Composite hydrogel dressing and application

Abstract

The invention belongs to the field of biomedical materials, and discloses a composite hydrogel dressing and application. The composite hydrogel dressing is prepared by the following steps: blending 4-aminophenylboronic acid grafted hyaluronic acid and gallic acid grafted collagen, adjusting the pH value, carrying out a cross-linking reaction to obtain a cross-linking product, and stirring and mixing the cross-linking product with a gallic acid reduced silver nanoparticle solution. The antibacterial and self-healing composite hydrogel dressing for promoting wound repair has proper adhesion, mechanical properties and free radical scavenging ability, and excellent antibacterial property, biocompatibility and self-healing ability.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and more specifically, relates to a composite hydrogel dressing and its application. Background Art

[0002] The increasing incidence of complex wounds resulting from burns, large tissue necrosis and defects, surgery, and chronic wounds caused by diabetic foot ulcers urges the development of functional dressings that can resist exogenous infection and promote tissue regeneration. Hydrogels, a typical three-dimensional network polymer material, possess high water content (>90%) and tissue-like elastic modulus, which not only maintain a moist wound environment but also provide mechanical support. Their unique freeze-drying and rehydration properties further enhance their portability and clinical applicability (the freeze-dried state facilitates storage and transportation, and upon activation with saline, they quickly form a bioadhesive dressing). Therefore, they have attracted extensive attention from biomedical materials researchers. Compared with traditional chemically synthesized polymer hydrogels, hyaluronic acid, a major component of the extracellular matrix, possesses superior water retention. Through sustained hydration, it can alleviate wound dryness and inhibit bacterial biofilm formation. Similar to collagen, which has a triple-helical structure, they can interact with multiple receptors to induce cellular responses, promoting cell migration and proliferation, angiogenesis, and tissue regeneration. Hydrogels constructed from these hydrogels exhibit unique bioactivity. However, its poor mechanical properties and lack of self-repair ability severely limit its service life and restrict its application in implantable flexible electrodes, strain sensing materials and wound repair.

[0003] Three-dimensional network hydrogels constructed based on non-covalent bonds such as molecular chain entanglement, hydrogen bonds, or hydrophobic bonds can significantly improve the mechanical properties of collagen-based gels (Liu H, Hu X, Li W, et al. A highly-stretchable and adhesive hydrogel for noninvasive joint wound closure driven by hydrogen bonds[J]. Chemical Engineering Journal, 2023, 452: 139368.). However, these hydrogels are unidirectional and irreversible. Under external stress, once the network is destroyed, it cannot self-repair the cracks and return to an integrated gel. In addition, to prevent reinfection during wound healing, antibacterial ability is also necessary. The introduction of antibiotics can significantly improve antibacterial ability (Tripathi S, Singh BN, Singh D, et al. Optimization and evaluation of ciprofloxacin-loaded collagen / chitosan scaffolds for skin tissue engineering [J]. 3Biotech, 2021, 11 (4): 160.). However, if absorbed through the skin or enters the blood circulation through the wound, it may increase the metabolic burden on the liver and kidneys. There is also a certain risk of drug resistance. Non-antibiotic hydrogels based on nanosilver, chitosan, etc. can effectively avoid the above safety hazards. In addition, suitable adhesion properties are conducive to fitting with body parts, thereby giving full play to its efficacy.

[0004] Given the inherent advantages of collagen and hyaluronic acid in biological properties, the development of hydrogels with suitable adhesion, sufficient mechanical strength, excellent antibacterial properties and self-healing properties has great biomedical application value. Summary of the Invention

[0005] The present invention aims to address the deficiencies of the prior art and propose a composite hydrogel dressing and its application. The composite hydrogel dressing of the present invention, which promotes wound repair and antibacterial self-healing, has appropriate adhesion, mechanical properties, free radical scavenging ability, and excellent antibacterial properties, biocompatibility, and self-healing ability.

[0006] In order to achieve the above objectives, the present invention provides a composite hydrogel dressing on one hand, which is prepared by blending hyaluronic acid grafted with 4-aminophenylboronic acid and collagen grafted with gallic acid, adjusting the pH value, and then performing a cross-linking reaction to obtain a cross-linked product, and stirring and mixing the cross-linked product with a solution of silver nanoparticles reduced with gallic acid.

[0007] The technical principles of the present invention are as follows:

[0008] First, existing collagen-based hydrogels lack repairability and have weak mechanical properties, which is determined by the formation mechanism of collagen-based hydrogels. Chemical cross-linking methods, such as chemical cross-linking with glutaraldehyde or EDC / NHS cross-linkers, can form a more stable network structure by generating covalent cross-links between active groups within or between collagen molecules. This method improves the mechanical properties and stability of the hydrogel network. However, under the action of external stress, once the network structure is destroyed and cracks appear, the gel system cannot self-repair, thereby losing the integrity of the hydrogel structure.

[0009] Therefore, in order to improve the mechanical properties of collagen-based hydrogels and impart good self-healing properties, the present invention uses chemical cross-linking to achieve this:

[0010] The present invention uses EDC / NHS cross-linking agents to mediate the formation of amide covalent bonds between the carboxylic acid groups of gallic acid and the amino groups of collagen, thereby enhancing the stability of the collagen network, improving mechanical properties, and also providing active sites for subsequent functionalization. In addition, the grafting modification of collagen also limits the self-assembly behavior of the collagen molecules due to their unique triple helical structure, thereby inhibiting the formation of a three-dimensional collagen fiber network. The hydrogel formed by this collagen three-fiber network is irreversible, that is, the gel has no self-repair ability after rupture. To further improve the mechanical properties, a certain amount of macromolecules are introduced into the modified collagen molecules to increase the cross-linking density and enhance the mechanical properties by covalent bonding. Among them, the reversible dynamic covalent bond can also give the hydrogel self-healing properties. The present invention selects hyaluronic acid molecules, one of the main components of the extracellular matrix, which has excellent biocompatibility and can also promote cell migration and reduce scar formation by maintaining a moist environment on the wound surface. After it is modified with 4-aminophenylboronic acid, the boronic acid bond formed between it and the catechol group on the collagen molecule modified with gallic acid is used to give the gel self-healing properties.

[0011] The composite hydrogel material of the present invention has a three-dimensional porous structure, which is obtained by chemical crosslinking of gallic acid-grafted collagen and 4-aminophenylboronic acid-grafted hyaluronic acid through boron ester bonds and intermolecular hydrogen bonding.

[0012] According to the present invention, preferably, the mass ratio of the hyaluronic acid grafted with 4-aminophenylboronic acid to the collagen grafted with gallic acid is (0.02-0.06): (0.01-0.02).

[0013] According to the present invention, preferably, the pH value of the blended system of hyaluronic acid grafted with 4-aminophenylboronic acid and collagen grafted with gallic acid is adjusted to 7-9.5.

[0014] According to the present invention, preferably, the preparation method of the composite hydrogel dressing comprises:

[0015] mixing the gallic acid-grafted collagen with an acetic acid aqueous solution to obtain a gallic acid-grafted collagen-acetic acid solution;

[0016] mixing hyaluronic acid grafted with 4-aminophenylboronic acid with water to obtain a hyaluronic acid-aqueous solution grafted with 4-aminophenylboronic acid;

[0017] Mixing a gallic acid-grafted collagen-acetic acid solution with a 4-aminophenylboronic acid-grafted hyaluronic acid-aqueous solution to obtain a blend system of 4-aminophenylboronic acid-grafted hyaluronic acid and gallic acid-grafted collagen, adjusting the pH value of the blend system with a first pH adjuster, and then performing a cross-linking reaction to obtain a cross-linked product;

[0018] The cross-linked product is stirred and mixed with a silver nanoparticle solution reduced with gallic acid to obtain the composite hydrogel dressing.

[0019] According to the present invention, preferably, the first pH adjuster is at least one of a NaHCO3-NaCl mixed solution, a boric acid-borax buffer solution and a Tris-HCl buffer solution.

[0020] According to the present invention, preferably, the preparation method of the 4-aminophenylboronic acid grafted hyaluronic acid comprises: mixing a hyaluronic acid aqueous solution with a first cross-linking agent and adjusting the pH value to obtain a dissolving system; mixing the dissolving system with 4-aminophenylboronic acid hydrochloride and reacting under stirring to obtain a product solution, and dialyzing and freeze-drying to obtain the 4-aminophenylboronic acid grafted hyaluronic acid.

[0021] In the present invention, as a preferred embodiment, in the step of obtaining the 4-aminophenylboronic acid grafted hyaluronic acid through dialysis and freeze-drying:

[0022] Dialysis was performed using a NaCl aqueous solution as the external dialysis solution, and the internal dialysis solution was taken out and freeze-dried to obtain hyaluronic acid grafted with 4-aminophenylboronic acid.

[0023] The molecular weight of the dialysis bag used in this step is 1000-3500 Da, and the concentration of the NaCl aqueous solution is 0.1-0.3 mol / L.

[0024] According to the present invention, preferably, the concentration of the hyaluronic acid aqueous solution is 5-8.5 g / L.

[0025] According to the present invention, preferably, the first cross-linking agent is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and the mass ratio of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide, hyaluronic acid and 4-aminophenylborate hydrochloride is (0.8-1.5): (0.4-0.75): (0.7-1.4): (0.6-1.2).

[0026] According to the present invention, preferably, the temperature at which the hyaluronic acid aqueous solution and the first cross-linking agent are mixed is 35-45° C., so that the first cross-linking agent is dissolved in the hyaluronic acid aqueous solution under a slightly warm environment.

[0027] According to the present invention, preferably, a second pH adjuster is used to adjust the pH value of the dissolution system to 4-6; the second pH adjuster is at least one of NaOH, Na2HPO4 and NH3·H2O.

[0028] According to the present invention, preferably, the time for reacting the dissolving system with 4-aminophenylborate hydrochloride under stirring is 12-36 hours and the temperature is 10-25°C.

[0029] According to the present invention, preferably, the preparation of the gallic acid-grafted collagen is carried out under light-proof conditions, including: mixing a gallic acid aqueous solution with a second cross-linking agent and reacting at a constant temperature to obtain an activated product; mixing the cooled activated product with a collagen acetic acid aqueous solution to obtain a mixed system, adjusting the pH value of the mixed system and reacting under a nitrogen atmosphere and stirring conditions to obtain a product solution, and dialyzing and freeze-drying to obtain the gallic acid-grafted collagen.

[0030] In the present invention, the gallic acid aqueous solution is mixed with the second cross-linking agent and reacted at a constant temperature in the dark, thereby activating the carboxyl groups in the gallic acid to obtain an activated product.

[0031] In the present invention, as a preferred embodiment, in the step of obtaining the gallic acid-grafted collagen through dialysis and freeze-drying:

[0032] The dialysis was performed using deionized water at a temperature of 2-8° C. as the dialysis external fluid, and the dialysis internal fluid was taken out and freeze-dried to obtain gallic acid-grafted collagen.

[0033] The dialysis bag used in this step has a molecular weight of 10,000-20,000 Da.

[0034] According to the present invention, preferably, the concentration of the gallic acid aqueous solution is 10-15 g / L.

[0035] According to the present invention, preferably, the second cross-linking agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and the mass ratio of gallic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is (0.5-0.7):(0.4-0.75):(0.25-0.45).

[0036] According to the present invention, preferably, the constant temperature is 35-45° C., and the reaction time at the constant temperature is 20-40 min.

[0037] According to the present invention, preferably, the collagen acetic acid aqueous solution is prepared by mixing collagen with acetic acid aqueous solution, the concentration of the acetic acid aqueous solution is 0.3-0.8 mol / L, and the concentration of the collagen in the collagen acetic acid aqueous solution is 5-8 g / L; the collagen is at least one of bovine Achilles tendon collagen, pig skin collagen and bullfrog skin collagen.

[0038] According to the present invention, preferably, the mass ratio of the collagen and gallic acid in the mixed system is (0.75-1.2):(0.5-0.7).

[0039] According to the present invention, preferably, a third pH adjuster is used to adjust the pH value of the mixed system to 4-6; the third pH adjuster is an acetic acid-sodium acetate buffer solution.

[0040] According to the present invention, preferably, the reaction time under nitrogen atmosphere and stirring conditions is 12-36 hours and the temperature is 2-8°C.

[0041] According to the present invention, preferably, the volume fraction of the gallic acid-reduced silver nanoparticle solution in the composite hydrogel dressing is 2.5-10%.

[0042] In the present invention, it is worth noting that the natural active molecule gallic acid is introduced into the present invention, which plays a dual function in the hydrogel. On the one hand, it participates in network construction as a cross-linking agent. On the other hand, it utilizes the reducing property of its polyphenol structure to reduce silver ions to silver nanoparticles in situ under mild conditions and uniformly load them into the hybrid hydrogel. This green synthesis strategy avoids the toxicity of traditional reducing agents while giving the hydrogel significant antibacterial properties. The resulting composite hydrogel not only has excellent biocompatibility, mechanical adaptability and self-repair properties, but also effectively inhibits wound infection by introducing Ag nanoparticles.

[0043] According to the present invention, preferably, the preparation of the gallic acid-reduced silver nanoparticle solution is carried out under light-proof conditions, comprising: mixing an aqueous silver nitrate solution and an aqueous gallic acid solution to obtain a mixed system, adjusting the pH value of the mixed system and reacting to obtain the gallic acid-reduced silver nanoparticle solution.

[0044] According to the present invention, preferably, a fourth pH adjuster is used to adjust the pH value of the mixed system to 7-11; the fourth pH adjuster is a Tris-HCl buffer solution and / or a Tris-NaOH buffer solution.

[0045] According to the present invention, preferably, the reaction time is 15-40 minutes.

[0046] According to the present invention, preferably, the concentration of the silver nitrate aqueous solution is 0.03-0.07 mol / L.

[0047] According to the present invention, preferably, the concentration of the gallic acid aqueous solution is 0.05-0.10 mol / L.

[0048] According to the present invention, preferably, the volume ratio of the silver nitrate aqueous solution to the gallic acid aqueous solution is (0.8-1.2): (0.8-1.2).

[0049] According to the present invention, preferably, the composite hydrogel dressing is prepared by blending hyaluronic acid grafted with 4-aminophenylboronic acid and collagen grafted with gallic acid, adjusting the pH value, and then performing a cross-linking reaction to obtain a cross-linked product, and then stirring and mixing the cross-linked product with a solution of silver nanoparticles reduced with gallic acid and other functional additives;

[0050] The other functional auxiliary agents are at least one of pharmaceutically active molecules, epidermal growth factor, gold nanoparticles and zinc oxide nanoparticles.

[0051] According to the present invention, preferably, the pharmaceutically active molecule is ciprofloxacin and / or curcumin.

[0052] In the present invention, generally, the raw material components of the antibacterial self-healing hydrogel dressing for promoting wound repair according to the present invention may also include functional adjuvants, thereby giving the hydrogel of the present invention more functionality. Those skilled in the art can add functional adjuvants according to the functional adjuvants conventionally used in the art, including pharmaceutically active molecules (such as ciprofloxacin, curcumin, etc.), functional molecules such as epidermal growth factor, nanoparticles such as gold and zinc oxide, according to the required addition ratio of the functional adjuvant. It is noted that when ensuring the function, it is generally recommended to add a lower proportion of functional adjuvants, so as to retain the proportion of the gel matrix to the greatest extent and ensure that the product has more beneficial properties.

[0053] In the present invention, the equipment used for freeze drying is a freeze dryer, LGJ-18C, purchased from Beijing Songyuan Co., Ltd.

[0054] Another aspect of the present invention provides use of the composite hydrogel dressing in preparing a medicine for treating wound healing.

[0055] According to the present invention, preferably, the wound is a diabetic foot ulcer wound, a knife wound or a bacterial infection wound.

[0056] The beneficial effects of the technical solution of the present invention are as follows: the present invention loads gallic acid-reduced silver nanoparticles into a blend of 4-aminophenylboronic acid-grafted hyaluronic acid and gallic acid-grafted collagen to form a composite hydrogel with appropriate adhesion, mechanical properties, free radical scavenging ability, excellent antibacterial properties, biocompatibility, and self-healing ability. Specifically:

[0057] (1) The present invention loads gallic acid-reduced silver nanoparticles onto a hydrogel, resulting in a composite hydrogel dressing with excellent antibacterial properties, with an inhibition rate exceeding 99% against Staphylococcus aureus and Escherichia coli. Furthermore, the composite hydrogel dressing prepared by the present invention can remove excess reactive oxygen species, promote wound edge contraction, and significantly accelerate the wound closure process.

[0058] (2) The composite hydrogel dressing prepared by the present invention has good adhesion properties and can adhere to plastic, metal, glass, wood, and skin.

[0059] (3) The composite hydrogel dressing prepared by the present invention has good mechanical strength, and its tensile strength is 4.58 MPa.

[0060] (4) The present invention blends hyaluronic acid grafted with 4-aminophenylboronic acid and collagen grafted with gallic acid, and a cross-linking reaction occurs through the formation of dynamically reversible boron ester bonds, so that the composite hydrogel dressing prepared by the present invention has excellent self-repairing ability.

[0061] (5) The composite hydrogel dressing prepared by the present invention has good biocompatibility. Using human umbilical vein endothelial cells (HUVEC) as model cells, the cell live-dead staining experiment, cytotoxicity experiment and scratch test showed that the hydrogel has good cell biocompatibility; the blood test showed that its hemolysis rate was less than 5%, confirming that there is no hemolysis risk.

[0062] (6) The composite hydrogel dressing prepared by the present invention has a simple preparation method, high repeatability, is easy to be produced on a large scale in industry, and the raw materials are readily available without causing environmental pollution.

[0063] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings, wherein like reference numerals generally represent like components throughout the exemplary embodiments of the present invention.

[0065] Figure 1 The graphs show the rheological properties of the composite hydrogel dressings obtained in Examples 1-4 of the present invention.

[0066] Figure 2 (a)-(c) shows the self-healing performance of the composite hydrogel dressing obtained in Example 4 of the present invention.

[0067] Figure 3 (a)-(b) shows the tensile and compressive strain diagrams of the composite hydrogel dressings obtained in Examples 1-4 of the present invention (Figure 3 (a) is the tensile strain diagram, and Figure 3 (b) is the compressive strain diagram).

[0068] Figures 4(a)-(f) show the adhesion performance diagrams of the composite hydrogel dressing obtained in Example 4 of the present invention (Figure 4(a) shows adhesion to plastic, Figure 4(b) shows adhesion to metal, Figure 4(c) shows adhesion to glass, Figure 4(d) shows adhesion to wood, Figure 4(e) shows adhesion to skin, and Figure 4(f) shows adhesion to glass bottle and skin).

[0069] Figures 5(a)-(b) show the comparison of the cell live-death staining experiment and CCK-8 cell proliferation experiment of the composite hydrogel dressing obtained in Example 4 of the present invention and the comparative example (Figure 5(a) is a diagram of the cell live-death staining experiment observed by a fluorescence inverted microscope; Figure 5(b) is the cell proliferation rate in hydrogel extracts of different concentrations).

[0070] Figure 6 (a)-(b) shows the comparative results of the antibacterial test of the composite hydrogel dressing obtained in Example 4 of the present invention and the comparative example.

[0071] Figures 7(a)-(c) show the therapeutic effects of the hydrogel dressing prepared in Example 4 of the present invention, commercial dressing, and sterile gauze as a control group on infected wounds in rats.

[0072] Figures 8(a)-(f) show the evaluation results of the wound healing and anti-inflammatory ability of rat wound skin tissue using the hydrogel dressing prepared in Example 4 of the present invention, commercial dressing and sterile gauze as control groups.

[0073] Figure 9 These are morphological photographs of the hydrogels obtained at pH 5.0 in Example 5 and at pH 7.4 in Example 2. DETAILED DESCRIPTION

[0074] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. Instead, these embodiments are provided to make the present invention more thorough and complete and to fully convey the scope of the present invention to those skilled in the art.

[0075] In the following embodiments and comparative examples:

[0076] The 4-aminophenyl boronate hydrochloride is from Anhui Zesheng Technology Co., Ltd. (A060259)

[0077] The hyaluronic acid was obtained from Shanghai MacLean Biochemical Technology Co., Ltd. (C16465404);

[0078] Gallic acid was obtained from Shanghai MacLean Biochemical Technology Co., Ltd. (C14653861);

[0079] The 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was obtained from Shanghai MacLean Biochemical Technology Co., Ltd. (C16007466);

[0080] The N-hydroxysuccinimide was obtained from Beijing Yikainuo Technology Co., Ltd. (KBCHL202).

[0081] Example 1

[0082] This embodiment provides a composite hydrogel dressing, the preparation method of which includes:

[0083] S1: Preparation of gallic acid-reduced silver nanoparticle solution was carried out under light-protected conditions, including:

[0084] Equal volumes of 0.05 mol / L AgNO3 aqueous solution and 0.07 mol / L gallic acid aqueous solution were mixed in the dark, and then reacted at pH = 9 (pH adjusted using Tris-NaOH solution) in the dark for 40 minutes to obtain a gallic acid-reduced silver nanoparticle solution.

[0085] S2: The preparation method of hyaluronic acid grafted with 4-aminophenylboronic acid comprises:

[0086] A hyaluronic acid aqueous solution (8 g / L, 0.15 L) was mixed with 1.2 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.72 g of N-hydroxysuccinimide (NHS) at 40°C to dissolve the first crosslinker in the hyaluronic acid aqueous solution under a slightly warm environment. The pH was then adjusted to 5.2 (using aqueous NaOH solution) to obtain a solution. The solution was then mixed with 0.92 g of 4-aminophenylboronic acid hydrochloride and reacted at 10°C for 24 hours to obtain a product solution. The solution was then dialyzed using a 0.15 mol / L NaCl aqueous solution as the external dialysis solution (dialysis bag molecular weight 1500 Da). The dialyzed solution was removed and freeze-dried to obtain hyaluronic acid grafted with 4-aminophenylboronic acid.

[0087] S3: Preparation of gallic acid grafted collagen is carried out under light-protected conditions, including:

[0088] Gallic acid aqueous solution (13 g / L, 5 mL) was mixed with 0.064 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.042 g of N-hydroxysuccinimide (NHS) and reacted at a constant temperature of 40°C in the dark for 30 min to obtain an activated product;

[0089] The cooled activated product was mixed with 15 mL of bovine Achilles tendon collagen acetic acid aqueous solution (the bovine Achilles tendon collagen acetic acid aqueous solution was prepared by mixing bovine Achilles tendon collagen with acetic acid aqueous solution, the concentration of the acetic acid aqueous solution was 0.5 mol / L, and the concentration of the bovine Achilles tendon collagen in the bovine Achilles tendon collagen acetic acid aqueous solution was 5 g / L) to obtain a mixed system, and the pH value of the mixed system was adjusted to 5 (pH adjustment was performed using HAc-NaAc buffer solution) and the reaction was carried out under nitrogen atmosphere, light-proof and stirring conditions (reaction time was 36 h, temperature was 4 ° C) to obtain a product solution. Then, dialysis was performed using 4 ° C deionized water as the dialysis external solution (the dialysis bag molecular weight was 14000 Da), the dialysis internal solution was taken out, and freeze-dried to obtain the gallic acid grafted collagen;

[0090] S4: Dissolving the gallic acid grafted collagen in a 0.5 mol / L acetic acid aqueous solution to obtain a gallic acid grafted collagen-acetic acid solution (the concentration of the gallic acid grafted collagen in the gallic acid grafted collagen-acetic acid solution is 5 g / L); mixing the hyaluronic acid grafted with 4-aminophenylboronic acid with water to obtain a 4-aminophenylboronic acid grafted hyaluronic acid-aqueous solution (the concentration of the hyaluronic acid grafted with 4-aminophenylboronic acid in the hyaluronic acid grafted with 4-aminophenylboronic acid-aqueous solution is 25 g / L); / L); the gallic acid-grafted collagen-acetic acid solution and the hyaluronic acid-aqueous solution grafted with 4-aminophenylboronic acid were mixed in equal volumes, and the pH of the solution was adjusted to 9.2 (pH adjustment was performed using a NaHCO3-NaCl mixed solution) to form a hyaluronic acid-collagen hydrogel; the gallic acid-reduced silver nanoparticle solution obtained in step S1 was added to the above-mentioned hyaluronic acid-collagen hydrogel to a volume fraction of 2.5%, and stirred to obtain a composite hydrogel dressing CG-HB-1.

[0091] The hydrogel prepared in this example is flexible and elastic, can be stretched and wound, and has self-healing properties. The stress-strain performance test of Test Example 3 shows that the tensile strength of the hydrogel prepared in this example is 2.52 MPa.

[0092] Example 2

[0093] This embodiment provides a composite hydrogel dressing, the preparation method of which includes:

[0094] S1: Preparation of gallic acid-reduced silver nanoparticle solution is the same as in Example 1;

[0095] S2: The preparation method of hyaluronic acid grafted with 4-aminophenylboronic acid comprises:

[0096] A hyaluronic acid aqueous solution (6.7 g / L, 0.15 L) was mixed with 0.9567 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.6532 g of N-hydroxysuccinimide (NHS) at 40°C to dissolve the first crosslinker in the hyaluronic acid aqueous solution under a slightly warm environment. The pH was then adjusted to 5.5 (using a Na2HPO4 solution) to obtain a solution. The solution was then mixed with 0.8672 g of 4-aminophenylboronic acid hydrochloride and reacted at 10°C for 24 hours to obtain a product solution. The solution was then dialyzed using a 0.15 mol / L NaCl aqueous solution as the external dialysis solution (dialysis bag molecular weight 1500 Da). The dialyzed solution was removed and freeze-dried to obtain hyaluronic acid grafted with 4-aminophenylboronic acid.

[0097] S3: Preparation of gallic acid grafted collagen is carried out under light-protected conditions, including:

[0098] Gallic acid aqueous solution (10 g / L, 5 mL) was mixed with 0.0402 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.0285 g of N-hydroxysuccinimide (NHS) and reacted at a constant temperature of 40°C in the dark for 30 min to obtain an activated product;

[0099] The cooled activated product was mixed with 15 mL of bovine Achilles tendon collagen acetic acid aqueous solution (the bovine Achilles tendon collagen acetic acid aqueous solution was prepared by mixing bovine Achilles tendon collagen with acetic acid aqueous solution, the concentration of the acetic acid aqueous solution was 0.5 mol / L, and the concentration of the bovine Achilles tendon collagen in the bovine Achilles tendon collagen acetic acid aqueous solution was 5 g / L) to obtain a mixed system, and the pH value of the mixed system was adjusted to 5.5 (pH adjustment was performed using HAc-NaAc buffer solution) and the reaction was carried out under nitrogen atmosphere, light-proof and stirring conditions (reaction time was 24 h, temperature was 4 ° C) to obtain a product solution. Then, dialysis was performed using 4 ° C deionized water as the dialysis external solution (the dialysis bag molecular weight was 15000 Da), the dialysis internal solution was taken out, and freeze-dried to obtain the gallic acid grafted collagen;

[0100] S4: Dissolving the gallic acid grafted collagen in a 0.5 mol / L acetic acid aqueous solution to obtain a gallic acid grafted collagen-acetic acid solution (the concentration of the gallic acid grafted collagen in the gallic acid grafted collagen-acetic acid solution is 4 g / L); mixing the hyaluronic acid grafted with 4-aminophenylboronic acid with water to obtain a 4-aminophenylboronic acid grafted hyaluronic acid-aqueous solution (the concentration of the hyaluronic acid grafted with 4-aminophenylboronic acid in the hyaluronic acid grafted with 4-aminophenylboronic acid-aqueous solution is 4 g / L). The gallic acid-grafted collagen-acetic acid solution and the 4-aminophenylboronic acid-grafted hyaluronic acid-aqueous solution were mixed in equal volumes, and the pH of the solution was adjusted to 7.4 (using Tris-HCl for pH adjustment) to form a hyaluronic acid-collagen hydrogel; the gallic acid-reduced silver nanoparticle solution obtained in step S1 was added to the hyaluronic acid-collagen hydrogel to a volume fraction of 4%, and stirred to obtain a composite hydrogel dressing CG-HB-2.

[0101] The hydrogel prepared in this example is flexible and elastic, can be stretched and wound, and has self-healing properties. The stress-strain performance test of Test Example 3 shows that the tensile strength of the hydrogel prepared in this example is 2.99 MPa.

[0102] Example 3

[0103] This embodiment provides a composite hydrogel dressing, the preparation method of which includes:

[0104] S1: Preparation of gallic acid-reduced silver nanoparticle solution is the same as in Example 2;

[0105] S2: The preparation method of hyaluronic acid grafted with 4-aminophenylboronic acid is the same as that in Example 2;

[0106] S3: Preparation of gallic acid grafted collagen is carried out under light-protected conditions, including:

[0107] Gallic acid aqueous solution (13 g / L, 5 mL) was mixed with 0.0587 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.0358 g of N-hydroxysuccinimide (NHS) and reacted at 40°C in the dark for 30 min to obtain the activated product;

[0108] The cooled activated product was mixed with 15 mL of bovine Achilles tendon collagen acetic acid aqueous solution (the bovine Achilles tendon collagen acetic acid aqueous solution was prepared by mixing bovine Achilles tendon collagen with acetic acid aqueous solution, the concentration of the acetic acid aqueous solution was 0.5 mol / L, and the concentration of the bovine Achilles tendon collagen in the bovine Achilles tendon collagen acetic acid aqueous solution was 5 g / L) to obtain a mixed system, and the pH value of the mixed system was adjusted to 5 (pH adjustment was performed using HAc-NaAc buffer solution) and the reaction was carried out under nitrogen atmosphere, light-proof and stirring conditions (reaction time was 36 h, temperature was 4 ° C) to obtain a product solution. Then, dialysis was performed using 4 ° C deionized water as the dialysis external solution (the dialysis bag molecular weight was 14000 Da), the dialysis internal solution was taken out, and freeze-dried to obtain the gallic acid grafted collagen;

[0109] S4: dissolving the gallic acid grafted collagen in a 0.5 mol / L acetic acid aqueous solution to obtain a gallic acid grafted collagen-acetic acid solution (the concentration of the gallic acid grafted collagen in the gallic acid grafted collagen-acetic acid solution is 5 g / L); mixing the hyaluronic acid grafted with 4-aminophenylboronic acid with water to obtain a 4-aminophenylboronic acid grafted hyaluronic acid-aqueous solution (the concentration of the hyaluronic acid grafted with 4-aminophenylboronic acid in the hyaluronic acid-aqueous solution is 5 g / L); The concentration is 35 g / L); the gallic acid-grafted collagen-acetic acid solution and the hyaluronic acid-aqueous solution grafted with 4-aminophenylboronic acid are mixed in equal volumes, and the pH of the solution is adjusted to 9 (pH adjustment is performed using a boric acid-borax mixed solution) to form a hyaluronic acid-collagen hydrogel; the gallic acid-reduced silver nanoparticle solution obtained in step S1 is added to the above-mentioned hyaluronic acid-collagen hydrogel to a volume fraction of 4%, and stirred to obtain a composite hydrogel dressing CG-HB-3.

[0110] The hydrogel prepared in this example is flexible and elastic, can be stretched and wound, and has self-healing properties. The stress-strain performance test of Test Example 3 shows that the tensile strength of the hydrogel prepared in this example is 3.73 MPa.

[0111] Example 4

[0112] This embodiment provides a composite hydrogel dressing, the preparation method of which includes:

[0113] S1: Preparation of gallic acid-reduced silver nanoparticle solution is the same as in Example 2;

[0114] S2: The preparation of hyaluronic acid grafted with 4-aminophenylboronic acid is the same as that in Example 2;

[0115] S3: Preparation of gallic acid grafted collagen is carried out under light-protected conditions, including:

[0116] Gallic acid aqueous solution (13 g / L, 5 mL) was mixed with 0.0587 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.0358 g of N-hydroxysuccinimide (NHS) and reacted at 40°C in the dark for 30 min to obtain the activated product;

[0117] The cooled activated product was mixed with 15 mL of bovine Achilles tendon collagen acetic acid aqueous solution (the bovine Achilles tendon collagen acetic acid aqueous solution was prepared by mixing bovine Achilles tendon collagen with acetic acid aqueous solution, the concentration of the acetic acid aqueous solution was 0.5 mol / L, and the concentration of the bovine Achilles tendon collagen in the bovine Achilles tendon collagen acetic acid aqueous solution was 5 g / L) to obtain a mixed system, and the pH value of the mixed system was adjusted to 5 (pH adjustment was performed using HAc-NaAc buffer solution) and the reaction was carried out under nitrogen atmosphere, light-proof and stirring conditions (reaction time was 24 h, temperature was 4 ° C) to obtain a product solution. Then, dialysis was performed using 4 ° C deionized water as the dialysis external solution (the molecular weight of the dialysis bag was 14000 Da), the dialysis internal solution was taken out, and freeze-dried to obtain the gallic acid-grafted collagen;

[0118] S4: dissolving the gallic acid grafted collagen in a 0.5 mol / L acetic acid aqueous solution to obtain a gallic acid grafted collagen-acetic acid solution (the concentration of the gallic acid grafted collagen in the gallic acid grafted collagen-acetic acid solution is 5 g / L); mixing the hyaluronic acid grafted with 4-aminophenylboronic acid with water to obtain a 4-aminophenylboronic acid grafted hyaluronic acid-aqueous solution (the concentration of the hyaluronic acid grafted with 4-aminophenylboronic acid in the hyaluronic acid-aqueous solution is 4 g / L); 0 g / L); mixing equal volumes of the gallic acid-grafted collagen-acetic acid solution and the hyaluronic acid-aqueous solution grafted with 4-aminophenylboronic acid, and adjusting the pH of the solution to 8 (using a NaHCO3-NaCl mixed solution for pH adjustment) to form a hyaluronic acid-collagen hydrogel; adding the gallic acid-reduced silver nanoparticle solution obtained in step S1 to the above-mentioned hyaluronic acid-collagen hydrogel to a volume fraction of 4%, and stirring to obtain a composite hydrogel dressing CG-HB-4.

[0119] The hydrogel prepared in this example is flexible and elastic, can be stretched and wound, and has self-healing properties. The stress-strain performance test of Test Example 3 shows that the tensile strength of the hydrogel prepared in this example is 4.55 MPa.

[0120] Example 5

[0121] This embodiment provides a composite hydrogel dressing, the preparation method of which includes:

[0122] S1: Preparation of gallic acid-reduced silver nanoparticle solution was carried out under light-protected conditions, including:

[0123] Equal volumes of 0.05 mol / L AgNO3 aqueous solution and 0.07 mol / L gallic acid aqueous solution were mixed in the dark, and then reacted at pH = 9 (pH adjusted using Tris-NaOH) in the dark for 40 minutes to obtain a gallic acid-reduced silver nanoparticle solution.

[0124] S2: The preparation method of hyaluronic acid grafted with 4-aminophenylboronic acid comprises:

[0125] A hyaluronic acid aqueous solution (6.7 g / L, 0.15 L) was mixed with 0.9567 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.6532 g of N-hydroxysuccinimide (NHS) at 40°C to dissolve the first crosslinker in the hyaluronic acid aqueous solution under a slightly warmed environment. The pH was then adjusted to 5.0 (using NH3·H2O) to obtain a solution. The solution was then mixed with 0.8672 g of 4-aminophenylboronic acid hydrochloride and reacted at 10°C with stirring for 24 hours to obtain a product solution. The solution was then dialyzed using a 0.15 mol / L NaCl aqueous solution as the external dialysis solution (dialysis bag molecular weight 1500 Da). The dialyzed solution was removed and freeze-dried to obtain hyaluronic acid grafted with 4-aminophenylboronic acid.

[0126] S3: Preparation of gallic acid grafted collagen is carried out under light-protected conditions, including:

[0127] Gallic acid aqueous solution (13 g / L, 5 mL) was mixed with 0.0587 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.0358 g of N-hydroxysuccinimide (NHS) and reacted at 40°C in the dark for 30 min to obtain the activated product;

[0128] The cooled activated product was mixed with 15 mL of bovine Achilles tendon collagen acetic acid aqueous solution (the bovine Achilles tendon collagen acetic acid aqueous solution was prepared by mixing bovine Achilles tendon collagen with acetic acid aqueous solution, the concentration of the acetic acid aqueous solution was 0.5 mol / L, and the concentration of the bovine Achilles tendon collagen in the bovine Achilles tendon collagen acetic acid aqueous solution was 5 g / L) to obtain a mixed system, and the pH value of the mixed system was adjusted to 5 (pH adjustment was performed using HAc-NaAc buffer solution) and the reaction was carried out under nitrogen atmosphere, light-proof and stirring conditions (reaction time was 24 h, temperature was 4 ° C) to obtain a product solution. Then, dialysis was performed using 4 ° C deionized water as the dialysis external solution (the molecular weight of the dialysis bag was 14000 Da), the dialysis internal solution was taken out, and freeze-dried to obtain the gallic acid-grafted collagen;

[0129] S4: dissolving the gallic acid grafted collagen in a 0.5 mol / L acetic acid aqueous solution to obtain a gallic acid grafted collagen-acetic acid solution (the concentration of the gallic acid grafted collagen in the gallic acid grafted collagen-acetic acid solution is 5 g / L); mixing the hyaluronic acid grafted with 4-aminophenylboronic acid with water to obtain a 4-aminophenylboronic acid grafted hyaluronic acid-aqueous solution (the concentration of the hyaluronic acid grafted with 4-aminophenylboronic acid in the hyaluronic acid grafted with 4-aminophenylboronic acid-aqueous solution is 5 g / L). The gallic acid-grafted collagen-acetic acid solution and the 4-aminophenylboronic acid-grafted hyaluronic acid-aqueous solution were mixed in equal volumes, and the pH of the solution was adjusted to 5 (using Tris-HCl for pH adjustment) to form a hyaluronic acid-collagen hydrogel; the gallic acid-reduced silver nanoparticle solution obtained in step S1 was added to the hyaluronic acid-collagen hydrogel to a volume fraction of 2.5%, and stirred to obtain a composite hydrogel dressing CG-HB-5.

[0130] The hydrogel prepared in Example 5 was in a fluid state and had adhesive properties, but could not be stretched or wound, and did not have self-healing properties.

[0131] It was found that the formation of dynamic boron ester bonds of the present invention has certain pH requirements. If the pH is too low, the dynamic boron ester bonds cannot form, the macromolecular components based on collagen and hyaluronic acid cannot be effectively cross-linked, and the formed gel exhibits a certain degree of fluidity but does not have self-healing properties, as shown in Example 5. In addition, Figure 9 The morphological photographs of the hydrogels obtained at pH 5.0 in Example 5 and at pH 7.4 in Example 2 are given.

[0132] Comparative Example

[0133] The difference between this comparative example and Example 4 is only that:

[0134] In this comparative example, only hyaluronic acid-collagen hydrogel (denoted as CG-HB) was obtained, and the gallic acid-reduced silver nanoparticle solution obtained in step S1 was not added to the hyaluronic acid-collagen hydrogel.

[0135] Test Example 1 Rheological Properties Analysis

[0136] The rheological properties of the hydrogels of Examples 1-4 were tested using a rotational rheometer (DHR-1, purchased from TA Instruments, USA). The storage modulus (G') and loss modulus (G") of the hydrogels were measured at a constant strain amplitude of 2.0% and a frequency range of 0.1-10 Hz. The changes in the storage modulus of the samples at 10 Hz were analyzed. Figure 1 shown.

[0137] It can be seen from this that although gallic acid-grafted collagen and 4-aminophenylboronic acid-grafted hyaluronic acid can achieve reversible cross-linking of collagen-based hydrogels through boron ester bonds and have self-healing properties, their composition ratio will have an important influence on the mechanical properties of the gel. If the proportion of 4-aminophenylboronic acid-grafted hyaluronic acid is too low, the formed composite material cannot present a gel state. Increasing the proportion of 4-aminophenylboronic acid-grafted hyaluronic acid can significantly improve the mechanical properties of the gel ( Figure 1 This is likely due to the multiple intermolecular interactions induced by the phenylboronic acid groups in the grafted hyaluronic acid macromolecules, including synergistic effects of dynamic phenylboronic acid ester bonds, hydrogen bonding networks, and polymer chain entanglement. However, if the proportion of gallic acid grafted to collagen is too low, the advantages of collagen's excellent physiological activity cannot be realized.

[0138] Test Example 2 Self-healing performance test

[0139] The self-healing properties of the hydrogel are mainly characterized by direct observation and instrumental testing (rotational rheometer). In this test example, the self-healing properties of the hydrogel of Example 4 were tested;

[0140] The direct observation method proceeds as follows: Two hydrogels are taken. One is dyed orange with sunset yellow, while the other remains untreated. The two hydrogels are then placed close together at room temperature. After self-healing, they are picked up with tweezers and the healing process observed. Figure 2(a) shows a photograph of two separate hydrogels repaired and fused together upon contact, demonstrating the excellent self-healing properties of the hydrogels presented herein.

[0141] The instrumental testing procedure is as follows: Continuous step strain sweep test parameters were determined using the oscillatory strain sweep mode of the rotational rheometer. Oscillatory strain sweep test conditions were: a fixed frequency of 1 Hz, and a strain range of 5%-1000%. The continuous step strain sweep test conditions were: a fixed frequency of 1 Hz; a test at 3% shear strain for 200 seconds; then a test at 800% shear strain for 200 seconds; and three alternating tests of G' and G" were repeated. Testing was performed using a PP-40 (40 mm) parallel plate detector.

[0142] As shown in Figure 2(b), the modulus changes of the hydrogel under different strains;

[0143] As shown in Figure 2(c), this is a periodic step strain scan of the hydrogel. The hydrogel undergoes a corresponding sol-gel transition under the strain action under repeated cycles, showing excellent self-healing properties.

[0144] Test Example 3 Stress-strain performance test

[0145] The mechanical properties (including the tensile strength of the hydrogel) of Examples 1-4 were measured using a universal material testing machine (WDD-5KN, purchased from Beijing Guance Precision Instrument Equipment Co., Ltd.). The hydrogels were made into long strip samples and cylindrical samples. -1 The sample was stretched or compressed at a speed of , and the stress-strain curve of the hydrogel was obtained, as shown in Figure 3(a)-(b).

[0146] Tensile or compressive strain (ε T , %) are calculated as follows:

[0147]

[0148] Where l and l0 are the real-time length and original length of the test sample, respectively (mm).

[0149] The stress (δ, MPa) in tension or compression is calculated as follows:

[0150]

[0151] Where F is the tensile stress (N); A0 represents the initial cross-sectional area of the test material (mm 2 ).

[0152] Test Example 4: Cell live-dead staining assay and CCK-8 cell proliferation assay

[0153] (1) Cell live-death staining experiment:

[0154] The sterilized hydrogel samples were extracted with DMEM medium in a cell culture incubator at 37 °C and 5% CO2 for 24 h to prepare 500 μg mL -1 The extract (the concentration of hydrogel in DMEM medium is 500 μg·mL -1 ) working concentration, and quantitative analysis was performed using a live / dead cell dual fluorescence labeling method, in which calcein-AM (green fluorescence) labels the integrity of live cell membranes, and propidium iodide (red fluorescence) specifically binds to necrotic cell nucleic acids. The cell concentration was 5×104 cells·mL -1 After culturing for 24 hours, 100 μL of the cell suspension was mixed with 100 μL of the hydrogel extract. The samples were then washed three times with 100 μL of PBS per well. A mixed cell staining solution of AM and PI was added and incubated at 37°C for 60 minutes. The cells were then gently washed three times with 100 μL of PBS per well to prevent excess stain from interfering with observation. Finally, an inverted fluorescence microscope was adjusted to photograph the cell fluorescence staining results.

[0155] Figure 5(a) shows live-dead staining images of HUVEC cells after co-culture of the blank group, CG-HB, and CG-HB-4 gel extracts with HUVEC cells for 24 hours, as observed using an inverted fluorescence microscope. The upper panel shows live cell staining, the middle panel shows dead cell staining, and the lower panel shows a combined live-dead staining image. Green represents live cells, and red represents dead cells. The blank group did not contain any gel sample.

[0156] (2) CCK-8 cell proliferation assay

[0157] CCK-8 kit was used to quantitatively analyze the proliferation of cells on the surface of conductive hydrogel. The cell seeding density was 5×104 cells·mL -1 , prepare a working solution containing 10% CCK-8 fresh culture medium, add it to the cell culture plate at 24h and 48h respectively, and incubate it in a constant temperature incubator at 37°C and 5% CO2 for 3h. The blank plate with CCK-8 reagent is the blank group. Pipette 100μL of culture medium from each well and transfer it to a 96-well plate. Measure the absorbance at 450nm with a microplate reader. Each group of samples contains 5 parallel samples, and calculate the mean and standard deviation. Calculate the cell proliferation rate V according to the following formula:

[0158]

[0159] Among them: A s is the absorbance value of the experimental group, A b is the absorbance value of the blank group, A c is the absorbance value of the control group. The results are shown in Figure 5(b).

[0160] Test Example 5 Antibacterial Performance Experiment

[0161] The antibacterial ability of the hydrogel was evaluated by the plate method. First, 20 μL of bacterial solution was added dropwise to 100 μL of PBS solution as a blank sample. The hydrogel was placed in a sterile test tube, and 20 μL of bacterial suspension was added to the surface of each hydrogel. The inoculated hydrogel was cultured in an incubator at 37°C and a relative humidity of not less than 90% for 2 hours. Then 980 μL of sterilized culture medium was added to each sterile tube and the stock bacteria were resuspended. At the same time, a negative control without hydrogel was prepared. Finally, 100 μL of each sample was spread on an agar plate, cultured in a 37°C incubator for 24 hours, and the number of bacterial colonies was recorded. The inhibition rate X was calculated based on the following formula:

[0162]

[0163] Where: A0 is the number of colonies in the control group, and A1 is the number of colonies in the sample group.

[0164] Test Example 6 Wound Healing Experiment

[0165] Rats were injected with 5% chloral hydrate solution and hair was removed. Full-thickness wounds with a diameter of 15 mm were cut with surgical scissors. The wounds were then covered with sterile gauze, commercially available dressings (sterile dressings from Kefu Medical Technology Co., Ltd.) and the CG-HB-4 (CG-HB@AgNPs) hydrogel of Example 4, bandaged with sterile gauze, and fixed with elastic plastic. The wounds treated with sterile gauze served as the control group. The wound dressings were changed every 2 days after surgery, and photos of the wounds were taken with a camera at predetermined time intervals. Image J software was used to accurately detect the wounds. All rats were killed after a certain period of time; the new skin was immediately removed and fixed with 10% paraformaldehyde. Skin tissue sections were stained with H&E and Masson to observe the pathological changes and collagen deposition of the regenerated tissue. Immunohistochemistry was used to analyze the inflammatory response of the regenerated wound tissue, and then the expression of tumor necrosis factor (TNF-α) and mannose receptor (CD206) was evaluated. The wound healing rate H was calculated according to the following formula:

[0166]

[0167] Where: A0 is the initial wound area, As is the current wound area.

[0168] As shown in Figure 7(a), the wound surface photos of rats on the 1st, 3rd, 7th, 10th and 14th days are shown; as shown in Figure 7(b), the wound traces during the healing process of rat wounds are superimposed; as shown in Figure 7(c), the wound healing rate calculated based on the wound healing area changes with healing time.

[0169] As shown in Figure 8(a), it is the H&E staining image of the rat wound on the 7th and 14th days; as shown in Figures 8(b) and (c), it is the Masson staining and collagen deposition in the tissue sections of the rat wound on the 7th and 14th days, respectively; as shown in Figure 8(d), it is the immunohistochemistry image of the tissue sections of the rat wound on the 14th day; as shown in Figures 8(e) and (f), it is the expression of CD206 and TNF-α in the tissue sections of the rat wound on the 14th day, respectively.

[0170] The composite hydrogel dressing of the present invention is prepared by mixing hyaluronic acid grafted with 4-aminophenylboronic acid, collagen grafted with gallic acid, and a solution of Ag nanoparticles reduced with gallic acid. The cross-linked network of the composite hydrogel is formed through the interaction of covalent bonds such as dynamic boron ester bonds and amide bonds, as well as non-covalent hydrogen bonds. The hydrogel dressing loaded with silver nanoparticles exhibits excellent antibacterial properties and can alleviate inflammatory reactions. The resulting hydrogel has suitable adhesion, sufficient mechanical strength, good biocompatibility, and self-healing properties, and can be used for wound healing of complex and chronic wounds.

[0171] While various embodiments of the present invention have been described above, the above description is intended to be illustrative, not exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A composite hydrogel dressing, characterized in that: The composite hydrogel dressing is prepared by mixing hyaluronic acid grafted with 4-aminophenylboronic acid and collagen grafted with gallic acid, adjusting the pH value, and then performing a cross-linking reaction to obtain a cross-linked product, and then stirring and mixing the cross-linked product with a silver nanoparticle solution reduced with gallic acid.

2. The composite hydrogel dressing according to claim 1, wherein The mass ratio of the hyaluronic acid grafted with 4-aminophenylboronic acid to the collagen grafted with gallic acid is (0.02-0.06): (0.01-0.02); The pH value of the blended system of hyaluronic acid grafted with 4-aminophenylboronic acid and collagen grafted with gallic acid is adjusted to 7-9.

5.

3. The composite hydrogel dressing according to claim 1 or 2, wherein: The preparation method of the composite hydrogel dressing comprises: mixing the gallic acid-grafted collagen with an acetic acid aqueous solution to obtain a gallic acid-grafted collagen-acetic acid solution; mixing hyaluronic acid grafted with 4-aminophenylboronic acid with water to obtain a hyaluronic acid-aqueous solution grafted with 4-aminophenylboronic acid; Mixing a gallic acid-grafted collagen-acetic acid solution with a 4-aminophenylboronic acid-grafted hyaluronic acid-aqueous solution to obtain a blend system of 4-aminophenylboronic acid-grafted hyaluronic acid and gallic acid-grafted collagen, adjusting the pH value of the blend system with a first pH adjuster, and then performing a cross-linking reaction to obtain a cross-linked product; stirring and mixing the cross-linked product with a silver nanoparticle solution reduced with gallic acid to obtain the composite hydrogel dressing; Preferably, the first pH regulator is at least one of a NaHCO3-NaCl mixed solution, a boric acid-borax buffer solution and a Tris-HCl buffer solution.

4. The composite hydrogel dressing according to claim 1, wherein The preparation method of the 4-aminophenylboronic acid grafted hyaluronic acid comprises: mixing a hyaluronic acid aqueous solution with a first cross-linking agent and adjusting the pH value to obtain a dissolving system; mixing the dissolving system with 4-aminophenylboronic acid hydrochloride and reacting them under stirring to obtain a product solution; dialyzing and freeze-drying the product solution to obtain the 4-aminophenylboronic acid grafted hyaluronic acid; Preferably, the concentration of the hyaluronic acid aqueous solution is 5-8.5 g / L; Preferably, the first cross-linking agent is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, and the mass ratio of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide, hyaluronic acid and 4-aminophenylborate hydrochloride is (0.8-1.5):(0.4-0.75):(0.7-1.4):(0.6-1.2); Preferably, the temperature of the mixture of the hyaluronic acid aqueous solution and the first cross-linking agent is 35-45°C; Preferably, a second pH regulator is used to adjust the pH value of the dissolution system to 4-6; the second pH regulator is at least one of NaOH, Na2HPO4 and NH3·H2O; Preferably, the reaction time of the dissolving system and 4-aminophenylborate hydrochloride under stirring is 12-36 hours and the temperature is 10-25°C.

5. The composite hydrogel dressing according to claim 1, wherein The preparation of the gallic acid-grafted collagen is carried out under light-proof conditions, comprising: mixing a gallic acid aqueous solution with a second cross-linking agent and reacting them at a constant temperature to obtain an activated product; mixing the cooled activated product with a collagen acetic acid aqueous solution to obtain a mixed system; adjusting the pH value of the mixed system and reacting them under a nitrogen atmosphere and stirring to obtain a product solution; dialyzing and freeze-drying the solution to obtain the gallic acid-grafted collagen; Preferably, the concentration of the gallic acid aqueous solution is 10-15 g / L; Preferably, the second cross-linking agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and the mass ratio of gallic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is (0.5-0.7):(0.4-0.75):(0.25-0.45); Preferably, the constant temperature is 35-45° C., and the reaction time at the constant temperature is 20-40 min; Preferably, the collagen acetic acid aqueous solution is prepared by mixing collagen with an acetic acid aqueous solution, the concentration of the acetic acid aqueous solution is 0.3-0.8 mol / L, and the concentration of the collagen in the collagen acetic acid aqueous solution is 5-8 g / L; the collagen is at least one of bovine Achilles tendon collagen, pig skin collagen, and bullfrog skin collagen; Preferably, the mass ratio of the collagen and gallic acid in the mixed system is (0.75-1.2):(0.5-0.7); Preferably, the pH value of the mixed system is adjusted to 4-6 by using a third pH adjuster; the third pH adjuster is an acetic acid-sodium acetate buffer solution; Preferably, the reaction time under nitrogen atmosphere and stirring conditions is 12-36 hours and the temperature is 2-8°C.

6. The composite hydrogel dressing according to claim 1, wherein The volume fraction of the gallic acid-reduced silver nanoparticle solution in the composite hydrogel dressing is 2.5-10%.

7. The composite hydrogel dressing according to claim 1, wherein The preparation of the silver nanoparticle solution reduced with gallic acid is carried out under light-proof conditions, comprising: mixing a silver nitrate aqueous solution and a gallic acid aqueous solution to obtain a mixed system, adjusting the pH value of the mixed system and reacting to obtain the silver nanoparticle solution reduced with gallic acid; Preferably, a fourth pH adjuster is used to adjust the pH value of the mixed system to 7-11; the fourth pH adjuster is a Tris-HCl buffer solution and / or a Tris-NaOH buffer solution; Preferably, the reaction time is 15-40 min; Preferably, the concentration of the silver nitrate aqueous solution is 0.03-0.07 mol / L; Preferably, the concentration of the gallic acid aqueous solution is 0.05-0.10 mol / L; Preferably, the volume ratio of the silver nitrate aqueous solution to the gallic acid aqueous solution is (0.8-1.2):(0.8-1.2).

8. The composite hydrogel dressing according to claim 1, wherein The composite hydrogel dressing is prepared by mixing hyaluronic acid grafted with 4-aminophenylboronic acid and collagen grafted with gallic acid, adjusting the pH value, and then performing a cross-linking reaction to obtain a cross-linked product, and then stirring and mixing the cross-linked product with a solution of silver nanoparticles reduced with gallic acid and other functional additives; The other functional adjuvant is at least one of pharmaceutically active molecules, epidermal growth factor, gold nanoparticles and zinc oxide nanoparticles; Preferably, the pharmaceutically active molecule is ciprofloxacin and / or curcumin.

9. Use of the composite hydrogel dressing according to any one of claims 1 to 8 in the preparation of a medicament for treating wound healing.

10. The use according to claim 9, wherein: The wound is a diabetic foot ulcer wound, a knife wound or a bacterial infection wound.

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