Enzyme-based microcapsule composite hydrogel with wound healing function as well as preparation method and application of enzyme-based microcapsule composite hydrogel

Abstract

The invention belongs to the technical field of biological materials and medicines, and discloses enzyme-based microcapsule composite hydrogel with a wound healing function, a preparation method and application, and the preparation method comprises the following steps: mixing and stirring a chitosan solution and a gelatin solution for 2 hours, adding a plasticizer solution, continuing to stir uniformly, then adding MOF-818 ALG microcapsules, and stirring uniformly to obtain the enzyme-based microcapsule composite hydrogel with the wound healing function. And finally, dropwise adding a cross-linking agent into the solution, completely mixing, pouring into a glass culture dish, keeping for 96 hours, completing the cross-linking process, and air-drying to obtain the microcapsule-loaded hydrogel. The preparation method comprises the following steps: preparing MOF-818 from copper, zirconium and 4-pyrazolecarboxylic acid as raw materials, synthesizing a copper alginate microcapsule by adopting free radical polymerization and ionic crosslinking methods, and preparing the hydrogel on the basis of chitosan and gelatin. The enzyme-based microcapsule composite hydrogel disclosed by the invention has good biocompatibility, air permeability, water binding capacity, degradability and swelling resistance, and is a multifunctional dressing for effectively coping with healing of infected wounds.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomaterials and medical technology, and particularly relates to an enzyme-based microcapsule composite hydrogel for wound healing, a preparation method and application thereof. BACKGROUND

[0002] Skin is the largest organ of the human body, which has multiple functions such as protection, sensation, temperature regulation, secretion and excretion. Skin damage mainly refers to the destruction of the integrity, continuity or function of the skin, which is specifically manifested as abnormal changes in the surface or deep layer of the skin. Mild skin damage can be restored through simple care, while severe skin damage requires professional medical intervention. Hydrogel is an ideal material for promoting wound healing due to its excellent moisturizing property, air permeability, biocompatibility and drug release capacity, and is particularly suitable for chronic infected wounds.

[0003] In order to enhance the efficacy of hydrogel, functional components such as anti-inflammatory and antibacterial components are usually introduced. Copper ions Cu 2+ Due to its broad-spectrum antibacterial, antiviral, anti-inflammatory and pro-angiogenic properties, copper ions Cu 2+ are widely incorporated into carriers such as hydrogel. The existing technology usually utilizes the pH responsiveness of Cu 2+ to achieve partial functional regulation. As a drug delivery carrier, microcapsules can improve the stability of drugs, achieve controllable release and potential targeting, and show good prospects in the application of wound healing, which helps to reduce toxic side effects and improve efficacy.

[0004] However, the existing copper-containing hydrogel mainly introduces Cu 2+ through simple physical mixing or ionic crosslinking, and its release mechanism mainly depends on passive diffusion or basic environmental response, which lacks intelligence and precision, and is difficult to efficiently solve the problems of persistent inflammation, low cell activity or abnormal protease environment in chronic wounds. When applied to hydrogel dressings, microcapsules often focus on simple drug encapsulation and release, and their release behavior is often independent of the actual biological state of the wound, and the functional synergy with hydrogel is insufficient. Chronic infected wounds often have multiple pathological factors, including persistent infection, excessive inflammation, protease imbalance, cell dysfunction and impaired angiogenesis, and existing single-function materials or simple combinations of materials are difficult to effectively integrate and synergistically exert the multiple functions of antibacterial, anti-inflammatory and tissue regeneration. Therefore, it is particularly important to develop a new intelligent hydrogel dressing that utilizes highly functional microcapsules to achieve intelligent response release of functional factors and efficiently integrate and synergize multiple treatment mechanisms.

[0005] Through retrieval, no patent publication related to the present patent application has been found. SUMMARY

[0006] The present application aims to overcome the deficiencies in the prior art, and provides an enzyme-based microcapsule composite hydrogel with wound healing, a preparation method and application.

[0007] The technical scheme adopted by the present application to solve its technical problems is:

[0008] A preparation method of an enzyme-based microcapsule composite hydrogel with wound healing, comprising the following steps:

[0009] The chitosan solution and the gelatin solution are mixed and stirred for 2 hours, the plasticizer solution is added and uniformly stirred, then the MOF-818@ALG microcapsules with a mass concentration of 1%-5%(w / w) are added, finally the crosslinking agent with a concentration of 8-12mg / mL is dropped into the above solution and fully mixed, then poured into a glass culture dish and kept for 96h to complete the crosslinking process and air dry, thereby obtaining the microcapsule-loaded hydrogel;

[0010] The ratio of chitosan: gelatin: plasticizer: crosslinking agent g:g:mL:mL is 1:4:1:1.

[0011] Further, the chitosan solution is obtained by dissolving chitosan in acetic acid, and the mass concentration of the chitosan solution is 1%-3%(w / v); the gelatin solution is obtained by dissolving gelatin in ultrapure water, and the mass concentration of the gelatin solution is 5%-12%(w / v); the mass concentration of the plasticizer solution is 5%-12%(w / v).

[0012] Further, the plasticizer is glycerol, polyethylene glycol or xylitol.

[0013] Further, the crosslinking agent is genipin, tannic acid or gallic acid.

[0014] Further, the preparation of the MOF-818@ALG microcapsules comprises the following steps:

[0015] (1) Preparation of MOF-818: Cu(NO3)2·3H2O, ZrOCl2·8H2O and 4-pyrazole carboxylic acid are dissolved in organic solvent one at a molar ratio of 0.5-0.7:0.1-0.3:0.15-0.25, organic solvent two is added to the above solution, ultrasonic mixing is performed, and then the solution is placed in a polytetrafluoroethylene-lined reaction kettle, naturally cooled at 100℃ for 8h, then resuspended in organic solvent one and organic solvent three, centrifuged and dried, thereby obtaining MOF-818;

[0016] (2) Preparation of MOF-818 copper alginate microcapsules MOF-818@ALG: weigh the sodium alginate powder and MOF-818 powder to prepare a sodium alginate aqueous solution, sterilize the solution and the copper chloride solution by high pressure, add the sodium alginate solution to the second centrifuge tube of the centrifugal sleeve device, add the copper chloride solution to the first centrifuge tube of the centrifugal sleeve device, set the needle of the second centrifuge tube at a distance of 0.45-1.5 cm from the top liquid surface of the copper chloride solution, place the centrifugal sleeve device in the centrifuge, and centrifuge the sodium alginate aqueous solution and the copper chloride solution at a volume ratio of 0.8-1.2:10.0-13.0 at a speed of 1800-2500 rpm / min for 8-12 min to obtain the copper alginate microcapsules MOF-818@ALG.

[0017] Further, the organic solvent one is N,N-dimethylacetamide, N,N-dimethylformamide or N-methylpyrrolidone; the organic solvent two is trifluoroacetic acid, formic acid or hydrochloric acid; and the organic solvent three is ethanol, methanol or acetonitrile.

[0018] Further, the final concentration of sodium alginate in the solution is 0.5%-2.0%, and the final concentration of MOF-818 in the solution in step (2) is 0.05 mg / mL; and the mass concentration of the copper chloride solution is 1.5%-2.5%.

[0019] Further, the centrifugal sleeve device comprises a first centrifuge tube and a second centrifuge tube, and the second centrifuge tube is coaxially and tightly detachably connected to the first centrifuge tube; the first centrifuge tube comprises a first centrifuge tube body and a first centrifuge tube cover, the first centrifuge tube body is arranged in a vertical direction and has a top open centrifuge tube shape, the first centrifuge tube cover is tightly and coaxially detachably arranged on the upper part of the opening of the first centrifuge tube body, and a second centrifuge tube mounting through hole is integrally formed on the first centrifuge tube cover; the first centrifuge tube body can contain the copper chloride solution;

[0020] The second centrifugal tube comprises a second centrifugal tube body, a second centrifugal tube cover and a needle, the second centrifugal tube body is arranged in a vertical direction, the second centrifugal tube body is in a centrifugal tube shape with open top and bottom, the second centrifugal tube cover is movably and detachably connected with the open top of the second centrifugal tube body, the second centrifugal tube body can contain sodium alginate aqueous solution, the open bottom of the second centrifugal tube body is movably and detachably connected with the needle, and the needle is arranged in a vertical direction; the shape of the outer surface of the upper part of the second centrifugal tube body is consistent with the shape of the inner surface of the second centrifugal tube mounting hole, the upper part of the second centrifugal tube body can be movably and detachably connected with the first centrifugal tube cover through the second centrifugal tube mounting hole, and the upper surface of the second centrifugal tube body is flush with the upper surface of the first centrifugal tube cover, and the middle and lower part of the second centrifugal tube body is arranged in the first centrifugal tube body in a downward extending manner;

[0021] Alternatively, the volume of the first centrifugal tube is 50 mL, the volume of the second centrifugal tube is 1.5 mL, and the needle is arranged at a position 0.45-1.5 cm above the copper chloride solution contained in the first centrifugal tube body.

[0022] The enzyme-based microcapsule composite hydrogel prepared by the preparation method.

[0023] The enzyme-based microcapsule composite hydrogel is used for preparing a drug for promoting wound healing.

[0024] The advantages and positive effects obtained by the application are as follows:

[0025] 1. The enzyme-based microcapsule composite hydrogel has good biocompatibility, air permeability, water retention, degradability and anti-swelling property, and is a multifunctional dressing for effectively treating infected wounds; the composite hydrogel contains copper alginate microcapsules, which mainly kill bacteria by slowly releasing copper ions, and the positive charge of chitosan can adsorb bacteria, so that copper can destroy the cell membrane of bacteria at a low concentration, has a rapid bactericidal effect, and avoids the occurrence of bacterial drug resistance; mouse wound experiments show that the enzyme-based microcapsule composite hydrogel can promote the regeneration of collagen, and the epidermis of the mouse wound is well remodeled. The composite hydrogel delivery system can cooperate with each other in antibiosis and wound healing, promote each other, and provide a new method and idea for the healing of infected wounds.

[0026] 2. The enzyme-based microcapsule composite hydrogel is prepared from copper, zirconium and 4-pyrazole carboxylic acid as raw materials to prepare MOF-818, and free radical polymerization and ionic crosslinking methods are used to synthesize copper alginate microcapsules, and chitosan and gelatin are used as the basis to prepare hydrogel. The enzyme-based microcapsule composite hydrogel has good biocompatibility, air permeability, water retention, degradability and anti-swelling property, and is a multifunctional dressing for effectively treating infected wounds. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The Fourier transform infrared spectrogram (FTIR) of the hydrogel in the application;

[0028] Figure 2 The X-ray photoelectron spectroscopy (XPS) of MOF-818 in the application (left picture) and the high-resolution spectrogram of Cu in MOF-818 (right picture);

[0029] Figure 3 The field emission scanning electron microscope image (SEM) of the hydrogel in the application; wherein, the left picture in the first row of pictures is the SEM image of the hydrogel (GeL), and the right picture is the SEM image of the MOF-818 microcapsule hydrogel (MAG); the left picture in the second row of pictures is the local magnified image of the SEM image of the hydrogel (GeL), and the right picture is the local magnified image of the SEM image of the MOF-818 microcapsule hydrogel (MAG);

[0030] Figure 4 The antibacterial performance diagram of the hydrogel in the application against E. coli and S. aureus; wherein, A is the antibacterial diagram of S. aureus and E. coli, and B is the antibacterial rate of GeL and MAG;

[0031] Figure 5 The hemolysis experiment diagram of the hydrogel in the application;

[0032] Figure 6 The skin wound healing situation diagram of mice in different treatment groups in the application; wherein, A is the wound healing experiment schematic diagram, and B is the wound healing photo;

[0033] Figure 7 The Masson collagen deposition content diagram of mice in different treatment groups in the application;

[0034] Figure 8 The structural connection schematic diagram of the centrifugal sleeve device in the application;

[0035] Figure 9 The structural connection schematic diagram of the second centrifugal tube of the centrifugal sleeve device in the application. DETAILED DESCRIPTION

[0036] The application will be further described below in conjunction with the examples, which are descriptive rather than limiting, and cannot be used to limit the protection scope of the application.

[0037] The various experimental operations involved in the specific examples are all conventional techniques in the art, and the parts not specially noted in the text can be implemented by referring to various commonly used tool books, scientific and technical literature or related instructions, manuals, etc. before the application date of the application.

[0038] A method for preparing an enzyme-based microcapsule composite hydrogel with wound healing, comprising the following steps:

[0039] The chitosan solution and the gelatin solution are mixed and stirred for 2 hours, a plasticizer solution is added and stirred until uniform, then 1%-5%(w / w) MOF-818@ALG microcapsules are added, and finally 8-12mg / mL crosslinking agent is added dropwise into the above solution and fully mixed, then poured into a glass culture dish and kept for 96h to complete the crosslinking process and air dried to obtain a microcapsule-loaded hydrogel;

[0040] The ratio of chitosan: gelatin: plasticizer: crosslinking agent is 1:4:1:1.

[0041] Preferably, the chitosan solution is obtained by dissolving chitosan in acetic acid, and the mass concentration of the chitosan solution is 1%-3%(w / v); the gelatin solution is obtained by dissolving gelatin in ultrapure water, and the mass concentration of the gelatin solution is 5%-12%(w / v); and the mass concentration of the plasticizer solution is 5%-12%(w / v).

[0042] Preferably, the plasticizer is glycerol, polyethylene glycol or xylitol.

[0043] Preferably, the crosslinking agent is genipin, tannic acid or gallic acid.

[0044] Preferably, the preparation of the MOF-818@ALG microcapsules comprises the following steps:

[0045] (1) Preparation of MOF-818: Cu(NO3)2·3H2O, ZrOCl2·8H2O and 4-pyrazole carboxylic acid are dissolved in organic solvent one at a molar ratio of 0.5-0.7:0.1-0.3:0.15-0.25, organic solvent two is added to the above solution, and then ultrasonic mixing is performed, and the mixture is placed in a polytetrafluoroethylene-lined reaction kettle, and then naturally cooled at 100℃ for 8h, and then resuspended in organic solvent one and organic solvent three, and then centrifuged and dried to obtain MOF-818;

[0046] (2) Preparation of MOF-818 alginate copper microcapsules MOF-818@ALG: weigh the sodium alginate powder and MOF-818 powder to prepare a sodium alginate aqueous solution, sterilize the solution and the copper chloride solution by high pressure, add the sodium alginate solution to the second centrifuge tube of the centrifugal sleeve device, add the copper chloride solution to the first centrifuge tube of the centrifugal sleeve device, set the needle of the second centrifuge tube at a distance of 0.45-1.5 cm from the top liquid surface of the copper chloride solution, place the centrifugal sleeve device in the centrifuge, and centrifuge the sodium alginate aqueous solution and the copper chloride solution at a volume ratio of 0.8-1.2:10.0-13.0 at a speed of 1800-2500 rpm / min for 8-12 min to obtain the alginate copper microcapsules MOF-818@ALG.

[0047] Preferably, the organic solvent one is N,N-dimethylacetamide, N,N-dimethylformamide or N-methylpyrrolidone; the organic solvent two is trifluoroacetic acid, formic acid or hydrochloric acid; and the organic solvent three is ethanol, methanol or acetonitrile.

[0048] Preferably, the final concentration of sodium alginate in the solution is 0.5%-2.0%, and the final concentration of MOF-818 in the solution in step (2) is 0.05 mg / mL; and the mass concentration of the copper chloride solution is 1.5%-2.5%.

[0049] Preferably, the centrifugal sleeve device comprises a first centrifuge tube 1 and a second centrifuge tube 2, the second centrifuge tube is coaxially and tightly detachably connected with the first centrifuge tube; the first centrifuge tube comprises a first centrifuge tube body 11 and a first centrifuge tube cover 12, the first centrifuge tube body is arranged in a vertical direction and has a top-open centrifuge tube shape, the first centrifuge tube cover is tightly coaxially and detachably arranged on the upper part of the opening of the first centrifuge tube body, and a second centrifuge tube mounting through hole (not labeled in the figure) is integrally formed on the first centrifuge tube cover; the first centrifuge tube body can contain the copper chloride solution;

[0050] The second centrifugal tube comprises a second centrifugal tube body 21, a second centrifugal tube cover 22 and a needle 3. The second centrifugal tube body is arranged in a vertical direction and has an open top and an open bottom. The second centrifugal tube cover is movably and detachably connected to the open top of the second centrifugal tube body. The second centrifugal tube body can contain a sodium alginate aqueous solution. The open bottom of the second centrifugal tube body is movably and detachably connected to the needle. The needle is arranged in a vertical direction. The upper outer surface of the second centrifugal tube body is shaped to match the inner surface of the second centrifugal tube mounting hole. The upper part of the second centrifugal tube body is movably and detachably connected to the first centrifugal tube cover through the second centrifugal tube mounting hole. The upper surface of the second centrifugal tube body is flush with the upper surface of the first centrifugal tube cover. The middle and lower parts of the second centrifugal tube body extend downward into the first centrifugal tube body.

[0051] Alternatively, the volume of the first centrifugal tube is 50 mL, the volume of the second centrifugal tube is 1.5 mL, and the needle is arranged at a position 0.45-1.5 cm above the copper chloride solution contained in the first centrifugal tube body.

[0052] The enzyme-based microcapsule composite hydrogel prepared by the preparation method described above.

[0053] The enzyme-based microcapsule composite hydrogel described above is used in the preparation of a drug for promoting wound healing.

[0054] Specifically, the preparation and detection are as follows:

[0055] Example 1: Preparation of enzyme-based microcapsule composite hydrogel (MAG) with wound healing

[0056] (1) Preparation of MOF-818: First, Cu(NO3)2·3H2O (124 mg), ZrOCl2·8H2O (42.5 mg), and 4-pyrazole carboxylic acid (32.5 mg) were dissolved in 20 mL of DMF. Then, trifluoroacetic acid (120 μL) was added to the above solution, which was thoroughly mixed and then ultrasonicated. Then, the solution was placed in a 50 mL polytetrafluoroethylene-lined reaction kettle and naturally cooled at 100°C for 8 h. After that, the MOF-818 was resuspended in DMF and ethanol and centrifuged three times, and then dried to obtain the MOF-818.

[0057] (2) MOF-818 alginate copper microcapsule (MOF-818@ALG) preparation: first, weigh 0.5 g of sodium alginate powder and 2.5 mg of MOF-818 powder, add 50 mL of deionized water, stir uniformly with a glass rod and place for 24 h to prepare a 1% sodium alginate solution. Accurately weigh 2 g of copper chloride powder, add 100 mL of deionized water, stir uniformly with a glass rod to prepare a 2% copper chloride solution. First, sterilize the prepared sodium alginate solution and copper chloride solution, add 1% sodium alginate solution to the second centrifuge tube 2 in the centrifuge sleeve device, and add 2% copper chloride solution to the first centrifuge tube 1 in the centrifuge sleeve device, the needle in the device is 0.45-1.5 cm away from the liquid level of the copper chloride solution, the centrifuge sleeve device is placed in the centrifuge, and the centrifuge is operated at a speed of 2100 rpm / min for 10 min, so that the 1% sodium alginate aqueous solution is uniformly dropped into the copper chloride aqueous solution, and the hydrogel beads (i.e. MOF-818 microcapsules) are completely soaked until the MOF-818@ALG microcapsules are completely solidified.

[0058] As shown in Figure 8 and Figure 9 , the centrifuge sleeve device comprises a first centrifuge tube 1 and a second centrifuge tube 2, and the second centrifuge tube can be coaxially and tightly detachably connected with the first centrifuge tube; the first centrifuge tube comprises a first centrifuge tube body 11 and a first centrifuge tube cover 12, the first centrifuge tube body is arranged in a vertical direction, and the first centrifuge tube body is in the shape of a centrifuge tube with an open top, the first centrifuge tube cover is tightly and coaxially detachably arranged on the upper part of the opening of the first centrifuge tube body, and a second centrifuge tube mounting hole (not labeled in the figure) is integrally formed on the first centrifuge tube cover; the first centrifuge tube body can contain copper chloride solution;

[0059] The second centrifuge tube comprises a second centrifuge tube body 21, a second centrifuge tube cover 22 and a needle 3, the second centrifuge tube body is arranged in a vertical direction, and the second centrifuge tube body is in the shape of a centrifuge tube with open top and bottom, the second centrifuge tube cover is movably and detachably connected with the open top of the second centrifuge tube body, the second centrifuge tube body can contain sodium alginate aqueous solution, and the needle is tightly and detachably connected with the open bottom of the second centrifuge tube body in a communicating manner, and the needle is arranged in a vertical direction; the upper surface of the second centrifuge tube body is flush with the upper surface of the first centrifuge tube cover, and the upper part of the second centrifuge tube body can be coaxially and tightly detachably connected with the first centrifuge tube cover through the second centrifuge tube mounting hole, and the upper surface of the second centrifuge tube body is flush with the upper surface of the first centrifuge tube cover, and the lower part of the second centrifuge tube body extends downward into the first centrifuge tube body;

[0060] Alternatively, the first centrifuge tube has a volume of 50 mL and the second centrifuge tube has a volume of 1.5 mL, and the needle is disposed 0.45-1.5 cm above the copper chloride solution contained within the first centrifuge tube body.

[0061] Accurately weigh 1 g of chitosan into 50 mL of 1% acetic acid to prepare a 2% chitosan (CS) solution, and accurately weigh 4 g of gelatin into 50 mL of ultrapure water to prepare an 8% gelatin solution. Stir the two solutions together for 2 h, then add 1 mL of 2% glycerol as a plasticizer and continue stirring until the solution is uniform. Then add the MOF-818@ALG microcapsules. Finally, dissolve 10 mg of genipin in 1 mL of ultrapure water, and add the genipin solution dropwise into the above solution as a crosslinking agent. Pour the solution into a glass culture dish (90 mm) and keep it for 96 h to complete the crosslinking process and air dry. The resulting product is an enzyme-based microcapsule composite hydrogel (MAG) with wound healing.

[0062] Comparative Example 1: Preparation of a microcapsule hydrogel (AG)

[0063] First, accurately weigh 0.5 g of sodium alginate powder into 50 mL of deionized water, stir the solution with a glass rod until it is uniform, and then let it stand for 24 h to prepare a 1% sodium alginate solution. Accurately weigh 2 g of copper chloride powder into 100 mL of deionized water, stir the solution with a glass rod until it is uniform, and then let it stand to prepare a 2% copper chloride solution. First, sterilize the prepared sodium alginate solution and copper chloride solution by autoclaving. Then, add the 1% sodium alginate solution and the 2% copper chloride solution to a microcapsule preparation device (i.e., the centrifuge sleeve device in Example 1) and centrifuge at 2100 rpm / min for 10 min. This causes the 1% sodium alginate solution to drip uniformly into the 2% copper chloride solution, and the copper alginate microcapsules are obtained after complete coagulation.

[0064] Accurately weigh 1 g of chitosan into 50 mL of 1% acetic acid to prepare a 2% chitosan (CS) solution, and accurately weigh 4 g of gelatin into 50 mL of ultrapure water to prepare an 8% gelatin solution. Stir the two solutions together for 2 h, then add 1 mL of 2% glycerol as a plasticizer and continue stirring until the solution is uniform. Then add the MOF-818@ALG microcapsules. Finally, dissolve 10 mg of genipin in 1 mL of ultrapure water, and add the genipin solution dropwise into the above solution as a crosslinking agent. Pour the solution into a glass culture dish (90 mm) and keep it for 96 h to complete the crosslinking process and air dry. The resulting product is an enzyme-based microcapsule composite hydrogel (MAG) with wound healing.

[0065] Comparative Example 2: Preparation of a hydrogel (GeL)

[0066] Accurately weigh 1g of chitosan and dissolve it in 50mL of 1% acetic acid to prepare a 2% chitosan (CS) solution. Weigh 4g of gelatin and dissolve it in 50mL of ultrapure water to prepare an 8% gelatin solution. Combine the two solutions and stir for 2 hours. Then, add 1mL of 2% glycerol as a plasticizer and continue stirring until homogeneous. Dissolve 10mg of genipin in 1mL of ultrapure water as a crosslinking agent. Add the genipin solution dropwise to the above solution and mix thoroughly. Pour the mixture into a 90mm glass petri dish and keep it in the petri dish for 96 hours to complete the crosslinking process. Then, air dry the mixture to obtain a hydrogel (GeL).

[0067] Example 2: Fourier Transform Infrared Spectroscopy Analysis of Hydrogels

[0068] Accurately weigh 1 mg each of MOF-818, lyophilized and thoroughly ground hydrogel (GeL), microencapsulated hydrogel (AG), and enzyme-based microencapsulated composite hydrogel (MAG) sample powders, and mix them separately with 99 mg of potassium bromide, then grind them into dense particles. These particles were then pressed into transparent sheets using a tablet press under 8 tons of pressure. Finally, the particles were observed at 4000-400 cm⁻¹ using a Fourier transform infrared spectrophotometer. -1 The absorption peak below. For example... Figure 1 As shown, the results demonstrate the successful synthesis of the metal-organic framework (MOF-818). In the infrared spectrum of MOF-818, at 3432 cm⁻¹... -1 The strong and broad peak at 3000 cm⁻¹ is due to the hydroxyl group. -1 The absorption peaks at the left and right edges originate from C, N, and O vibrations and are located in the 2500-2000 cm⁻¹ range. -1 The characteristic absorption peak at 1636 cm⁻¹ is attributed to the CH stretching vibration of the imidazole ring. -1 and 1384cm -1 The absorption peak at 1503 cm⁻¹ corresponds to the C=N and CN bonds of imidazole. -1 The characteristic peak at 1080 cm⁻¹ is due to the bending vibration of the CH₃ group in 2-methylimidazole. -1 and 1284cm -1 The characteristic peak at 603 cm⁻¹ is due to the stretching vibration of the imidazole ring. -1 The strong peak matches the Ni-N tensile vibration, indicating coordination between the metal nodes and organic ligands in MOF-818. In GeL, ALG-GeL, and MOF-818@ALG-Gel, the 3400 cm⁻¹ peak... -1 The nearby absorption peaks originate from the NH stretching vibration in chitosan, while the amide I band, mainly caused by the CO stretching vibration, is located at 1634 cm⁻¹. -1 The amide II band is caused by the combined stretching vibration of CN and the bending vibration of NH, and is located at 1534 cm⁻¹. -1; amide III band is caused by CH2wagging vibration, N-H bending vibration and C-N stretching vibration, located at 1234 cm -1 ; and 1150 cm -1 ; is the characteristic peak of the amino group after cross-linking of chitosan and genipin. Comparing MOF-818 and MOF-818@ALG-Gel with the above characteristic peaks shows that the samples are successfully synthesized.

[0069] Example 3: X-ray photoelectron spectroscopy (XPS) of MOF-818

[0070] The measured MOF-818 powder was pressed into a 3x3mm tablet with a weight greater than 10mg. The prepared sample was placed in the XPS instrument, and the scanning range was set to 0-1200eV with a scanning step of 0.05-2eV. During the test, the sample charge needs to be controlled to ensure the accuracy of the test results. The results are shown in Figure 2 A, through the complete XPS spectrum, clear signals of C, O, N, Cu and Zr were obtained, and the N element was from 4-pyrazole carboxylic acid. The XPS high-resolution spectrum of copper is shown in Figure 2 B, it was found that the fitting peaks Cu2p1 / 2(952eV) and Cu2p3 / 2(933eV) of Cu2p spectrum were accompanied by two satellite peaks, indicating that the main form of copper element in MOF-818 was Cu 2+ , indicating that Cu 2+ in the MAG has antibacterial, anti-inflammatory and tissue regeneration promoting effects.

[0071] Example 4: Scanning electron microscope image (SEM) of hydrogel

[0072] The hydrogel (GeL) and enzyme-based microcapsule composite hydrogel (MAG) were quickly frozen in liquid nitrogen and then freeze-dried in a freeze dryer overnight. The sample surface was sprayed with gold before scanning electron microscope observation and photography. Cross-section and surface photos were taken to capture their structure and morphology. As shown in Figure 3 , both groups of hydrogels have numerous pores. Comparison found that the pore size of GeL hydrogel is more chaotic, while the introduction of copper alginate MOF-818 microcapsules (MA) forms uniform pores, indicating that the structure of the hydrogel is also closely related to the hydrogen bonds formed between copper alginate and alginate. Strong intermolecular interaction can directly affect the microstructure of the hydrogel. The addition of copper alginate mainly promotes the formation of uniform voids and the integrity of the overall structure in the chitosan hydrogel network through intermolecular interaction.

[0073] Example 5: Antibacterial experiment of hydrogel on E. coli and S. aureus

[0074] Antibacterial experiments were carried out using S. aureus and E. coli, respectively. Single colonies of streak culture were cultured in liquid medium for 24 h, and then centrifuged and diluted with PBS to 10 6 CFU / mL. 125, 250 and 500 μL of the completely cross-linked hydrogel were measured and added to a 24-well plate, and then an equal amount of bacterial suspension was added as an experimental group. An equal volume of PBS was used as a control group. The mixture was incubated in a 37 °C incubator for 4 h, and then 50 μL of the bacterial suspension was plated and incubated in a 37 °C incubator for 24 h. The formation of colonies was observed. Figure 4 A is the antibacterial graph of S. aureus and E. coli, Figure 4 B is the antibacterial rate of the hydrogel and enzyme-based microcapsule composite hydrogel. The results show that the resistance rate of the hydrogel (GeL) to the two bacteria is greater than 70%, which proves that the hydrogel has good antibacterial activity. The enzyme-based microcapsule composite hydrogel (MAG) added with MA (MOF-818 microcapsule) has antibacterial activity to S. aureus and E. coli, and the antibacterial rate is greater than 90%. The synergistic antibacterial effect is also very prominent. The antibacterial rate of the hydrogel in the MAG experimental group to S. aureus and E. coli is close to 100%. It is proved that the MOF-818@ALG microcapsule and chitosan have a synergistic effect, which can synergistically improve the performance of the enzyme-based microcapsule composite hydrogel (MAG) prepared. It is also proved that the hydrogel and the MOF-818@ALG microcapsule in the present application have a synergistic effect, which can synergistically improve the performance of the enzyme-based microcapsule composite hydrogel (MAG) prepared.

[0075] Example 6: Hemolysis experiment of hydrogel

[0076] The blood was taken from the eyeball of the anesthetized mouse, and the red blood cells (RBCS) were obtained by centrifuging the whole blood at 1000 rpm for 10 min. The precipitated red blood cells were washed 2-3 times with physiological saline (0.9% NaCl solution) solution according to the above method until the supernatant was not red. The physiological saline was diluted to a mass concentration of 5%. The diluted red blood cell suspension (1.0 mL) and 1.0 mL of physiological saline (0.9% NaCl solution) were mixed as a negative control, the red blood cell suspension (1.0 mL) + 1.0 mL of PBS mixture was used as a positive control, and the red blood cell suspension (1.0 mL) and 1 mL of hydrogel leaching solution were used as an experimental group. Incubate at 37 °C on a shaker at 60 rpm for 1 h, and then take the supernatant (1000 rpm / 10 min). The ultraviolet absorbance at 540 nm was recorded by optical density (OD). The hemolysis rate calculation formula is as follows:

[0077] Hemolysis rate (%) = [(OD h - OD n )] / [(ODt -OD n )] x 100%

[0078] ODh, ODn, ODt are the absorbance values of the sample, negative control and positive control, respectively.

[0079] As shown in Table 2, the hemolysis rates of each group were less than 5%, indicating that the hydrogels had high safety and good blood compatibility, and could be used for further biomedical applications. Figure 5

[0080] Example 7: Mouse skin wound healing experiment of different treatment groups

[0081] 6-8 week old Kunming male mice weighing 20-30 g were selected for the experiment. The animals were bred and operated in the animal center of Tianjin University of Science and Technology. After 7 days of adaptive feeding, the mice were anesthetized by intraperitoneal injection of 15 mg / mL 2,2,2-tribromoethanol (150 mg / kg) before the experiment. After shaving the back of the mice, a full-thickness wound with a diameter of 8 mm was made on the midline, Figure 6 A is a schematic diagram of the wound healing experiment. The experimental animals were divided into 3 groups, including the model group (PBS, n = 9), the hydrogel group (GeL, n = 9), and the MOF-818 microcapsule hydrogel group (MAG, n = 9). After the modeling was completed, 50 μL of sterile PBS was added to the wound of the PBS group, and 50 μL of 10 6 CFU / mL S. aureus was added to the wounds of the GeL and MAG groups to induce inflammatory wounds. On days 0, 3, 7, 10, and 14, the healing process was recorded with a camera and random samples were taken. The wound area was calculated by Figure 6 B can be seen that on the 3rd day, no obvious wound healing trend was observed in the three groups, as they were all in the inflammatory outbreak period. On the 7th day, the GeL and MAG groups had already shown wound healing, while the control group was still in the first stage of wound healing due to the lack of treatment. On the 10th day, the MAG group showed a clear trend of wound area reduction, while the GeL group healed slowly, which may be related to the addition of copper ions in the MAG group, consistent with the above experimental trend. On the 14th day, the control group showed a clear trend of wound reduction, indicating that the mouse had resisted the inflammatory infection with its own immune system, while the MAG experimental group had already shown phenomena such as scab shedding, complete hair follicle development, and new hair growth. This indicates that the MOF-818 microcapsule hydrogel (MAG) significantly accelerates the healing of infected wounds and promotes functional repair of the skin, such as hair follicle regeneration, through the triple action of antibacterial, anti-inflammatory, and promoting tissue regeneration, and its efficacy is much higher than that of ordinary hydrogel (GeL) and natural healing (PBS). 2+

[0082] ​​Example 8: Masson staining of the skin of mice in different treatment groups

[0083] After the experiment of Example 7 was completed, the experimental mice were subjected to euthanasia procedures, and the model excision wound regeneration skin samples were trimmed to the appropriate size and placed in 4% paraformaldehyde. Subsequently, the tissues were wrapped and dehydrated, and then embedded using an embedding machine. The tissue-embedded paraffin blocks were removed and placed in a microtome for sectioning. The paraffin sections were placed in a drying machine and deparaffinized using different concentrations of ethanol (including anhydrous ethanol, 95%, 90%, 80%, and 70%). Cell nuclei were stained with hematoxylin: weigert iron hematoxylin staining for 5 min in the Masson staining kit, and then washed with water without interruption to remove the free hematoxylin stain. Differentiation solution (1% hydrochloric acid ethanol) was used for differentiation for 30 s, and then washed with water without interruption for about several minutes to remove the differentiation solution attached to the surface of the section. Blue reversal solution (saturated lithium carbonate) was used for blue reversal, and the sections were observed under a microscope to determine the staining. If the staining was not sufficient, the operation was repeated until the desired result was achieved. Ponceau staining: ponceau pink staining solution in the Masson staining kit was used for staining for about 6 min, and then 2% glacial acetic acid was used for washing without interruption to remove the free ponceau pink stain. Phosphomolybdate treatment: the paraffin sections were immersed in the aqueous phosphomolybdate solution in the Masson staining kit for about 4 min, and then washed with 2% glacial acetic acid without interruption for 1 min. Aniline blue staining: the paraffin sections were not washed with water, but were directly recolored with aniline blue solution in the Masson staining kit for 5 min. Differentiation: the paraffin sections were placed in a culture dish filled with 1% ice acetic acid for 1 min.

[0084] Mounting: the paraffin sections were dehydrated by being placed in anhydrous ethanol and xylene in turn for 3 times, and then mounted using neutral balsam. Microscopic observation: the color change of collagen deposition during wound healing was observed under a microscope, and the collagen fibers appeared blue.

[0085] Collagen production and maturation is one of the indicators of wound healing. The amount of collagen production was determined by Masson staining, and the results are shown in Figure 7 Figure 8. The control group formed a small amount of collagen fibers (green arrow) on day 14, the GeL group formed a small amount of loosely arranged collagen fibers on day 14, and the MAG group continued to form clear, continuous, regular, and tightly interwoven collagen fibers on day 14. Therefore, we concluded that the enzyme-based microcapsule composite hydrogel (MAG) group can accelerate wound healing and promote collagen deposition.

[0086] The material obtained by compounding MOF-818, microcapsules, and hydrogel is mainly highlighted in the present application, which can accelerate the healing of infected wounds and promote functional repair of the skin.

[0087] Although the embodiments of the present application have been disclosed for illustrative purposes, those skilled in the art can understand that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present application and the appended claims, and thus the scope of the present application is not limited to the disclosed embodiments.

Claims

1. A method for preparing an enzyme-based microcapsule composite hydrogel with wound healing properties, characterized in that: Includes the following steps: Chitosan solution and gelatin solution were mixed and stirred for 2 hours. Plasticizer solution was added and stirred until homogeneous. Then, 1%-5% (w / w) of MOF-818@ALG microcapsules were added. Finally, 8-12 mg / mL of crosslinking agent was added dropwise to the above solution and mixed thoroughly. The mixture was then poured into a glass petri dish and kept for 96 hours to complete the crosslinking process. The mixture was then air-dried to obtain a hydrogel loaded with microcapsules. The ratio of chitosan: gelatin: plasticizer: crosslinking agent (g:g:mL:mL) is 1:4:1:

1.

2. The preparation method according to claim 1, characterized in that: The chitosan solution is obtained by dissolving chitosan in acetic acid, and the mass concentration of the chitosan solution is 1%-3% (w / v). The gelatin solution is obtained by dissolving gelatin in ultrapure water, and the mass concentration of the gelatin solution is 5%-12% (w / v). The mass concentration of the plasticizer solution is 5%-12% (w / v).

3. The preparation method according to claim 1, characterized in that: The plasticizer is glycerin, polyethylene glycol, or xylitol.

4. The preparation method according to claim 1, characterized in that: The crosslinking agent is genipin, tannic acid, or gallic acid.

5. The preparation method according to claim 1, characterized in that: The preparation of the MOF-818@ALG microcapsules includes the following steps: (1) Preparation of MOF-818: Cu(NO3)2·3H2O, ZrOCl2·8H2O and 4-pyrazole carboxylic acid were dissolved in organic solvent one in a molar ratio of 0.5-0.7:0.1-0.3:0.15-0.

25. Organic solvent two was added to the above solution, and the mixture was thoroughly mixed and sonicated. The mixture was placed in a reaction vessel lined with polytetrafluoroethylene and cooled naturally at 100℃ for 8 hours. The mixture was then resuspended in organic solvent one and organic solvent three, centrifuged and dried to obtain MOF-818. (2) Preparation of MOF-818 copper alginate microcapsules MOF-818@ALG: Sodium alginate powder and MOF-818 powder were weighed to prepare sodium alginate aqueous solution. The solution was autoclaved with copper chloride solution. The sodium alginate solution was added to the second centrifuge tube of the centrifuge tube device, and the copper chloride solution was added to the first centrifuge tube of the centrifuge tube device. The needle of the second centrifuge tube was set at a distance of 0.45-1.5 cm from the top liquid surface of the copper chloride solution. The centrifuge tube device was placed in a centrifuge. The volume ratio of sodium alginate aqueous solution to copper chloride solution was 0.8-1.2:10.0-13.

0. The centrifuge was centrifuged at 1800-2500 rpm / min for 8-12 min to obtain copper alginate microcapsules MOF-818@ALG.

6. The preparation method according to claim 5, characterized in that: The first organic solvent is N,N-dimethylacetamide, N,N-dimethylformamide, or N-methylpyrrolidone; the second organic solvent is trifluoroacetic acid, formic acid, or hydrochloric acid; and the third organic solvent is ethanol, methanol, or acetonitrile.

7. The preparation method according to claim 5, characterized in that: The final mass concentration of sodium alginate in the solution is 0.5%-2.0%, and the final mass concentration of MOF-818 in the solution in step (2) is 0.05 mg / mL; the mass concentration of the copper chloride solution is 1.5%-2.5%.

8. The preparation method according to any one of claims 5 to 7, characterized in that: The centrifuge tube device includes a first centrifuge tube and a second centrifuge tube. The second centrifuge tube is coaxially and detachably connected to the first centrifuge tube. The first centrifuge tube includes a first centrifuge tube body and a first centrifuge tube cap. The first centrifuge tube body is arranged vertically and is shaped like a centrifuge tube with an open top. The first centrifuge tube cap is coaxially and detachably disposed above the opening of the first centrifuge tube body, and a second centrifuge tube mounting through hole is integrally formed on the first centrifuge tube cap. The first centrifuge tube body can contain a copper chloride solution. The second centrifuge tube includes a second centrifuge tube body, a second centrifuge tube cap, and a needle. The second centrifuge tube body is arranged vertically and is shaped like a centrifuge tube with openings at both the top and bottom. The second centrifuge tube cap is movably and detachably connected to the top opening of the second centrifuge tube body. The second centrifuge tube body can hold an aqueous solution of sodium alginate. The needle is detachably and detachably connected to the bottom opening of the second centrifuge tube body and is arranged vertically. The shape of the upper outer surface of the second centrifuge tube body matches the shape of the inner surface of the second centrifuge tube mounting through hole. The upper part of the second centrifuge tube body can be coaxially and detachably connected to the first centrifuge tube cap through the second centrifuge tube mounting through hole. The upper surface of the second centrifuge tube body is flush with the upper surface of the first centrifuge tube cap. The lower middle part of the second centrifuge tube body extends downward into the first centrifuge tube body. Alternatively, the first centrifuge tube has a volume of 50 mL, the second centrifuge tube has a volume of 1.5 mL, and the needle is positioned 0.45-1.5 cm above the copper chloride solution contained in the first centrifuge tube.

9. The enzyme-based microcapsule composite hydrogel prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the enzyme-based microcapsule composite hydrogel as described in claim 9 in the preparation of a wound-healing medicament.

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