A recombinant natural enzyme nanogel as well as a preparation method and application thereof

Abstract

The present application belongs to the field of diabetic foot wound treatment, and particularly relates to a recombinant natural enzyme nanogel, a preparation method and application thereof. The recombinant natural enzyme nanogel comprises a recombinant natural nanoenzyme, a carrier material, a gel matrix, a humectant and a buffer, and has a good clinical effect on promoting the healing of a diabetic foot wound. The recombinant natural enzyme nanogel, the preparation method and the application thereof have the advantages that the remodeling of a microenvironment of a diabetic foot wound and tissue regeneration can be realized through an intelligent cascade reaction of a recombinant natural enzyme, and the recombinant natural enzyme nanogel is a new treatment method which is non-invasive, simple, safe and highly compliant, and is expected to become an auxiliary treatment method for the healing of a diabetic foot wound in the future, and is also helpful to tissue regeneration and rapid healing of a foot ulcer patient.

Description

Technical Field

[0001] This invention belongs to the field of diabetic foot wound treatment, specifically relating to a recombinant natural enzyme nanogel, its preparation method, and its application. Background Technology

[0002] Diabetic wounds are a complication of diabetes, characterized by delayed healing or persistent failure to heal. They often ulcerate due to bacterial infection or other complications, posing a risk of amputation. Diabetic wounds are chronic wounds that are difficult to heal and have a high recurrence rate. The difficulty in healing diabetic wounds is closely related to increased advanced glycation end products (AGEs), long-term chronic inflammation, and peripheral nerve dysfunction.

[0003] The mechanisms underlying diabetic wounds are complex and have not yet been fully elucidated.

[0004] ① Advanced Glycation End Products (AGEs): Long-term hyperglycemia leads to non-enzymatic glycation of proteins and lipids, forming AGEs, which inhibit angiogenesis and exacerbate inflammatory responses. A chronically high-glycemic environment causes arteriosclerosis and sluggish nerve conduction, resulting in ischemia and necrosis of distal tissues. Sensation of pain and temperature is significantly reduced. High blood glucose levels hinder the phagocytic ability of macrophages to clear apoptotic neutrophils, thus creating a persistent pro-inflammatory state.

[0005] ② Hypoxic microenvironment: Poor tissue perfusion in wounds directly causes local tissue ischemia, hypoxia, and reperfusion injury. Studies have found that, compared with normally healed wounds, the variation range of tissue oxygen partial pressure in diabetic wounds is 5–15 mmHg, while that in normally healed wounds is 30–50 mmHg; diabetic wounds are in a "hypoxic" state, indicating that improving local oxygen supply and utilization in chronic wounds is also key to the treatment of diabetic wounds.

[0006] ③ Oxidative damage from reactive oxygen species (ROS): During normal wound healing, reactive oxygen species play a positive role in sterilization, regulation of cytokine expression, and regulation of cell proliferation. At the same time, they are an unavoidable byproduct of cellular oxidative phosphorylation. In the early stages of chronic wounds, the concentration of reactive oxygen species increases, but the activity of antioxidant enzymes does not increase. The local oxidative and antioxidant systems are out of balance. Excessive levels of reactive oxygen species can damage components such as proteins, nucleic acids, and lipids, thereby negatively impacting wound healing.

[0007] ④ Disruption of the microbial environment: These microorganisms compete with the granulation tissue and other repair tissues of the ulcer for nutrients and consume oxygen, making the already difficult-to-heal ulcer wound even more difficult to heal. At the same time, the consumption of oxygen provides a better growth environment for anaerobic bacteria, allowing them to multiply rapidly. However, the widespread overuse of antibiotics in recent years has led to antibiotic resistance and tolerance in bacteria, resulting in more drug-resistant bacteria.

[0008] Traditional treatments (such as debridement, antibiotic dressings, and hyperbaric oxygen therapy) have limited effectiveness, necessitating novel treatment methods to improve the wound microenvironment and promote healing. While existing medical dressings (such as chitosan-based dressings) provide a physical barrier and some antibacterial effects, they cannot regulate core pathological factors such as high glucose, hypoxia, and oxidative stress. Currently, no similar blood glucose-lowering hydrogels for wounds are available on the market; most wound hydrogels primarily focus on moisturizing and anti-infection effects, lacking products that lower blood glucose and eliminate oxidative stress products.

[0009] Existing patent CN119454920A discloses a composite enzyme nanogel, its preparation method and application, but its stability is generally poor and it cannot be used directly as a complete formulation. Summary of the Invention

[0010] The purpose of this invention is to overcome the shortcomings of existing clinical treatment technologies and to provide a recombinant natural enzyme nanogel, its preparation method and application, which solves the problems of slow healing, poor anti-infection and tissue regeneration effects of diabetic foot wounds in the above-mentioned background technology.

[0011] The technical solution adopted by this invention to solve its technical problem is:

[0012] A recombinant natural enzyme nanogel, comprising, by weight, the following components:

[0013]

[0014]

[0015] The recombinant natural enzyme includes at least one of glucose oxidase, superoxide dismutase, catalase, or horseradish peroxidase.

[0016] The carrier material includes one or more of polyethylene glycol-40 stearate, polyethylene glycol-40 hydrogenated castor oil, polyethylene glycol-100 glyceryl stearate, polyethylene glycol-6 caprylic / caprylic glycerate, polyglycerol-3 stearate, or polyglycerol-4 diisostearate.

[0017] The mass ratio of the recombinant natural enzyme to the carrier material is 0.5:2-20; wherein, the mass ratio of the recombinant natural enzyme to polyethylene glycol-40 stearate is 0.5:2-20; the mass ratio of the recombinant natural enzyme to polyethylene glycol-40 hydrogenated castor oil is 0.5:0.5-15; the mass ratio of the recombinant natural enzyme to polyethylene glycol-100 glyceryl stearate is 0.5:0.5-10; the mass ratio of the recombinant natural enzyme to polyethylene glycol-6 caprylic / caprylic acid glycerate is 0.5:2-20; the mass ratio of the recombinant natural enzyme to polyglycerol-3 stearate is 0.5:2-10; and the mass ratio of the recombinant natural enzyme to polyglycerol-4 diisostearate is 0.5:0.5-20.

[0018] The gel matrix includes acrylic polymers or cellulose derivatives.

[0019] The moisturizer is one or more of glycerin, propylene glycol, hyaluronic acid, and sorbitol.

[0020] The buffer is at least one of boric acid, citric acid, borax, sodium citrate, and sodium dihydrogen phosphate.

[0021] A method for preparing recombinant natural enzyme nanogels includes the following steps:

[0022] Step 1: Add the gel matrix to water for injection, heat to prepare a slurry, and cool for later use;

[0023] Step 2: Dissolve or disperse the carrier material in water for injection, stir until homogeneous, and then add the recombinant natural enzyme while stirring to dissolve it.

[0024] Step 3: Add the buffer to the water for injection and stir until dissolved, then add the humectant and stir until dissolved;

[0025] Step 4: Add the solution from Step 2 to the solution from Step 1 and mix well. Then add the solution from Step 3, adjust the pH value, add water for injection, and continue stirring until uniform. Filter with a microporous membrane to obtain recombinant natural enzyme nanogel.

[0026] Recombinase is a protein with poor stability. Encapsulating it in a carrier material forms a protective layer on the surface of the recombinase, thus preventing it from coming into contact with other materials (including air) and stabilizing it. During the slow dissociation of the carrier material, the recombinase is continuously and slowly released, thereby achieving a long-term therapeutic effect.

[0027] The heating temperature in step one is 60-80℃.

[0028] In step four, adjust the pH value to 5.0–6.5.

[0029] A recombinant natural enzyme nanogel is applied to accelerate the healing of diabetic foot wounds. Furthermore, the recombinant natural enzyme nanogel GAT@F of this invention can be used as an adjunct therapy for the healing of diabetic foot wounds, downregulating local blood glucose levels and oxidative stress in the wound tissue, reducing inflammatory infiltration, promoting cell proliferation and migration, and regenerating granulation tissue, thus facilitating the acceleration of diabetic wound healing.

[0030] The recombinant natural enzyme nanogel provided by this invention can reshape the microenvironment and regenerate tissue in diabetic foot wounds through the intelligent cascade reaction of recombinant natural enzymes. It is a novel treatment method that is non-invasive, simple, safe, and has high compliance. It is expected to become an adjunctive treatment method for the healing of diabetic foot wounds in the future, and it can also help patients with foot ulcers to regenerate tissue and heal quickly.

[0031] The present invention has the following advantages:

[0032] 1. The recombinant natural enzyme nanogel of the present invention overcomes the shortcomings of the prior art, such as easy enzyme leakage, lack of continuous oxygen supply, easy secondary infection, and inability to match the dynamic changes of the wound microenvironment. Through the spatial proximity effect of recombinant natural enzyme, gel carrier optimization and dynamic circulation oxygen supply mechanism, the problems of poor gaseous oxygen permeability and single function of traditional enzyme products are systematically solved.

[0033] 2. The recombinant natural enzyme nanogel formulation of the present invention overcomes the limitations of traditional enzyme formulations. The sustained-release properties of the carrier material combined with the gel structure give the recombinant natural enzyme a longer half-life, which can continuously and effectively release the recombinant natural enzyme, greatly reducing the frequency of administration, reducing the burden on patients, and increasing medication compliance.

[0034] 3. The preparation process of this invention is relatively simple and the preparation cost is low, which facilitates industrial production. Moreover, compared with the existing patents in the prior art, this invention has undergone further clinical testing and has better results. Attached Figure Description

[0035] Figure 1 The results show the reduction in local tissue blood glucose levels in Example 2 and Comparative Example 2.

[0036] Figure 2 This is the treatment result for granulation bed coverage in Example 2. Detailed Implementation

[0037] To enable those skilled in the art to better understand the present invention, 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 should fall within the scope of protection of the present invention. The present invention uses recombinant natural enzymes (GOD and CAT) purchased from the market (Hunan Yuanchuang Juhe Pharmaceutical Co., Ltd.) as the main components to formulate a recombinant natural enzyme nanogel preparation, and conducts clinical trials using this preparation to evaluate its efficacy and safety.

[0038] The carrier materials used in the following examples are polyethylene glycol-40 stearate, polyethylene glycol-40 hydrogenated castor oil, polyethylene glycol-100 glyceryl stearate, polyethylene glycol-6 caprylic / caprylic glycerate, polyglycerol-3 stearate, and polyglycerol-4 diisostearate.

[0039] [Enzyme Activity Test]

[0040] To test the activity of the recombinant natural enzyme in the above-mentioned carrier material, the titers of GOD and CAT in the recombinant natural enzyme were determined by spectrophotometry and ultraviolet spectrophotometry, respectively.

[0041] GOD titer assay: Take 10 mL of recombinant natural enzyme nanogel, centrifuge at low temperature (2000 RCF) for 20 min, dilute 20 times with purified water, then add 1 μmol each of PBS solution (pH 7.4), phenol, 4-aminoantipyrine, peroxidase (POD) and glucose substrate, mix for 10 min, measure absorbance at 500 nm wavelength, and calculate enzyme titer according to the following formula.

[0042] formula:

[0043] Among them, A 500 12.9 is the millimolecular extinction coefficient of quinone imine (L·mmol). -1 ·cm -1 ), where 0.5 is the molar ratio of H2O2 to the colorimetric reagent.

[0044] CAT potency assay: Take 10 mL of recombinant natural enzyme nanogel, centrifuge at low temperature (2000 RCF) for 20 min, dilute 20 times with purified water, then add 0.1 mol / L, measure the absorbance at 240 nm wavelength, and calculate the enzyme potency according to the following formula.

[0045] formula:

[0046] Among them, A240 The absorbance change rate is 0.1, which is the defined unit (absorbance decrease of 0.1 = 1U), and W is the sample mass (g).

[0047] Example 1

[0048] The ingredients are as follows: 5 parts recombinant natural enzyme, 10 parts carrier material (polyethylene glycol-40 stearate), 15 parts carbomer (gel matrix), 2 parts propylene glycol (humectant), and 0.5 parts sodium citrate (buffer).

[0049] Preparation method:

[0050] Step 1: Add the gel matrix to water for injection, heat to 60-80℃ to prepare a slurry, and cool for later use;

[0051] Step 2: Dissolve or disperse the carrier material in water for injection, stir until homogeneous, and then add the recombinant natural enzyme while stirring to dissolve it.

[0052] Step 3: Add the buffer to the water for injection and stir until dissolved, then add the humectant and stir until dissolved;

[0053] Step 4: Add the solution from Step 2 to the solution from Step 1 and mix well. Then add the solution from Step 3 and adjust the pH to 5.0–6.5. Add water for injection and continue stirring until homogeneous. Filter through a 0.22 μm microporous membrane to obtain recombinant natural enzyme nanogel.

[0054] Examples 2-6

[0055] Examples 2-6 are identical to Example 1 in all other respects, except for the carrier, as detailed in Table 1 below:

[0056] Table 1. Ratios of recombinant natural enzymes to carrier materials in Examples 1-6

[0057]

[0058] Comparative Example 1

[0059] Recombinant natural enzyme nanogels without added carrier materials.

[0060] Experimental results:

[0061] The enzyme activity test results are shown in the table below.

[0062] Table 2. Results of GOD activity assay (unit: U / mL)

[0063]

[0064]

[0065] Table 3. Results of CAT activity assay (unit: U / g)

[0066]

[0067] As shown in Tables 2 and 3, the enzyme activity of Example 2 was the strongest. Therefore, Example 2 was selected for sample preparation and clinical efficacy and safety studies were conducted.

[0068] Comparative Example 2: Commercially available chitosan gel; the blank group was a gel without recombinant natural enzymes.

[0069] Clinical trial protocol

[0070] Granulation coverage was the primary clinical endpoint, analyzed at visit 4 after the last treatment for each patient. Similarly, wound healing rate, a secondary clinical endpoint, was analyzed using the same procedure, with scoring across multiple dimensions including the Wlfl grading scale (wound depth, infection, ischemia) and the IDSA / IWGDF grading scale (wound infection severity). At each visit, the physician determined the wound size and took photographs with a ruler, which were then evaluated in a blinded manner by a third party. Retrospective wound size measurements were cross-checked with visit records. Data from the blinded evaluations were analyzed by a third-party statistician. Safety follow-up was conducted on days 30 and 60 after the last treatment, collecting safety information.

[0071] Number of cases: 49 wounds from 43 patients. After randomization, 20 wounds from 18 patients were assigned to the GAT@F group, 20 wounds from 17 patients were assigned to the medical chitosan group, and 9 wounds from 8 patients were assigned to the blank hydrogel group. The statistical results of the experiment are shown in Table 4.

[0072] Table 4. Statistical results of clinical trial data

[0073]

[0074] a 18 cases were treated with GAT@F hydrogel dressings, 17 cases with medical chitosan hydrogel dressings, and 8 cases with blank hydrogel dressings.

[0075] b Sixteen cases were treated with GAT@F hydrogel dressings, 16 cases with medical chitosan hydrogel dressings, and 8 cases with blank hydrogel dressings.

[0076] A p-value < 0.05 is considered statistically significant.

[0077] Chi-square test Mann-Whitney U test

[0078] Blood glucose levels in local tissues after treatment in both Example 2 and Comparative Example 2 were measured using a blood glucose meter. Results are shown below. Figure 1 Furthermore, the coverage of the granulation bed in the local tissue was observed, and the results are shown in […]. Figure 2 The results showed that the wound healing rate of Example 2 was 42.36±25.85%, the wound healing rate of Comparative Example 2 was 38.37±21.63%, and the wound healing rate of the blank control group was 12.66±4.10%. In addition, the granulation tissue coverage of Example 2 was increased by 7.22±16.08%, which was significantly higher than that of Comparative Example 2, proving that the therapeutic efficacy of the recombinant natural enzyme nanogel of the present invention is superior to that of commercially available products.

[0079] More importantly, no serious adverse events (SAEs) occurred in the treatment group of Example 2, while there were 2 SAEs in Comparative Example 2.

[0080] The above embodiments illustrate that the present invention achieves a paradigm shift from "passive coverage" to "intelligent response," comprehensively addressing the shortcomings of existing technologies in wound adaptability, drug retention, and environmental adaptability. These innovations systematically overcome the three major technical bottlenecks of poor enzyme stability, low substrate delivery efficiency, and weak environmental responsiveness in existing technologies, providing a transformative solution for the treatment of diabetic foot ulcers (DFU) that combines metabolic regulation, oxidative stress relief, and tissue regeneration.

[0081] This invention significantly improves the stability of natural enzymes through the effective encapsulation of carrier materials, thereby ensuring their continued enzyme activity. According to the data results in Tables 2 and 3 of the examples, the enzyme activity of the nanogel formulation without carrier materials (Comparative Example 1) is more than half less than that of the enzyme activity in Example 2. This is the core difference between this invention and the published patent CN119454920A.

[0082] Furthermore, the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant natural enzyme nanogel, characterized in that: Based on parts by weight, it comprises the following components: The recombinant natural enzyme includes at least one of glucose oxidase, superoxide dismutase, catalase, or horseradish peroxidase.

2. The recombinant natural enzyme nanogel as described in claim 1, characterized in that: The carrier material includes one or more of polyethylene glycol-40 stearate, polyethylene glycol-40 hydrogenated castor oil, polyethylene glycol-100 glyceryl stearate, polyethylene glycol-6 caprylic / caprylic glycerate, polyglycerol-3 stearate, or polyglycerol-4 diisostearate.

3. The recombinant natural enzyme nanogel as described in claim 2, characterized in that: The mass ratio of the recombinant natural enzyme to the carrier material is 0.5:2-20; wherein, the mass ratio of the recombinant natural enzyme to polyethylene glycol-40 stearate is 0.5:2-20; the mass ratio of the recombinant natural enzyme to polyethylene glycol-40 hydrogenated castor oil is 0.5:0.5-15; the mass ratio of the recombinant natural enzyme to polyethylene glycol-100 glyceryl stearate is 0.5:0.5-10; the mass ratio of the recombinant natural enzyme to polyethylene glycol-6 caprylic / caprylic acid glycerate is 0.5:2-20; the mass ratio of the recombinant natural enzyme to polyglycerol-3 stearate is 0.5:2-10; and the mass ratio of the recombinant natural enzyme to polyglycerol-4 diisostearate is 0.5:0.5-20.

4. The recombinant natural enzyme nanogel as described in claim 1, characterized in that: The gel matrix includes acrylic polymers or cellulose derivatives.

5. The recombinant natural enzyme nanogel as described in claim 1, characterized in that: The moisturizer is one or more of glycerin, propylene glycol, hyaluronic acid, and sorbitol.

6. The recombinant natural enzyme nanogel as described in claim 1, characterized in that: The buffer is at least one of boric acid, citric acid, borax, sodium citrate, and sodium dihydrogen phosphate.

7. A method for preparing recombinant natural enzyme nanogels as described in any one of claims 1-6, characterized in that, Includes the following steps: Step 1: Add the gel matrix to water for injection, heat to prepare a slurry, and cool for later use; Step 2: Dissolve or disperse the carrier material in water for injection, stir evenly, and then add the recombinant natural enzyme while stirring to dissolve it; Step 3: Add the buffer to the water for injection and stir until dissolved, then add the humectant and stir until dissolved; Step 4: Add the solution from Step 2 to the solution from Step 1 and mix well. Then add the solution from Step 3. Adjust the pH value, add water for injection, continue stirring until homogeneous, filter with a microporous membrane to obtain recombinant natural enzyme nanogel.

8. The method for preparing a recombinant natural enzyme nanogel as described in claim 7, characterized in that: The heating temperature in step one is 60-80℃.

9. The method for preparing a recombinant natural enzyme nanogel as described in claim 7, characterized in that: In step four, adjust the pH value to 5.0–6.

5.

10. A recombinant natural enzyme nanogel as described in any one of claims 1-6, used to accelerate the healing of diabetic foot wounds.

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