Renewable boundary lubrication natural polyphenol anti-inflammatory hydrogel as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedical anti-inflammatory gels, and discloses a renewable boundary lubrication natural polyphenol anti-inflammatory hydrogel and a preparation method and application thereof.The hydrogel is of a hydrophilic viscoelastic sea island structure, gallic acid grafted gelatin blended with hyaluronic acid serves as a base material, celecoxib lipidosome is entrapped in the hydrogel, and the hydrogel is prepared from natural polyphenols, an articular cavity is injected through an enzyme crosslinking technology to form gel, hyaluronic acid and lipidosome form a hydrated lubricating layer on a cartilage-gel interface, and after the interface is abraded, internal lipidosome and hyaluronic acid are exposed to supplement lubrication so as to form sustainable lubrication; the liposome responds to friction shear crushing to release celecoxib and cooperates with gallic acid at an interface to resist inflammation progress and repair cartilage, the injectable hydrogel is developed based on gallic acid, hyaluronic acid, gelatin and celecoxib liposome to treat early osteoarthritis, mechanical lubrication and inflammation resistance are combined, and the preparation method has the advantages that the preparation method is simple, the preparation cost is low, and the application prospect is wide. The problems of large-dose toxicity, short lubrication period and the like caused by independent treatment are effectively solved, and the preparation has important clinical application significance.

Description

Technical Field

[0001] The present application belongs to the technical field of biomedical anti-inflammatory gels, and specifically relates to a renewable boundary-lubricating natural polyphenol anti-inflammatory hydrogel, and its preparation method and application. Background Art

[0002] Osteoarthritis is a chronic degenerative disease characterized by progressive degeneration of articular cartilage, synovial inflammation, and abnormal bone remodeling. WHO statistics show that osteoarthritis has a high incidence rate. Currently, there are more than 300 million patients worldwide, among which the elderly population over 65 years old is the susceptible group, and knee inflammation accounts for more than 80%, and it is showing a trend of younger age. Studies have shown that obesity, sports injuries and genetic mutations can lead to an earlier age of onset. The friction coefficient of normal joints is close to that of ice (μ≈0.03). The lubrication function of joints depends on the key lubricating molecules of synovial fluid and cartilage surface. Their failure is the core pathological mechanism of osteoarthritis. At the same time, due to the failure of lubrication, the friction coefficient of the joints increases by more than 10 times, and the wear of cartilage is accelerated, forming a vicious cycle of "tissue damage-inflammation-decreased lubrication-secondary damage-intensified inflammation".

[0003] Currently, the main clinical treatments for osteoarthritis are oral nonsteroidal anti-inflammatory drugs and intra-articular injections of hyaluronic acid. However, exogenous hyaluronic acid has a short half-life and a short mechanical lubrication effect. In addition, oral nonsteroidal anti-inflammatory drugs require large doses and pose a risk of gastrointestinal toxicity and cardiovascular damage.

[0004] Therefore, developing a new drug for treating osteoarthritis has great market prospects. Summary of the Invention

[0005] The purpose of this application is to solve the problems of the prior art and to provide a renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel and its preparation method and application.

[0006] In order to solve the technical problem, the technical solution of the present application is: a renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel, comprising gallic acid, hyaluronic acid, gelatin, celecoxib liposomes and a solvent, wherein the weight percentages of the gallic acid, hyaluronic acid, gelatin and celecoxib liposomes are 1~2wt%, 0.3~0.8wt%, 8~12wt% and 1~5wt% respectively, and the solvent makes up the balance; the celecoxib liposomes are composed of lecithin, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol and celecoxib, wherein the weight ratio of lecithin, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol and celecoxib is 15~25:2~6:1~5:1~5.

[0007] Preferably, the weight percentages of the gallic acid, hyaluronic acid, gelatin and celecoxib liposomes are 1.2wt%, 0.5wt%, 10wt% and 2wt% respectively, and the solvent makes up the balance.

[0008] Preferably, the weight ratio of lecithin, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol and celecoxib is 20:4:2:2.

[0009] Preferably, the solvent is sodium chloride solution, phosphate buffer or pure water.

[0010] Preferably, the preparation method of the renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel is used to prepare the above-mentioned renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel, comprising the following steps: Step 1: Activation of gallic acid: Ultrapure water and N,N-dimethylformamide are mixed evenly and fully released heat, the volume ratio of ultrapure water to N,N-dimethylformamide being 1.2-1.6:1, gallic acid and N-hydroxysuccinimide or 1-ethyl-(3-dimethylaminopropyl)carbodiimide are added, and then activated in an environment of 4-6°C for 4-12 hours under stirring conditions of 300-800 rpm. A nitrogen atmosphere is provided throughout the activation process to obtain activated gallic acid. The concentration of gallic acid in the activated gallic acid is 2-3 wt %, the concentration of N-hydroxysuccinimide is 2.5-3.5 wt %, and the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 2-2.5 wt %; Step 2: Preparation of gallic acid-modified gelatin: A gelatin solution with a concentration of 5 wt% was mixed with the gallic acid activated in step 1 at a volume ratio of 1 to 2:1. The mixture was reacted at 40° C. under a nitrogen atmosphere for 12 to 24 hours with a stirring speed of 800 to 1000 rpm. The reaction solution was collected and dialyzed at room temperature for 6 to 10 days to remove unreacted molecules. The mixture was then freeze-dried and collected to obtain gallic acid-modified gelatin GAG. Step 3: Preparation of natural polyphenol anti-inflammatory hydrogel: Gallic acid-modified gelatin (GAG) and hyaluronic acid are dissolved in a solvent at a weight ratio of 8-12:0.3-0.8, and the dissolution temperature is <55°C. 0.5 wt% transaminase is added to the mixed system, and gelation is carried out at 37°C for 3-5 minutes. Unreacted molecules are removed by dialysis at room temperature for 4-14 days, and the mixture is freeze-dried and collected to obtain the natural polyphenol anti-inflammatory hydrogel (GAGgel). Step 4: Preparation of celecoxib liposomes: Phosphatidylcholine, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol, and celecoxib were dissolved in chloroform at a weight ratio of 15-25:2-6:1-5:1-5; chloroform was removed by rotary evaporation at 37°C, and the mixture was hydrated with solvent and ultrasonically disrupted to obtain a celecoxib liposome dispersion CLP, which was then concentrated to a final concentration of 1-5 wt% by ultrafiltration and centrifugation; Step 5: Preparation of renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel; dissolve the natural polyphenol anti-inflammatory hydrogel GAGgel into the celecoxib liposome dispersion CLP and mix thoroughly, the dissolution temperature is <45°C, then add 0.5wt% transaminase, and heat at 37°C for 3~5 minutes to obtain the renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel GAGgel@CLP, wherein the weight percentages of gallic acid, hyaluronic acid, gelatin and celecoxib liposomes are 1~2wt%, 0.3~0.8wt%, 8~12wt% and 1~5wt%, respectively, and the balance is solvent, which is sodium chloride solution, phosphate buffer or pure water.

[0011] Compared with the prior art, the advantages of this application are: (1) This application proposes a renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel with a hydrophilic viscoelastic island structure. The substrate is a gallic acid grafted gelatin blended with hyaluronic acid, and celecoxib liposomes are encapsulated inside. It is injected into the joint cavity to form a gel through enzyme crosslinking technology. The hyaluronic acid and liposomes form a hydrated lubricating layer at the cartilage-gel interface. After the interface is worn, the internal liposomes and hyaluronic acid are exposed to replenish lubrication to form sustainable lubrication. The liposomes respond to friction shear and break to release celecoxib, which cooperates with gallic acid at the interface to resist the progression of inflammation and repair cartilage. This application develops an injectable hydrogel for the treatment of early osteoarthritis based on gallic acid, hyaluronic acid, gelatin and celecoxib liposomes, which combines mechanical lubrication and inflammation resistance, effectively overcoming the problems of high-dose toxicity and short lubrication cycle caused by single treatment, and has important clinical application significance. (2) The surface of the celecoxib liposomes of the present application contains a large number of phosphatidyl hydrophilic groups, which can tightly bind to water molecules to form a hydration film. Water molecules can move relatively freely within the hydration film, thereby producing a lubricating effect at the interface. At the same time, the celecoxib liposomes respond to friction and are exposed to the cartilage surface, thereby achieving the anti-inflammatory purpose; (3) This application uses enzyme-crosslinked gallic acid gelatin as a substrate to encapsulate high-concentration liposomes and hyaluronic acid. The hyaluronic acid and liposomes form a hydrated lubricating layer at the cartilage-gel interface, and after the interface is worn, the internal liposomes and hyaluronic acid are exposed to replenish the hydration layer to form a regenerative lubrication layer; (4) The renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel prepared in this application has good anti-inflammatory function and lubricating effect. The hydrogel first exposes gallic acid to eliminate reactive oxygen species to avoid oxidative stress, and then responds to friction shear to release anti-inflammatory drugs in a sequential treatment strategy. This strategy is consistent with the pathogenesis of early osteoarthritis, especially traumatic osteoarthritis. The hydrogel has good biocompatibility and injectability, and is suitable for the treatment of early osteoarthritis, especially traumatic osteoarthritis. (5) The preparation method of the present application is simple in process and low in cost. The use of transaminase as a cross-linking agent avoids the cytotoxicity caused by traditional photocuring agents and improves injectability. The use of liposome-loaded non-steroidal anti-inflammatory drug celecoxib in conjunction with gallic acid-gelatin in situ injection greatly reduces the dosage of the drug and improves the utilization rate, avoiding gastrointestinal damage and cardiovascular harm caused by oral administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a process flow chart of a method for preparing a renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel for this application; Figure 2 This is the H NMR spectrum of the gallic acid modified gelatin GAG in Example 1 of the present application. 1 HNMR and FTIR spectroscopy results; Figure 3 The transmission electron microscope image and particle size and distribution diagram of the celecoxib liposome dispersion CLP in Example 2 of the present application; Figure 4 Scanning electron microscope images, mechanical property diagrams, and swelling diagrams of the natural polyphenol anti-inflammatory hydrogel GAGgel and the renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel GAGgel@CLP in Examples 1 and 3 of the present application; Figure 5 Graphs showing the friction coefficients of the natural polyphenol anti-inflammatory hydrogel GAGgel and the renewable boundary lubrication natural polyphenol anti-inflammatory hydrogel GAGgel@CLP in Examples 1 and 3 of the present application; Figure 6 The cytotoxicity and hemolysis detection graphs of the natural polyphenol anti-inflammatory hydrogel GAGgel and the renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel GAGgel@CLP in Examples 1 and 3 of the present application are shown; Figure 7 Flow cytometry images of the natural polyphenol anti-inflammatory hydrogel GAGgel and the renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel GAGgel@CLP in Examples 1 and 3 of the present application. DETAILED DESCRIPTION

[0013] The present application is described in detail below with reference to the accompanying drawings and specific examples, but the present application is not limited to these examples. This application covers any substitutions, modifications, equivalent methods, and solutions made within the spirit and scope of this application. To provide a thorough understanding of this application, specific details are described in detail in the following examples of this application, but those skilled in the art can fully understand this application without these detailed descriptions.

[0014] This application proposes a renewable boundary-lubricating natural polyphenol anti-inflammatory hydrogel, which has a hydrophilic viscoelastic island structure. Gallic acid molecules are coupled to the gelatin side chains through acylation, and celecoxib liposomes are exposed to the cartilage surface in response to friction to achieve the anti-inflammatory purpose. At the same time, hyaluronic acid and liposomes form a hydrated lubricating layer at the cartilage-gel interface, which plays a lubricating role.

[0015] The natural polyphenol compound gallic acid proposed in this application can efficiently scavenge ROS to resist oxidative stress, and target multiple targets of the NF-κB / MAPK pathway to inhibit the inflammatory cascade reaction; gelatin contains an arginine-glycine-aspartic acid sequence, which is conducive to cell attachment and growth, and the presence of a large number of amino groups also provides the possibility for gallic acid modification; celecoxib is a non-steroidal anti-inflammatory drug that specifically inhibits COX-2 enzyme activity and reduces prostaglandin E2 production; liposomes are assembled from phospholipids, cholesterol and other components, and have a composition, drug loading capacity and hydration and lubrication potential similar to cell membranes.

[0016] The present application discloses a renewable boundary-lubricating natural polyphenol anti-inflammatory hydrogel, comprising gallic acid, hyaluronic acid, gelatin, celecoxib liposomes and a solvent, wherein the weight percentages of the gallic acid, hyaluronic acid, gelatin and celecoxib liposomes are 1-2wt%, 0.3-0.8wt%, 8-12wt% and 1-5wt%, respectively, and the solvent makes up the balance; the celecoxib liposomes are composed of lecithin, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol and celecoxib, wherein the weight ratio of lecithin, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol and celecoxib is 15-25:2-6:1-5:1-5.

[0017] Preferably, the weight percentages of the gallic acid, hyaluronic acid, gelatin and celecoxib liposomes are 1.2wt%, 0.5wt%, 10wt% and 2wt% respectively, and the solvent makes up the balance.

[0018] Preferably, the weight ratio of lecithin, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol and celecoxib is 20:4:2:2.

[0019] Preferably, the solvent is sodium chloride solution, phosphate buffer or pure water.

[0020] like Figure 1 As shown, preferably, the preparation method of the renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel is used to prepare the above-mentioned renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel, comprising the following steps: Step 1: Activation of gallic acid: Ultrapure water and N,N-dimethylformamide are mixed evenly and fully released heat, the volume ratio of ultrapure water to N,N-dimethylformamide being 1.2-1.6:1, gallic acid and N-hydroxysuccinimide or 1-ethyl-(3-dimethylaminopropyl)carbodiimide are added, and then activated in an environment of 4-6°C for 4-12 hours under stirring conditions of 300-800 rpm. A nitrogen atmosphere is provided throughout the activation process to obtain activated gallic acid. The concentration of gallic acid in the activated gallic acid is 2-3 wt %, the concentration of N-hydroxysuccinimide is 2.5-3.5 wt %, and the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 2-2.5 wt %; Step 2: Preparation of gallic acid-modified gelatin: A gelatin solution with a concentration of 5 wt% was mixed with the gallic acid activated in step 1 at a volume ratio of 1 to 2:1. The mixture was reacted at 40° C. under a nitrogen atmosphere for 12 to 24 hours with a stirring speed of 800 to 1000 rpm. The reaction solution was collected and dialyzed at room temperature for 6 to 10 days to remove unreacted molecules. The mixture was then freeze-dried and collected to obtain gallic acid-modified gelatin GAG. Step 3: Preparation of natural polyphenol anti-inflammatory hydrogel: Gallic acid-modified gelatin (GAG) and hyaluronic acid are dissolved in a solvent at a weight ratio of 8-12:0.3-0.8, and the dissolution temperature is <55°C. 0.5 wt% transaminase is added to the mixed system, and gelation is carried out at 37°C for 3-5 minutes. Unreacted molecules are removed by dialysis at room temperature for 4-14 days, and the mixture is freeze-dried and collected to obtain the natural polyphenol anti-inflammatory hydrogel (GAGgel). Step 4: Preparation of celecoxib liposomes: Phosphatidylcholine, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol, and celecoxib were dissolved in chloroform at a weight ratio of 15-25:2-6:1-5:1-5; chloroform was removed by rotary evaporation at 37°C, and the mixture was hydrated with solvent and ultrasonically disrupted to obtain a celecoxib liposome dispersion CLP, which was then concentrated to a final concentration of 1-5 wt% by ultrafiltration and centrifugation; Step 5: Preparation of renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel; dissolve the natural polyphenol anti-inflammatory hydrogel GAGgel into the celecoxib liposome dispersion CLP and mix thoroughly, the dissolution temperature is <45°C, then add 0.5wt% transaminase, and heat at 37°C for 3~5 minutes to obtain the renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel GAGgel@CLP, wherein the weight percentages of gallic acid, hyaluronic acid, gelatin and celecoxib liposomes are 1~2wt%, 0.3~0.8wt%, 8~12wt% and 1~5wt%, respectively, and the balance is solvent, which is sodium chloride solution, phosphate buffer or pure water.

[0021] Preferably, the renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel is used, and the renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel is used for the treatment of osteoarthritis.

[0022] Example 1: Preparation of natural polyphenol anti-inflammatory hydrogel GAGgel; Activation of Gallic Acid: Mix 38 ml of ultrapure water and 25 ml of N,N-dimethylformamide until fully exothermic. Add gallic acid and N-hydroxysuccinimide or 1-ethyl-(3-dimethylaminopropyl)carbodiimide at concentrations of 2.5 wt%, 2.7 wt%, and 2.3 wt%, respectively. Activate at 4°C with stirring at 600 rpm for 12 h. A nitrogen atmosphere was maintained throughout the activation process.

[0023] Preparation of gallic acid modified gelatin GAG: A gelatin solution with a concentration of 5 wt % was mixed with the activated gallic acid at a volume ratio of 2:1, and reacted at 40° C. for 10 hours under a nitrogen atmosphere with a stirring speed of 800 rpm.

[0024] The reaction solution was collected, dialyzed at room temperature for 6-10 min to remove unreacted molecules, and then freeze-dried and collected.

[0025] like Figure 2 As shown, Figure 2 (a) and (b) are blank gelatin and gallic acid modified gelatin respectively. 1 HNMR results, where Figure 2 In (b), a characteristic peak generated by the ortho-position hydrogen of the gallic acid benzene ring can be observed at 7.0 ppm, proving that the modification was successful. Figure 2 (c) is the Fourier transform infrared spectrum result at 3500cm -1 A broad peak due to the stretching vibration of phenolic hydroxyl groups can be observed, further confirming the successful modification of gelatin by gallic acid.

[0026] The gallic acid-modified gelatin (10 wt%) and hyaluronic acid (0.5 wt%) were dissolved in a 0.9% sodium chloride solution at a temperature <55°C. 0.5 wt% transaminase was added to the mixture, and the mixture was gelled at 37°C for 3-5 minutes to obtain the natural polyphenol anti-inflammatory hydrogel GAGgel.

[0027] like Figure 4 As shown, Figure 4 The left image in (a) is a scanning electron microscope image of the natural polyphenol anti-inflammatory hydrogel GAGgel. The hydrogel shows a continuous porous structure with a pore size of 5~50μm, which will provide sufficient internal space for cell growth and penetration as well as drug transport. Figure 4The black line shown in (b) is the mechanical properties of the natural polyphenol anti-inflammatory hydrogel GAGgel. After the hydrogel system is doped with hyaluronic acid, a special semi-interpenetrating structure of linear molecules running through the three-dimensional network is formed. At the same time, the strongly hydrated hyaluronic acid molecules shorten the distance between the polymer molecular chains, strengthen the hydrogen bonding effect, and increase the storage modulus of the natural polyphenol anti-inflammatory hydrogel GAGgel to 10kPa. Figure 4 As shown in (c), the swelling rate of the natural polyphenol anti-inflammatory hydrogel GAGgel is shown. The hydrogel reaches swelling equilibrium within 24 hours, with a maximum swelling rate of approximately 15%. This low swelling behavior is conducive to maintaining the gel structure and providing stable drug release in the joints.

[0028] like Figure 5 As shown in the figure, the friction behavior of the natural polyphenol anti-inflammatory hydrogel GAGgel is demonstrated. The gel block is loaded into a liquid pool of 3cm*3cm*0.5cm in size, and the probe is loaded with a polyethylene ball with a diameter of 8mm to perform a linear reciprocating friction test under a load of 1N. Figure 5 It is shown that the natural polyphenol anti-inflammatory hydrogel GAGgel described in the present application reduces the friction coefficient from 0.23 (blank group) to 0.1 (Example 1), and has excellent lubrication performance.

[0029] like Figure 6 As shown, Figure 6 (a) shows that the cells still maintained a survival rate of more than 90% after being co-incubated with the natural polyphenol anti-inflammatory hydrogel GAGgel. Figure 6 Middle (b) shows that there is no obvious hemolysis after the natural polyphenol anti-inflammatory hydrogel GAGgel is co-incubated with 2% packed red blood cells, indicating that the natural polyphenol anti-inflammatory hydrogel GAGgel has good biocompatibility.

[0030] like Figure 7 As shown, in the Raw 264.7 macrophage inflammation model constructed with lipopolysaccharide, compared with the blank group, LPS inflammation group and inflammation + GAGgel treatment, co-incubation with the natural polyphenol anti-inflammatory hydrogel GAGgel significantly promoted the downregulation of the proportion of M1 type macrophages and avoided inflammatory polarization.

[0031] Example 2: Preparation of Celecoxib Liposome Dispersion CLP for Knee Joint Injection; Celecoxib liposomes were prepared by a thin film hydration method. Phosphatidylcholine, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG2000), and celecoxib were dissolved in chloroform at a mass ratio of 20:4:2:2. The chloroform was removed by rotary evaporation at 37°C and the liposomes were hydrated with 0.9% sodium chloride solution. Celecoxib liposome dispersion CLP was obtained by ultrasonication and concentrated to a final concentration of 1-5 wt% by ultrafiltration and centrifugation.

[0032] like Figure 3 As shown, Figure 3 (a) is a transmission electron microscope image of celecoxib liposome dispersion CLP. Figure 3 (b) shows the particle size distribution of celecoxib liposome dispersion CLP.

[0033] Example 3: Preparation of renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel GAGgel@CLP; The above-mentioned natural polyphenol anti-inflammatory hydrogel GAGgel (10wt%) and hyaluronic acid (0.5wt%) were dissolved in the celecoxib liposome dispersion CLP and mixed thoroughly. The dissolution temperature was <45°C. 0.5wt% transaminase was added to the above-mentioned mixed system and gelled at 37°C for 3-5 minutes.

[0034] like Figure 4 As shown, Figure 4 The right image of (a) is an electron microscope image of the renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel GAGgel@CLP. Figure 4 The red line shown in (b) represents the mechanical properties of the renewable boundary lubricated natural polyphenol anti-inflammatory hydrogel GAGgel@CLP.

[0035] contrast Figure 4 In (b), GAGgel and GAGgel@CLP, liposomes are uniformly incorporated into the hydrogel as nanoparticles to disperse stress when subjected to force, thereby avoiding damage to the hydrogel structure caused by local stress concentration. As a result, the storage modulus of the renewable boundary lubricated natural polyphenol anti-inflammatory hydrogel GAGgel@CLP is further increased to 12kPa, which can meet the mechanical support requirements of the cartilage surface.

[0036] like Figure 5 The figure shows the tribological behavior of the renewable boundary-lubricating natural polyphenol anti-inflammatory hydrogel GAGgel@CLP. The gel block was loaded into a 3cm*3cm*0.5cm liquid pool. A linear reciprocating friction test was performed under a 1N load using an 8mm diameter polyethylene ball attached to the probe. As shown in the figure, the renewable boundary-lubricating natural polyphenol anti-inflammatory hydrogel GAGgel@CLP described in this application reduced the friction coefficient from 0.23 (blank group) to 0.02 (Example 3), demonstrating significant lubrication properties and potential for joint lubrication.

[0037] like Figure 6 As shown, Figure 6 (a) shows that the cells still maintained a survival rate of more than 85% after being co-incubated with the renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel GAGgel@CLP. Figure 6Middle (b) shows that there is no obvious hemolysis after the renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel GAGgel@CLP is co-incubated with 2% packed red blood cells, indicating that the renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel GAGgel@CLP has good biocompatibility.

[0038] like Figure 7 As shown, in the Raw 264.7 macrophage inflammation model constructed with lipopolysaccharide, compared with the blank group, LPS inflammation group, inflammation + GAGgel treatment and inflammation + GAGgel@CLP treatment, the renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel GAGgel@CLP significantly promoted the downregulation of the proportion of M1 type macrophages and avoided inflammatory polarization after co-incubation, which is suitable for the treatment of osteoarthritis, especially traumatic osteoarthritis.

[0039] Experimental verification shows that the renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel of the present application has anti-inflammatory and lubricating effects in the range of 1-2wt% gallic acid, 0.3-0.8wt% hyaluronic acid, 8-12wt% gelatin, 1-5wt% celecoxib liposomes and the remainder of the solvent.

[0040] The present application proposes a renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel with a hydrophilic viscoelastic island structure. The substrate is gallic acid grafted gelatin blended with hyaluronic acid, and celecoxib liposomes are encapsulated inside. The hydrogel is injected into the joint cavity through enzyme cross-linking technology to form a gel. The hyaluronic acid and liposomes form a hydrated lubricating layer at the cartilage-gel interface. After the interface is worn, the internal liposomes and hyaluronic acid are exposed to supplement the lubrication to form sustainable lubrication. The liposomes respond to friction and shear to break and release celecoxib to cooperate with the gallic acid at the interface to resist the progression of inflammation and repair cartilage. The present application develops an injectable hydrogel for the treatment of early osteoarthritis based on gallic acid, hyaluronic acid, gelatin and celecoxib liposomes, which combines mechanical lubrication and inflammation resistance to effectively overcome the problems of high-dose toxicity and short lubrication cycle caused by single treatment, and has important clinical application significance.

[0041] The surface of the celecoxib liposomes of the present application contains a large number of phosphatidyl hydrophilic groups, which can tightly bind to water molecules to form a hydration film. Water molecules can move relatively freely within the hydration film, thereby producing a lubricating effect at the interface. At the same time, the celecoxib liposomes respond to friction and are exposed to the cartilage surface, thereby achieving the anti-inflammatory purpose.

[0042] This application uses enzyme-cross-linked gallic acid gelatin as a base material to encapsulate high-concentration liposomes and hyaluronic acid. The hyaluronic acid and liposomes form a hydrated lubricating layer at the cartilage-gel interface, and after the interface is worn, the internal liposomes and hyaluronic acid are exposed to replenish the hydration layer to form renewable lubrication.

[0043] The renewable boundary-lubricating natural polyphenol anti-inflammatory hydrogel prepared in this application has good anti-inflammatory function and lubricating effect. The hydrogel first exposes gallic acid to eliminate reactive oxygen to avoid oxidative stress, and then responds to friction shear to release anti-inflammatory drugs in a sequential treatment strategy. This strategy is consistent with the pathogenesis of early osteoarthritis, especially traumatic osteoarthritis. The hydrogel has good biocompatibility and injectability, and is suitable for the treatment of early osteoarthritis, especially traumatic osteoarthritis.

[0044] The preparation method of the present application has a simple process and low cost. The use of transaminase as a cross-linking agent avoids the cytotoxicity caused by traditional photocuring agents and improves injectability. The use of liposome-loaded non-steroidal anti-inflammatory drug celecoxib in conjunction with gallic acid-gelatin in situ injection greatly reduces the dosage of the drug and improves the utilization rate, avoiding gastrointestinal damage and cardiovascular hazards caused by oral administration.

[0045] The preferred embodiments of the present application have been described in detail above, but the present application is not limited to the above embodiments. Various changes can be made within the scope of knowledge possessed by ordinary technicians in this field without departing from the purpose of the present application.

[0046] Many other changes and modifications can be made without departing from the concept and scope of the present application. It should be understood that the present application is not limited to the specific embodiments, and the scope of the present application is defined by the appended claims.

Claims

1. Renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel, characterized by: The invention comprises gallic acid, hyaluronic acid, gelatin, celecoxib liposomes and a solvent, wherein the weight percentages of the gallic acid, hyaluronic acid, gelatin and celecoxib liposomes are 1-2wt%, 0.3-0.8wt%, 8-12wt% and 1-5wt% respectively, and the solvent makes up the balance. The celecoxib liposomes are composed of lecithin, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol and celecoxib, wherein the weight ratio of lecithin, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol and celecoxib is 15-25:2-6:1-5:1-5.

2. The renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel according to claim 1, characterized in that: The weight percentages of the gallic acid, hyaluronic acid, gelatin and celecoxib liposomes are 1.2wt%, 0.5wt%, 10wt% and 2wt% respectively, and the solvent makes up the balance.

3. The renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel according to claim 1, characterized in that: The weight ratio of the lecithin, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol and celecoxib is 20:4:2:

2.

4. The renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel according to claim 1, characterized in that: The solvent is sodium chloride solution, phosphate buffer or pure water.

5. A method for preparing a renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel, characterized in that: The method for preparing the renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel according to any one of claims 1 to 4 comprises the following steps: Step 1: Activation of gallic acid: Ultrapure water and N,N-dimethylformamide are mixed evenly and fully released heat, the volume ratio of ultrapure water to N,N-dimethylformamide being 1.2-1.6:1, gallic acid and N-hydroxysuccinimide or 1-ethyl-(3-dimethylaminopropyl)carbodiimide are added, and then activated in an environment of 4-6°C for 4-12 hours under stirring conditions of 300-800 rpm. A nitrogen atmosphere is provided throughout the activation process to obtain activated gallic acid. The concentration of gallic acid in the activated gallic acid is 2-3 wt %, the concentration of N-hydroxysuccinimide is 2.5-3.5 wt %, and the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 2-2.5 wt %; Step 2: Preparation of gallic acid-modified gelatin: A gelatin solution with a concentration of 5 wt% was mixed with the gallic acid activated in step 1 at a volume ratio of 1 to 2:

1. The mixture was reacted at 40° C. under a nitrogen atmosphere for 12 to 24 hours with a stirring speed of 800 to 1000 rpm. The reaction solution was collected and dialyzed at room temperature for 6 to 10 days to remove unreacted molecules. The mixture was then freeze-dried and collected to obtain gallic acid-modified gelatin GAG. Step 3: Preparation of natural polyphenol anti-inflammatory hydrogel: Gallic acid-modified gelatin (GAG) and hyaluronic acid are dissolved in a solvent at a weight ratio of 8-12:0.3-0.8, and the dissolution temperature is <55°C. 0.5 wt% transaminase is added to the mixed system, and gelation is carried out at 37°C for 3-5 minutes. Unreacted molecules are removed by dialysis at room temperature for 4-14 days, and the mixture is freeze-dried and collected to obtain the natural polyphenol anti-inflammatory hydrogel (GAGgel). Step 4: Preparation of celecoxib liposomes: Phosphatidylcholine, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol, and celecoxib were dissolved in chloroform at a weight ratio of 15-25:2-6:1-5:1-5; chloroform was removed by rotary evaporation at 37°C, and the mixture was hydrated with solvent and ultrasonically disrupted to obtain a celecoxib liposome dispersion CLP, which was then concentrated to a final concentration of 1-5 wt% by ultrafiltration and centrifugation; Step 5: Preparation of renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel; dissolve the natural polyphenol anti-inflammatory hydrogel GAGgel into the celecoxib liposome dispersion CLP and mix thoroughly, the dissolution temperature is <45°C, then add 0.5wt% transaminase, and heat at 37°C for 3~5 minutes to obtain the renewable boundary lubricating natural polyphenol anti-inflammatory hydrogel GAGgel@CLP, wherein the weight percentages of gallic acid, hyaluronic acid, gelatin and celecoxib liposomes are 1~2wt%, 0.3~0.8wt%, 8~12wt% and 1~5wt%, respectively, and the balance is solvent, which is sodium chloride solution, phosphate buffer or pure water.

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