Anti-inflammatory injectable enzyme-containing hydrogel as well as preparation method and application thereof

Abstract

The invention discloses an anti-inflammatory injectable enzyme-containing hydrogel and a preparation method and application thereof.The enzyme-containing hydrogel takes cascade enzyme catalytic reaction of galactose oxidase and catalase as an initiation system, takes guar gum and ethylene glycol chitosan as gelling units, and takes a cross-linking agent as a cross-linking agent to prepare the anti-inflammatory injectable enzyme-containing hydrogel. The enzyme-containing hydrogel is prepared by a gel forming method combining cascade enzyme catalysis and polysaccharide molecule dynamic crosslinking. The enzyme-containing hydrogel prepared by the preparation method disclosed by the invention has the capabilities of removing and inhibiting various active oxygen and active nitrogen, can play an effective anti-inflammatory role in an inflammatory microenvironment, further has the advantages of simple preparation method, mild reaction conditions, good biocompatibility and self-repairing capability, and is a bioactive gel material with great prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of hydrogels, and in particular relates to an anti-inflammatory injectable enzyme-containing hydrogel and a preparation method and application thereof. Background Art

[0002] Hydrogel is a type of soft and wet material that swells in water and contains a large amount of water. It has a three-dimensional porous network structure and good biocompatibility. It has been widely used in the fields of tissue engineering, wound repair, drug sustained release, bioimaging, biosensing, etc. Hydrogels can be generally divided into physical hydrogels and chemical hydrogels. Chemical hydrogels are a type of hydrogel that is cross-linked by forming covalent bonds in the network, including traditional covalent bonds and dynamic covalent bonds. Due to the difference in bond energy, the hydrogel formed by dynamic covalent cross-linking is different from the covalent bond hydrogel in the traditional sense. The reversible dynamic covalent bond gives the gel many functionalities such as injectability, self-healing, and stimulus responsiveness. Injectable hydrogels are a type of hydrogel that can be extruded through tiny needles and micropores and quickly formed in situ at the target site. They often have the property of shear thinning and are often used as ideal tissue filling materials. They can also be applied to 3D printing technology, which is of great significance.

[0003] Enzymes are a class of active macromolecules with biocatalytic capabilities. They can dynamically regulate the synthesis of substances, energy conversion and immune defense in cell metabolism by specifically catalyzing target molecules to generate the desired products. They are very suitable for conducting treatment research on many diseases. Especially for diseases related to redox homeostasis imbalance such as oxidative stress and cytokine storm, the levels of reactive oxygen and reactive nitrogen free radicals in the pathological environment can be flexibly changed through the action of oxidoreductases to reverse oxidative damage, thereby achieving the effect of treating the disease. Some oxidoreductase cascade systems, such as D-amino acid oxidase / catalase, urate oxidase / catalase, xanthine oxidase / catalase, etc., can remove excess free radicals, regulate the redox balance of the microenvironment of living organisms, directly or indirectly protect tissues and organs from oxidative stress damage, and can also regulate a variety of cell signaling pathways to achieve anti-inflammatory effects.

[0004] In enzyme therapy, since the enzyme is directly injected into the tissue, the enzyme will be quickly degraded by substances in the surrounding environment and lose its activity, which greatly reduces its therapeutic effect. It will also trigger an immune response and pose a risk to the living organism. Therefore, it is necessary to make a suitable carrier scaffold for the enzyme. Among the existing biomaterials, hydrogels are similar to extracellular matrix in structure and function, and have been used in clinical research fields such as regenerative medicine and drug therapy. Hydrogels can provide enzymes with a biomimetic cell membrane and organelle membrane structure, which is very suitable for encapsulation, protection and stabilization of enzymes, and is therefore an excellent carrier for enzyme immobilization. Enzyme-containing hydrogels have shown great application value in biomedical fields such as tissue engineering, drug sustained release and disease diagnosis and treatment due to their unique biosafety, bioresponsiveness and functionality to the physiological environment. In addition, enzyme-containing hydrogels also have many potential characteristics and values ​​waiting for people to develop and use.

[0005] Jin Woo Bae et al. mixed phenol-rich polymers with horseradish peroxidase (HRP) and hydrogen peroxide in an aqueous medium to form a hydrogel in situ. By changing the concentrations of HRP and hydrogen peroxide, the various properties of the hydrogel can be controlled (Jin Woo Bae, Jong Hoon Choi, Yunki Lee, et al,. Journal of TissueEngineering and Regenerative Medicine ( IF 3.1 ) Pub Date : 2014-06-12).

[0006] Chinese patent CN110404083B discloses an injectable hydrogel material and its preparation method and application. The injectable hydrogel material is generated in situ by injecting components A and B in equal volumes using a double injection process; the component A is carboxymethyl chitosan, and the component B is obtained by reacting a dodecylated polysaccharide with an auxiliary cross-linking agent and an aldehyde-containing contrast agent. The material has the functions of good motion tolerance, biocompatibility and biodegradability, multimodal development, and controllable degradation.

[0007] However, the current injectable hydrogels have complex ingredients and complicated preparation steps, are prone to side reactions, and produce byproducts that affect production efficiency, and cannot take into account both antioxidant and anti-inflammatory properties. Therefore, it is necessary to develop an injectable hydrogel that can take into account both antioxidant and anti-inflammatory effects. Summary of the invention

[0008] In view of the above technical problems, the present invention provides an anti-inflammatory injectable enzyme-containing hydrogel and a preparation method and application thereof. The enzyme-containing hydrogel is prepared by a gelling method using a cascade enzyme catalysis reaction of galactose oxidase and catalase as an initiating system, guar gum and ethylene glycol chitosan as gelling units, and a cascade enzyme catalysis combined with dynamic cross-linking of polysaccharide molecules. The enzyme-containing hydrogel has the advantages of antioxidant regulation, anti-inflammatory, and injectability.

[0009] In order to achieve the above object, the present invention provides an anti-inflammatory injectable enzyme-containing hydrogel, which comprises the following raw materials in percentage by mass concentration: 0.10-0.75w / v% guar gum, 0.65-2.60w / v% ethylene glycol chitosan, 0.03-0.43w / v% complex enzyme, and the balance is solvent.

[0010] Preferably, the complex enzyme consists of galactose oxidase and catalase.

[0011] Further preferably, the mass ratio of the galactose oxidase to the catalase is 3:4.

[0012] More preferably, the activity of the catalase is 100-110 times that of the galactose oxidase; and the activity of the galactose oxidase is 5-10 U.

[0013] Preferably, the solvent is water or PBS solution.

[0014] Further preferably, the concentration of the PBS solution is 0.01 mol / L.

[0015] Preferably, the polymerization degree of the ethylene glycol chitosan is greater than 400.

[0016] The present invention also provides a method for preparing an anti-inflammatory injectable enzyme-containing hydrogel, comprising the following steps: (1) dissolving guar gum and ethylene glycol chitosan in a solvent to obtain a mixed solution; (2) Add the complex enzyme into the mixed solution, mix well and react to obtain the enzyme-containing hydrogel.

[0017] Preferably, the reaction temperature in step (2) is 25-37°C.

[0018] The present invention also provides an application of an anti-inflammatory injectable enzyme-containing hydrogel, which is an in vivo anti-inflammatory support material and 3D printing biological ink.

[0019] Preferably, the in vivo anti-inflammatory support material is injected into the body by injection; and the 3D printing bio-ink is formed by extruding the enzyme-containing hydrogel through micropores.

[0020] The beneficial effects of the present invention are: 1. The hydrogel prepared with guar gum and ethylene glycol chitosan as gelling units has a three-dimensional network structure whose cross-linking point is a Schiff base bond, which is a dynamic covalent bond. It breaks when subjected to external force, and can be reformed in a new equilibrium state after the gel is deformed. Therefore, the prepared hydrogel is injectable, self-healing, and stimuli-responsive. It can be injected into the human body to form a hydrogel in the body as a supporting material.

[0021] 2. Galactose oxidase and catalase are used as the cascade enzyme catalytic initiation system to initiate the formation of Schiff base dynamic covalent bonds between guar gum and glycol chitosan, thereby constructing a three-dimensional network structure of the hydrogel at 25-37°C, achieving room temperature gelation and in vivo gelation, thereby achieving local injection and in situ molding, and at the same time having self-repair capabilities.

[0022] 3. After the hydrogel is formed, the complex enzyme (galactose oxidase and catalase) is fixed in situ in the three-dimensional network, so it can remove and inhibit a variety of reactive oxygen and reactive nitrogen, and downregulate the level of cellular inflammatory factors, and has antioxidant regulation and anti-inflammatory properties.

[0023] 4. The enzyme-containing hydrogel prepared by the present invention has a certain resistance to external influences. When biological fluids such as experimental mouse blood, synovial fluid, etc. are added before the enzyme-containing hydrogel is gelled, the gelation process is not affected, and the biological fluid can be evenly dispersed in the hydrogel.

[0024] 5. The present invention can directly prepare enzyme-containing hydrogel by a one-pot method after mixing various raw materials, and has the advantages of simple raw materials, few by-products, controllable gelation process and oxidation process, mild reaction conditions and good enzyme-containing hydrogel effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of the gelling method of the present invention.

[0026] Figure 2 G prepared in Example 10 0.50 C 1.30 Fourier transform infrared spectrum of @GAO / CAT hydrogel.

[0027] Figure 3 G prepared in Example 2 0.10 C 1.30 @GAO / CAT hydrogel and G prepared in Example 10 0.50 C 1.30 @GAO / CAT hydrogel microscopic morphology, A in the figure is G 0.10 C 1.30 @GAO / CAT hydrogel, B is G 0.50 C 1.30@GAO / CAT hydrogel.

[0028] Figure 4 G prepared in Example 10 0.50 C 1.30 @GAO / CAT hydrogel rheological diagram of the gelation process. The intersection of G' and G'' in the figure represents the gelation time.

[0029] Figure 5 G prepared in Example 10 0.50 C 1.30 @GAO / CAT hydrogel and ABTS in solution •+ UV-visible absorption spectrum of free radical action.

[0030] Figure 6 G prepared in Example 10 0.50 C 1.30 @The fluorescence spectrum of GAO / CAT hydrogel's removal and inhibition of active substances, where A is reactive oxygen and B is reactive nitrogen.

[0031] Figure 7 G prepared in Example 10 0.50 C 1.30 @Bar chart of the regulation of inflammatory factors by GAO / CAT hydrogel, in which A is IL-6 and B is TNF-α.

[0032] Figure 8 G prepared in Example 10 0.50 C 1.30 @GAO / CAT 3D printing of hydrogel.

[0033] Fig. 9 G prepared in Example 10 0.50 C 1.30 @GAO / CAT hydrogel gelation after mixing with biological fluids. A in the figure is the blood of experimental mice and B is the synovial fluid. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further explained below in conjunction with the accompanying drawings and specific embodiments. It is worth noting that the following embodiments are only preferred embodiments of the present invention and should not be understood as limiting the present invention. The protection scope of the present invention shall be based on the contents recorded in the claims. Any modification or replacement made by those skilled in the art to the technical solution of the present invention without creative work shall fall within the protection scope of the present invention.

[0035] In the following examples and comparative examples, the activity of galactose oxidase was 7.2 U, and the activity of catalase was 770 U.

[0036] Examples 1-16 (1) 0.03 w / v% galactose oxidase and 0.04 w / v% catalase were mixed to obtain a composite enzyme; (2) Dissolve guar gum and ethylene glycol chitosan in 0.01 mol / L PBS solution and mix well to obtain a mixed solution; the concentration of guar gum is 0.10, 0.25, 0.50 and 0.75 w / v%, and the concentration of ethylene glycol chitosan is 0.65, 1.30, 1.95 and 2.60 w / v%; (3) Add the complex enzyme to the mixed solution, stir and mix evenly, and then gel in an air atmosphere at 25°C to obtain enzyme-containing hydrogel G x C y @GAO / CAT, where x represents the feed concentration of guar gum and y represents the feed concentration of glycol chitosan.

[0037] Comparative Example 1 The method and steps are the same as those in Example 10, except that ethylene glycol chitosan is replaced by carboxymethyl chitosan to prepare the gel. It is found that after ethylene glycol chitosan is replaced by carboxymethyl chitosan, no gel can be formed, and the final product is still in liquid state.

[0038] Comparative Example 2 The method and steps are the same as those in Example 10, except that guar gum is replaced by sodium alginate to prepare the gel. It is found that after guar gum is replaced by sodium alginate, no gel can be formed, and the final product is still in liquid state.

[0039] Comparative Example 3 The method and steps are the same as those in Example 10, except that the feed concentration of ethylene glycol chitosan is changed to 0.50 w / v% to prepare G 0.50 C 0.50 @GAO / CAT. The results show that when the concentration of ethylene glycol chitosan is lower than 0.65 w / v%, although it can form a gel, it is not self-supporting and has no fixed shape.

[0040] Comparative Example 4 The method and steps were the same as those in Example 10, except that the feed concentration of ethylene glycol chitosan was changed to 3.00 w / v% to prepare G 0.50 C 3.00 @GAO / CAT.

[0041] Comparative Example 5 The method and steps are the same as those in Example 10, except that the concentration of guar gum was changed to 0.05 w / v% to prepare G 0.05 C 1.30@GAO / CAT. The results showed that when the concentration of guar gum was lower than 0.10 w / v%, no gel could be formed and the final product was still liquid.

[0042] Comparative Example 6 The method and steps are the same as those in Example 10, except that the concentration of guar gum was changed to 1.00 w / v% to prepare G 1.00 C 1.30 @GAO / CAT.

[0043] Results detection: The enzyme-containing hydrogels prepared in the above examples and comparative examples were tested for microstructure, mechanical properties, injectability, antioxidant regulation, and scavenging and inhibiting properties of active oxygen and active nitrogen. The detection method is as follows. The test results are shown in Table 1; Microstructure: The enzyme-containing hydrogel was immersed in liquid nitrogen for quick freezing for 30 seconds, then taken out and quickly placed in a freeze dryer for drying. After the sample was completely dry, it was fixed on the test sample table with double-sided carbon conductive tape, and then the sample surface was sprayed with gold using a vacuum coating machine. Finally, it was observed using a scanning electron microscope with an applied voltage of 5 KV. Mechanical properties: The enzyme-containing hydrogel was made into a cylinder with a diameter of 14 mm and a height of 6 mm, and placed on an electronic universal testing machine (Suzhou Farui Electronics Co., Ltd.). The stress-strain curve was obtained at a compression rate of 2 mm / min. The strain range was 0-10%, and the initial linear slope was calculated and recorded as the compression modulus of the hydrogel; Injectable performance: 200 μL of unformed enzyme-containing hydrogel solution was dropped onto the parallel plate platform of a rotational rheometer (Thermo Fisher Scientific Inc.), and the rotor was lowered to fill the gap between the parallel plates. The test temperatures were set to 25°C and 37°C, the rotation frequency was 1 Hz, the rotation stress was 1 Pa, and the gelation time was recorded. Antioxidant regulation properties: 2,2'-Azobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS 2- ABTS was prepared by reacting 2.45 mM potassium persulfate (7 mM) with 16 h in dark conditions. •+ The free radical solution (30 μL) was then mixed with the enzyme-containing hydrogel and diluted to 2 mL with PBS. After standing in the dark for 10 min, the supernatant was removed and tested by UV-visible absorption spectroscopy (Shimadzu Corporation, Japan). The wavelength range of 500-900 nm was scanned and the absorbance at the peak of 734 nm was recorded. sample ; Among them, ABTS •+ The free radical solution (30 μL) was diluted with PBS to a mixture of 2 mL as the control group, and its absorbance at the peak of 734 nm was measured. sample; The calculation formula of antioxidant regulation performance (AOR) is: AOR = (A control - A sample ) / A control ×100%; Reactive oxygen scavenging and inhibition performance: Weigh 2',7'-dichlorodifluorofluorescein diacetate (DCFH-DA) (5 mg) and dissolve it in dimethyl sulfoxide (DMSO) (1.026 mL), then dilute it 200 times with PBS to obtain DCFH-DA solution; add the enzyme-containing hydrogel (100 mg) to a solution (1 mL) rich in reactive oxygen free radicals (including superoxide anions, hydrogen peroxide, hydroxyl free radicals, singlet oxygen, etc.), then add DCFH-DA solution (10 μL), keep it at 37°C for 30 min, and then perform fluorescence spectrum detection (Hitachi, Japan); use Ex=488 nm as the excitation wavelength, scan the fluorescence intensity in the emission wavelength range of 500-600 nm, and record the fluorescence intensity F of the experimental group and the control group at the peak position of 520 nm. sample,520 and F control,520 ; The active oxygen inhibition rate was calculated by the following formula: Inhibition = (F control,520 -F sample,520 ) / F control,520 ×100%; Reactive nitrogen scavenging and inhibition properties: from peroxynitrite anion (ONOO - ) A certain amount of O71 fluorescent probe was taken from the detection kit and diluted 10 times with PBS to obtain O71 solution. 0.50 C 1.30 @GAO / CAT gel (100 mg) was added to a solution (1 mL) rich in active nitrogen free radicals (including peroxynitrite anions, etc.), and then the above-mentioned O71 solution (10 μL) was added thereto. After being kept at a constant temperature of 37°C for 30 min, fluorescence spectrum detection was performed; using Ex = 488 nm as the excitation wavelength, the fluorescence intensity in the emission wavelength range of 500-650 nm was scanned, and the fluorescence intensity F of the experimental group and the control group was recorded at the peak position of 515 nm. sample,515 and F control,515 ; The active nitrogen inhibition rate was calculated by the following formula: Inhibition = (F control,515 -F sample,515 ) / F control,515 ×100%; Table 1 Formula and performance parameters of enzyme-containing hydrogel

[0044] Note: G is guar gum, C is glycol chitosan, GAO is galactose oxidase, and CAT is catalase.

[0045] It can be seen from Table 1 that when the concentrations of G and C change, the compression modulus of the enzyme-containing hydrogel is affected to a certain extent. When the concentration of guar gum remains unchanged, the compression modulus gradually increases with the increase of the concentration of ethylene glycol chitosan; when the concentration of ethylene glycol chitosan remains unchanged, the compression modulus also shows a gradually increasing trend with the increase of the concentration of guar gum (Examples 1-16).

[0046] When any of the raw materials of guar gum and glycol chitosan was replaced, the enzyme-containing hydrogel could not be formed, and the final product was in the form of a solution (Comparative Examples 1 and 2).

[0047] When an appropriate amount of guar gum and too low a concentration (such as 0.50 w / v%) of ethylene glycol chitosan are used to prepare enzyme-containing hydrogel, although gelation can be achieved, the mechanical properties of the gel are too weak, it is not self-supporting, and it has no fixed shape, so its compression modulus cannot be measured (Comparative Example 3); when an appropriate amount of guar gum and an excess amount (such as 3.00 w / v%) of ethylene glycol chitosan are combined to prepare enzyme-containing hydrogel, its compression modulus is 5859.0±238.3 (Comparative Example 4); when an enzyme-containing hydrogel is prepared by too low a concentration (such as 0.05 w / v%) of guar gum and an appropriate amount of ethylene glycol chitosan, gel cannot be formed, the final product is still in liquid form, and its compression modulus cannot be measured (Comparative Example 5); when an excess amount (such as 1.00 w / v%) of guar gum and an appropriate amount of ethylene glycol chitosan are used to prepare enzyme-containing hydrogel, its compression modulus is 2899.7±366.3 (Comparative Example 6).

[0048] Depend on Figure 2 It can be seen that the Fourier transform infrared spectrum shows the characteristic peak of imine (C=N) stretching vibration (1557 cm -1 ), indicating the formation of Schiff base bonds and G 0.50 C 1.30 @GAO / CAT gel was successfully prepared; Figure 3 It can be seen that, through scanning electron microscopy, G 0.10 C 1.30 @GAO / CAT Gel and G 0.50 C 1.30 @GAO / CAT gels both present a three-dimensional porous microstructure. 0.10 C 1.30 The average pore size of GAO / CAT gel is 42 μm. 0.50 C 1.30 The average pore size of @GAO / CAT gel is 5μm.

[0049] In addition, the experiment found that no matter how the concentrations of G and C changed, the gelation time, AOR, reactive oxygen and reactive nitrogen inhibition rates of the enzyme-containing hydrogel at different temperatures did not change significantly. The gelation time at 25°C was about 17 min, and the gelation time at 37°C was about 11 s ( Figure 4 ); AOR increases with the increase of enzyme hydrogel concentration. When the concentration of enzyme hydrogel is 200 mg / mL, AOR is about 92.04% ( Figure 5 ); When the concentration of enzyme-containing hydrogel was 100 mg / mL, its inhibition rate of active oxygen in the solution was 60.3±2.3% ( Figure 6 ), the inhibition rate of active nitrogen was 32.5±8.3% ( Figure 7 ).

[0050] Embodiment 17 The method and steps were the same as those in Example 10, except that the amount of galactose oxidase was changed from 0.03 w / v% to 0.18 w / v%, and the amount of catalase was changed from 0.04 w / v% to 0.25 w / v%, to prepare G 0.50 C 1.30 @GAO / CAT.

[0051] Comparative Example 7 The method and steps were the same as those in Example 10, except that the amount of galactose oxidase was changed from 0.03 w / v% to 0.30 w / v% to prepare G 0.50 C 1.30 @GAO / CAT.

[0052] Comparative Example 8 The method and steps were the same as those in Example 10, except that the amount of galactose oxidase was changed from 0.03 w / v% to 0.015 w / v% to prepare G 0.50 C 1.30 @GAO / CAT.

[0053] Comparative Example 9 The method and steps were the same as those in Example 10, except that galactose oxidase was replaced by glucose oxidase to prepare the enzyme-containing hydrogel. It was found that after galactose oxidase was replaced by glucose oxidase, gel could not be formed and the final product was still liquid.

[0054] Comparative Example 10 The method and steps were the same as those in Example 10, except that the amount of catalase was changed from 0.04 w / v% to 0.50 w / v% to prepare G 0.50 C 1.30 @GAO / CAT.

[0055] Comparative Example 11 The method and steps were the same as those in Example 10, except that the amount of catalase was changed from 0.04 w / v% to 0, to prepare G 0.50 C 1.30 @GAO.

[0056] Results: The performance of the enzyme-containing hydrogel was tested according to the above method. The results are shown in Table 3: Table 2 Effects of enzyme type and dosage on the properties of enzyme-containing hydrogels

[0057] It can be seen from Table 2 that when the dosage of galactose oxidase and catalase is changed, the compression modulus, gelation time at 25°C, gelation time at 37°C, AOR, active oxygen inhibition rate and active nitrogen inhibition rate of the enzyme-containing hydrogel are affected to varying degrees. When the dosage of galactose oxidase and catalase increases at the same time, the compression modulus, AOR, active oxygen inhibition rate and active nitrogen inhibition rate of the enzyme-containing hydrogel significantly increase, while the 25°C gelation time and 37°C gelation time significantly decrease (Examples 10 and 17); when the concentration of catalase remains unchanged, the compression modulus, AOR, active oxygen inhibition rate and active nitrogen inhibition rate of the enzyme-containing hydrogel increase with the increase of the galactose oxidase concentration, while the 25°C gelation time and 37°C gelation time decrease accordingly (Example 10, Comparative Examples 7 and 8); when the concentration of galactose oxidase remains unchanged, the compression modulus, AOR, active oxygen inhibition rate and active nitrogen inhibition rate of the enzyme-containing hydrogel increase with the increase of the catalase concentration, but the 25°C gelation time and 37°C gelation time do not change significantly (Example 10, Comparative Examples 10 and 11).

[0058] When galactose oxidation was replaced by glucose oxidase, the enzyme-containing hydrogel could not be formed and the final product was in the form of a solution (Comparative Example 9).

[0059] Example 18 Anti-inflammatory performance detection Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) are two important inflammatory cytokines produced and released by macrophages, which can induce inflammatory response and cell apoptosis, thereby accelerating the progression of inflammatory diseases. Therefore, the anti-inflammatory effect of enzyme-containing hydrogel can be analyzed by lipopolysaccharide (LPS) stimulating the changes of the above two pro-inflammatory factors produced by mouse mononuclear macrophage leukemia cells (RAW264.7) regulated by enzyme-containing hydrogel. The specific steps are as follows: (1) LPS (1 μg / mL) was added to the culture medium of adherent RAW264.7 macrophages (purchased from the Chinese Academy of Sciences Cell Bank), followed by the addition of G 0.50 C 1.30 @GAO / CAT gel to a final concentration of 10, 30, and 100 mg / mL; Negative control: adherent RAW264.7 macrophages without any treatment; Positive control: LPS (1 μg / mL) was added to the culture medium of adherent RAW264.7 macrophages, followed by the addition of 0.50 C 1.30 @GAO / CAT gel with an equal volume of PBS.

[0060] (2) placing the system obtained in step (1) in a cell culture incubator at 37°C and 5% CO2 for 24 h, and then centrifuging and filtering to collect the cells; (3) extracting total RNA from the cells collected in step (2) using Trizol reagent, and reverse transcribing to obtain cDNA; (4) Using primers to perform qPCR reaction on cDNA to detect the relative mRNA expression levels of IL-6 and TNF-α. The qPCR primer sequences are shown in Table 3: Table 3 qPCR primer sequences

[0061] The results are as follows Figure 7 As shown in Figure 2, LPS stimulation increased the expression of proinflammatory cytokines IL-6 and TNF-α in macrophages, while G 0.50 C 1.30 The addition of GC@GAO / CAT gel can weaken the production of IL-6 and TNF-α caused by LPS stimulation, and in the concentration range of 10-100 mg / mL, as the amount of this enzyme-containing hydrogel increases, the inhibitory effect on IL-6 and TNF-α is significantly improved, but when the concentration is further increased, the rate of increase in the inhibitory effect gradually decreases. This shows that GC@GAO / CAT gel can effectively inhibit the inflammatory factors produced during cell inflammation and has a certain anti-inflammatory effect.

[0062] Example 19 3D printing application For the microporous extrusion of enzyme-containing hydrogels and 3D printing of gel scaffolds, the enzyme-containing hydrogel precursor printing ink is pneumatically extruded and deposited on a receiving platform through program control, printed layer by layer, and stacked in layers. The inner diameter of the nozzle used is 600 μm.

[0063] Depend on Figure 8 It can be seen that the G prepared in Example 10 0.50 C 1.30 @GAO / CAT gel can be extruded through micropores and made into a hydrogel scaffold using 3D printing technology.

[0064] Example 20 Influence of external factors A hydrogel precursor solution was prepared according to the formula of Example 10, biological liquid was added thereto, and the mixture was evenly mixed to obtain an enzyme-containing hydrogel. The gelation process and the distribution of the biological liquid in the hydrogel were observed. The biological liquid was blood and synovial fluid of experimental mice, and the mass ratio of the biological liquid to the hydrogel precursor solution was 1:4.

[0065] The results are as follows Fig. 9 As shown, experimental mouse blood ( Fig. 9 A) and synovial fluid ( Fig. 9 B) G 0.50 C 1.30 @GAO / CAT gel precursors can both form stable hydrogels, and the blood and synovial fluid of experimental mice are evenly distributed in the hydrogels.

Claims

1. An anti-inflammatory injectable enzyme-containing hydrogel, characterized in that: The enzyme-containing hydrogel comprises the following raw materials in percentage by mass concentration: 0.10-0.75 w / v % guar gum, 0.65-2.60 w / v % ethylene glycol chitosan, 0.03-0.43 w / v % complex enzyme, and the remainder is solvent.

2. The anti-inflammatory injectable enzyme-containing hydrogel according to claim 1, characterized in that: The complex enzyme consists of galactose oxidase and catalase.

3. The anti-inflammatory injectable enzyme-containing hydrogel according to claim 2, characterized in that: The mass ratio of the galactose oxidase to the catalase is 3:

4.

4. The anti-inflammatory injectable enzyme-containing hydrogel according to claim 3, characterized in that: The activity of the catalase is 100-110 times that of the galactose oxidase; the activity of the galactose oxidase is 5-10 U.

5. The anti-inflammatory injectable enzyme-containing hydrogel according to claim 1, characterized in that: The solvent is water or PBS solution.

6. The anti-inflammatory injectable enzyme-containing hydrogel according to claim 5, characterized in that: The concentration of the PBS solution is 0.01 mol / L.

7. The anti-inflammatory injectable enzyme-containing hydrogel according to claim 1, characterized in that: The polymerization degree of the ethylene glycol chitosan is greater than 400.

8. A method for preparing the anti-inflammatory injectable enzyme-containing hydrogel according to any one of claims 1 to 7, characterized in that: The steps include: (1) dissolving guar gum and ethylene glycol chitosan in a solvent to obtain a mixed solution; (2) Add the complex enzyme to the mixed solution, mix well and react to obtain the enzyme-containing hydrogel.

9. The preparation method according to claim 8, characterized in that: The reaction temperature in step (2) is 25-37°C.

10. A use of the anti-inflammatory injectable enzyme-containing hydrogel according to any one of claims 1 to 7, characterized in that: The application is in vivo anti-inflammatory support material and 3D printing biological ink.

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