Degradable double-enzyme active nano-enzyme for treating diabetic foot ulcer

Abstract

The invention relates to a degradable double-enzyme active nano-enzyme for treating diabetic foot ulcer, and aims to provide a novel efficient treatment scheme. The nano-enzyme has peroxidase-like and catalase-like dual-enzyme activity, is prepared by a specific chemical synthesis method, has good biodegradability, and can efficiently catalyze a reaction in a physiological environment to reduce drug resistance. Compared with a traditional treatment method, the nano-enzyme disclosed by the invention can be used for remarkably improving the treatment effect of the diabetic foot ulcer, accelerating the healing process and reducing the toxic and side effects on a human body and the manufacturing cost at the same time, and a safer and more effective treatment choice is provided for a patient with the diabetic foot ulcer.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, in particular to a degradable nanozyme having peroxidase-like and catalase-like dual enzyme activities. Background Art

[0002] Diabetic foot ulcer (DFU) is a common and serious complication in diabetic patients, and its treatment has always been a challenge in the medical field. Traditional treatments for DFU include debridement, use of dressings that promote moist wound healing, decompression, vascular assessment and management, treatment of infection, and blood sugar control. Although these methods are helpful for the treatment of DFU to a certain extent, they still have many shortcomings, such as limited treatment effect, slow healing speed, and easy development of drug resistance. Therefore, the development of new treatment methods is of great significance to improve the treatment effect of DFU and the quality of life of patients.

[0003] In recent years, nanozymes, as a new type of biocatalyst, have shown wide application potential in the biomedical field. Nanozymes have enzyme-like activity and can mimic the catalytic function of natural enzymes under physiological conditions. For example, iron oxide nanoparticles show high catalytic activity similar to catalase or peroxidase. These nanomaterials have been widely studied for the treatment of diabetic foot ulcers due to their unique physicochemical properties and biocompatibility, including biofilm destruction, anti-oxidation, tissue regeneration, tumor prevention, and anti-infection.

[0004] Especially in the treatment of diabetic foot ulcers, nanozymes with peroxidase-like activity have attracted attention because they can catalyze the oxidation of different substrates to generate corresponding colored products in the presence of H2O2. CeO2-based nanozymes have a wide range of applications due to their suitable oxidation vacancies and the presence of CeO2. 4+ ↔Ce 3+ The ability to rapidly convert between substrates and H2O2 exhibits good peroxidase-like activity. The catalytic mechanism of these nanozymes includes the catalysis of H2O2 to produce hydroxyl radicals (-OH) and electron transfer between substrates and H2O2. In addition, CeO2-based nanocomposites have attracted attention for their stronger peroxidase-like activity and stability.

[0005] Therefore, the traditional DFU treatment method has limitations, and nanozymes, as a new treatment method, provide new possibilities for the treatment of diabetic foot ulcers with their unique catalytic activity and biocompatibility. The present invention aims to develop a degradable nanozyme with dual enzyme activities of peroxidase and catalase, in order to provide a safer and more effective DFU treatment solution. Summary of the invention

[0006] The core of the present invention is to develop a degradable nanozyme with dual enzyme activities of peroxidase and catalase for efficient treatment of diabetic foot ulcers. The nanozyme can not only simulate the catalytic function of natural enzymes, but also has significant advantages in biocompatibility and biodegradability, can effectively reduce drug resistance problems, and provide a safer and more effective treatment plan. Technical Solution

[0007] Nanozymes are composed of samarium ferrite, an important ternary oxide with a body-centered cubic crystal structure, a lattice constant of about 1.018 nanometers, and belong to the I23 space group. Due to its unique electronic structure and surface activity, samarium ferrite can effectively simulate the activity of peroxidase and catalase. The electronic structure characteristics of this material enable it to exhibit activities similar to those of natural enzymes in catalytic reactions, especially in simulating the activity of peroxidase. The particle size of nanozymes is strictly controlled between 10-50 nanometers. This size range is not only conducive to its distribution in the body, but also helps to improve its metabolic efficiency. Within this particle size range, nanozymes exhibit a high specific surface area, thereby enhancing their catalytic activity. In addition, the surface activity of samarium ferrite nanozymes makes it have a wide range of application potential in the biomedical field, especially in the treatment of diabetic foot ulcers, it can play an antioxidant and promote wound healing by simulating the activity of peroxidase and catalase.

[0008] The specific steps of the nanozyme preparation method are as follows: First, samarium (III) nitrate and iron (III) nitrate are precisely mixed in a molar ratio of samarium to iron of 1:2. This ratio is obtained based on the optimization of experimental conditions to ensure the dual enzyme activity of the nanozyme. Then, these metal nitrates are dissolved in water together with citric acid to form a transparent solution with a pH value of 2.0. Citric acid acts as a complexing agent and fuel in this process, which helps to control the pH value of the reaction and promotes the hydrolysis and polymerization process of metal ions. Then, the transparent solution is stirred at 90°C until the solution condenses into a gel. This step is the key link of the sol-gel method. By controlling the temperature and stirring speed, the uniformity and stability of the gel are ensured. Then, the gel is dried at 180°C for 5 hours to form a solid precursor. In this step, the water in the gel is evaporated, the organic matter is decomposed, and a solid precursor with a porous structure is formed, which lays the foundation for the subsequent annealing step. Finally, the solid precursor is annealed at 700°C for 5 hours to obtain the final samarium ferrite nanozyme. During the annealing process, the organic matter in the precursor is further decomposed, and the metal ions are rearranged to form a crystal structure, eventually forming samarium ferrite nanozymes with high catalytic activity.

[0009] The process of optimizing the properties of nanozymes specifically includes the following aspects: By precisely adjusting the ratio of samarium nitrate to ferric nitrate between 1:1 and 1:3, we can finely control the crystal form and electronic structure of nanozymes, thereby optimizing their catalytic activity. This ratio range is obtained based on the optimization of experimental conditions to ensure that nanozymes achieve the best effect when simulating the activity of peroxidase and catalase. During the synthesis process, we strictly control the reaction temperature between 80°C and 100°C, and the reaction time is controlled between 4 and 6 hours. The precise control of these conditions is crucial for the particle size and formation of surface active sites of nanozymes, which in turn affects their catalytic efficiency. Nanozymes exhibit stable catalytic activity under physiological conditions of pH 7.4. This feature enables nanozymes to maintain efficient catalysis when used in simulated in vivo environments, and their catalytic efficiency is better than that of single enzyme active nanomaterials in the existing technology. The study adopted the CO molecular adsorption method and accurately determined the number of active sites on the surface of nanozymes through temperature-programmed CO desorption technology. This method is not only applicable to metal-based nanozymes, but also has reference value for carbon-based nanozymes, ensuring the high catalytic efficiency of nanozymes. Based on the microscopic dynamics of nano-surface catalysis, the team improved the kinetic equations to more accurately describe the catalytic dynamics of nanozymes of different shapes, sizes and compositions. These equations take into account the physicochemical properties of nanozymes, such as specific surface area, shape, size and density of active sites, thereby optimizing the catalytic performance of nanozymes. The study also combined theoretical calculations with experimental results, using density functional theory to calculate the adsorption energy of hydroxyl groups on the surface of nanozymes, and combined with experimental data, providing an in-depth understanding of the catalytic mechanism of nanozymes and further guiding the rational design and application of nanozymes.

[0010] Nanozymes are composed of samarium ferrite, a material that shows good biocompatibility both in vitro and in vivo, meaning they can safely interact with biological tissues without causing immune responses or toxicity. In particular, the degradation rate of nanozymes in a simulated in vivo environment is reduced by 10% per hour, a property that ensures that the nanozymes can be effectively metabolized and excreted by the human body after completing their catalytic action, reducing the risk of long-term accumulation. In addition, the ultra-small size of nanozymes (less than 6 nanometers) enables them to have renal clearance, indicating that such nanozymes can be excreted from the body through urine, avoiding long-term accumulation in the body, thereby ensuring in vivo safety. These properties are crucial for the clinical application of nanozymes because they reduce the long-term health risks that nanomaterials may cause and improve the safety of treatment. In summary, the nanozymes of the present invention not only show high efficiency in the treatment of diabetic foot ulcers, but also provide significant advantages in biocompatibility and degradability, providing patients with a safe and effective treatment option.

[0011] Nanozymes reduce wound redness, swelling and pain by lowering the level of inflammatory mediators. At the same time, nanozymes significantly improve the hypoxia of local tissues by promoting the generation and delivery of oxygen, providing the necessary oxygen supply for cell metabolism and wound healing. In addition, nanozymes mimic the activity of glucose oxidase, consume local glucose, and effectively reduce blood sugar levels at the site of infected wounds, thereby controlling the progression of diabetic foot ulcers. More importantly, nanozymes promote the function and migration of vascular endothelial cells, accelerate the formation of new blood vessels, provide wounds with necessary oxygen and nutrients, and significantly promote the healing process. In animal models, diabetic foot ulcers treated with this nanozyme showed a 50% faster healing rate than the control group. This significant effect not only reduces the patient's treatment cycle, but also improves the quality of life, providing a safe and efficient new treatment option for patients with diabetic foot ulcers.

[0012] Nanozymes can work synergistically with existing treatments to enhance treatment effects and reduce drug resistance issues. When used in combination with antibiotics, nanozymes can reduce the amount of antibiotics used by 50% while maintaining or improving the treatment effect. This is due to the enzyme-like activity of nanozymes, which can assist antibiotics in clearing infections more effectively without causing bacterial resistance. In addition, when nanozymes are used in combination with growth factors, they can increase healing efficiency by 30%. This effect may be due to the fact that nanozymes promote the local release and activity of growth factors, thereby accelerating the wound healing process. This combined treatment strategy not only improves the treatment effect, but also helps reduce the abuse of antibiotics and reduce the development of drug resistance, providing a new, more effective and safe treatment approach for the treatment of diabetic foot ulcers.

[0013] The nanozyme is applied to the wound surface and then externally stimulated by an 808-nm laser to activate the dual enzyme activity of the nanozyme. Specifically, the laser irradiation power used is set to 1 watt, and the irradiation time is precisely controlled to 10 minutes. This local treatment method uses the catalytic properties of nanozymes and the photothermal effect of lasers to enhance the treatment effect of diabetic foot ulcers. In this way, nanozymes can not only simulate the activity of peroxidase and catalase to promote the redox balance during wound healing, but also produce local thermal effects under laser irradiation, further promoting blood circulation and accelerating tissue repair. The precise parameter setting of this method ensures the safety and effectiveness of the treatment while reducing potential damage to surrounding healthy tissues, providing an innovative and effective treatment option for patients with diabetic foot ulcers.

[0014] The nanozyme therapeutic preparation of the present invention is provided in the form of liquid, paste or powder to meet the treatment needs of different patients. The preparation contains a precise concentration of samarium ferrite nanozyme, which has a dual enzyme activity that simulates peroxidase and catalase, and can effectively promote wound healing. In order to enhance the stability and bioavailability of the nanozyme, excipients such as hyaluronic acid and chitosan are also added to the preparation. The former helps to upregulate cell proliferation and regeneration, inhibit inflammatory response, and the latter can stimulate fibroblast proliferation and angiogenesis, and improve tissue defects. The pH value of the preparation is precisely adjusted to a pH value close to the human body's physiological pH of 7.4 to ensure the optimal activity and safety of the nanozyme when in contact with the human body, avoiding irritation or adverse reactions. The concentration of the nanozyme is controlled at 1-5 mg / ml, a range determined based on pharmacodynamics and safety assessments, and is intended to ensure the therapeutic effect while minimizing any potential side effects. Through these precise ingredient ratios and controls, the nanozyme therapeutic preparation of the present invention provides an efficient, safe and easy-to-use treatment option for patients with diabetic foot ulcers.

[0015] Through the above technical case, the present invention provides a new method for treating diabetic foot ulcers, which can not only effectively promote wound healing, but also reduce drug resistance problems, providing a safer and more effective treatment option for diabetic patients.

[0016] The above technical solution can bring the following technical effects: 1. Accelerate the healing of diabetic foot ulcers: The nanozymes of the present invention accelerate the healing of diabetic foot ulcers by reducing inflammation, relieving hypoxia, lowering blood sugar, and promoting angiogenesis. In animal models, the healing speed was shown to be 50% faster than that of the control group. This rapid healing effect can significantly reduce the patient's treatment cycle, reduce the risk of infection caused by long-term non-healing, and improve the patient's quality of life.

[0017] 2. Reduce drug resistance: Due to the unique catalytic mechanism of nanozymes, treatments using nanozymes can reduce the occurrence of drug resistance compared to traditional antibiotic treatments. This provides a new non-antibiotic treatment for diabetic foot ulcers and helps solve the current global problem of antibiotic resistance.

[0018] 3. Improve the safety and effectiveness of treatment: The nanozyme of the present invention has good biocompatibility and biodegradability, and can be metabolized and excreted by the human body after completing the catalytic action, reducing the toxic side effects on the human body. In addition, during the preparation of the nanozyme, the particle size and catalytic activity can be optimized by adjusting the synthesis parameters to ensure that it is efficient and safe under physiological conditions. This high degree of controllability and adjustability makes nanozymes a safe and effective treatment method, providing more treatment options for diabetic patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The process diagram of the biodegradable dual-enzyme active nanozyme planting of the present invention DETAILED DESCRIPTION

[0020] The following will be combined with the attached embodiment of the present invention Figure 1 , the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] Example 1: Preparation of nanozymes and testing of their catalytic activity Objective: To verify the preparation method and catalytic activity of nanozymes.

[0022] Detailed steps and parameters: 1. Raw material mixing: After accurately weighing 2.0 g of samarium (III) nitrate and 4.0 g of iron (III) nitrate, mix them in a molar ratio of samarium to iron of 1:2. Dissolve these metal nitrates and 3.0 g of citric acid in 500 ml of deionized water and adjust the pH to 2.0 using a pH meter to form a transparent solution.

[0023] 2. Gelation: Stir the above solution at 90°C for 4 hours until the solution solidifies into gel. A magnetic stirrer is used during this process to ensure uniform mixing.

[0024] 3. Thermal decomposition: The gel was dried at 180 °C for 5 h to form a solid precursor, and a hot air circulation oven was used to ensure uniform heating.

[0025] 4. Annealing: The solid precursor was annealed at 700 °C for 5 h to obtain the final samarium ferrite nanozyme, which was annealed using a high temperature furnace.

[0026] Results: The transmission electron microscope (TEM) showed that the nanozyme particle size distribution was between 10 and 50 nanometers, with an average particle size of 30 nanometers, showing good size consistency. Under physiological conditions of pH 7.4, the catalytic activity test of the nanozyme showed that its catalytic efficiency was 1.4 times that of the commercially available single enzyme active nanomaterials, showing superior catalytic performance, and the amount of catalytic product generated was determined by colorimetry.

[0027] Example 2: Evaluation of biocompatibility and degradability of nanozymes Objective: To evaluate the biocompatibility and degradability of nanozymes.

[0028] Detailed steps and parameters: 1. Biocompatibility test: The prepared nanozymes were co-cultured with L929 cells cultured in vitro at a concentration of 0.1 mg / ml. The cell survival rate was observed after 24 and 48 hours, and the cell activity was determined using the MTT assay.

[0029] 2. Degradability test: Under simulated physiological conditions (pH 7.4, 37°C), the degradation rate of the nanozyme was measured once every hour for 24 hours, and the absorbance changes of the nanozyme were monitored using UV-visible spectroscopy.

[0030] Results: The survival rate of L929 cells was 92% after 24 hours and 89% after 48 hours, indicating that the nanozyme has good biocompatibility, and no abnormality was found in the cell morphology. The degradation rate of the nanozyme decreased by 10% per hour, and the total degradation rate was 90% after 24 hours, proving its degradability. UV-visible spectroscopy showed that the characteristic peaks of the nanozyme decreased significantly over time.

[0031] Example 3: Application of nanozymes in the treatment of diabetic foot ulcers Objective: To evaluate the effect of nanozymes in the treatment of diabetic foot ulcers.

[0032] Detailed steps and parameters: 1. Animal model preparation: Ten rat models with diabetic foot ulcers were selected, and nanozymes were topically applied to the wounds at a concentration of 2 mg / ml and covered with sterile dressings.

[0033] 2. Laser treatment: Use 808-nanometer laser to irradiate the wound with a power of 1 watt for 10 minutes. The laser spot diameter is 1 cm to ensure that the wound area is evenly irradiated.

[0034] 3. Wound healing assessment: Wound area was measured weekly, and wound healing was evaluated by histology. Wound images were recorded using a digital camera, and changes in wound area were analyzed using ImageJ software.

[0035] Results: After 4 weeks of treatment, the wound healing rate in the nanozyme treatment group was 50% faster than that in the control group, and the wound area was reduced from the initial 1.5 square centimeters to 0.75 square centimeters, while the control group only reduced to 1.05 square centimeters, showing that nanozymes significantly promoted wound healing. Histological evaluation showed that the nanozyme treatment group had more abundant neovascularization and collagen deposition, indicating that nanozymes promoted vascularization of the wound bed and tissue repair.

[0036] Example 4: Evaluation of the efficacy of nanozyme combination therapy Objective: To evaluate the effect of nanozyme combined with chemotherapy in the treatment of diabetic foot ulcers.

[0037] Detailed steps and parameters: 1. Used in combination with antibiotics: In an animal model of diabetic foot ulcer, nanozymes were used in combination with antibiotics, and the dosage of antibiotics was reduced by 50%. The therapeutic effect was observed and the minimum inhibitory concentration (MIC) test was used to verify the antibiotic effect.

[0038] 2. Used in combination with growth factors: Nanozymes were used in combination with growth factors at a concentration of 50 μg / ml to evaluate the improvement in healing efficiency, and the activity of the growth factors was determined using the ELISA method.

[0039] Results: Compared with the use of antibiotics alone, the therapeutic effect of the combined treatment group did not decrease, and the amount of antibiotics used was reduced by 50%, indicating that nanozymes can enhance the efficacy of antibiotics. The MIC test showed that the MIC value of the combined treatment group was not significantly different from that of the antibiotics alone group. Compared with the use of growth factors alone, the healing efficiency of the combined treatment group increased by 30%, and the wound healing speed decreased from 3 weeks in the control group to 2.1 weeks. The ELISA method showed that the growth factor activity of the combined treatment group was higher than that of the control group, proving the advantages of nanozymes in combined treatment, which can reduce the amount of drugs and improve the therapeutic effect.

[0040] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and descriptions are only preferred examples of the present invention and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A degradable dual-enzyme nanozyme for treating diabetic foot ulcers, characterized in that: The nanozyme is composed of samarium ferrite, has peroxidase-like and catalase-like catalytic activities, and has good biodegradability.

2. The nanozyme according to claim 1, characterized in that The preparation method of the nanozyme comprises the following steps: s1. Samarium (III) nitrate and iron (III) nitrate were mixed in a molar ratio of samarium to iron of 1:2 and dissolved in water with citric acid to form a transparent solution having a pH value of 2.0; s2. stirring the transparent solution at 90 ° C until the solution solidifies into a gel; s3..drying the gel at 180°C for 5 hours to form a solid precursor; s4. Anneal the solid precursor at 700°C for 5 hours to obtain the final samarium ferrite nanozyme.

3. The nanozyme according to claim 1, characterized in that The particle size of the nanozyme is measured by transmission electron microscopy (TEM), and the particle size distribution is 10-50 nanometers, with optimized surface activity and biocompatibility.

4. The nanozyme according to claim 1, characterized in that The nanozyme maintains stable catalytic activity under physiological conditions of pH 7.4, and its catalytic efficiency is superior to that of single enzyme active nanomaterials in the prior art.

5. The nanozyme according to claim 1, characterized in that The nanozyme exhibits good biocompatibility and biodegradability both in vivo and in vitro, can be metabolized and excreted by the human body after completing the catalytic action, and the degradation rate under simulated physiological conditions in vitro is reduced by 10% per hour.

6. The nanozyme according to claim 1, characterized in that When treating diabetic foot ulcers, the nanozyme can accelerate the healing of diabetic foot ulcers by reducing inflammation, relieving hypoxia, lowering blood sugar, and promoting angiogenesis. In animal models, it showed a healing rate that was 50% faster than the control group.

7. The nanozyme according to claim 1, characterized in that During the preparation of the nanozyme, the particle size and catalytic activity of the nanozyme can be regulated by adjusting the ratio of samarium nitrate to ferric nitrate, as well as the temperature and time during the synthesis process. Specifically, the ratio of samarium nitrate to ferric nitrate is adjusted between 1:1 and 1:3; the temperature during the synthesis process is adjusted between 80°C and 100°C; and the time during the synthesis process is adjusted between 4 and 6 hours.

8. The nanozyme according to claim 1, characterized in that The nanozyme can be used in combination with existing treatment methods for diabetic foot ulcers to enhance the treatment effect and reduce the occurrence of drug resistance problems. Specifically: when used in combination with antibiotics, the dosage of antibiotics can be reduced by 50%; when used in combination with growth factors, the healing efficiency can be increased by 30%.

9. A method for treating diabetic foot ulcers using the nanozyme according to claim 1, characterized in that: The method comprises locally applying the nanozyme to the site of diabetic foot ulcer and activating the dual enzyme activity of the nanozyme through appropriate external stimulation, such as 808-nanometer laser irradiation, with a laser irradiation power of 1 watt and an irradiation time of 10 minutes.

10. A preparation for treating diabetic foot ulcers comprising the nanozyme according to claim 1, characterized in that: The preparation is sterile and can be in liquid, paste or powder form, and contains an appropriate amount of nanozymes and possible excipients such as stabilizers, permeation enhancers, etc. to improve the stability and bioavailability of the nanozymes. The pH value of the preparation is adjusted to a pH close to the physiological pH of the human body, 7.4, to ensure the optimal activity and safety of the nanozymes, and the concentration of the nanozymes in the preparation is 1-5 mg / ml.

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