Method for preparing a metal-loaded polyphenol hydrogel dressing and use thereof

Abstract

The application discloses a kind of metal-polyphenol hydrogel dressings preparation method and its application, it is related to the technical field of biomedical materials, first preparation specified proportion ethanol aqueous solution, gallus acid is dissolved with magnesium chloride ultrasonic, ammonia water is stirred and reacted, and solid crude product is collected by centrifugation.Washing and drying are carried out to obtain purified magnesium-gallus acid nanoparticles, which are dispersed and then added to methylacrylated gelatin and photoinitiator, filtered after constant-temperature dissolution.The filtrate is blue light crosslinking solidification, washing and ultraviolet double-side sterilization, then extracted by culture medium and filtered, to obtain metal-polyphenol hydrogel dressing and corresponding extract.The application can stabilize the structure of polyphenol, realize the controlled release of active substances, and avoid the toxicity caused by the rapid release of ions.The dressing can scavenge free radicals, inhibit inflammation, promote angiogenesis and tissue regeneration, has good biocompatibility, can maintain a moist environment for the wound, accelerate wound healing and reduce scar formation.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a method for preparing a metal-loaded polyphenol hydrogel dressing and its application. Background Technology

[0002] The skin is the largest organ in the human body, undertaking core physiological functions such as protection, sensory perception, and temperature regulation. Skin defects are common clinical conditions, encompassing various types including trauma, burns, and chronic ulcers, seriously affecting patients' health and quality of life. Traditional dressings, mainly gauze and bandages, can only achieve basic hemostasis and fluid absorption, lacking anti-inflammatory and tissue regeneration functions. Furthermore, dressings tend to dry and adhere to the wound surface, and changing them can damage newly formed tissue, failing to meet the core needs of modern wound repair.

[0003] The theory of moist wound healing has driven the development of novel wound dressings, as a moist environment can accelerate wound epithelialization and reduce scar formation. Hydrogels, with their three-dimensional porous structure, high biocompatibility, and moisturizing properties, have become ideal carriers for wound repair, mimicking the extracellular matrix environment, maintaining wound moisture, and reducing the risk of infection. However, conventional hydrogels lack functional active components, making it difficult to address the oxidative stress damage and insufficient angiogenesis in chronic wounds, resulting in limited repair efficacy.

[0004] Gallic acid, a natural polyphenol, possesses antioxidant, anti-inflammatory, and antibacterial activities, which can scavenge free radicals in wounds and alleviate inflammation. Magnesium ions participate in physiological regulation and can promote cell proliferation and tissue repair. The combination of the two can exert a synergistic repair effect. However, free gallic acid is easily oxidized and inactivated, and direct application of magnesium ions can release them too quickly and easily cause local toxicity. Current loading technologies cannot achieve stable coordination and controllable release of the two, making it difficult to balance activity protection, biosafety, and repair efficacy. Therefore, the development of high-performance functional hydrogel dressings has become an urgent clinical need. Summary of the Invention

[0005] This invention proposes a method for preparing a metal-loaded polyphenol hydrogel dressing and its application, in order to solve the problems mentioned in the prior art.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a method for preparing a metal-loaded polyphenol hydrogel dressing, comprising the following steps: S1 prepares an ethanol aqueous solution with a volume fraction of 30%. Gallic acid solid and magnesium chloride solid are added to the prepared ethanol aqueous solution in sequence. The ultrasonic equipment is turned on for dispersion treatment, and the ultrasonic treatment is continued until the solid raw materials are completely dissolved. S2 Slowly add a 10% (v / v) ammonia solution to the mixed reaction solution, turn on the stirrer and stir continuously, and carry out the metal-polyphenol coordination reaction under normal temperature and pressure conditions. After the coordination reaction is completed, start the centrifugation device to obtain crude magnesium-gallic acid metal-polyphenol nanoparticles. S3 uses anhydrous ethanol as the washing solvent to centrifuge and wash the crude magnesium-gallic acid metal-polyphenol nanoparticles multiple times to remove unreacted free raw materials and soluble byproducts. After washing, the solid product is placed in a vacuum drying device to obtain purified magnesium-gallic acid metal-polyphenol nanoparticles. S4 Weigh a certain amount of purified magnesium-gallic acid metal-polyphenol nanoparticles, add them to ultrapure deionized water, turn on the ultrasonic device to homogenize and disperse them until the nanoparticles are evenly distributed in the liquid phase system, and obtain a stable nanoparticle dispersion. S5 adds methacrylamide gelatin and lithium phenyl-2,4,6-trimethylbenzoylphosphonate to the nanoparticle dispersion, and places the mixture in a constant temperature incubator to form a homogeneous hydrogel precursor mixture; S6 uses a microporous membrane to pressurize and filter the hydrogel precursor mixture to remove undissolved impurities, large aggregates and microbial contaminants from the system, resulting in a clear and sterile hydrogel precursor solution. S7 slowly injects the hydrogel precursor liquid into a sterile custom mold, places the mold under a blue light irradiation device, turns on the blue light to carry out a photocrosslinking and curing reaction, and obtains a metal-loaded polyphenol hydrogel dressing semi-finished product. S8 uses sterile phosphate buffer to repeatedly soak and wash the semi-finished metal-polyphenol hydrogel dressing to remove uncrosslinked free monomers and residual initiators. After washing, the front and back of the semi-finished hydrogel are sterilized separately using ultraviolet light to obtain sterile metal-polyphenol hydrogel dressing. S9 completely immerses the sterilized metal-polyphenol hydrogel dressing in cell culture medium and places it in a constant temperature incubator for extraction. After extraction, the extraction system is filtered and sterilized using a microporous membrane to obtain a pure hydrogel extract.

[0007] Furthermore, the volume of the ethanol-water solution in S1 is 10 mL to 50 mL, the concentration of gallic acid in the mixed reaction solution is 20 mM, the concentration of magnesium chloride in the mixed reaction solution is 7 mM, the power of the ultrasonic treatment is set to 100 W to 300 W, the ultrasonic treatment time is set to 10 min to 30 min, and the system temperature is kept in the range of 20 °C to 25 °C during the ultrasonic treatment process.

[0008] Furthermore, the ammonia solution in S2 was added dropwise, with the dropping rate controlled at 1 mL / min to 3 mL / min. The total time for the coordination reaction was 8 h, and the centrifugation time was set to 10 min to 20 min, while maintaining a low temperature environment during the centrifugation process.

[0009] Furthermore, the number of anhydrous ethanol washings in S3 was set to 3 to 5 times, with a liquid-to-solid volume ratio of 10:1 to 20:1 for each wash. The vacuum drying temperature was set to 40°C to 60°C, and the vacuum drying time was set to 12h to 24h. The prepared magnesium-gallic acid metal-polyphenol nanoparticles had a spherical or near-spherical structure with a particle size distribution of 90nm to 110nm. The particles were well dispersed and showed no obvious agglomeration.

[0010] Furthermore, in S4, the mass-to-volume ratio of magnesium-gallic acid metal-polyphenol nanoparticles to deionized water was 10 mg: 1 mL, the ultrasonic dispersion power was 200 W to 400 W, the dispersion time was 15 min to 40 min, and the dispersed nanoparticle dispersion remained uniform and stable within 24 h.

[0011] Furthermore, the added mass of methacrylamide gelatin in S5 was 80 mg, and the added mass of lithium phenyl-2,4,6-trimethylbenzoylphosphonate was 2.5 mg. The temperature of the constant temperature incubator was set to 37°C, and the mixture was allowed to stand for 30 minutes to dissolve. The entire dissolution process was carried out in a sterile environment.

[0012] Furthermore, in S6, the microporous filter membrane has a pore size of 0.22μm, and the filtration method is positive pressure filtration with a filtration pressure of 0.1MPa to 0.2MPa. In S7, the blue light irradiation wavelength is 405nm, the blue light irradiation power is 5W to 15W, the photocrosslinking curing time is 30s, and the mold material is medical-grade polypropylene.

[0013] Furthermore, the pH value of the sterile phosphate buffer in S8 is 7.2 to 7.4, the number of washes is 3 to 5, the power of the ultraviolet light source is 30W to 40W, the single-sided ultraviolet sterilization time is 2 hours, and the microbial limit of the sterilized hydrogel dressing meets the requirements of the national standard for medical dressings.

[0014] Furthermore, the cell culture medium used in S9 was DMEM high-glucose medium, the extraction temperature was 37℃, and a 5% CO2 gas environment was maintained during the extraction process. Sterilization was achieved using a 0.22μm microporous membrane, and the storage temperature was 4℃. The shelf life was 7 days. The DPPH free radical scavenging efficiency of the hydrogel extract was calculated using a professional formula. , in the formula This represents the DPPH free radical scavenging efficiency. This represents the absorbance value of the blank group. The absorbance value represents the sample group, and the absorbance detection wavelength is fixed at 517nm.

[0015] Furthermore, the application is directly used for the repair of skin defects and wounds in humans or animals, including mechanical trauma wounds, thermal burn wounds, chronic diabetic ulcer wounds, and pressure injury wounds. During application, the metal-polyphenol hydrogel dressing continuously releases magnesium ions and gallic acid active components, clearing excess reactive oxygen free radicals in the wound microenvironment and promoting endothelial cell migration and angiogenesis. When applied to full-thickness skin defects in mice, the wound healing rate is higher than 95% after 12 days. The dressing has good biocompatibility, a survival rate of more than 90% for L929 fibroblasts, and is non-cytotoxic and non-irritating to tissues.

[0016] Compared with existing technologies, the beneficial effects of this invention are: This invention forms a stable nanostructure through the coordination assembly of metal and polyphenols, effectively inhibiting the oxidative deactivation of polyphenols, significantly improving the chemical stability of active components, and extending the efficacy period.

[0017] The nanoparticle loading system enables the slow and controllable release of active substances, avoiding the local toxicity caused by the rapid release of metal ions and ensuring the biosafety of the dressing throughout its use.

[0018] Dressings can effectively remove excess reactive oxygen free radicals in the wound microenvironment, block persistent inflammation caused by oxidative stress, and create a stable physiological environment for wound repair.

[0019] The active components released by the dressing exert a dual synergistic effect, effectively stimulating endothelial cell migration and angiogenesis while scavenging free radicals and inhibiting inflammation, thus improving blood supply to the wound.

[0020] All raw materials used in the dressing have excellent biocompatibility, are non-cytotoxic and non-irritating to tissues, and will not cause adverse reactions when in direct contact with the wound, meeting the safety standards for medical dressings.

[0021] The three-dimensional structure of hydrogel can closely adhere to the wound, continuously maintain a moist healing environment, reduce adhesion damage between the dressing and the wound, and lower the risk of secondary wound injury.

[0022] Dressings can effectively regulate the expression of inflammatory factors in wounds, inhibit excessive inflammatory responses, and promote fibroblast proliferation and epithelial tissue regeneration, thereby comprehensively accelerating wound healing.

[0023] The dressing is suitable for repairing various skin defects and wounds, taking into account the needs of both acute and chronic wounds, reducing scar tissue formation, improving the quality of wound repair, and has broad clinical application value. Attached Figure Description

[0024] Figure 1 This is a scanning electron microscope image of the Mg@GAMPNs nanoparticles proposed in this invention; Figure 2 Comparison of Fourier transform infrared spectra of Mg@GAMPNs; Figure 3 X-ray photoelectron spectroscopy (XPS) spectrum of Mg@GAMPNs; Figure 4 Photographs of Mg@GAMPNs photocrosslinked on GelMA hydrogel; Figure 5 Scanning electron microscope image of Mg@GA MPNs loaded on GelMA hydrogel; Figure 6 A graph showing the efficiency of hydrogels in scavenging DPPH free radicals; Figure 7 These are fluorescence micrographs of live and dead cells double-stained with Calcein AM / PI. Figure 8 The effect of hydrogel extract on L929 cell viability; Figure 9 Image showing cell scratch healing under hydrogel extract intervention; Figure 10 The effect of hydrogel extract on inflammatory factors; Figure 11 Macroscopic photographs of healing at different time points in a mouse model of full-thickness skin defects. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] In the description of this invention, it should be understood that the terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," and "counterclockwise," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0027] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of the stated features. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified. Furthermore, the terms "installed," "connected," and "linked" should be interpreted broadly; for example, they may refer to a fixed connection, a detachable connection, or an integral connection; they may refer to a mechanical connection or an electrical connection; they may refer to a direct connection or an indirect connection through an intermediate medium; and they may refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances. The invention will now be described in further detail with reference to the accompanying drawings.

[0028] Reference Figures 1 to 11 A method for preparing a metal-loaded polyphenol hydrogel dressing includes the following steps: S1 prepares an ethanol aqueous solution with a volume fraction of 30%. Gallic acid solid and magnesium chloride solid are added to the prepared ethanol aqueous solution in sequence. The ultrasonic equipment is turned on for dispersion treatment. The ultrasonic treatment is continued until the solid raw materials are completely dissolved to form a uniform and transparent mixed reaction solution.

[0029] S2 Slowly adds a 10% (v / v) ammonia solution to the mixed reaction solution, turns on the stirrer and stirs continuously, and carries out the metal-polyphenol coordination reaction under normal temperature and pressure conditions. After the coordination reaction is completed, the centrifuge is started and the solid product in the system is collected by centrifugation to obtain crude magnesium-gallic acid metal-polyphenol nanoparticles.

[0030] S3 uses anhydrous ethanol as the washing solvent to perform multiple centrifugal washings on the crude magnesium-gallic acid metal-polyphenol nanoparticles to remove unreacted free raw materials and soluble byproducts. After washing, the solid product is placed in a vacuum drying device for constant temperature vacuum drying to obtain purified magnesium-gallic acid metal-polyphenol nanoparticles.

[0031] S4 Weigh a fixed amount of purified magnesium-gallic acid metal-polyphenol nanoparticles, add them to ultrapure deionized water, turn on the ultrasonic equipment for homogenization and dispersion, and continue to disperse until the nanoparticles are uniformly distributed in the liquid phase system and there are no visible agglomerates, thus obtaining a stable nanoparticle dispersion.

[0032] S5 adds methacrylamide gelatin and lithium phenyl-2,4,6-trimethylbenzoylphosphonate to the nanoparticle dispersion, places the mixture in a constant temperature incubator, and stirs continuously until the solid components are completely dissolved to form a homogeneous hydrogel precursor mixture.

[0033] S6 uses a microporous membrane to pressurize and filter the hydrogel precursor mixture to remove undissolved impurities, large aggregates and microbial contaminants from the system, resulting in a clear and sterile hydrogel precursor solution.

[0034] S7 slowly injects the hydrogel precursor solution into a sterile custom mold, places the mold under a blue light irradiation device, turns on the blue light to carry out the photocrosslinking and curing reaction, and after the curing reaction is completed, completely demolds the hydrogel from the mold to obtain a semi-finished metal-polyphenol hydrogel dressing.

[0035] S8 uses sterile phosphate buffer to repeatedly soak and wash the semi-finished metal-loaded polyphenol hydrogel dressing to remove uncrosslinked free monomers and residual initiators. After washing, the front and back of the semi-finished hydrogel are sterilized separately using ultraviolet light to obtain sterile metal-loaded polyphenol hydrogel dressing.

[0036] S9 completely immerses the sterilized metal-polyphenol hydrogel dressing in cell culture medium and places it in a constant temperature incubator for long-term extraction. After extraction, the extraction system is filtered and sterilized using a microporous membrane to obtain a pure hydrogel extract. The hydrogel extract is then transferred to a sterile centrifuge tube and stored in a low-temperature refrigeration device for later use.

[0037] In this invention, the volume of the ethanol aqueous solution in S1 is 10 mL to 50 mL, the concentration of gallic acid in the mixed reaction solution is 20 mM, the concentration of magnesium chloride in the mixed reaction solution is 7 mM, the power of the ultrasonic treatment is set to 100 W to 300 W, the ultrasonic treatment time is set to 10 min to 30 min, and the system temperature is kept in the range of 20°C to 25°C during the ultrasonic treatment process to avoid the raw materials from oxidizing and decomposing due to temperature rise.

[0038] In this invention, the ammonia solution in S2 is added dropwise, with the dropping rate controlled at 1 mL / min to 3 mL / min. The total time for the coordination reaction is 8 h. The centrifugation speed is set to 8000 r / min to 12000 r / min, and the centrifugation time is set to 10 min to 20 min. The centrifugation process is carried out in a low-temperature environment to prevent damage to the nanoparticle structure.

[0039] In this invention, the number of anhydrous ethanol washings in S3 is set to 3 to 5 times, the liquid-to-solid volume ratio of each washing is 10:1 to 20:1, the vacuum drying temperature is set to 40°C to 60°C, the vacuum drying time is set to 12h to 24h, and the vacuum degree is controlled to 0.08MPa to 0.095MPa. The prepared magnesium-gallic acid metal-polyphenol nanoparticles have a spherical or near-spherical structure, a particle size distribution of 90nm to 110nm, and good particle dispersibility without obvious agglomeration.

[0040] In this invention, the mass-volume ratio of magnesium-gallic acid metal-polyphenol nanoparticles to deionized water in S4 is 1 mg:5 mL to 1 mg:20 mL, the ultrasonic dispersion power is 200 W to 400 W, the dispersion time is 15 min to 40 min, and the dispersed nanoparticle dispersion remains uniform and stable within 24 h, without sedimentation or stratification.

[0041] In this invention, the added mass of methacrylamide gelatin in S5 is 80 mg, the added mass of lithium phenyl-2,4,6-trimethylbenzoylphosphonate is 2.5 mg, the temperature of the constant temperature incubation device is set to 37°C, and the dissolution is allowed to stand for 30 min. The entire dissolution process is kept in a sterile environment to avoid contamination by exogenous microorganisms.

[0042] In this invention, the microporous filter membrane in S6 has a pore size of 0.22 μm, and the filtration method is positive pressure filtration with a filtration pressure of 0.1 MPa to 0.2 MPa. In S7, the blue light irradiation wavelength is 405 nm, the blue light irradiation power is 5 W to 15 W, the photocrosslinking curing time is 30 s, the mold material is medical-grade polypropylene, and the internal dimensions of the mold are customized according to the clinical wound requirements.

[0043] In this invention, the pH value of the sterile phosphate buffer in S8 is 7.2 to 7.4, the number of washes is 3 to 5, the soaking time for each wash is 10 to 20 minutes, the power of the ultraviolet light source is 30W to 40W, the single-sided ultraviolet sterilization time is 2 hours, and the microbial limit of the sterilized hydrogel dressing meets the requirements of the national standard for medical dressings.

[0044] In this invention, the cell culture medium in S9 is DMEM high-glucose medium, the extraction temperature is 37℃, the extraction time is 72h, the extraction process is maintained in a 5% CO2 gas environment, the filtration sterilization is performed using a 0.22μm microporous membrane, the low-temperature storage temperature is 4℃, the storage shelf life is 7d, and the DPPH free radical scavenging efficiency of the hydrogel extract is calculated using a professional formula, which is η=(A0-As) / A0×100%, where η represents the DPPH free radical scavenging efficiency, A0 represents the absorbance value of the blank group, As represents the absorbance value of the sample group, and the absorbance detection wavelength is fixed at 517nm.

[0045] In this invention, the application is directly used for the repair of skin defects and wounds in humans or animals. These skin defects and wounds include mechanical trauma wounds, thermal burn wounds, chronic diabetic ulcer wounds, and pressure injury wounds. During application, the metal-loaded polyphenol hydrogel dressing continuously releases magnesium ions and gallic acid active components, clearing excess reactive oxygen free radicals in the wound microenvironment, reducing the expression levels of inflammatory factors such as IL-1β, IL-6, and TNF-α, promoting endothelial cell migration and angiogenesis, and accelerating fibroblast proliferation and epithelial tissue regeneration. When applied to full-thickness skin defects in mice, the wound healing rate is higher than 95% after 12 days. The dressing has good biocompatibility, with a survival rate of over 90% for L929 fibroblasts. It is non-cytotoxic and non-irritating to tissues, adheres to the wound surface to maintain a moist healing environment, reduces scar tissue formation, and meets the clinical needs for the repair of chronic and acute wounds.

[0046] The following three examples further illustrate the specific implementation of this system: This specific embodiment details a method for preparing a metal-polyphenol hydrogel dressing and its application. The core innovation of this invention is the preparation of magnesium-gallic acid metal-polyphenol nanoparticles via coordination reaction, which are then loaded onto a methacrylamide gelatin hydrogel system. After photocrosslinking, purification, and sterilization, a multifunctional wound repair dressing is obtained, achieving stable loading and controlled release of active ingredients, solving the technical problems of easy oxidation of polyphenols and excessively rapid release of metal ions. This embodiment includes three preferred embodiments and one comparative example of conventional technology. All preparation steps are fully elaborated, and process parameters are comprehensively detailed, fully covering all technical contents of claims 1 to 10. The prepared dressing can meet the repair needs of both acute and chronic wounds. (See attached document.) Figures 1 to 11 The characterization results are a perfect match.

[0047] Core coordination chemical reaction formula The carboxyl and hydroxyl functional groups in gallic acid molecules undergo multi-component coordination with magnesium ions to generate stable magnesium-gallic acid metal-polyphenol nanoparticles, as shown in the following reaction formula: C7H6O5 (gallic acid) + Mg²⁺ → Mg(C7H5O5)2 (magnesium-gallic acid coordination nanoparticles) + H⁺ Example 1 S1. Prepare a 30% (v / v) ethanol aqueous solution. Measure 20 mL of this ethanol aqueous solution and place it in a clean reaction vessel. Add gallic acid solid and magnesium chloride solid in sequence. Control the concentration of gallic acid to 20 mM and the concentration of magnesium chloride to 7 mM. Turn on the ultrasonic device, set the power to 200 W, and ultrasonically treat for 20 min until the solid is completely dissolved, forming a homogeneous and transparent mixed reaction solution.

[0048] S2 added a 10% ammonia solution dropwise to the mixed reaction solution at a rate of 2 mL / min, started the centrifuge, set the speed to 10000 r / min, centrifuged for 15 min, collected the solid product, and obtained crude magnesium-gallic acid metal-polyphenol nanoparticles.

[0049] S3 used anhydrous ethanol to centrifuge and wash the crude product four times, with a liquid-to-solid volume ratio of 15:1 each time. After washing, the solid was placed in a vacuum drying device with a temperature of 50℃ and a vacuum of 0.09MPa for 18 hours to obtain purified magnesium-gallic acid metal-polyphenol nanoparticles.

[0050] S4 Weigh 10mg of purified nanoparticles, add 1mL of ultrapure deionized water, turn on the ultrasonic device, set the power to 300W, and ultrasonically disperse for 30min until the particles are uniformly dispersed without agglomeration, thus obtaining a stable nanoparticle dispersion. Then, use a pipette to measure 50μL of the mother liquor and add 950μL of deionized water to mix well.

[0051] S5 added 80 mg of methacrylamide gelatin and 2.5 mg of lithium phenyl-2,4,6-trimethylbenzoylphosphonate to the dispersion, placed the system in a 37°C constant temperature incubator, and allowed it to stand for 30 min to dissolve, forming a homogeneous hydrogel precursor mixture.

[0052] S6 uses a 0.22μm microporous membrane for positive pressure filtration at a pressure of 0.15MPa to remove impurities and contaminants, resulting in a clear and sterile hydrogel precursor solution.

[0053] S7 injects the precursor liquid into a medical polypropylene mold, places it under a 405nm blue light irradiation device, sets the power to 10W, and irradiates for 30 seconds to complete photocrosslinking and curing. The hydrogel is then completely demolded to obtain a dressing semi-finished product.

[0054] S8 uses sterile phosphate buffer with a pH of 7.4 to wash the semi-finished product 4 times, soaking for 15 minutes each time to remove free monomers and initiators. The dressing is then sterilized on both sides for 2 hours using a 35W ultraviolet light source to obtain a sterile metal-polyphenol hydrogel dressing.

[0055] S9 immersed the sterile dressing in DMEM high-glucose medium and extracted it at 37°C and 5% CO2 for 72 hours. The extract was then filtered through a 0.22μm microporous membrane for sterilization, transferred to a sterile centrifuge tube, and stored at 4°C for later use.

[0056] Example 2 S1. Prepare a 30% (v / v) ethanol aqueous solution. Measure 20 mL of this ethanol aqueous solution and place it in a clean reaction vessel. Add gallic acid solid and magnesium chloride solid in sequence. Control the concentration of gallic acid to 20 mM and the concentration of magnesium chloride to 7 mM. Turn on the ultrasonic device, set the power to 200 W, and ultrasonically treat for 20 min until the solid is completely dissolved, forming a homogeneous and transparent mixed reaction solution.

[0057] S2 added a 10% ammonia solution dropwise to the mixed reaction solution at a rate of 2 mL / min, started the centrifuge, set the speed to 10000 r / min, centrifuged for 15 min, collected the solid product, and obtained crude magnesium-gallic acid metal-polyphenol nanoparticles.

[0058] S3 used anhydrous ethanol to centrifuge and wash the crude product four times, with a liquid-to-solid volume ratio of 15:1 each time. After washing, the solid was placed in a vacuum drying device with a temperature of 50℃ and a vacuum of 0.09MPa for 18 hours to obtain purified magnesium-gallic acid metal-polyphenol nanoparticles.

[0059] S4 Weigh 10mg of purified nanoparticles, add 1mL of ultrapure deionized water, turn on the ultrasonic device, set the power to 300W, and ultrasonically disperse for 30min until the particles are uniformly dispersed without agglomeration, thus obtaining a stable nanoparticle dispersion. Then, use a pipette to measure 100μL of the mother liquor and add 900μL of deionized water to mix well.

[0060] S5 added 80 mg of methacrylamide gelatin and 2.5 mg of lithium phenyl-2,4,6-trimethylbenzoylphosphonate to the dispersion, placed the system in a 37°C constant temperature incubator, and allowed it to stand for 30 min to dissolve, forming a homogeneous hydrogel precursor mixture.

[0061] S6 uses a 0.22μm microporous membrane for positive pressure filtration at a pressure of 0.15MPa to remove impurities and contaminants, resulting in a clear and sterile hydrogel precursor solution.

[0062] S7 injects the precursor liquid into a medical polypropylene mold, places it under a 405nm blue light irradiation device, sets the power to 10W, and irradiates for 30 seconds to complete photocrosslinking and curing. The hydrogel is then completely demolded to obtain a dressing semi-finished product.

[0063] S8 uses sterile phosphate buffer with a pH of 7.4 to wash the semi-finished product 4 times, soaking for 15 minutes each time to remove free monomers and initiators. The dressing is then sterilized on both sides for 2 hours using a 35W ultraviolet light source to obtain a sterile metal-polyphenol hydrogel dressing.

[0064] S9 immersed the sterile dressing in DMEM high-glucose medium and extracted it at 37°C and 5% CO2 for 72 hours. The extract was then filtered through a 0.22μm microporous membrane for sterilization, transferred to a sterile centrifuge tube, and stored at 4°C for later use.

[0065] Example 3 S1. Prepare a 30% (v / v) ethanol aqueous solution. Measure 20 mL of this ethanol aqueous solution and place it in a clean reaction vessel. Add gallic acid solid and magnesium chloride solid in sequence. Control the concentration of gallic acid to 20 mM and the concentration of magnesium chloride to 7 mM. Turn on the ultrasonic device, set the power to 200 W, and ultrasonically treat for 20 min until the solid is completely dissolved, forming a homogeneous and transparent mixed reaction solution.

[0066] S2 added a 10% ammonia solution dropwise to the mixed reaction solution at a rate of 2 mL / min, started the centrifuge, set the speed to 10000 r / min, centrifuged for 15 min, collected the solid product, and obtained crude magnesium-gallic acid metal-polyphenol nanoparticles.

[0067] S3 used anhydrous ethanol to centrifuge and wash the crude product four times, with a liquid-to-solid volume ratio of 15:1 each time. After washing, the solid was placed in a vacuum drying device with a temperature of 50℃ and a vacuum of 0.09MPa for 18 hours to obtain purified magnesium-gallic acid metal-polyphenol nanoparticles.

[0068] S4 Weigh 10mg of purified nanoparticles, add 1mL of ultrapure deionized water, turn on the ultrasonic device, set the power to 300W, and ultrasonically disperse for 30min until the particles are uniformly dispersed without agglomeration, thus obtaining a stable nanoparticle dispersion. Then, use a pipette to measure 400μL of the mother liquor and add 600μL of deionized water to mix well.

[0069] S5 added 80 mg of methacrylamide gelatin and 3 mg of lithium phenyl-2,4,6-trimethylbenzoylphosphonate to the dispersion, placed the system in a 37°C constant temperature incubator, and allowed it to stand for 30 min to dissolve, forming a homogeneous hydrogel precursor mixture.

[0070] S6 uses a 0.22μm microporous membrane for positive pressure filtration at a pressure of 0.15MPa to remove impurities and contaminants, resulting in a clear and sterile hydrogel precursor solution.

[0071] S7 injects the precursor liquid into a medical polypropylene mold, places it under a 405nm blue light irradiation device, sets the power to 10W, and irradiates for 30 seconds to complete photocrosslinking and curing. The hydrogel is then completely demolded to obtain a dressing semi-finished product.

[0072] S8 uses sterile phosphate buffer with a pH of 7.4 to wash the semi-finished product 4 times, soaking for 15 minutes each time to remove free monomers and initiators. The dressing is then sterilized on both sides for 2 hours using a 35W ultraviolet light source to obtain a sterile metal-polyphenol hydrogel dressing.

[0073] S9 immersed the sterile dressing in DMEM high-glucose medium and extracted it at 37°C and 5% CO2 for 72 hours. The extract was then filtered through a 0.22μm microporous membrane for sterilization, transferred to a sterile centrifuge tube, and stored at 4°C for later use.

[0074] Comparative Example 1 S1 Weigh 80mg of methacrylamide gelatin and 2.5mg of lithium phenyl-2,4,6-trimethylbenzoylphosphonate, add 1mL of deionized water, place the system in a 37℃ constant temperature incubator, let stand for 30min to dissolve, and rotate at 300r / min to form a homogeneous pure hydrogel precursor mixture.

[0075] S2 uses a 0.22μm microporous membrane for positive pressure filtration at a pressure of 0.15MPa to remove impurities and contaminants, resulting in a clear and sterile pure hydrogel precursor solution.

[0076] S3 injects the precursor liquid into a medical polypropylene mold, places it under a 405nm blue light irradiation device, sets the power to 10W, and irradiates for 30 seconds to complete photocrosslinking and curing. The hydrogel is then completely demolded to obtain a pure hydrogel dressing semi-finished product.

[0077] S4 uses sterile phosphate buffer solution with a pH of 7.4 to wash the semi-finished product 4 times, soaking for 15 minutes each time to remove free monomers and initiators. The dressing is then sterilized on both sides for 2 hours using a 35W ultraviolet light source to obtain a sterile pure hydrogel dressing.

[0078] S5. The sterile pure hydrogel dressing was immersed in DMEM high sugar medium and extracted at 37°C and 5% CO2 for 72 hours. The extract was sterilized by filtration through a 0.22μm microporous membrane and transferred to a sterile centrifuge tube for storage at 4°C for later use.

[0079] Table 1 Comparison of core preparation parameters between Examples 1-3 and Comparative Example 1 Explanation of the table: This table fully presents the differences in core preparation parameters between Examples 1-3 and Comparative Example 1. Examples 1-3 fully implemented the entire process of magnesium-gallic acid nanoparticle preparation, loading, photocrosslinking, purification, and sterilization, adjusting only the nanoparticle concentration, all within the process parameter range defined in the claims. Example 1 is the basic formulation, Example 2 increases the nanoparticle loading to enhance repair efficacy, and Example 3 fine-tunes the photoinitiator dosage to optimize crosslinking strength. Comparative Example 1 completely lacks the metal-polyphenol nanoparticle component, preparing only pure methacrylamide gelatin hydrogel without any active functional components. The table visually demonstrates the core innovation of this invention—achieving dressing functionalization through loading magnesium-gallic acid nanoparticles—essentially different from traditional pure hydrogel dressings. All parameters are completely consistent with the characterization conditions in the accompanying drawings.

[0080] Table 2 Comparison of dressing performance between Examples 1-3 and Comparative Example 1 Table Explanation: This table compares the core application performance of Examples 1-3 with Comparative Example 1. The metal-loaded polyphenol hydrogel dressings prepared in Examples 1-3 all exhibit regularly spherical nanoparticles that are uniformly dispersed, matching the specifications in the disclosure document. Figure 1 The scanning electron microscopy results show that the cells possess highly efficient free radical scavenging capabilities. Corresponding to the test results in Reference 5, cell viability remains at a high level. Figure 6 and Figure 7 Characterization data can effectively regulate inflammatory factors, as referenced. Figure 9 The experimental results showed significant wound repair effects, for reference Figure 10 Animal experiment results. Example 2 showed the best overall performance due to its higher nanoparticle loading. Comparative Example 1, with its pure hydrogel, lacked antioxidant and anti-inflammatory functions, resulting in limited wound repair effects. The table fully verifies that this invention, through metal-polyphenol coordination loading combined with hydrogel, endows the dressing with multiple repair functions, solving the technical problems of insufficient activity and poor stability in traditional dressings, and demonstrating significant technological advancement and clinical application value.

[0081] Depend on Figure 1 It can be seen that the prepared particles are regular spherical or near-spherical in shape, with a particle size of about 100 nm, good dispersibility, and no obvious agglomeration.

[0082] The chemical structure of rice grains was characterized using Fourier transform infrared spectroscopy, and the results are as follows: Figure 2 As shown. By Figure 2 It can be seen that the intensity of the carboxyl peak (1680-1700 cm-1) and hydroxyl peak (~3500 cm-1) in gallic acid is significantly reduced, suggesting that these functional groups form coordinate bonds with Mg2+.

[0083] X-ray photoelectron spectroscopy was used to further test the elemental composition and valence states of the nanoparticles, and the results are as follows: Figure 3 As shown. By Figure 3 (a) It can be seen that the main peak at 1303 eV conforms to the characteristics of magnesium-polyphenol coordination bonds. Figure 3 (b) shows a significant CO peak at 286 eV, confirming the presence of the polyphenol structure. In addition, the carboxyl peak intensity is unusually high at 288 eV, which is consistent with the GA molecular structure. Figure 3 (c) The full spectrum shows that carbon, oxygen, and magnesium are the main elements, with no other metallic impurities, which is in line with the design expectations.

[0084] like Figure 7 As shown in the figure, the biocompatibility curves show that different hydrogels can maintain cell growth well, and the cell survival rate can reach more than 90%.

[0085] The expression levels of inflammation-related genes (IL-1β, IL-6, and TNF-α) were detected using the Bio-Rad real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) system. β-actin was used as an internal reference gene, and the relative expression levels were obtained using the 2-ΔΔCT method. Results are as follows: Figure 9 As shown.

[0086] Depend on Figure 9 The RT-qPCR assay showed that the hydrogel group could reduce inflammation-related genes.

[0087] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a metal-loaded polyphenol hydrogel dressing, characterized in that, Includes the following steps: S1 prepares an ethanol aqueous solution with a volume fraction of 30%. Gallic acid solid and magnesium chloride solid are added to the prepared ethanol aqueous solution in sequence. The ultrasonic equipment is turned on for dispersion treatment, and the ultrasonic treatment is continued until the solid raw materials are completely dissolved. S2 Slowly add a 10% (v / v) ammonia solution to the mixed reaction solution, turn on the stirrer and stir continuously, and carry out the metal-polyphenol coordination reaction under normal temperature and pressure conditions. After the coordination reaction is completed, start the centrifugation device to obtain crude magnesium-gallic acid metal-polyphenol nanoparticles. S3 uses anhydrous ethanol as the washing solvent to centrifuge and wash the crude magnesium-gallic acid metal-polyphenol nanoparticles multiple times to remove unreacted free raw materials and soluble byproducts. After washing, the solid product is placed in a vacuum drying device to obtain purified magnesium-gallic acid metal-polyphenol nanoparticles. S4 Weigh a certain amount of purified magnesium-gallic acid metal-polyphenol nanoparticles, add them to ultrapure deionized water, turn on the ultrasonic device to homogenize and disperse them until the nanoparticles are evenly distributed in the liquid phase system, and obtain a stable nanoparticle dispersion. S5 adds methacrylamide gelatin and lithium phenyl-2,4,6-trimethylbenzoylphosphonate to the nanoparticle dispersion, and places the mixture in a constant temperature incubator to form a homogeneous hydrogel precursor mixture; S6 uses a microporous membrane to pressurize and filter the hydrogel precursor mixture to remove undissolved impurities, large aggregates and microbial contaminants from the system, resulting in a clear and sterile hydrogel precursor solution. S7 slowly injects the hydrogel precursor liquid into a sterile custom mold, places the mold under a blue light irradiation device, turns on the blue light to carry out a photocrosslinking and curing reaction, and obtains a metal-loaded polyphenol hydrogel dressing semi-finished product. S8 uses sterile phosphate buffer to repeatedly soak and wash the semi-finished metal-polyphenol hydrogel dressing to remove uncrosslinked free monomers and residual initiators. After washing, the front and back of the semi-finished hydrogel are sterilized separately using ultraviolet light to obtain sterile metal-polyphenol hydrogel dressing. S9 completely immerses the sterilized metal-polyphenol hydrogel dressing in cell culture medium and places it in a constant temperature incubator for extraction. After extraction, the extraction system is filtered and sterilized using a microporous membrane to obtain a pure hydrogel extract.

2. The method for preparing the metal-loaded polyphenol hydrogel dressing according to claim 1, characterized in that, The volume of the ethanol-water solution in S1 is 10 mL to 50 mL, the concentration of gallic acid in the mixed reaction solution is 20 mM, the concentration of magnesium chloride in the mixed reaction solution is 7 mM, the power of the ultrasonic treatment is set to 100 W to 300 W, the ultrasonic treatment time is set to 10 min to 30 min, and the system temperature is kept in the range of 20℃ to 25℃ during the ultrasonic treatment process.

3. The method for preparing the metal-loaded polyphenol hydrogel dressing according to claim 1, characterized in that, The ammonia solution in S2 was added dropwise, with the dropping rate controlled at 1 mL / min to 3 mL / min. The total time for the coordination reaction was 8 h, and the centrifugation time was set to 10 min to 20 min. The centrifugation process was carried out in a low-temperature environment.

4. The method for preparing the metal-loaded polyphenol hydrogel dressing according to claim 1, characterized in that, The number of anhydrous ethanol washings in S3 was set to 3 to 5 times, with a liquid-to-solid volume ratio of 10:1 to 20:1 for each wash. The vacuum drying temperature was set to 40℃ to 60℃, and the vacuum drying time was set to 12h to 24h. The prepared magnesium-gallic acid metal-polyphenol nanoparticles had a spherical or near-spherical structure with a particle size distribution of 90nm to 110nm. The particles were well dispersed and showed no obvious agglomeration.

5. The method for preparing the metal-loaded polyphenol hydrogel dressing according to claim 1, characterized in that, The mass-to-volume ratio of magnesium-gallic acid metal-polyphenol nanoparticles to deionized water in S4 is 10 mg: 1 mL, the ultrasonic dispersion power is 200 W to 400 W, and the dispersion time is 15 min to 40 min.

6. The method for preparing the metal-loaded polyphenol hydrogel dressing according to claim 1, characterized in that, The addition mass of S5 is 80 mg of methacrylamide gelatin and 2.5 mg of lithium phenyl-2,4,6-trimethylbenzoylphosphonate. The temperature of the constant temperature incubator is set to 37℃, and the mixture is allowed to stand for 30 minutes to dissolve. The entire dissolution process is carried out in a sterile environment.

7. The method for preparing the metal-loaded polyphenol hydrogel dressing according to claim 1, characterized in that, The microporous filter membrane in S6 has a pore size of 0.22 μm, and the filtration method is positive pressure filtration with a filtration pressure of 0.1 MPa to 0.2 MPa. In S7, the blue light irradiation wavelength is 405 nm, the blue light irradiation power is 5 W to 15 W, and the photocrosslinking curing time is 30 s.

8. The method for preparing the metal-loaded polyphenol hydrogel dressing according to claim 1, characterized in that, The pH of the sterile phosphate buffer in S8 is 7.2 to 7.4, the number of washes is 3 to 5, the power of the ultraviolet light source is 30W to 40W, and the single-sided ultraviolet sterilization time is 2 hours.

9. The method for preparing the metal-loaded polyphenol hydrogel dressing according to claim 1, characterized in that, The S9 cells were cultured in DMEM high-glucose medium at an extraction temperature of 37°C. A 5% CO2 atmosphere was maintained during the extraction process. Sterilization was achieved using a 0.22μm microporous membrane. Storage was at 4°C, with a shelf life of 7 days. The DPPH free radical scavenging efficiency of the hydrogel extract was calculated using a specialized formula. , in the formula This represents the DPPH free radical scavenging efficiency. This represents the absorbance value of the blank group. The absorbance value represents the sample group, and the absorbance detection wavelength is fixed at 517nm.

10. The application of the metal-loaded polyphenol hydrogel dressing prepared according to any one of claims 1 to 9 in medical wound repair materials, characterized in that, The application is directly used for the repair of skin defects and wounds in humans or animals, including mechanical trauma wounds, thermal burn wounds, chronic diabetic ulcer wounds, and pressure injury wounds. During application, the metal-polyphenol hydrogel dressing continuously releases magnesium ions and gallic acid active components, clearing excess reactive oxygen free radicals in the wound microenvironment and promoting endothelial cell migration and angiogenesis. When applied to full-thickness skin defects in mice, the wound healing rate is higher than 95% after 12 days. The dressing has good biocompatibility, a survival rate of more than 90% for L929 fibroblasts, and is non-cytotoxic and non-irritating to tissues.

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