 3 consecutive freeze-thaw cycles to obtain a polyvinyl alcohol-chitosan pre-gel; 2) Remove the contained moisture from the above pre-gel by freeze-drying to obtain a polyvinyl alcohol-chitosan aerogel; 3) Immerse the above aerogel completely in an aqueous solution of grape seed protein that can form ionic bonds with chitosan for 3 h to prepare a high-strength anti-swelling precursor hydrogel (PCG) with coordinated cross-linking of multiple non-covalent bonds such as hydrogen bonds and ionic bonds, which constitutes the tough layer of the target hydrogel.

[0009] S02. Surface modification to introduce double bonds Freeze-dry the high-strength anti-swelling precursor hydrogel obtained in step S01, then float it in a mixed solution of dichloromethane and dimethyl sulfoxide containing acyl chloride for 12 h to introduce double bonds to the surface. After reacting at room temperature for 24 h, dialyze it in pure water for 3-7 days to remove residual chemicals to obtain a hydrogel with double bonds introduced by surface modification (M-PCG).

[0010] S03. Interfacial polymerization to introduce poly-SBMA molecular brushes Coat the top layer of the hydrogel with double bonds introduced by surface modification obtained in step S02 with an aqueous solution of SBMA, add a photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, and irradiate the bottom of the hydrogel with ultraviolet light for 40 min for photoinitiation, and remove the excess SBMA with deionized water to obtain a hydrogel with poly-SBMA molecular brushes introduced by interfacial polymerization (SM-PCG).

[0011] S04. Loading drugs 1) Remove the moisture from the hydrogel obtained in step S03 by freeze-drying to obtain an aerogel; 2) Preparation of lecithin encapsulating hydrophobic drugs by thin film hydration method: Dissolve lecithin and hydrophobic drugs in a conical flask containing dichloromethane, and stir and mix at room temperature for 3 h. Then, evaporate dichloromethane in a water bath at 45 °C until a coating is formed. Place the flask in a vacuum oven at 45 °C overnight to remove the residual solvent and form a dry film. Dissolve the dry film with deionized water and sonicate to obtain a uniform aqueous solution. Then, centrifuge in a centrifuge for 15 minutes to remove the unencapsulated drugs successfully. The supernatant after centrifugation is the lecithin aqueous dispersion; 3) Dissolve the hydrophilic drug in the lecithin aqueous dispersion, and then soak the above-mentioned aerogel to obtain a hydrogel (SD-PCG) containing both hydrophilic and hydrophobic drugs.

[0012] S05. Preparation of the adhesion layer Dissolve 10% gelatin aqueous solution at 45 °C for 1 h, then inject the glue, and then freeze in the refrigerator for 30 min to obtain a gelatin hydrogel; soak the gelatin hydrogel in 1% plant polyphenol aqueous solution for 24 h to obtain a gelatin-plant polyphenol hydrogel. Melt the gelatin-plant polyphenol hydrogel in an oven at 60 °C to obtain an adhesive of gelatin and plant polyphenol, and apply it to the bottom surface of the hydrogel containing both hydrophilic and hydrophobic drugs obtained in step S04 near the tough layer to obtain a cartilage-mimicking multifunctional drug release hydrogel (SD-PCG-A).

[0013] Further, the acyl chloride described in step S02 is any one or more of acryloyl chloride, methacryloyl chloride, crotonyl chloride, pentenoyl chloride, and cinnamoyl chloride.

[0014] Further, the volume ratio of the two solvents, dichloromethane and dimethyl sulfoxide, used to prepare the mixed solution in step S02 is 1:1 - 9:1; the density of the mixed solution is greater than the density of the precursor hydrogel to ensure that surface modification only occurs on the surface.

[0015] Further, the hydrophobic drug described in step S04 is any one of ciprofloxacin and norfloxacin.

[0016] Further, the hydrophilic drug described in step S04 is any one of sodium salicylate and ascorbic acid.

[0017] Further, the mass of the hydrophobic drug in step S04 is 0.5 - 6% of the mass of lecithin.

[0018] Further, the mass fraction of the hydrophilic drug in step S04 is 5 - 10%.

[0019] Further, the plant polyphenol in step S05 is any one or more of tannic acid, grape polyphenol, and tea polyphenol.

[0020] The grape seed protein used in the present invention contains abundant phenolic substances such as proanthocyanidins, endowing the hydrogel with excellent antioxidant properties, which can control the level of reactive oxygen species in the body and thus prevent the occurrence and progression of osteoarthritis.

[0021] The method for extracting and purifying grape seed protein used in the present invention is disclosed in Chinese Patent Application CN201810129805.4, specifically as follows: Extraction and purification of grape seed protein: 1) Pretreatment: Weigh grape seeds, soak them in 0.05 mol / L NaOH solution for 10 - 20 min, rinse, dry, and pulverize. 2) Degreasing: Degrease grape seeds with petroleum ether and dry. 3) Decolorization and dephenolization: Decolorize with polyvinylpyrrolidone, dephenolize with acidified ethanol, centrifuge to remove the supernatant, dry, and refrigerate. 4) Alkaline solution extraction: Dissolve the decolorized and dephenolized powder in 0.1 mol / L NaOH solution, place it in a water bath at 40 - 50 °C for 40 - 60 min, centrifuge to remove the residue, and obtain the supernatant. 5) Acid solution deposition: Dropwise add 1.0 mol / L HCl to the supernatant to adjust the pH value to 3 - 4 for precipitation, centrifuge to remove the supernatant, and obtain a flocculent precipitate. 6) Purification: Wash the flocculent precipitate with deionized water until neutral, and freeze - dry to obtain grape seed protein.

[0022] The present invention relies on the synergistic action of ionic bonds, crystalline microdomains, and hydrogen bonds to prepare a hydrogel precursor. Its compressive strength is 35 - 42 MPa, the water content is greater than 70%, and it has the ability to scavenge free radicals (8.7% - 43.0% for OH free radicals and 16.0% - 46.5% for DPPH free radicals); introducing an amphoteric ion anti - fouling layer on the top of the precursor hydrogel can not only resist protein adsorption but also reduce the friction coefficient; self - assemble lecithin and hydrophobic drugs (ciprofloxacin, norfloxacin) to form vesicles or micelles to encapsulate the hydrophobic drugs, soak the above - mentioned hydrogel in a lecithin aqueous dispersion and an aqueous solution containing hydrophilic drugs (such as sodium salicylate, ascorbic acid) to provide biological activity, and lecithin will form a boundary layer on the surface of the hydrogel to further reduce the friction coefficient of the hydrogel (0.023 - 0.018); construct an adhesion layer at the bottom to obtain a large number of groups that can interact with the skin and bones, such as amino groups, carboxyl groups in gelatin and phenolic hydroxyl groups in tannic acid, which can form a large number of hydrogen bonds, ionic complexation and other interactions with the skin and bones, making the hydrogel exhibit good adhesion performance (75 - 82 kPa); use biocompatible polymer materials such as polyvinyl alcohol, chitosan, and grape seed protein as the matrix and construct it by physical cross - linking, avoiding the use of toxic chemical cross - linkers and ensuring the biological safety of the material.

[0023] In the present invention, a cartilage-mimicking multifunctional drug-releasing hydrogel adheres usually through the interaction between adjacent surfaces. However, low friction repels this interaction. Therefore, achieving both low friction and self-adhesion is a huge challenge. Based on the relationship between material structure and properties, the inventors prepared a cartilage-mimicking multifunctional drug-releasing hydrogel using a bionic concept. The obtained hydrogel has excellent lubricating performance, anti-swelling performance, anti-fouling ability, free radical scavenging ability, compressive strength, self-adhesion performance, drug controlled-release ability, and biocompatibility. However, achieving high strength, anti-swelling, wear resistance, anti-fouling, free radical scavenging, self-adhesion, drug controlled-release, and biocompatibility simultaneously has always been a technical problem faced in the field of cartilage replacement materials. Inspired by the lubricating and anti-fouling properties of natural cartilage, zwitterions are grafted onto the hydrogel surface. The anti-fouling is achieved by forming a hydration layer as a physical barrier, using electrostatic repulsion to prevent charged pollutants from approaching, and relying on the flexible sliding of zwitterions to reduce pollutant adsorption sites, and the friction coefficient can be effectively reduced. The middle part of the hydrogel mimics the tough layer of cartilage, and a variety of non-covalent bonds are used for synergistic crosslinking to provide strength support. Drugs are encapsulated into the hydrogel by using the porous structure of the hydrogel or the self-assembly of lecithin to form vesicles or micelles, which can achieve antibacterial effects while reducing the friction coefficient. At the bottom of the hydrogel, an adhesive is added to mimic the high integration of cartilage and the underlying bone to achieve self-adhesion. Using biocompatible polymer materials such as polyvinyl alcohol, chitosan, and grape seed protein as the matrix, physical crosslinking methods are used to avoid the use of toxic chemical crosslinking agents, ensuring the biological safety of the material.

[0024] Compared with the hydrogel loaded with lecithin alone and grafted with zwitterions alone, the friction coefficient is significantly reduced after loading zwitterions and adding lecithin. It shows that the dual lubricating effect of lecithin and SBMA makes the friction coefficient of the hydrogel lower.

[0025] In the present invention, due to the three-dimensional network structure and hydrophilic functional groups of the hydrogel, its highly porous structure enables hydrophilic small molecule drugs to be adsorbed in the pores of the hydrogel network through non-covalent bonds such as ionic bonds and hydrogen bonds. When it comes to hydrophobic drugs, lecithin can self-assemble in aqueous solution to form vesicles or micelles to encapsulate the drugs in the hydrophobic core, and the hydrophobic drugs slowly diffuse from the lecithin vesicles or micelles, which can not only improve the solubility of hydrophobic drugs but also control the drug release rate.

[0026] In the present invention, double bonds are selected to be introduced on the hydrogel surface, which can enhance the grafting density and stability of the SBMA molecular brush, and at the same time form a stronger interaction with the hydrogel matrix.

[0027] Advantages of the present invention: The present invention has the following prominent substantive features and significant progress compared with the prior art.

[0028] (1) The hydrogel obtained in the present invention simultaneously has adhesion performance and a low friction coefficient, better meeting the requirements of cartilage tissue engineering.

[0029] (2) The dual lubrication effect of lecithin and zwitterions in the hydrogel obtained in the present invention enables the hydrogel to have an extremely low friction coefficient.

[0030] (3) The inventor team of the present invention based on the relationship between material structure and performance, and prepared a cartilage-mimicking multifunctional drug-release hydrogel using a bionic concept. During the preparation of the hydrogel, soaking in grape seed protein solution endows the hydrogel with excellent anti-swelling and free radical scavenging abilities; introducing zwitterions on the surface of the anti-fouling layer endows the hydrogel with excellent anti-fouling ability and friction coefficient; due to the synergistic cross-linking effect of multiple non-covalent bonds in the tough layer, the hydrogel has excellent compressive strength; at the bottom of the hydrogel, the high integration of cartilage and subchondral bone is simulated to achieve self-adhesion performance; vesicles or micelles formed by the self-assembly of lecithin and hydrophobic drugs encapsulate hydrophobic drugs, and the hydrogel is successively soaked in a lecithin aqueous dispersion and an aqueous solution containing hydrophilic drugs to provide biological activity, and lecithin will form a boundary layer on the surface of the hydrogel, further reducing the friction coefficient of the hydrogel.

[0031] (4) The cartilage-mimicking multifunctional drug-release hydrogel obtained in the present invention simultaneously has excellent lubrication performance, anti-swelling performance, anti-fouling performance, compressive strength, free radical scavenging ability, self-adhesion performance, biocompatibility and drug controlled release ability. The water content of the obtained cartilage-mimicking multifunctional drug-release hydrogel is about 72%, the compressive strength can reach 42 MPa, the friction coefficient is as low as 0.018, and the adhesion strength to bone can reach 82 kPa; the cell survival rate is greater than about 95%; this provides a guarantee for the strength and durability of the material implanted into the organism. The inventor summarized the performance parameters of the hydrogels obtained in Examples 14-18 of the present invention and compared them with the relevant literature data of existing cartilage replacement hydrogel materials, as shown in Table 1 for details.

[0032] 。

[0033] (5) In the present invention, vesicles or micelles formed by the self-assembly of lecithin and hydrophobic drugs encapsulate hydrophobic drugs, and the hydrogel is successively soaked in a lecithin aqueous dispersion and an aqueous solution containing hydrophilic drugs to provide biological activity, and lecithin will form a boundary layer on the surface of the hydrogel, further reducing the friction coefficient of the hydrogel.

[0034] (6) In the present invention, by introducing double bonds on the top layer of the hydrogel, active sites are provided for subsequent SBMA polymerization, enabling it to undergo a polymerization reaction with SBMA, grafting polySBMA on the surface of the hydrogel to achieve ultra-low friction and the effect of anti-fouling, making it difficult for pollutants such as proteins and bacteria to adsorb on the surface of the hydrogel.

[0035] (7) By applying an adhesion layer to the bottom of the hydrogel, the present invention enables the bottom surface of the hydrogel to obtain a large number of hydrophilic groups, which can form a large number of hydrogen bonds, ion complexations and other interactions with the skin and bones, providing good bone and sub-bone adhesion performance for the hydrogel. The hydrogel of the present invention has excellent adhesion strength, effectively avoiding various adverse effects that may be caused by poor bonding. At the same time, this method of applying the adhesion layer at the bottom also protects the low friction performance of the upper surface of the hydrogel from being affected.

[0036] (8) The preparation method of the cartilage-mimicking multifunctional drug-releasing hydrogel of the present invention is simple. During the preparation process, biocompatible polymer materials such as polyvinyl alcohol, chitosan and grape seed protein are used as matrices and constructed by physical cross-linking, avoiding the use of toxic chemical cross-linking agents and ensuring the biological safety of the materials.

[0037] (9) The present invention not only provides a preparation method for the cartilage-mimicking multifunctional drug-releasing hydrogel, providing a new method for the preparation of hydrogel materials, but also provides a new idea for the integration of various conflicting properties of hydrogel materials during the preparation process, which is of great significance for the research, development and application of hydrogel materials in the field of artificial cartilage. Description of the Drawings

[0038] Figure 1 is a synthesis schematic diagram of the cartilage-mimicking multifunctional drug-releasing hydrogel obtained from Example 14 of the present invention; Figure 2 is the release curves of the hydrophobic drug ciprofloxacin (a) and the hydrophilic drug sodium salicylate (b) of the cartilage-mimicking multifunctional drug-releasing hydrogel obtained from Example 14 of the present invention; Figure 3 is the inhibition zones of the cartilage-mimicking multifunctional drug-releasing hydrogel SD-PCG-A obtained from Example 14 of the present invention and the high-strength anti-swelling precursor hydrogel PCG obtained from Example 1 against Escherichia coli (a) and Staphylococcus aureus (b). Embodiments The technical solutions of the present invention will be described in detail below with reference to the accompanying drawings, but the implementation of the present invention is not limited thereto.

[0040] Example 1 Preparation of High-Strength Anti-Swelling Precursor Hydrogel Weigh 20 parts of polyvinyl alcohol and 1 part of chitosan by weight, add them to 180 parts of deionized water, dissolve at 90 °C for 6 h to form a uniform solution, then inject the solution into a mold and let it stand for 24 h to remove air bubbles. Place it in a -20 °C freezer for 8 h, then transfer it to room temperature to thaw for 4 h, and repeat the cycle three times to synthesize a physically crosslinked polyvinyl alcohol-chitosan pre-gel. After freeze-drying and dehydrating the polyvinyl alcohol-chitosan pre-gel, soak it in a 25 wt% grape seed protein solution for 3 h to obtain a high-strength anti-swelling precursor hydrogel PCG-1.

[0041] Test the properties of the high-strength anti-swelling precursor hydrogel obtained in Example 1. The test results show that: the water content of the hydrogel obtained in Example 1 is 74%, the compressive strength is 44 MPa, the friction coefficient is 0.028, the scavenging rate of OH radicals is 43.0%, the scavenging rate of DPPH radicals is 46.5%, and the cell viability is greater than 95%, showing biocompatibility.

[0042] Example 2 Preparation of high-strength anti-swelling precursor hydrogel Weigh 10 parts of polyvinyl alcohol and 5 parts of chitosan by weight, add them to 190 parts of deionized water, dissolve at 90 °C for 6 h to form a uniform solution, then inject the solution into a mold and let it stand for 24 h to remove air bubbles. Place it in a -20 °C freezer for 12 h, then transfer it to room temperature to thaw for 6 h, and repeat the cycle three times to synthesize a physically crosslinked polyvinyl alcohol-chitosan pre-gel. After freeze-drying and dehydrating the polyvinyl alcohol-chitosan pre-gel, soak it in a 25 wt% grape seed protein solution for 3 h to obtain a high-strength anti-swelling precursor hydrogel PCG-2.

[0043] Test the properties of the high-strength anti-swelling precursor hydrogel obtained in Example 2. The test results show that: the water content of the hydrogel obtained in Example 2 is 72%, the compressive strength is 40 MPa, the friction coefficient is 0.028, the scavenging rate of OH radicals is 42.0%, the scavenging rate of DPPH radicals is 44.2%, and the cell viability is greater than 95%, showing biocompatibility.

[0044] Example 3 Surface modification to introduce double bonds Freeze-dry the high-strength anti-swelling precursor hydrogel PCG-1 obtained in Example 1, then float it in a mixed solution of dichloromethane and dimethyl sulfoxide containing methacryloyl chloride (the volume ratio of the two solvents dichloromethane and dimethyl sulfoxide is 9:1) for 12 h to introduce double bonds onto the surface. After reacting at room temperature for 24 h, dialyze it in pure water for 3 days to remove residual chemicals to obtain a surface-modified hydrogel M-PCG-11 with double bonds introduced.

[0045] Example 4 Surface modification to introduce double bonds The high-strength anti-swelling precursor hydrogel PCG-1 obtained in Example 1 was freeze-dried, and then floated in a mixed solution of dichloromethane and dimethyl sulfoxide containing acryloyl chloride (the volume ratio of the two solvents dichloromethane and dimethyl sulfoxide was 8:1) for 12 h to introduce double bonds onto the surface. After reacting at room temperature for 24 h, it was dialyzed in pure water for 3 days to remove residual chemicals, and the hydrogel M-PCG-12 with double bonds introduced by surface modification was obtained.

[0046] Example 5 Surface modification to introduce double bonds The high-strength anti-swelling precursor hydrogel PCG-1 obtained in Example 1 was freeze-dried, and then floated in a mixed solution of dichloromethane and dimethyl sulfoxide containing pentenoyl chloride (the volume ratio of the two solvents dichloromethane and dimethyl sulfoxide was 5:1) for 12 h to introduce double bonds onto the surface. After reacting at room temperature for 24 h, it was dialyzed in pure water for 3 days to remove residual chemicals, and the hydrogel M-PCG-13 with double bonds introduced by surface modification was obtained.

[0047] Example 6 Surface modification to introduce double bonds The high-strength anti-swelling precursor hydrogel PCG-2 obtained in Example 2 was freeze-dried, and then floated in a mixed solution of dichloromethane and dimethyl sulfoxide containing methacryloyl chloride (the volume ratio of the two solvents dichloromethane and dimethyl sulfoxide was 3:1) for 12 h to introduce double bonds onto the surface. After reacting at room temperature for 24 h, it was dialyzed in pure water for 7 days to remove residual chemicals, and the hydrogel M-PCG-21 with double bonds introduced by surface modification was obtained.

[0048] Example 7 Surface modification to introduce double bonds The high-strength anti-swelling precursor hydrogel PCG-2 obtained in Example 2 was freeze-dried, and then floated in a mixed solution of dichloromethane and dimethyl sulfoxide containing methacryloyl chloride (the volume ratio of the two solvents dichloromethane and dimethyl sulfoxide was 1:1) for 12 h to introduce double bonds onto the surface. After reacting at room temperature for 24 h, it was dialyzed in pure water for 7 days to remove residual chemicals, and the hydrogel M-PCG-22 with double bonds introduced by surface modification was obtained.

[0049] Example 8 Interfacial polymerization to introduce poly-SBMA molecular brush The top layer of the hydrogel M-PCG-11 with surface modification introducing double bonds obtained in Example 3 was coated with an aqueous solution of SBMA. A photoinitiator, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, was added, and the bottom of the hydrogel was irradiated with ultraviolet light for 40 min for photoinitiation. Excess SBMA was removed with deionized water to obtain the hydrogel SM-PCG-11 with polySBMA molecular brushes introduced by interfacial polymerization. The layer with polySBMA grafted on the surface is the anti-fouling layer.

[0050] In the same manner, using the hydrogel M-PCG-12 with surface modification introducing double bonds obtained in Example 4, the hydrogel SM-PCG-12 with polySBMA molecular brushes introduced by interfacial polymerization was prepared.

[0051] In the same manner, using the hydrogel M-PCG-13 with surface modification introducing double bonds obtained in Example 5, the hydrogel SM-PCG-13 with polySBMA molecular brushes introduced by interfacial polymerization was prepared.

[0052] In the same manner, using the hydrogel M-PCG-21 with surface modification introducing double bonds obtained in Example 6, the hydrogel SM-PCG-21 with polySBMA molecular brushes introduced by interfacial polymerization was prepared.

[0053] In the same manner, using the hydrogel M-PCG-22 with surface modification introducing double bonds obtained in Example 7, the hydrogel SM-PCG-22 with polySBMA molecular brushes introduced by interfacial polymerization was prepared.

[0054] Example 9 Preparation of drug-loaded hydrogel 1) The hydrogel SM-PCG-11 obtained in Example 8 was freeze-dried to remove moisture to obtain an aerogel; 2) Lecithin and ciprofloxacin (the mass of ciprofloxacin was 0.5% of the mass of lecithin) were added to a conical flask containing dichloromethane for dissolution, and stirred and mixed at room temperature for 3 h. Then, dichloromethane was evaporated in a water bath at 45 °C until a coating was formed. The conical flask was then placed in a vacuum oven at 45 °C overnight to remove the residual solvent, forming a dry film. The dry film was dissolved in deionized water and sonicated to obtain a uniform aqueous solution, and then centrifuged in a centrifuge for 15 minutes to remove the unsuccessfully encapsulated drug. The supernatant after centrifugation was the lecithin aqueous dispersion loaded with the drug; 3) Sodium salicylate with a mass fraction of 5% was dissolved in the lecithin aqueous dispersion, and then the above aerogel was soaked to obtain the hydrogel SD-PCG-11 containing both the hydrophilic drug sodium salicylate and the hydrophobic drug ciprofloxacin.

[0055] Example 10 Preparation of drug-loaded hydrogel 1) Freeze-dry the hydrogel SM-PCG-12 obtained in Example 8 to remove moisture and obtain an aerogel; 2) Add lecithin and ciprofloxacin (the mass of ciprofloxacin is 6% of the mass of lecithin) into a conical flask containing dichloromethane for dissolution, stir and mix at room temperature for 3 h, then evaporate dichloromethane in a water bath at 45 °C until a coating is formed. Then place the conical flask in a vacuum oven at 45 °C overnight to remove the residual solvent and form a dry film. Dissolve the dry film with deionized water and ultrasonicate to obtain a uniform aqueous solution, and then centrifuge in a centrifuge for 15 minutes to remove the unsuccessfully encapsulated drug. The supernatant after centrifugation is the lecithin aqueous dispersion loaded with the drug; 3) Dissolve sodium salicylate with a mass fraction of 7% in the lecithin aqueous dispersion, and then soak the above aerogel to obtain a hydrogel SD-PCG-12 containing both the hydrophilic drug sodium salicylate and the hydrophobic drug ciprofloxacin.

[0056] Example 11 Preparation of the drug-loaded hydrogel 1) Freeze-dry the hydrogel SM-PCG-13 obtained in Example 8 to remove moisture and obtain an aerogel; 2) Add lecithin and norfloxacin (the mass of norfloxacin is 0.5% of the mass of lecithin) into a conical flask containing dichloromethane for dissolution, stir and mix at room temperature for 3 h, then evaporate dichloromethane in a water bath at 45 °C until a coating is formed. Place the flask in a vacuum oven at 45 °C overnight to remove the residual solvent and form a dry film. Dissolve the dry film with deionized water and ultrasonicate to obtain a uniform aqueous solution, and then centrifuge in a centrifuge for 15 minutes to remove the unsuccessfully encapsulated drug. The supernatant after centrifugation is the lecithin aqueous dispersion loaded with the drug; 3) Dissolve sodium salicylate with a mass fraction of 10% in the lecithin aqueous dispersion, and then soak the above aerogel to obtain a hydrogel SD-PCG-13 containing both the hydrophilic drug sodium salicylate and the hydrophobic drug norfloxacin.

[0057] Example 12 Preparation of the drug-loaded hydrogel 1) Freeze-dry the hydrogel SM-PCG-21 obtained in Example 8 to remove moisture and obtain an aerogel; 2) Add lecithin and norfloxacin (the mass of norfloxacin is 6% of the mass of lecithin) into a conical flask containing dichloromethane for dissolution, and stir and mix at room temperature for 3 h. Then, evaporate dichloromethane in a water bath at 45 °C until a coating is formed. Place the conical flask in a vacuum oven at 45 °C overnight to remove the residual solvent and form a dry film. Dissolve the dry film with deionized water and ultrasonicate to obtain a homogeneous aqueous solution. Then, centrifuge in a centrifuge for 15 minutes to remove the unsuccessfully encapsulated drugs. The supernatant after centrifugation is the lecithin aqueous dispersion loaded with drugs; 3) Dissolve ascorbic acid with a mass fraction of 5% in the lecithin aqueous dispersion, and then soak the above-mentioned aerogel to obtain a hydrogel SD-PCG-21 containing both hydrophilic drug ascorbic acid and hydrophobic drug norfloxacin.

[0058] Example 13 Preparation of drug-loaded hydrogel 1) Freeze-dry the hydrogel SM-PCG-22 obtained in Example 8 to remove water and obtain an aerogel; 2) Add lecithin and norfloxacin (the mass of norfloxacin is 0.5% of the mass of lecithin) into a conical flask containing dichloromethane for dissolution, and stir and mix at room temperature for 3 h. Then, evaporate dichloromethane in a water bath at 45 °C until a coating is formed. Place the flask in a vacuum oven at 45 °C overnight to remove the residual solvent and form a dry film. Dissolve the dry film with deionized water and ultrasonicate to obtain a homogeneous aqueous solution. Then, centrifuge in a centrifuge for 15 minutes to remove the unsuccessfully encapsulated drugs. The supernatant after centrifugation is the lecithin aqueous dispersion loaded with drugs; 3) Dissolve ascorbic acid with a mass fraction of 10% in the lecithin aqueous dispersion, and then soak the above-mentioned aerogel to obtain a hydrogel SD-PCG-22 containing both hydrophilic drug ascorbic acid and hydrophobic drug norfloxacin.

[0059] Example 14 Preparation of chondroid multi-functional drug release hydrogel Inject a 10% aqueous gelatin solution after heating at 45 °C for 1 h, and then freeze in a refrigerator for 30 min to obtain a gelatin hydrogel; Soak the gelatin hydrogel in a 1% aqueous tannic acid solution for 24 h to obtain a gelatin-tannic acid hydrogel. Melt the gelatin-tannic acid hydrogel in an oven at 60 °C to obtain an adhesive of gelatin and tannic acid, and apply it to the bottom layer of the prepared hydrogel SD-PCG-11, that is, apply it to the bottom surface of the hydrogel close to the tough layer, to obtain a chondroid multi-functional drug release hydrogel SD-PCG-A-11.

[0060] The properties of the cartilage-mimicking multifunctional drug-releasing hydrogel obtained in Example 14 were tested. The test results showed that the water content of the hydrogel obtained in Example 14 was 72%, the compressive strength was 42 MPa, the friction coefficient was 0.018, the adhesion strengths to bone and skin in the adhesion strength test were 82 kPa and 40 kPa respectively, it had the ability to scavenge free radicals and antibacterial ability, and the cell viability was greater than 95%, showing biocompatibility.

[0061] The synthesis schematic diagram of the cartilage-mimicking multifunctional drug-releasing hydrogel obtained in Example 14 is shown in Figure 1 。

[0062] The release curves of the hydrophobic drug ciprofloxacin (a) and the hydrophilic drug sodium salicylate (b) of the cartilage-mimicking multifunctional drug-releasing hydrogel obtained in Example 14 are shown in Figure 2 。

[0063] The antibacterial zones of the cartilage-mimicking multifunctional drug-releasing hydrogel SD-PCG-A obtained in Example 14 and the high-strength anti-swelling precursor hydrogel PCG obtained in Example 1 against Escherichia coli (a) and Staphylococcus aureus (b) are shown in Figure 3 。

[0064] Example 15 Preparation of cartilage-mimicking multifunctional drug-releasing hydrogel Inject the 10% gelatin aqueous solution after heating at 45 °C for 1 h, and then freeze it in the refrigerator for 30 min to obtain the gelatin hydrogel; soak the gelatin hydrogel in 1% tannic acid aqueous solution for 24 h to obtain the gelatin-tannic acid hydrogel. Melt the gelatin-tannic acid hydrogel in an oven at 60 °C to obtain the adhesive of gelatin and tannic acid, and apply it to the bottom layer of the prepared hydrogel SD-PCG-12, that is, apply it to the bottom surface of the hydrogel close to the tough layer, to obtain the cartilage-mimicking multifunctional drug-releasing hydrogel SD-PCG-A-12.

[0065] The properties of the cartilage-mimicking multifunctional drug-releasing hydrogel obtained in Example 15 were tested. The test results showed that the water content of the hydrogel obtained in Example 15 was 70%, the compressive strength was 38 MPa, the friction coefficient was 0.02, the adhesion strengths to bone and skin in the adhesion strength test were 75 kPa and 30 kPa respectively, it had the ability to scavenge free radicals and antibacterial ability, and the cell viability was greater than 95%, showing biocompatibility.

[0066] Example 16 Preparation of cartilage-mimicking multifunctional drug-releasing hydrogel Inject glue after heating a 10% gelatin aqueous solution at 45 °C for 1 h, and then freeze it in a refrigerator for 30 min to obtain a gelatin hydrogel; soak the gelatin hydrogel in a 1% polyphenol aqueous solution for 24 h to obtain a gelatin-polyphenol hydrogel. Melt the gelatin-polyphenol hydrogel in an oven at 60 °C to obtain an adhesive of gelatin and polyphenol, and apply it to the bottom layer of the prepared hydrogel SD-PCG-13, that is, apply it to the bottom surface of the hydrogel close to the tough layer, to obtain a cartilage-mimicking multifunctional drug-releasing hydrogel SD-PCG-A-13.

[0067] Test the properties of the cartilage-mimicking multifunctional drug-releasing hydrogel obtained in Example 16. The test results show that: the water content of the hydrogel obtained in Example 16 is 74%, the compressive strength is 35 MPa, the friction coefficient is 0.019, the adhesion strengths to bone and skin in the adhesion strength test are 78 kPa and 35 kPa respectively, it has the ability to scavenge free radicals and antibacterial ability, and the cell survival rate is greater than 95%, showing biocompatibility.

[0068] Example 17 Preparation of cartilage-mimicking multifunctional drug-releasing hydrogel Inject glue after heating a 10% gelatin aqueous solution at 45 °C for 1 h, and then freeze it in a refrigerator for 30 min to obtain a gelatin hydrogel; soak the gelatin hydrogel in a 1% tea polyphenol aqueous solution for 24 h to obtain a gelatin-tea polyphenol hydrogel. Melt the gelatin-tea polyphenol hydrogel in an oven at 60 °C to obtain an adhesive of gelatin and tea polyphenol, and apply it to the bottom layer of the prepared hydrogel SD-PCG-21, that is, apply it to the bottom surface of the hydrogel close to the tough layer, to obtain a cartilage-mimicking multifunctional drug-releasing hydrogel SD-PCG-A-21.

[0069] Test the properties of the cartilage-mimicking multifunctional drug-releasing hydrogel obtained in Example 17. The test results show that: the water content of the hydrogel obtained in Example 17 is 72%, the compressive strength is 40 MPa, the friction coefficient is 0.018, the adhesion strengths to bone and skin in the adhesion strength test are 80 kPa and 37 kPa respectively, it has the ability to scavenge free radicals and antibacterial ability, and the cell survival rate is greater than 95%, showing biocompatibility.

[0070] Example 18 Preparation of cartilage-mimicking multifunctional drug-releasing hydrogel Inject glue after heating a 10% gelatin aqueous solution at 45 °C for 1 h, and then freeze it in a refrigerator for 30 min to obtain a gelatin hydrogel; soak the gelatin hydrogel in a 1% grape polyphenol aqueous solution for 24 h to obtain a gelatin-grape polyphenol hydrogel. Melt the gelatin-grape polyphenol hydrogel in an oven at 60 °C to obtain an adhesive of gelatin and grape polyphenol, and apply it to the bottom layer of the prepared hydrogel SD-PCG-22, that is, apply it to the bottom surface of the hydrogel close to the tough layer, to obtain a cartilage-mimicking multifunctional drug-releasing hydrogel SD-PCG-A-22.

[0071] Test the properties of the cartilage-mimicking multifunctional drug-releasing hydrogel obtained in Example 18. The test results show that: the water content of the hydrogel obtained in Example 18 is 71%, the compressive strength is 40 MPa, the friction coefficient is 0.023, the adhesion strengths to bone and skin in the adhesion strength test are 77 kPa and 34 kPa respectively, it has the ability to scavenge free radicals and antibacterial ability, and the cell survival rate is greater than 95%, showing biocompatibility.

[0072] To facilitate the comparison of the properties of the cartilage-mimicking multifunctional drug-releasing hydrogel material obtained in the present invention with those of the precursor hydrogel, the performance data of the precursor hydrogels obtained in Examples 1 and 2 are summarized with the performance data of the cartilage-mimicking multifunctional drug-releasing hydrogels obtained in Examples 14-18, as shown in Table 2.

[0073] 。

[0074] The inventor team of the present invention adopted the above very ingenious method to integrate the conflicting properties of the hydrogel material into a hydrogel with a multi-layer structure, ensuring its water content while improving the strength of the hydrogel, achieving excellent lubrication performance while making it have excellent adhesion performance, and introducing drugs into the hydrogel through the method of hydrogel porosity or lecithin self-assembly. The preparation method is simple, and the hydrogel prepared in this study uses biocompatible polymer materials such as polyvinyl alcohol, chitosan and grape seed protein as the matrix and is constructed by physical cross-linking, avoiding the use of toxic chemical cross-linking agents and ensuring the biosafety of the material. The properties of the obtained hydrogel are superior to the comprehensive level of existing cartilage replacement hydrogel materials (see Tables 1 and 2 for details), which is of great significance for broadening the application of hydrogels in the field of artificial cartilage.

[0075] The above are only preferred embodiments of the present invention, and are not intended to limit the scope of the rights of the present invention. Any technical solution implemented within the scope of the rights covered by this patent application, or any person skilled in the art, using the disclosed method content to make many possible changes and modifications, all fall within the protection scope of the present invention.

Claims

1. A cartilage-mimicking multifunctional drug-releasing hydrogel material, characterized in that, Using polyvinyl alcohol, chitosan, grape seed protein, acyl chloride, sulfobetaine methacrylate, and lecithin as raw materials, a multi-layered hydrogel is prepared by mimicking the cartilage structure, with a mixture of gelatin and plant polyphenols used as an adhesive to load drugs.

2. A preparation method of a cartilage-mimicking multifunctional drug-release hydrogel material, characterized in that, It includes five steps: preparation of a high-strength anti-swelling precursor hydrogel, surface modification to introduce double bonds, interfacial polymerization to introduce poly-SBMA molecular brushes, drug loading, and preparation of the adhesion layer.

3. The preparation method of a cartilage-mimicking multifunctional drug-releasing hydrogel material according to claim 2, characterized in that, The specific operations are carried out according to the following steps: S01. Preparation of a high-strength anti-swelling precursor hydrogel 1) Heat 10 - 20 parts of polyvinyl alcohol, 1 - 5 parts of chitosan, and 180 - 190 parts of deionized water at 90 °C for 6 hours to obtain a homogeneous solution. Then, inject the obtained solution into a mold and let it stand at room temperature to remove air bubbles. Place it in a -20 °C freezer for 8 - 12 h, transfer it to room temperature and let it stand for 4 - 6 h, and perform freeze-thaw cycles 3 times to obtain a polyvinyl alcohol-chitosan pre-gel. 2) Remove the contained moisture from the above pre-gel by freeze-drying to obtain a polyvinyl alcohol-chitosan aerogel. 3) Immerse the above aerogel completely in an aqueous solution of grape seed protein that can form ionic bonds with chitosan for 3 h to prepare a high-strength anti-swelling precursor hydrogel crosslinked by multiple non-covalent bonds such as hydrogen bonds and ionic bonds, which constitutes the tough layer of the target hydrogel. S02. Surface modification to introduce double bonds Freeze-dry the high-strength anti-swelling precursor hydrogel obtained in step S01, and then float it in a mixed solution of dichloromethane and dimethyl sulfoxide containing acyl chloride for 12 h to introduce double bonds to the surface. After reacting at room temperature for 24 h, dialyze it in pure water for 3 - 7 days to remove residual chemicals to obtain a hydrogel with double bonds introduced by surface modification; the density of the mixed solution is greater than the density of the precursor hydrogel. S03. Interfacial polymerization to introduce poly-SBMA molecular brushes Coat the top layer of the hydrogel with double bonds introduced by surface modification obtained in step S02 with an aqueous solution of SBMA, add the photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, and irradiate the bottom of the hydrogel with ultraviolet light for 40 min for photoinitiation. Remove the excess SBMA with deionized water to obtain a hydrogel with poly-SBMA molecular brushes introduced by interfacial polymerization. S04. Drug loading 1) Remove the moisture from the hydrogel obtained in step S03 by freeze-drying to obtain an aerogel. 2) Prepare lecithin encapsulating hydrophobic drugs by the thin film hydration method: Dissolve lecithin and hydrophobic drugs in a conical flask containing dichloromethane and stir and mix them at room temperature for 3 h. Then, evaporate dichloromethane in a water bath at 45 °C until a coating is formed. Place the flask in a vacuum oven at 45 °C overnight to remove the residual solvent to form a dry film. Dissolve the dry film in deionized water and sonicate it to obtain a homogeneous aqueous solution, and then centrifuge it in a centrifuge for 15 minutes to remove the unsuccessfully encapsulated drugs. The supernatant after centrifugation is the lecithin aqueous dispersion. 3) Dissolve hydrophilic drugs in the lecithin aqueous dispersion, and then soak the above aerogel to obtain a hydrogel containing both hydrophilic drugs and hydrophobic drugs. S05. Preparation of the adhesion layer Dissolve the 10% gelatin aqueous solution at 45 °C for 1 h, then inject the glue, and then freeze it in the refrigerator for 30 min to obtain a gelatin hydrogel; soak the gelatin hydrogel in a 1% aqueous solution of plant polyphenols for 24 h to obtain a gelatin-plant polyphenol hydrogel; melt the gelatin-plant polyphenol hydrogel in an oven at 60 °C to obtain an adhesive of gelatin and plant polyphenols, and apply it to the bottom surface of the hydrogel containing both hydrophilic and hydrophobic drugs obtained in step S04 close to the tough layer to obtain a cartilage-mimicking multifunctional drug-releasing hydrogel.

4. The preparation method of a cartilage-mimicking multifunctional drug-releasing hydrogel material according to claim 3, characterized in that, The acyl chloride described in step S02 is any one or more of acryloyl chloride, methacryloyl chloride, crotonyl chloride, pentenoyl chloride, and cinnamoyl chloride.

5. The preparation method of a cartilage-mimicking multifunctional drug-releasing hydrogel material according to claim 3, characterized in that, The volume ratio of the two solvents, dichloromethane and dimethyl sulfoxide, used to prepare the mixed solution in step S02 is 1:1 - 9:

1.

6. The preparation method of a cartilage-mimicking multifunctional drug-releasing hydrogel material according to claim 3, characterized in that The hydrophobic drug described in step S04 is any one of ciprofloxacin and norfloxacin.

7. The preparation method of a cartilage-mimicking multifunctional drug-releasing hydrogel material according to claim 3, wherein The hydrophilic drug described in step S04 is any one of sodium salicylate and ascorbic acid.

8. The preparation method of a cartilage-mimicking multifunctional drug-releasing hydrogel material according to claim 3, wherein, The mass of the hydrophobic drug in step S04 is 0.5 - 6% of the mass of lecithin.

9. The preparation method of a cartilage-mimicking multifunctional drug-releasing hydrogel material according to claim 3, wherein, The mass fraction of the hydrophilic drug in step S04 is 5 - 10%.

10. The preparation method of a cartilage-mimicking multifunctional drug-releasing hydrogel material according to claim 3, characterized in that, The plant polyphenol described in step S05 is any one or more of tannic acid, grape polyphenols, and tea polyphenols.

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