Hydrogel powder loaded with bimetallic nano-enzyme and MG53 protein as well as preparation method and application of hydrogel powder

Abstract

The invention relates to hydrogel powder loaded with bimetallic nano-enzyme and MG53 protein as well as a preparation method and application of the hydrogel powder, and relates to the technical field of biological medicines. PEG / SF hydrogel is used as a drug carrier, ZnCeO2 nano-enzyme and MG53 protein are encapsulated in hydrogel powder, and the problems that in-situ administration is prone to inactivation, controlled release is difficult and the like are effectively solved; the invention discloses a synthesis method of a ZnCeO2 nano-enzyme, zinc atoms are introduced to directionally regulate and control a complex catalytic reaction in the nano-enzyme, and released Zn < 2 + > can be combined with a specific binding site of MG53 protein, so that the activity of the MG53 protein is enhanced, and plateau wound healing is synergistically promoted. The hydrogel powder efficiently promotes plateau wound healing from inflammation control and tissue repair, provides a new thought for plateau wound dressing development, and also has important clinical significance on plateau wound management.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a hydrogel powder loaded with bimetallic nanozymes and MG53 protein, its preparation method and application. Background Technology

[0002] my country has a vast plateau region, making it one of the countries with the largest plateau area and the largest population in the world. The unique climate and geographical conditions of the plateau region can significantly affect human physiological functions, especially wound healing. Studies have shown that wound healing time in plateau environments is 30% longer than in plains areas. Furthermore, the dry climate and strong ultraviolet radiation in plateau regions increase the risk of wound infection. Currently, treatment methods for high-origin wounds mainly focus on keeping the wound moist, avoiding wound exposure, and providing appropriate nutrition, which can promote wound healing to some extent, but still have certain limitations. Therefore, developing novel high-origin wound dressings is of significant clinical importance for the management of high-origin wounds.

[0003] MG53 (also known as TRIM 72) is a muscle factor secreted by skeletal muscle during exercise, and it plays a repairing role in damage to various organs such as the kidneys, brain, lungs, and heart. Currently, research teams have extended the therapeutic effects of MG53 protein to the field of diabetic wound repair. Their research results demonstrate that MG53 protein can promote the survival and differentiation of hair follicle stem cells and repair diabetic wounds in the environment of oxidative stress imbalance. This research suggests that exogenous administration of MG53 protein may be useful for repairing highly original wounds. However, achieving the repair of highly original tissues with exogenous MG53 protein still faces several challenges, such as a short half-life and difficulty in controlled release.

[0004] Under normal physiological conditions, cells rely on a series of natural antioxidant enzymes to maintain the balance of their own oxidation system and ensure the normal functioning of mitochondria. However, enzyme activity in cells with high originality is usually inhibited, making it difficult for cells to effectively regulate ROS levels. Currently, although therapeutic strategies based on natural enzymes have made some research progress, they face multiple challenges such as potential immunogenicity, unstable enzyme activity, and high cost. In contrast, nanozymes have become a new solution due to their excellent stability, lower cost, and significant antioxidant and anti-inflammatory effects. Among them, cerium dioxide (CeO2) can mimic the functions of various natural antioxidant enzymes, such as peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD), and has shown therapeutic potential in multiple disease models. However, when these complex catalytic reactions coexist, their reaction pathways and outcomes may antagonize other reactions, leading to a decrease in enzyme-like activity and affecting therapeutic efficacy. Summary of the Invention

[0005] The purpose of this invention is to address the above-mentioned problems by providing a hydrogel powder loaded with bimetallic nanozymes and MG53 protein, its preparation method, and its application.

[0006] The first aspect of this invention provides a method for preparing hydrogel powder loaded with bimetallic nanozymes and MG53 protein, employing the following technical solution:

[0007] A method for preparing a hydrogel powder loaded with bimetallic nanozymes and MG53 protein involves using PEG / SF hydrogel as a drug carrier to encapsulate ZnCeO2 nanozymes and MG53 protein in the hydrogel to obtain a hydrogel PEG / SF@MG53@ZnCeO2 loaded with bimetallic nanozymes and MG53 protein. The hydrogel is then ground into powder to obtain PEG / SF@MG53@ZnCeO2 hydrogel powder.

[0008] By employing the above-mentioned technical solution, using polyethylene glycol (PEG) and silk fibroin (SF) as the main body of the hydrogel, MG53 protein and ZnCeO2 nanozymes are encapsulated in the hydrogel powder. Based on the advantages of the PEG / SF hydrogel, such as its hydrophilic three-dimensional network structure, high biocompatibility, low liquid surface tension, and the existence of osmotic pressure difference for sustained drug release, the problems of protein aggregation, easy inactivation, and difficulty in controlled release during in-situ drug delivery can be effectively solved. Furthermore, by introducing zinc atoms to directionally regulate the complex catalytic reaction inside the nanozyme, the released Zn... 2+ It can bind to specific binding sites (RING, B-box) of the MG53 protein, enhancing its activity and helping to regulate its oligomerization, vesicle transport, and interaction with the damaged membrane, synergistically promoting the healing of highly original surfaces. Compared with traditional hydrogels, hydrogel powder is easier to store and has better environmental adaptability. The physical cross-linking method allows the hydrogel powder to quickly gel upon contact with tissue exudate or blood, promptly sealing the wound. This provides a new approach for the development of highly original surface dressings and has important clinical significance for the management of highly original surfaces.

[0009] Preferably, the preparation method includes the following steps:

[0010] S1. Preparation of bimetallic (ZnCeO2) nanozymes: Cerium acetate hexahydrate and zinc sulfate were dissolved in a mixed solvent, mixed and precipitated, the precipitate was centrifuged at high speed, washed with water and dried to obtain ZnCeO2 nanozymes. The mixed solvent was composed of water, acetic acid and ethylene glycol.

[0011] S2. Preparation of pure silk fibroin (SF) solution: Select silkworm cocoon raw material and mix with sodium carbonate and boil. Stir to remove sericin to obtain degummed silk fibers. Wash the degummed silk fibers to completely remove sericin and sodium carbonate, and then immerse them in lithium bromide solution for water bath. After the water bath is completed, dialyze to remove lithium bromide to obtain SF solution.

[0012] Preparation of S3 and PEG / SF@MG53@ZnCeO2 hydrogel powder: MG53 protein and ZnCeO2 nanozyme prepared in S1 were first mixed with polyethylene glycol solution, and then mixed evenly with SF solution prepared in S2 to obtain PEG / SF@MG53@ZnCeO2 hydrogel colloid. The colloid was freeze-dried and then ground into powder to obtain PEG / SF@MG53@ZnCeO2 hydrogel powder.

[0013] Preferably, in the above preparation method, in step S1, the ratio of cerium acetate hexahydrate to zinc sulfate is 2:1.

[0014] Preferably, in the above preparation method, step S1 specifically comprises:

[0015] S1. Preparation of bimetallic (ZnCeO2) nanozymes: 1.0 g of cerium acetate hexahydrate and 0.5 g of zinc sulfate were dissolved in a mixed solvent prepared by 1 mL of water, 1 mL of acetic acid and 25 mL of ethylene glycol. After stirring evenly, the mixture was stirred in an oil bath at 180℃ for 20 min. The precipitate obtained from the reaction was centrifuged at 18000 rpm / min for 10 min. After centrifugation, the precipitate was washed several times with water. After washing, the precipitate was freeze-dried. The dried product was ZnCeO2 nanozyme.

[0016] Preferably, in the above preparation method, step S2 specifically comprises:

[0017] S2. Preparation of pure silk fibroin (SF) solution: Select silkworm cocoons as raw materials, cut them into small pieces, mix them with a 0.02 M sodium carbonate solution, boil them, maintain the boiling state for 60 min and continuously stir to remove sericin, and obtain degummed silk fibers. Wash the degummed silk fibers repeatedly to completely remove sericin and sodium carbonate, and then immerse the silk fibers in a 9.3 M lithium bromide solution and water bath at 60℃ for 4 h. After the water bath, dialyze them for 3 days using a standard grade regenerated cellulose dialysis membrane (3500 kD) to remove lithium bromide, and obtain SF solution.

[0018] Preferably, in the above preparation method, step S3 specifically comprises:

[0019] Preparation of S3 and PEG / SF@MG53@ZnCeO2 hydrogel powder: MG53 protein and ZnCeO2 nanozyme prepared in S1 were first mixed with 6 wt% polyethylene glycol solution to obtain a mixture. Then, the mixture was mixed with an equal volume and mass fraction of SF solution prepared in S2 to obtain PEG / SF@MG53@ZnCeO2 hydrogel colloid. The colloid was freeze-dried and then ground into powder to obtain PEG / SF@MG53@ZnCeO2 hydrogel powder. The particle size of ZnCeO2 nanozyme was 2-8 nm, and the ratio of MG53 protein to ZnCeO2 nanozyme was 0.03:400.

[0020] Preferably, in the above preparation method, the concentration of ZnCeO2 nanozyme in the mixture is ρ, where 0 < ρ ≤ 400 μg / mL.

[0021] Preferably, the concentration of ZnCeO2 nanozyme in the mixture is 400 μg / mL.

[0022] By adopting the above technical solutions, optimizing the preparation method, step parameters, and drug concentration, the performance of hydrogel powder is improved while its biocompatibility is also enhanced.

[0023] A second aspect of the present invention provides a hydrogel powder loaded with bimetallic nanozymes and MG53 protein, which is prepared by any of the preparation methods described above.

[0024] A third aspect of the present invention provides the application of the above-mentioned hydrogel powder loaded with bimetallic nanozymes and MG53 protein in the field of highly original surface repair.

[0025] In summary, this application includes at least one of the following beneficial technical effects:

[0026] 1. Using polyethylene glycol (PEG) and silk fibroin (SF) as the main body of the hydrogel, MG53 protein and ZnCeO2 nanozymes are encapsulated in the hydrogel powder. Leveraging the advantages of the PEG / SF hydrogel, such as its hydrophilic three-dimensional network structure, high biocompatibility, low surface tension, and osmotic pressure difference for sustained drug release, this method effectively solves problems such as protein aggregation, easy inactivation, and difficulty in controlled release during in-situ drug delivery. By introducing zinc atoms to directionally regulate the complex catalytic reaction within the nanozyme, the released Zn... 2+It can bind to specific binding sites (RING, B-box) of the MG53 protein, enhancing its activity and helping to regulate its oligomerization, vesicle transport, and interaction with the damaged membrane, synergistically promoting the healing of highly original surfaces. Compared with traditional hydrogels, hydrogel powder is easier to store and has better environmental adaptability. The physical cross-linking method allows the hydrogel powder to quickly gel upon contact with tissue exudate or blood, promptly sealing the wound. This provides a new approach for the development of highly original surface dressings and has important clinical significance for the management of highly original surfaces.

[0027] 2. The hydrogel powder provided by this method has good biocompatibility and can continuously release ZnCeO2 nanozymes and MG53 protein into the wound tissue, clearing excess ROS (H2O2, OH-). - and O2 - This promotes the repair of wound tissue.

[0028] 3. The hydrogel powder provided by this method can achieve complete healing within 8 days in a high-altitude environment. At the same time, H&E staining sections show that the hydrogel powder group has better epithelial regeneration and granulation tissue area, which can significantly reduce excessive reactive oxygen species in the wound, balance the inflammatory response, repair damaged tissue, and accelerate the healing of high-altitude wounds.

[0029] 4. The hydrogel powder provided by this method can be widely used to treat other refractory wounds caused by bacterial infection, inflammatory imbalance, and tissue damage, including those caused by diabetic foot, pressure ulcers, and venous ulcers. Attached Figure Description

[0030] Figure 1 This is a structural characterization diagram of the ZnCeO2 nanozyme in this application, in which... Figure 1 A is the XRD pattern. Figure 1 B represents the XPS plot. Figure 1 C is the particle size distribution diagram. Figure 1 D represents the TEM and elemental energy spectrum;

[0031] Figure 2 This is a structural characterization diagram of the PEG / SF@MG53@ZnCeO2 hydrogel in this application, in which... Figure 2 A is an SEM image. Figure 2 B is the rheological analysis structure diagram. Figure 2 C represents the FTIR spectrum;

[0032] Figure 3 This is a graph showing the biosafety test results of ZnCeO2 nanozymes at different concentrations and different material groups. Figure 3 A and Figure 3 B is the hemolysis rate result graph. Figure 3 C and Figure 3 D represents the cell viability results;

[0033] Figure 4 The graph shows the clear verification results of reactive oxygen species (ROS) with different concentrations of ZnCeO2 nanozymes and different material groups. Figure 4 A and Figure 4 B is the CAT activity result graph. Figure 4 C and Figure 4 D is the graph showing the hydroxyl radical scavenging rate. Figure 4 E and Figure 4 F represents the SOD activity diagram;

[0034] Figure 5 This figure shows the results of investigating the drug release behavior of ZnCeO2 nanozymes and PEG / SF@MG53@ZnCeO2 hydrogels. Figure 5 A represents the concentration of ZnCeO2 in PBS at different time points. 2+ The release curve, Figure 5 B represents the release curves of ZnCeO2 from the hydrogel in PBS at different time points. Figure 5 C and Figure 5 D represents the standard curve and release curve of BSA in hydrogel at different time points in PBS;

[0035] Figure 6 The image shows the results of the high-originality surface restoration experiment. Figure 6 Image A shows the healing process after treatment with different materials. Figure 6 B shows the H&E staining patterns of different sample slices;

[0036] Figure 7 This application provides a schematic diagram of the preparation process of hydrogel powder and the principle of high-originality surface repair. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Modifications or equivalent substitutions made by those skilled in the art based on their understanding of the technical solutions of this invention, without departing from the spirit and scope of the invention, should be covered within the protection scope of this invention.

[0038] The reagent information used in the following examples is as follows. Unless otherwise specified, all reagents and chemicals are used as is without further processing. Other specific conditions not specified are performed according to standard conditions or the manufacturer's recommendations. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0039] Main reagents and materials:

[0040] Cerium acetate hexahydrate: (CH3CO2)3Ce·6H2O, CAS Registry No.: 206996-60-3;

[0041] Zinc sulfate: ZnSO4, CAS Registry Number: 7446-19-7;

[0042] Polyethylene glycol: HO(CH2CH2O)nH, CAS Registry Number: 25322-68-3;

[0043] Lithium bromide: LiBr, CAS Registry No.: 7550-35-8;

[0044] Sodium carbonate: Na2CO3, CAS Registry Number: 497-19-8;

[0045] Hydrogen peroxide: H2O2, CAS Registry Number: 7722-84-1 (solution);

[0046] Potassium iodide: KI, CAS Registry Number: 7681-11-0;

[0047] Ferrous sulfate heptahydrate: FeSO4·6H2O, CAS Registry No.: 7782-63-0;

[0048] Salicylic acid: C7H6O3, CAS Registry Number: 69-72-7;

[0049] Superoxide dismutase (SOD) Activity Assay Kit: Beijing Solarbio Science & Technology Co., Ltd., China;

[0050] MG53 protein: Novoprotein, China.

[0051] The chemical abbreviations and their definitions in this application are as follows:

[0052] Zinc-cerium bimetallic nanozymes (ZnCeO2 nanozymes); pure silk fibroin (SF); polyethylene glycol (PEG); hydrogels loaded with bimetallic nanozymes and MG53 protein using polyethylene glycol and pure silk fibroin as carriers (PEG / SF@MG53@ZnCeO2); polyethylene glycol and pure silk fibroin hydrogels (PEG / SF); other unspecified terms shall be interpreted according to industry conventions.

[0053] I. Preparation Example

[0054] Preparation Example 1

[0055] Preparation of ZnCeO2 nanozymes: Cerium acetate hexahydrate and zinc sulfate are dissolved in a mixed solvent, with a preferred ratio of 2:1. After mixing, the mixture is precipitated, the precipitate is centrifuged at high speed, washed with water, and dried to obtain ZnCeO2 nanozymes. The mixed solvent is composed of water, acetic acid, and ethylene glycol.

[0056] Specifically, 1.0 g of cerium acetate hexahydrate and 0.5 g of zinc sulfate were dissolved in a mixed solvent prepared from 1 mL of water, 1 mL of acetic acid and 25 mL of ethylene glycol. After stirring evenly, the mixture was stirred in an oil bath at 180°C for 20 min. The precipitate obtained from the reaction was centrifuged at 18000 rpm / min for 10 min. After centrifugation, the precipitate was washed three times with water. After washing, the precipitate was freeze-dried (-60°C, 48 h). The dried product was ZnCeO2 nanozyme.

[0057] Preparation Example 2

[0058] Preparation of SF solution: Select silkworm cocoons and mix them with sodium carbonate and boil them. Stir to remove sericin to obtain degummed silk fibers. Wash the degummed silk fibers to completely remove sericin and sodium carbonate, and then immerse them in a lithium bromide solution for a water bath. After the water bath is completed, dialyze to remove lithium bromide to obtain SF solution.

[0059] Specifically, silkworm cocoons are selected, cut into small pieces, and mixed with a 0.02 M sodium carbonate solution and boiled. The mixture is kept boiling for 60 minutes with continuous stirring to remove sericin, resulting in degummed silk fibers. The degummed silk fibers are then repeatedly washed to completely remove sericin and sodium carbonate. The silk fibers are then immersed in a 9.3 M lithium bromide solution and bathed in a water bath at 60°C for 4 hours. After the water bath, the solution is dialyzed for 3 days using a standard grade regenerated cellulose dialysis membrane (3500 kD) to remove lithium bromide, resulting in an SF solution. In this step, "M" refers to molar concentration (now called molar concentration), M = number of solute moles / number of liters of solution, i.e., mol / L.

[0060] II. Implementation Examples

[0061] Example 1

[0062] A method for preparing hydrogel powder loaded with bimetallic nanozymes and MG53 protein involves using PEG / SF hydrogel as a drug carrier to encapsulate ZnCeO2 nanozymes and MG53 protein within the hydrogel, thus obtaining a hydrogel PEG / SF@MG53@ZnCeO2 loaded with bimetallic nanozymes and MG53 protein. The hydrogel is then ground into powder to obtain PEG / SF@MG53@ZnCeO2 hydrogel powder. The specific steps are as follows:

[0063] S1. Preparation of bimetallic (ZnCeO2) nanozymes: ZnCeO2 nanozymes were prepared according to the preparation method provided in Preparation Example 1, and the products were stored at room temperature.

[0064] S2. Preparation of pure silk fibroin (SF) solution: Prepare SF solution according to the preparation method provided in Preparation Example 2.

[0065] Preparation of S3 and PEG / SF@MG53@ZnCeO2 hydrogel powder: MG53 protein and ZnCeO2 nanozyme prepared in S1 were first mixed with polyethylene glycol solution, and then mixed evenly with SF solution prepared in S2 to obtain PEG / SF@MG53@ZnCeO2 hydrogel colloid. The colloid was freeze-dried and ground into powder to obtain PEG / SF@MG53@ZnCeO2 hydrogel powder. In this step, the ratio of MG53 protein to ZnCeO2 nanozyme was 0.03:400.

[0066] Specifically, the ZnCeO2 nanozymes prepared in S1 were first screened, and ZnCeO2 nanozymes with a particle size of 2-8 nm were selected for later use. A 6 wt% polyethylene glycol (PEG) solution was prepared, and a 6 wt% SF solution was prepared from the SF solution prepared in S2.

[0067] 0.75 μg of MG53 protein and 100 mg of screened ZnCeO2 nanozyme were first mixed with 25 mL of 6 wt% polyethylene glycol solution to obtain a mixture. The concentration of ZnCeO2 nanozyme in the mixture was ρ, which was 400 μg / mL. The mixture was then mixed with an equal volume (25 mL) of 6 wt% SF solution to obtain PEG / SF@MG53@ZnCeO2 hydrogel. The colloid was freeze-dried (-45℃, 72 h) and then ground into powder to obtain PEG / SF@MG53@ZnCeO2 hydrogel powder.

[0068] Example 2

[0069] A method for preparing hydrogel powder loaded with bimetallic nanozymes and MG53 protein differs from Example 1 in that the amount of ZnCeO2 nanozyme in S3 is different. The amount of ZnCeO2 nanozyme is 12.5 mg, and the concentration ρ of ZnCeO2 nanozyme in the mixture is 50 μg / mL.

[0070] Example 3

[0071] A method for preparing hydrogel powder loaded with bimetallic nanozymes and MG53 protein differs from Example 1 in that the amount of ZnCeO2 nanozyme in S3 is different, the amount of ZnCeO2 nanozyme is 25 mg, and the concentration ρ of ZnCeO2 nanozyme in the mixture is 100 μg / mL.

[0072] Example 4

[0073] A method for preparing hydrogel powder loaded with bimetallic nanozymes and MG53 protein differs from Example 1 in that the amount of ZnCeO2 nanozyme in S3 is different, the amount of ZnCeO2 nanozyme is 50 mg, and the concentration ρ of ZnCeO2 nanozyme in the mixture is 200 μg / mL.

[0074] III. Comparative Example

[0075] Comparative Example 1

[0076] A method for preparing PEG / SF hydrogel powder: SF solution is prepared according to the method in S2 of Example 1. 25 mL of 6 wt% polyethylene glycol solution is mixed with 25 mL of 6 wt% SF to obtain PEG / SF hydrogel. The colloid is freeze-dried and then ground into powder to obtain PEG / SF hydrogel powder.

[0077] IV. Performance Testing Experiments and Results

[0078] 1. Structural characterization

[0079] 1.1 Structural Characterization of ZnCeO2 Nanozymes

[0080] The ZnCeO2 nanozyme prepared in Example 1 was scanned, and the results are as follows: Figure 1 As shown, where Figure 1 A is the XRD pattern of the nanozyme. Figure 1 B is the XPS spectrum of the nanozyme. Figure 1 C represents the particle size distribution of the nanozyme. Figure 1 D shows the TEM and elemental energy spectrum of the nanozyme. As can be seen from the figure, the crystal diffraction peaks of the nanozyme are consistent with the standard card JCPDS No. 34-0394. The XPS spectrum also shows three elements: Ce, Zn, and C. Observing its morphology under a transmission electron microscope, obvious lattice fringes were found. Further energy spectrum results also proved the successful incorporation of Zn.

[0081] 1.2 Structural characterization of PEG / SF@MG53@ZnCeO2 hydrogel powder

[0082] The structure of the PEG / SF@MG53@ZnCeO2 hydrogel powder prepared in Example 1 was characterized, and the results are as follows: Figure 2 As shown, where, Figure 2 A is a SEM image of the hydrogel powder. Figure 2 Figure B shows the rheological analysis results of the hydrogel powder. Figure 2 C is the FTIR spectrum of the hydrogel powder.

[0083] As shown in the figure, the hydrogel powder exhibits a loose and porous structure inside the hydrogel under scanning electron microscopy. Rheological analysis results show that the storage modulus (G') of the PEG / SF@MG53@ZnCeO2 hydrogel is greater than the loss modulus (G') after four minutes of mixing, indicating that the hydrogel has a rapid gelation capability. Furthermore, in the FTIR spectrum, the characteristic absorption peak of the α-helical structure of PEG / SF appears at 1652 cm⁻¹, while the β-sheet structure shows a peak at 1500 cm⁻¹. -1 and 1240 cm -1 Compared to SF, PEG / SF hydrogel exhibits increased absorption strength due to its β-sheet structure, consistent with the gelation principle of PEG / SF hydrogel.

[0084] 2. Biosafety Testing

[0085] Since the hydrogel powder provided by this method is mainly used for wound repair, it must have good biocompatibility. Biocompatibility testing is divided into two aspects: hemolysis rate determination and cell viability detection.

[0086] 2.1 Determination of hemolysis rate of ZnCeO2 nanozyme and PEG / SF@MG53@ZnCeO2 hydrogel powder

[0087] First, approximately 1 mL of fresh blood was obtained from the eyes of C57 mice. Red blood cells (RBCs) were separated after centrifugation at 1500 rpm. The RBCs were washed and prepared as an RBCs / saline solution. The ZnCeO2 nanozyme prepared in Example 1 was added to the saline solution to prepare solutions of different concentrations: 0, 50, 100, 200, 400, 800, and 1000 μg / mL. 1.1 mL of each solution was taken, and 100 μL of the RBCs / saline solution was added to each solution. The solutions were incubated at 37°C for 2 h. The OD value of the supernatant was then measured at 540 nm using a UV spectrophotometer.

[0088] Test results are as follows Figure 3 As shown in A, where Figure 3Figure A shows the test results of ZnCeO2 nanozymes at different concentrations. As can be seen from the figure, when the concentration of ZnCeO2 nanozymes is within the range of 0-400 μg / mL, its hemolysis rate is less than 5%. As the concentration increases, the hemolysis rate increases and exceeds the safe value. The optimal concentration is 400 μg / mL.

[0089] Equal masses of ZnCeO2 nanozyme, MG53 protein, PEG / SF hydrogel powder prepared in Example 1, and PEG / SF@MG53@ZnCeO2 hydrogel powder prepared in Examples 1-4 were taken and added to equal volumes of physiological saline to prepare physiological saline solutions for different material groups. 1.1 mL of each solution was taken, and 100 μL of RBCs / physiological saline mixture was added to each solution. The solutions were incubated at 37°C for 2 h. Afterwards, the OD value of the supernatant was measured at 540 nm using a UV spectrophotometer.

[0090] Since the concentrations of ZnCeO2 nanozymes in the PEG / SF@MG53@ZnCeO2 hydrogel powders prepared in Examples 1-4 were 400, 50, 100, and 200 μg / mL, respectively, and the test results were consistent with... Figure 3 The concentration results of the ZnCeO2 nanozyme in A were consistent; therefore, only the test results of the PEG / SF@MG53@ZnCeO2 hydrogel powder prepared in Example 1 were compared with those of other materials. Figure 3 B is a comparison chart of test results for different material groups. As can be seen from the chart, the hemolysis rate is below 5%, indicating good biocompatibility.

[0091] 2.2 Cell viability assay of ZnCeO2 and PEG / SF@MG53@ZnCeO2 hydrogel powder

[0092] Mouse epithelial fibroblasts (L929) were cultured at 2 × 10⁶ cells per well. 3 Appropriate densities of cells were seeded into 96-well plates. The ZnCeO2 nanozyme prepared in Example 1 was added to DMEM high-glucose medium to prepare different concentrations of ZnCeO2 nanozyme, with concentrations of 0, 50, 100, 200, 400, 800, and 1000 μg / mL. After stable cell attachment, 100 μL of DMEM high-glucose medium containing different concentrations of ZnCeO2 was added to each culture plate. After co-incubation for 1, 2, and 3 days, the original medium containing the material was replaced with 100 μL of fresh medium containing 10% CCK-8. After 2 h, the cells were removed from the incubator, and the OD value (450 nm) was measured.

[0093] Similarly, cell viability was assessed for different material groups (ZnCeO2, MG53, PEG / SF, PEG / SF@MG53@ZnCeO2), and the results are as follows: Figure 3 As shown in C and D.

[0094] in, Figure 3 C represents the CCK-8 results after incubating L929 cells with different concentrations of ZnCeO2 nanozymes. Figure 3 D shows the CCK-8 results after incubation of L929 cells with different material groups. As can be seen from the figure, after incubation of L929 cells with different concentrations of nanozymes and different material groups for 1-3 days, the cell activity was higher than 80% and gradually increased, indicating that the materials have good cell safety.

[0095] 3. Verification of reactive oxygen species scavenging performance of ZnCeO2 nanozymes and PEG / SF@MG53@ZnCeO2 hydrogel powder

[0096] 3.1 Assay of catalase (CAT) activity in ZnCeO2 and PEG / SF@MG53@ZnCeO2

[0097] CAT activity was measured for nanozymes of different concentrations and different materials. First, the H2O2 stock solution was diluted to 1 mM H2O2 solution. Then, different masses (100, 200, 300, 400, 600, 700, 800 μg) of ZnCeO2 (or different material groups: ZnCeO2, MG53, PEG / SF, PEG / SF@MG53@ZnCeO2) were rapidly mixed with 300 μL of H2O2 dilution to form different mixtures. After reacting in the dark for 30 min, 300 μL of 1 M KI solution was added to the mixture, mixed well, and reacted for 5 min. Subsequently, the absorbance of the final mixture was measured at 350 nm using a UV spectrophotometer.

[0098] Test results are as follows Figure 4 As shown in A and B, where... Figure 4 A shows the UV absorption curves of ZnCeO2 nanozymes of different concentrations reacted with H2O2 / KI. Figure 4 B shows the UV absorption curves of different material groups after reacting with H2O2 / KI. As can be seen from the figure, the absorbance at 350 nm gradually decreases with increasing nanozyme concentration, indicating a positive correlation between the CAT activity and concentration of the nanozyme. Furthermore, the MG53 and PEG / SF hydrogels exhibited lower CAT activity and less effective H2O2 scavenging. Simultaneously, due to the slow release of the nanozyme caused by the hydrogel encapsulation, the scavenging rate of PEG / SF@MG53@ZnCeO2 was slightly lower than that of the ZnCeO2 group.

[0099] 3.2 Evaluation of the hydroxyl radical scavenging ability (•OH) of ZnCeO2 and PEG / SF@MG53@ZnCeO2

[0100] The hydroxyl radical scavenging capacity (•OH) of nanozymes at different concentrations and different materials was evaluated. 9 mM ferrous sulfate was mixed with 8.8 mM H2O2, and different masses (100, 200, 300, 400, 500, 600, 700, 800 μg) of ZnCeO2 (or different material groups: ZnCeO2, MG53, PEG / SF, PEG / SF@MG53@ZnCeO2) were added to this mixture to eliminate •OH generated from the mixture. To quantify the remaining •OH, a 9 mM salicylic acid / ethanol solution was added. During this process, salicylic acid oxidized •OH to 2,3-dihydroxybenzoic acid (purple color). The absorption value was measured at a wavelength of 510 nm using a UV spectrophotometer to calculate the •OH generation rate.

[0101] The test results are as follows Figure 4 As shown in C and D, where... Figure 4 C is the UV absorption curve of ZnCeO2 nanozymes at different concentrations for scavenging hydroxyl radicals (•OH). Figure 4 D is the UV absorption curve of different material groups for scavenging hydroxyl radicals (•OH).

[0102] As shown in the figure, the absorbance at 510 nm gradually decreased with the increase of nanozyme concentration, indicating that the scavenging ability of nanozymes for hydroxyl radicals (•OH) is positively correlated with concentration. In addition, MG53 and SF / PEG hydrogels have a relatively small scavenging effect on hydroxyl radicals (•OH). At the same time, due to the slow release of nanozymes caused by the encapsulation of hydrogels, the scavenging rate of PEG / SF@MG53@ZnCeO2 is slightly lower than that of the ZnCeO2 group.

[0103] 3.3 Determination of superoxide dismutase (SOD) activity in ZnCeO2 and PEG / SF@MG53@ZnCeO2

[0104] We purchased an SOD activity assay kit to verify the simulated SOD activity of different concentrations of ZnCeO2 nanozymes and different material groups. All steps were performed according to the instructions, and the OD value at 560 nm was finally measured on a microplate reader.

[0105] Test results are as follows Figure 4 As shown in E and F, where... Figure 4 E represents the standard curve and measurement results for the SOD activity assay of nanozymes at different concentrations. Figure 4F is a comparison of the SOD activity measurement results of different material groups. The results show that the concentration gradient nanozymes exhibit good linearity. The SOD activity measurement results of different materials are the same as those of CAT and hydroxyl radicals. The SOD activities of MG53 and SF / PEG hydrogels are lower. At the same time, due to the slow release of nanozymes caused by the encapsulation of hydrogels, the activity of PEG / SF@MG53@ZnCeO2 is slightly lower than that of ZnCeO2 group.

[0106] 4. Investigation into the drug release behavior of ZnCeO2 and PEG / SF@MG53@ZnCeO2

[0107] 4.1 Zn in ZnCeO2 2+ In vitro release behavior investigation

[0108] First, 200 mg of ZnCeO2 nanozyme was soaked in 2 mL of PBS at 37.0℃. At different time points (2, 6, 16, 24, 48, 96, and 128 h), 200 µL of the solution was taken out and the same volume of PBS was added simultaneously. Zn was then determined using an inductively coupled plasma atomic emission spectrometer. 2+ The amount released.

[0109] Figure 5 A represents the concentration of ZnCeO2 in PBS at different time points. 2+ The release curves showed that ZnCeO2 can continuously release Zn in PBS. 2+ And it reaches equilibrium in 100 hours.

[0110] 4.2 Investigation on the in vitro release behavior of ZnCeO2 in PEG / SF@MG53@ZnCeO2

[0111] First, 1 g of PEG / SF@MG53@ZnCeO2 hydrogel powder was immersed in 5 mL of PBS at 37.0℃. At different time points (0, 2, 4, 6, 8, 10, 12, and 14 days), 200 µL of the solution was taken out and the same volume of PBS was added simultaneously. The Ce content in the solution was determined using inductively coupled plasma atomic emission spectrometry.

[0112] Figure 5 B shows the release curves of ZnCeO2 in the hydrogel at different time points in PBS. The results show that ZnCeO2 in the hydrogel is released rapidly in the first 4 days, the release rate slows down significantly after 4 days and tends to stabilize after 6 days.

[0113] 4.3 Investigation on the in vitro release behavior of protein drugs in PEG / SF@MG53@ZnCeO2

[0114] To construct a BSA standard curve: Weigh 100 mg of Coomassie Brilliant Blue G-250 and dissolve it in 50 mL of 95% anhydrous ethanol. Add 100 mL of 85% phosphoric acid and dilute to 1 L with deionized water. Filter, store in the dark, and allow to stabilize for 24 h before use. Prepare BSA standard solutions of 0, 10, 20, 40, 60, 80, and 100 µg / mL. React 1 mL of BSA standard solution with 5 mL of Coomassie Brilliant Blue G-250 solution at a 1:5 ratio for 5 min, and then measure the absorbance at 595 nm using a UV spectrophotometer.

[0115] Prepare a 1500 ug / mL BSA solution. Add 0.1 mL of BSA solution to each 1 mL of gel precursor solution (i.e., the solution before colloid freeze-drying in step S3 of Example 1) to prepare a hydrogel. Immerse the hydrogel in PBS. At different time points (0, 2, 4, 6, 8, 10, 12, and 14 days), take 1 mL of the immersion solution and react it with 5 mL of Coomassie Brilliant Blue solution in the dark for 5 min. Measure the absorbance of the reacted solution using a microplate reader.

[0116] Figure 5 C and D show the release curves of BSA in hydrogels at different time points in PBS. The results show that the rapid release period of BSA is in the first 6 days, after which the release rate slows down significantly, tends to stabilize, and shows a slight decrease. It is speculated that this phenomenon is related to protein degradation.

[0117] V. Application Examples

[0118] The PEG / SF@MG53@ZnCeO2 hydrogel powder prepared in Example 1 was applied to high-originality surface repair for testing, and compared with other materials. The testing methods are as follows:

[0119] Six-week-old male C57BL / 6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were purchased and fed in a hypobaric chamber (simulating an altitude of 5000 m) for one week. Subsequently, the mice were anesthetized, all the hair on their backs was shaved, and a circular full-thickness wound of the same diameter and thickness was formed on the back of the mice using a disposable 5 mm diameter skin biopsy punch.

[0120] Treatment was administered using physiological saline (control group) and different materials (experimental group): 20 µL of physiological saline was dripped onto the wound in the control group, while hydrogel powder was evenly applied to the wound in the hydrogel group. Mice were then placed back into a hypobaric oxygen chamber. To observe the healing process, wound repair was recorded and wound size measured using a digital camera on days 0, 2, 4, 6, 8, 10, 12, and 14 post-surgery. On day 14 of wound treatment, mice were euthanized, and wound tissue was excised and fixed overnight in tissue fixation solution. The tissue was then dehydrated using an automatic dehydrator, embedded in paraffin to prepare specimens, and cut into 5 µm sections for final H&E staining.

[0121] Figure 6 A shows high-quality, original surface healing photographs after treatment with different materials. Figure 6 B shows the H&E staining results of sample sections after treatment with different materials. As can be seen from the figure, PEG / SF@MG53@ZnCeO2 exhibited the best healing-promoting effect. MG53, ZnCeO2, and PEG / SF groups all had some effect on healing of highly original surfaces. The H&E staining results show that, except for the Control group, the remaining experimental groups all had intact epithelial tissue. The PEG / SF@MG53@ZnCeO2 group showed the best degree of epithelial repair and also had the largest granulation tissue area.

[0122] In summary, the PEG / SF@MG53@ZnCeO2 hydrogel powder provided by this invention offers advantages such as the hydrophilic three-dimensional network structure of PEG / SF hydrogel, high biocompatibility, low liquid surface tension, and the existence of osmotic pressure difference for sustained drug release. It effectively solves problems such as protein aggregation, easy inactivation, and difficulty in controlled release during in-situ drug delivery, exhibits good biocompatibility, and allows for the continuous release of ZnCeO2, enabling long-term treatment. The oxygen generated by ZnCeO2 during the scavenging of reactive oxygen species is crucial for cell proliferation and angiogenesis. The ZnCeO2 released in tissues... 2+ It can bind to specific binding sites of MG53 protein, enhancing its activity. Furthermore, PEG / SF@MG53@ZnCeO2 hydrogel powder can continuously release ZnCeO2 nanozymes and MG53 protein into wound tissue, clearing excess ROS and promoting wound tissue repair.

[0123] Compared to traditional hydrogels, hydrogel powder is easier to store and has better environmental adaptability. Its physical cross-linking allows it to rapidly gel upon contact with tissue exudate or blood, effectively sealing the wound. This invention provides a novel method for in-situ delivery of MG53 protein, potentially offering a new dressing and effective protective measure for highly original wound repair. It has significant clinical implications for the management of highly original wounds and can be further extended to the treatment of other refractory wounds caused by bacterial infection, inflammatory imbalance, and tissue damage, including diabetic foot ulcers, pressure ulcers, and venous ulcers.

Claims

1. A method for preparing a hydrogel powder loaded with bimetallic nanozymes and MG53 protein, characterized in that: Using PEG / SF hydrogel as a drug carrier, ZnCeO2 nanozymes and MG53 protein were encapsulated in the hydrogel to prepare hydrogel PEG / SF@MG53@ZnCeO2 loaded with bimetallic nanozymes and MG53 protein. After grinding the hydrogel into powder, PEG / SF@MG53@ZnCeO2 hydrogel powder was obtained.

2. The preparation method according to claim 1, characterized in that: Includes the following steps: S1. Preparation of bimetallic (ZnCeO2) nanozymes: Cerium acetate hexahydrate and zinc sulfate were dissolved in a mixed solvent, mixed and precipitated, the precipitate was centrifuged at high speed, washed with water and dried to obtain ZnCeO2 nanozymes. The mixed solvent was composed of water, acetic acid and ethylene glycol. S2. Preparation of pure silk fibroin (SF) solution: Select silkworm cocoon raw material and mix with sodium carbonate and boil. Stir to remove sericin to obtain degummed silk fibers. Wash the degummed silk fibers to completely remove sericin and sodium carbonate, and then immerse them in lithium bromide solution for water bath. After the water bath is completed, dialyze to remove lithium bromide to obtain SF solution. Preparation of S3 and PEG / SF@MG53@ZnCeO2 hydrogel powder: MG53 protein and ZnCeO2 nanozyme prepared in S1 were first mixed with polyethylene glycol solution, and then mixed evenly with SF solution prepared in S2 to obtain PEG / SF@MG53@ZnCeO2 hydrogel colloid. The colloid was freeze-dried and then ground into powder to obtain PEG / SF@MG53@ZnCeO2 hydrogel powder.

3. The preparation method according to claim 2, characterized in that: In S1, the ratio of cerium acetate hexahydrate to zinc sulfate is 2:

1.

4. The preparation method according to claim 2, characterized in that: S1. Preparation of bimetallic (ZnCeO2) nanozymes: 1.0 g of cerium acetate hexahydrate and 0.5 g of zinc sulfate were dissolved in a mixed solvent prepared by 1 mL of water, 1 mL of acetic acid and 25 mL of ethylene glycol. After stirring evenly, the mixture was stirred in an oil bath at 180 ℃ for 20 min. The precipitate obtained from the reaction was centrifuged at 18000 rpm / min for 10 min. After centrifugation, the precipitate was washed several times with water. After washing, the precipitate was freeze-dried. The dried product was ZnCeO2 nanozyme.

5. The preparation method according to claim 2, characterized in that: S2. Preparation of pure silk fibroin (SF) solution: Select silkworm cocoons as raw materials, cut them into small pieces, mix them with a 0.02 M sodium carbonate solution, boil them, maintain the boiling state for 60 min and continuously stir to remove sericin, and obtain degummed silk fibers. Wash the degummed silk fibers repeatedly to completely remove sericin and sodium carbonate, and then immerse the silk fibers in a 9.3 M lithium bromide solution and water bath at 60℃ for 4 h. After the water bath, dialyze them for 3 days using a standard grade regenerated cellulose dialysis membrane (3500 kD) to remove lithium bromide, and obtain SF solution.

6. The preparation method according to claim 2, characterized in that: Preparation of S3 and PEG / SF@MG53@ZnCeO2 hydrogel powder: MG53 protein and ZnCeO2 nanozyme prepared in S1 were first mixed with 6wt% polyethylene glycol solution to obtain a mixture. Then, the mixture was mixed with an equal volume and mass fraction of SF solution prepared in S2 to obtain PEG / SF@MG53@ZnCeO2 hydrogel colloid. The colloid was freeze-dried and then ground into powder to obtain PEG / SF@MG53@ZnCeO2 hydrogel powder. The particle size of ZnCeO2 nanozyme was 2-8 nm, and the ratio of MG53 protein to ZnCeO2 nanozyme was 0.03:

400.

7. The preparation method according to claim 6, characterized in that: In the mixture, the concentration of ZnCeO2 nanozyme is ρ, where 0 < ρ ≤ 400 μg / mL.

8. The preparation method according to claim 7, characterized in that: The concentration of ZnCeO2 nanozyme in the mixture is 400 μg / mL.

9. A hydrogel powder loaded with bimetallic nanozymes and MG53 protein, characterized in that: It is prepared by the preparation method according to any one of claims 1-8.

10. The application of the hydrogel powder loaded with bimetallic nanozymes and MG53 protein according to claim 9 in the field of high-originality surface repair.

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