Preparation method of dihydroquercetin preparation and application of dihydroquercetin preparation in treating bedsore

Abstract

The invention belongs to the field of biological medicine, and relates to a preparation method of a dihydroquercetin preparation and application of the dihydroquercetin preparation in bedsore treatment, and the method comprises the following specific steps: dissolving ferric trichloride hexahydrate in methanol, uniformly mixing, carrying out ultrasonic treatment, dropwise adding into a polyvinylpyrrolidone methanol solution, and uniformly mixing to obtain a mixed solution; dissolving dihydroquercetin in methanol, carrying out ultrasonic treatment, dropwise adding into the mixed solution, reacting at room temperature, dialyzing the reacted solution in a dialysis bag, and freeze-drying the dialyzed solution to obtain iron-dihydroquercetin nanoparticles; the preparation method comprises the following steps: dissolving iron-dihydroquercetin nanoparticles in an oxidized dextran aqueous solution, adding H2O2, and then mixing with an aminated gelatin aqueous solution to obtain the FTH-NO hydrogel. The hydrogel is good in photo-thermal stability, has good self-repairing performance, swelling property and degradation effect, has a good hemostatic effect, can accelerate healing of bedsores, and provides a new strategy for treatment of bedsore wounds, and the bacteriostasis rate of the hydrogel to escherichia coli and staphylococcus aureus is higher than 98%.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to wound dressings, and in particular to a preparation method of a dihydroquercetin preparation and application of the preparation in treating bedsores. Background Art

[0002] Pressure ulcers (PUs) are complex lesions caused by prolonged mechanical pressure, shear, or friction on bony prominences, leading to localized tissue ischemia, hypoxia, necrosis, and subsequent infection. Sustained pressure causes vascular occlusion and impaired cellular metabolism, triggering excessive accumulation of reactive oxygen species (ROS), exacerbating tissue oxidative damage and the inflammatory cascade. These conditions are characterized by high recurrence rates, long treatment cycles, and a heavy medical burden, posing a significant challenge to today's society and global development.

[0003] Traditional treatment methods mainly focus on local antibacterial and anti-inflammatory treatments, but are ineffective in repairing deep tissues and controlling chronic infections, have poor penetration into biofilms, and focus on surface treatment of wounds, making it difficult to regulate the wound microenvironment. This results in a long healing cycle and easy recurrence. Some dressings (such as gauze) are prone to adhesion to the wound, causing pain upon removal, and insufficient air permeability may aggravate local moisture and infection.

[0004] Compared with traditional methods, medical hydrogel dressings have a high water content that can keep wounds moist. Their polymer network structure can quickly reduce wound temperature, reduce inflammatory reactions and pain, and are particularly suitable for bedsores accompanied by burning pain or infection. Gelatin is a collagen hydrolysis product that is similar to the components of the human extracellular matrix (ECM). Its natural origin and degradability avoid foreign body reactions caused by long-term implantation. The hydrogel system prepared by combining gelatin with dextran through the Schiff base reaction can inhibit inflammatory signaling pathways by loading drugs such as growth factors or antibiotics, thereby achieving precise targeted treatment of the wound site. However, in the treatment of bedsores, relying solely on antibacterial and anti-inflammatory or promoting repair cannot achieve the ideal therapeutic effect.

[0005] Flavonoids are widely used in the treatment of several chronic diseases. Dihydroquercetin (TAX), a representative natural flavonoid, exhibits multi-target pharmacological activities, including antioxidant, anti-inflammatory, and antibacterial properties, as well as promoting cell proliferation and tissue regeneration. Studies have shown that TAX is widely used in promoting tissue repair, effectively and rapidly promoting the healing of acute trauma and diabetic wounds. However, TAX's low water solubility and rapid metabolism in the body significantly limit its effectiveness, significantly limiting its application.

[0006] Chinese patent CN 116570760 A discloses a "multifunctional sustained-release dressing for promoting chronic wound healing, its preparation method, and application." TAX is loaded into the lumen of HKUST-1 and then into a hydrogel, resulting in sustained release of dihydroquercetin (TAX). This overcomes the problem of TAX's rapid metabolism in the body, which makes it difficult to maintain efficacy. The resulting hydrogel accelerates the healing of diabetic wounds. However, the hydrogel's antibacterial effect is limited, and it lacks a photothermal effect, making it unsatisfactory for treating pressure sores. Summary of the Invention

[0007] In view of the above technical problems and defects, the present invention designs a dihydroquercetin preparation, which uses hydrogel as a carrier and adds Fe with peroxidase activity (POD) 3+ -TAX NZs, and use NIR photothermal therapy to treat bedsore wounds. The experimental results show that due to Fe 3+ -TAX NZs can catalyze hydrogen peroxide (H2O2) to generate •OH and activate Fe in hydrogels through NIR under stable near-infrared periodic irradiation. 3+ -Photothermal properties of TAX NZs, intelligent release of TAX and Fe 3+ Through the synergistic effect of the two, the sustained-release behavior of the drug is regulated, Escherichia coli and Staphylococcus aureus are inhibited (inhibition rate ≥ 98%), while promoting cell proliferation and differentiation, and performing precise targeted treatment on the wound microenvironment, thereby accelerating the repair process of pressure sores in mice and providing a new strategy for the treatment of pressure sore wounds.

[0008] To achieve the above object, the present invention adopts the following technical solutions: A method for preparing a dihydroquercetin preparation, comprising the following steps: Step 1. Preparation of iron-dihydroquercetin nanoparticles: Step 1.1. Dissolve ferric chloride hexahydrate in methanol, mix thoroughly using a vortexer, and sonicate. Then, add dropwise to a solution of polyvinylpyrrolidone in methanol and mix thoroughly at room temperature to obtain a mixed solution. Step 1.2. Dihydroquercetin was dissolved in methanol, sonicated, and then added dropwise to the mixed solution from step 1. The reaction was allowed to react at room temperature. The resulting solution was placed in a dialysis bag and dialyzed against ultrapure water overnight. The dialyzed solution was lyophilized to obtain iron-dihydroquercetin nanoparticles. Step 2. Preparation of hydrogel: The iron-dihydroquercetin nanoparticles prepared in step 1 were dissolved in an oxidized dextran aqueous solution, followed by addition of H2O2, and then mixed with an amino-treated gelatin aqueous solution to obtain a FTH-NO hydrogel.

[0009] As a preferred embodiment of the present invention, in step 1.1, 20 mg of ferric chloride hexahydrate is dissolved in 1 mL of methanol, and the concentration of the polyvinyl pyrrolidone methanol solution is 10 mg / mL; in step 1.2, 10 mg of dihydroquercetin is dissolved in 1 mL of methanol.

[0010] As a preferred embodiment of the present invention, the preparation method of amino gelatin is as follows: 5.0 g of gelatin is weighed and placed in 50 mL of phosphate buffer solution, heated at 50°C to dissolve, then 30 mL of ethylenediamine is added for modification, the pH is adjusted to 6.5 with hydrochloric acid, 2.0 g of DMTMM is added for dissolution, and the mixture is reacted at room temperature in the dark overnight. After the reaction is completed, the mixed solution is added to a dialysis bag, dialyzed in deionized water for three days, and freeze-dried to obtain amino gelatin.

[0011] As a preferred embodiment of the present invention, the preparation method of oxidized dextran is as follows: 8.0 g of dextran is weighed and dissolved in 200 mL of distilled water. After dissolution, 26 g of sodium periodate is added for oxidation, and the reaction is carried out in the dark at room temperature for 5 h. Subsequently, 16 mL of ethylene glycol is added to terminate the oxidation. The mixture after the reaction is placed in a dialysis bag and dialyzed for 72 h. The oxidized dextran is obtained after freeze-drying.

[0012] As a preferred embodiment of the present invention, in step 2, the concentration of the amino gelatin aqueous solution is 10%, the concentration of the oxidized dextran aqueous solution is 5%, and the ratio of amino gelatin to oxidized dextran is 2:1; the concentration of iron-dihydroquercetin nanoparticles in the configured hydrogel is 250 μg / mL, and the final concentration of H2O2 is 1 mmol / mL.

[0013] The FTH-NO hydrogel prepared by the above method showed good hemostatic effect in the liver tail bleeding model, so it can be used as a hemostatic material for in vivo or in vitro wound hemostasis to control bleeding volume.

[0014] The FTH-NO hydrogel prepared by the above method showed good repair effect in the skin repair test of the pressure sore mouse model. The wound healing rate reached more than 80% on the 14th day, and the regenerated epidermis could completely cover the wound. Therefore, it can be used in the preparation of materials to promote the healing of pressure sores.

[0015] Advantages and beneficial effects of the present invention: (1) The present invention prepares Fe-dihydroquercetin nanoparticles for the first time, which have peroxidase activity (POD) and photothermal properties, and can catalyze H2O2 to generate ·OH. The hydrogel loaded with Fe-dihydroquercetin nanoparticles reaches a certain temperature value after being irradiated by near-infrared light, which can play a role in high-temperature sterilization. At the same time, NIR irradiation can activate Fe in the hydrogel. 3+ -Photothermal properties of TAX NZs, intelligent release of TAX and Fe 3+, thus significantly enhancing the antibacterial effect. The antibacterial rates of the hydrogel against Escherichia coli and Staphylococcus aureus are both higher than 98%.

[0016] (2) The hydrogel provided by the present invention has a three-dimensional network structure, which is conducive to the circulation of oxygen and water. It has good moisture retention and swelling properties. The structural stability in the later stage of swelling can ensure that a moist environment is provided for the wound during the wound healing stage, which is more conducive to promoting the healing of bedsores.

[0017] (3) The hydrogel provided by the present invention has good self-repairing properties, good elasticity and degradation effect, and can reach the maximum degradation rate in a short time. The hydrogel releases the drug rapidly within 12 hours and reaches a slow and steady state after 12 hours. This drug release method can ensure that the drug can reach an effective concentration in a short time during the wound anti-inflammatory and hemostatic stages, thereby exerting a therapeutic effect.

[0018] (4) The hydrogel provided by the present invention has good photothermal stability. In the environment of the organism, it can still maintain its structure and performance when subjected to photothermal stimulation, providing a stable microenvironment for cell growth and differentiation. The present invention can precisely control the temperature to 40-45°C, thereby promoting the healing of bedsore wounds and avoiding high temperature damage to normal tissues.

[0019] (5) The hydrogel provided by the present invention can effectively reduce tissue inflammation, reduce inflammatory response, and better promote the repair of tissue wounds.

[0020] (6) The FTH-NO hydrogel provided by the present invention has a good hemostatic effect, and the hemostatic performance is enhanced with the assistance of NIR. Therefore, the provided hydrogel can be used as a hemostatic material for internal or external wound hemostasis, which can effectively control the amount of bleeding.

[0021] (7) The FTH-NO hydrogel provided by the present invention promoted angiogenesis under the irradiation of NIR light and accelerated the healing of bedsores. The wound healing rate reached more than 80% on the 14th day, and the regenerated epidermis could completely cover the wound, reducing the risk of secondary infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The Fe synthesized by the present invention 3+ -Characterization of TAX NZs; where a is Fe 3+ -TAX NZs transmission electron microscope image; b is Fe 3+ -TAX NZs diameter distribution; c is Fe 3+ -TAX NZs Fourier transform infrared spectrum; d is TAX XRD pattern; e is Fe 3+ -XRD pattern of TAX NZs; f is Fe 3+-TAX NZs XPS spectrum; g is Fe 3+ -POD-like activity of TAX NZs; Figure 2 is a characterization of the hydrogel of the present invention; wherein, a is the swelling behavior of the hydrogel; b is the degradation behavior of the hydrogel; c is the drug release of the hydrogel; d is the rheological behavior of the hydrogel; Figure 3 is the photothermal effect of the hydrogel of the present invention; wherein a is the infrared thermal spectrum; b is the different concentrations of Fe 3+ c. Photothermal heating curve of FTH-NO+NIR hydrogel; c. Photothermal heating and cooling curve of FTH-NO+NIR; d. Photothermal stability curve of FTH-NO+NIR; e. Linear time data obtained from the cooling cycle of lnθ panel b; Figure 4 is the in vitro activity of the hydrogel; where a is the inhibition rate against Staphylococcus aureus and Escherichia coli; b is the inhibition image against Staphylococcus aureus and Escherichia coli; c is the scanning electron microscopy image of Staphylococcus aureus and Escherichia coli; Figure 5 This is the hemolysis and hemostasis experiment of the hydrogel; a is the image of the liver tail bleeding model in mice; b is the amount of liver bleeding; c is the amount of tail bleeding; Figure 6 This is a skin repair experiment of a pressure ulcer mouse model using hydrogels; a is an image of wound healing on days 0, 3, 7, and 14; b is a graph of wound growth area on days 3, 7, and 14; c is the wound healing rate on days 3, 7, and 14 (bar graph represents SD ± mean, n = 3, * p <0.05,** p <0.01); Figure 7 Immunohistochemical analysis: a is the quantitative analysis of CD31; b is the quantitative analysis of CD68; c is the quantitative analysis of VEGF (bar graphs represent SD ± mean. n = 3, * p <0.05,** p <0.01); Figure 8 This is the infrared thermal spectrum of the mouse body. DETAILED DESCRIPTION

[0023] In order to enable those skilled in the art to better understand the technical solutions and advantages of the present invention, the present application is described in detail below with reference to the accompanying drawings, but this is not intended to limit the scope of protection of the present invention.

[0024] 1. Experiment: 1.1. Materials: Dihydroquercetin (TAX) was purchased from the China Food and Drug Administration (batch number: 111816–201102, purity: 98.0%). Ferric chloride hexahydrate (FeCl3·6H2O) and polyvinylpyrrolidone (PVP) were purchased from BASF Biotechnology Co., Ltd. (Hefei, China). Gelatin (Gel), dextran (DEX), methanol, ethanol, ethylenediamine, and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM) were purchased from Shanghai MacLean Biochemical Co., Ltd. (Shanghai, China). Sodium periodate (NaIO4) and phosphate buffer solution (PBS) were purchased from Sunshine Biotechnology Co., Ltd. 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH) and 2,2′-nitro-bis(3-ethylbenzothiazole-6-sulfonic acid) (ABTS) were purchased from Fuzhou Bilin Biotechnology Co., Ltd.

[0025] 1.2.Fe 3+ Preparation of TAX NZs (Iron-dihydroquercetin Nanoparticles): 20 mg of ferric chloride hexahydrate (FeCl3•6H2O) was dissolved in 1 mL of methanol, mixed using a vortex instrument, ultrasonicated for 5 min, and then slowly added dropwise to a 10 mg / mL polyvinyl pyrrolidone (PVP) methanol solution and mixed at room temperature for 10 min to obtain a mixed solution; then 10 mg of dihydroquercetin (TAX) was dissolved in 1 mL of methanol, ultrasonicated for 10 min, and slowly added dropwise to the above mixed solution. The reaction was carried out at room temperature for 1 h. The reaction solution was placed in a dialysis bag and dialyzed with ultrapure water overnight. The dialyzed solution was lyophilized to obtain iron-dihydroquercetin nanoparticles (Fe 3+ -TAX NZs).

[0026] 1.3.Fe 3+ -Characterization of TAX NZs: The morphology and shape of the nanoparticles were observed by transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FT-IR) at a wavelength of 4000–400 cm -1 The structure and functional groups of the nanoparticles were determined, the crystal structure of the nanoparticles was analyzed by X-ray diffraction (XRD), the size distribution of the nanoparticles was analyzed by dynamic light scattering analyzer (DLS), and the elemental analysis of the nanoparticles was performed by X-ray photoelectron spectroscopy (XPS).

[0027] 1.4. Peroxidase activity: Using TMB as the chromogenic substrate, in the presence of H2O2, the Fe 3+-TAX NZs peroxidase activity. TMB solution was prepared according to the existing method, and 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, and 250 μg / mL of Fe 3 + -TAX NZs and H2O2 were prepared into a solution with a final concentration of 1 mMol, mixed with the prepared TMB solution, and the color change of the solution was observed at room temperature, and then its UV-visible absorption spectrum was measured.

[0028] 1.5. Preparation of hydrogel: 1.5.1. Preparation of amino-modified gelatin: 5.0 g of gelatin (Gel) was weighed and placed in 50 mL of phosphate buffer solution (PBS, pH = 7.4), heated at 50°C to dissolve, then 30 mL of ethylenediamine was added for modification, and the pH was adjusted to 6.5 with hydrochloric acid (HCl). 2.0 g of DMTMM (4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride) was added and dissolved. The above mixture was reacted at room temperature in the dark overnight. After the reaction was completed, the mixed solution was added to a dialysis bag, dialyzed in deionized water for three days, and freeze-dried to obtain amino-modified gelatin (N-Gel).

[0029] 1.5.2. Preparation of oxidized dextran: 8.0 g of dextran (DEX) was weighed and dissolved in 200 mL of distilled water. After dissolution, 26 g of sodium periodate was added for oxidation. The reaction was allowed to react at room temperature in the dark for 5 h. Subsequently, 16 mL of ethylene glycol was added to terminate the oxidation. The reaction mixture was placed in a dialysis bag and dialyzed for 72 h. After lyophilization, oxidized dextran (ODEX) was obtained.

[0030] 1.5.3. Preparation of different hydrogels: Blank hydrogel (NO): prepared by mixing 10% (w / v) N-Gel aqueous solution with 5% (w / v) ODEX aqueous solution, with the ratio of N-Gel to ODEX being 2:1.

[0031] Dihydroquercetin-loaded hydrogel (T-NO): Dihydroquercetin was dissolved in a 5% (w / v) ODEX aqueous solution and then mixed with a 10% (w / v) N-Gel aqueous solution to achieve a dihydroquercetin concentration of 250 μg / mL in the prepared hydrogel.

[0032] Hydrogel loaded with iron-dihydroquercetin nanoparticles (FT-NO): Iron-dihydroquercetin nanoparticles were dissolved in a 5% (w / v) ODEX aqueous solution and then mixed with a 10% (w / v) N-Gel aqueous solution to achieve a concentration of 250 μg / mL of iron-dihydroquercetin nanoparticles in the prepared hydrogel.

[0033] Hydrogel loaded with iron-dihydroquercetin nanoparticles + H2O2 (FTH-NO): Iron-dihydroquercetin nanoparticles were dissolved in 5% (w / v) ODEX solution, followed by the addition of H2O2, and then mixed with 10% (w / v) N-Gel aqueous solution to achieve an iron-dihydroquercetin nanoparticle concentration of 250 μg / mL in the prepared hydrogel and a final H2O2 concentration of 1 mmol / mL.

[0034] Hydrogel loaded with iron-dihydroquercetin nanoparticles + H2O2 + near infrared (FTH-NO+NIR): Iron-dihydroquercetin nanoparticles were dissolved in 5% (w / v) ODEX solution, followed by addition of H2O2, and then mixed with 10% (w / v) N-Gel aqueous solution to make the concentration of iron-dihydroquercetin nanoparticles in the prepared hydrogel 250 μg / mL and the final concentration of H2O2 at 1 mmol / mL. The prepared hydrogel was observed under near infrared (1.5 W / cm 2 , 808 nm) for 10 min.

[0035] 1.6. Hydrogel Characterization 1.6.1. Swelling and degradation The swelling and in vitro degradation properties of the hydrogel were tested by the weighing method. Equal volumes of hydrogel were taken and completely immersed in PBS (pH=7.4). The hydrogel was taken out at different time points, the surface moisture was wiped off, and the hydrogel was weighed. The swelling rate (Swelling) and degradation rate (Degradation) of the hydrogel were calculated by the weight ratio before and after.

[0036] ; Wherein, Ma is the weight of freeze-dried hydrogel, and Mb is the weight of surface water after absorbing water; ; Among them, M0 is the initial weight, and M is the weight of the surface moisture after absorbing water.

[0037] 1.6.2. Drug release: 1 mL of hydrogel was immersed in 3 mL of PBS (pH = 7.4) and placed in a constant temperature shaker at 37°C. The extract was taken out at different time points to study the release behavior of the hydrogel. The TAX release amount was measured by UV–VIS method at 290 nm.

[0038] 1.6.3. Rheological behavior: The hydrogel was dynamically evaluated using a rheometer. The prepared hydrogel was placed on the flat plate of the rheometer, and the storage modulus (G') and loss modulus (G") of the hydrogel were tested using the frequency sweep mode. The thickness of the hydrogel sample was 2 mm, the distance between the plates was 1.0 mm, and the test conditions were 25°C, 1% strain, and a frequency of 1 Hz.

[0039] 1.7. Photothermal characteristics: In order to test the photothermal properties of the hydrogel, the photothermal effect of the hydrogel was analyzed. First, FTH-NO hydrogels with different concentrations (50, 100, 150, 200, 250 μg / mL) were prepared and exposed to near-infrared light (1.5 W / cm 2 The hydrogel samples were irradiated at 808 nm (100 nm) for approximately 10 minutes. The hydrogel temperature was measured and photographed using an infrared thermal imager. To test the hydrogel's thermal stability, the hydrogel was subjected to temperature increases and decreases over a period of time. This irradiation cycle was repeated three times, and the temperature was recorded using an infrared thermal imager.

[0040] 1.8. In vitro activity studies: 1.8.1 Antibacterial properties: The plate count method was used to determine the antibacterial ability of the hydrogels against Escherichia coli and Staphylococcus aureus. The hydrogels of each group were prepared and placed in a 24-well plate. The diluted bacterial suspension (800 μL, 1×10 7 CFU / mL, the FTH-NO group was exposed to NIR laser (1.5 W / cm 2 The suspension was irradiated with a wavelength of 808 nm (100 nm) for 10 minutes. The suspension was then incubated with the other groups at 37°C for 2 hours. 10 μL of the incubated suspension was inoculated onto solid culture medium and incubated at 37°C for 12 hours. The inhibition rate was then calculated. Furthermore, SEM was used to observe the morphological changes of Escherichia coli and Staphylococcus aureus in response to the hydrogels from each group.

[0041] ; Among them, A is the number of colonies in the control group, and B is the number of colonies after co-culture of hydrogels and bacteria in each group.

[0042] 1.8.2. Hemostasis test The hemostatic properties of the hydrogel were evaluated using a mouse liver bleeding model and a tail bleeding model. ICR mice were randomly divided into different groups and anesthetized with 0.5% sodium pentobarbital. A scalpel was used to dissect the mouse chest and abdomen, completely exposing the liver to the abdominal cavity. A wound was punctured in the liver with a syringe needle to allow blood to flow out. The wound was covered with hydrogels from different groups, and the blood flow was observed. Similarly, in the mouse tail hemostasis model, the mouse tail was placed vertically and cut short 5 cm from the tail end. The blood flow from the wound after covering it with hydrogel was observed, and the amount of bleeding before and after the application of the hydrogel was calculated.

[0043] 1.9. Bedsore Wound Repair Test: 1.9.1 Establishment of bedsore model: Male ICR mice (purchased from Yisi Laboratory Animal Technology Co., Ltd., Changchun, China) were housed in a standard experimental environment (temperature 22±2°C, humidity 60±5%) for model establishment. Mice were anesthetized with 0.5% sodium pentobarbital, and their backs were depilated with depilatory cream and wiped with saline. The mid-back region of the mice was gently lifted manually, and externally placed neodymium magnets (12 mm diameter, 5 mm height) were clamped on both sides of the skin. The mice were subjected to a 24-hour decubitus cycle (12-hour ischemia cycle and 12-hour perfusion cycle) for three cycles. Afterward, the magnets were removed. Successful model establishment was achieved when the wound surface exhibited an ulcer. The prepared hydrogels were then applied to the wounds (the FTH-NO+NIR group was illuminated for approximately ten minutes on days 1, 3, 7, and 14). Wound healing was observed on days 0, 7, and 14. Wound area was measured using Image J software to calculate wound healing rates.

[0044] 1.9.2 Immunohistochemical analysis: To more accurately assess the degree of wound healing in mice with decubitus ulcers, this study quantitatively analyzed the immunohistochemical expression levels of CD31, CD68, and VEGF proteins, based on existing immunohistochemical assays. Tissue samples were fixed in 4% paraformaldehyde for 24 hours, then dehydrated with a gradient of ethanol, embedded, and sectioned. Antigen retrieval was performed, and a 3% H₂O₂ solution was added dropwise. The cells were incubated in the dark for 10 minutes at room temperature, rinsed three times with PBS, blocked with serum for 30 minutes, and incubated with antibodies overnight at 4°C before color development. Protein expression levels were calculated using Image J software.

[0045] 2. Experimental results: 2.1. Fe 3+ -Characterization of TAX NZs 2.1.1. Transmission electron microscopy, infrared analysis, X-ray diffraction, and X-ray photoelectron spectroscopy: To verify the Fe 3+The performance of -TAX NZs was characterized in this paper. First, transmission electron microscopy (TEM) showed that Fe 3+ -TAX NZs are irregular in shape ( Figure 1 a), Fe 3+ -TAX NZs size, further analysis showed that the particle size range was 56.82 ± 9.13 nm ( Figure 1 b). Fourier transform infrared spectroscopy (FT-IR) shows that the C=O tensile strength is between 1150-1200 cm -1 , indicating that the C=O group of dihydroquercetin is successfully coordinated with trivalent iron ( Figure 1 c). Fe 3+ -TAX NZs XRD patterns showed Fe 3+ -TAX NZs are amorphous and no Fe was detected in the crystals ( Figure 1 XPS analysis also confirmed that Fe 3+ -TAX NZs were successfully synthesized. XPS analysis results showed that the nanozyme (FTH-NO) was mainly composed of Fe2p, O1s, N1s and C1s, which can be used to observe the metal elements in the nanoparticles. After detection, it was found that the XPS spectrum showed peaks corresponding to Fe2p, O1s, N1s and C1s at 714, 527, 399 and 283 eV respectively ( Figure 1 f), confirmed that Fe 3+ -TAX NZs were successfully synthesized. In addition, the present invention used ICP-OES to determine the ratio of Fe in Fe-dihydroquercetin nanozymes. The results showed that Fe 3+ The weight of Fe in -TAXNZs is approximately 25.511 μg / kg. The above results demonstrate the successful synthesis of iron-dihydroquercetin nanozymes (nanoparticles).

[0046] 2.1.2. Peroxidase activity: The present invention uses TMB as a chromogenic substrate to investigate the Fe 3+ -TAX NZs POD (peroxidase) activity. The results showed that increasing Fe 3+ The concentration of -TAX NZs leads to an increase in absorbance, and absorption peaks are observed at 370 nm and 652 nm, indicating that Fe 3+ -TAX NZs have POD mimetic activity, which can be attributed to the Fe 3+ -TAX NZs unique nanostructure ensures that it will not cause loss of enzyme activity due to environmental influences ( Figure 1 g).

[0047] 2.2. Characterization of hydrogels In the present invention, in order to study the effect of the addition of nanozymes on the hydrogels, each group of hydrogels was analyzed by scanning electron microscopy (SEM). It was found that the hydrogels with the addition of iron-dihydroquercetin nanozymes still maintained a three-dimensional network structure while showing a tighter pore structure, which facilitated the circulation of oxygen and water. In addition, the present invention conducted a 24-hour performance test on each group of hydrogels and found that each group of hydrogels reached an equilibrium state in about 10 hours and had good moisture retention and water absorption swelling properties, showing a relatively stable state ( Figure 2 a) The structural stability in the late swelling stage can ensure a moist environment for the wound during the wound healing stage, which is more conducive to promoting wound healing. The hydrogels of each group were placed in a PBS (pH=7.4) solution at 37°C to observe their degradation. As time went by, the hydrogels showed a state of continuous degradation. When a certain dissolution time was reached, the degradation of the hydrogels reached a balanced state. It can be seen that the FTH-NO and FTH-NO+NIR groups had the most significant effects. On the seventh day, the degradation state exceeded 80%, which may be due to the Fe 3+ The addition of -TAXNZs accelerated the degradation rate of the hydrogel under the influence of nanozyme activity and photothermal effect ( Figure 2 b). Overall, the hydrogel has a good degradation effect and can reach the maximum degradation rate in a short time.

[0048] In order to evaluate the drug release of the hydrogel with added nanozymes on wound healing, the present invention used the UV-VIS method to analyze the drug content of the extracts at different times. From the drug release curve, it can be seen that the drug is rapidly released within 12 hours and reaches a slowly flat state after 12 hours. 3+ -TAX NZs and the drug showed a stable release trend under near-infrared light irradiation ( Figure 2 c) The release behavior of TAX was enhanced and improved to a certain extent.

[0049] The present invention conducted a rheological test on the hydrogel and evaluated the performance of the hydrogel through step-by-step oscillation. The experimental results show that under a strain of 1%, the hydrogel can maintain its gel state well, but when the strain increases to 200%, its gel structure is destroyed. After removing the high strain, the hydrogel gradually restored its integrity due to the self-repairing effect of the Schiff base and hydrogen bonds possessed by the hydrogel itself. It was observed that the storage modulus (G') successfully returned to the initial value, which fully demonstrated that the hydrogel has good self-repairing properties. At the same time, it can be seen that within the angular velocity range of 1-100 rad / s, the storage modulus (G') of each group of hydrogels is higher than the loss modulus (G"), indicating that each group of hydrogels has good elastic properties and a high cross-linking density and a stable structure ( Figure 2d).

[0050] 2.3. Photothermal effect: To verify the photothermal effect of hydrogels, the present invention placed hydrogel samples of different concentrations under near-infrared light at 808 nm (1.5 W / cm 2 ) light, and measured the temperature change of the sample. At the same time, the sample was exposed to near-infrared light for a long time to test the photothermal stability of the sample to ensure that it can still maintain good photothermal performance after long-term exposure. Through precise temperature control, the local temperature is controlled at 40-45°C, which can promote wound healing and avoid high temperature damage to normal tissue. The results showed that under the same conditions, the temperature of FTH-NO+NIR hydrogel with a concentration of 250 µg / mL rose by 16.8°C, and the temperature of blank hydrogel (NO) of the same volume only rose by 4.5°C, and the infrared thermal imager can clearly observe the Fe 3+ -TAX NZs warming state, indicating that Fe 3+ -TAX NZs can be heated up quickly in a short time to reach the required temperature ( Figure 3 Subsequently, the hydrogel was observed to heat up and cool down within a fixed time, and it was found to have a certain degree of photothermal stability, indicating that it can still maintain its structure and performance when subjected to photothermal stimulation in the biological environment, providing a stable microenvironment for cell growth and differentiation ( Figure 3 cd). In addition, according to the negative natural logarithm of the cooling time lnθ, the ts value is 425.2389 (ts is the slope of the linear regression equation of -lnθ and T, T is the time of heating and cooling before and after irradiation, , Tmax is the maximum temperature under irradiation, Tsurr is the ambient temperature), and thus the photothermal conversion efficiency is 58.23% ( Figure 3 e), indicating that the sustained release of drugs can be regulated while maintaining photothermal stability, achieving precise targeted therapy.

[0051] 2.4. In vitro activity study of hydrogels: 2.4.1. In vitro antibacterial test: When the wound surface is exposed, it is accompanied by a series of inflammation and bacterial infection. Having antibacterial properties is an important characteristic of wound dressings. In order to evaluate whether the prepared wound dressing has certain antibacterial properties, common Staphylococcus aureus and Escherichia coli (Staphylococcus aureus and Escherichia coli were purchased from Shanghai Yaji Biotechnology Co., Ltd.) were selected and tested using the plate count method. According to the experimental results, it can be found that compared with other groups, especially the control group, after irradiation with 808 nm near-infrared light, the antibacterial effect is very significant. Compared with the control group, the inhibition rates of the other five groups on Staphylococcus aureus are 32.89±12.57%, 52.96±10.32%, 71.13±3.19%, 90.62±0.63%, and 98.07±0.6%, respectively. Compared with the same period last year, the inhibition rate on Escherichia coli is almost the same, and the inhibition results are 50.12±2.64%, 85.48±1.39%, 92.66±0.39%, 97.14±0.27%, and 99.39±0.17%, respectively. From the results of the antibacterial experiment, it can be seen that compared with Staphylococcus aureus, the FTH-NO+NIR group hydrogel has a more significant inhibitory effect on Escherichia coli. It can be seen that the hydrogels in the FTH-NO+NIR group reached a certain temperature after being irradiated with near-infrared light, which played a role in high-temperature sterilization, and the antibacterial rate was higher than 98% ( Figure 4 ab). To study the effect of near-infrared light on bacteria, the present invention observed the morphological changes of bacteria by scanning electron microscopy. The results showed that compared with the control group, the dead bacteria in the FTH-NO+NIR group after irradiation with 808 nm near-infrared light showed obvious shrinkage, with the bacterial cell wall and cell membrane concave inward and the overall shape becoming irregular ( Figure 4 c). Based on the above experimental results, it is shown that the FTH-NO+NIIR group hydrogel has a good antibacterial effect and can inhibit the infection of bacteria and microorganisms, while the metal nanozyme (Fe 3+ -TAX NZs) can efficiently destroy bacterial structures by producing a large amount of reactive oxygen species and photothermal effects, and has a significant antibacterial effect on a variety of bacteria, thereby improving the targeting and synergistic effect of drugs, enhancing the therapeutic effect and promoting wound healing.

[0052] 2.4.2. Hemostasis test: In order to test the hemostatic properties of the hydrogel on the body, the present invention adopted two mouse liver bleeding and tail bleeding models for analysis ( Figure 5a), the liver bleeding and tail bleeding of mice can be clearly observed. Compared with the control group, the two blood loss amounts in the FTH-NO+NIR hydrogel group were the lowest, and the hemostatic effect was significantly better than the first four groups. After calculation, the liver blood loss of mice was 136.37±3.75 mg, 118.2±4.2 mg, 88.3±7.5 mg, 71.43±6.04 mg, 59.97±7.65 mg, and 46.1±4.82 mg, respectively, according to the grouping order. The liver blood loss amount in the FTH-NO+NIR hydrogel group was less than 35% of that in the control group, showing good hemostatic performance ( Figure 5 b), the tail blood loss of mice was 20.67±0.51 mg, 15.5±0.92 mg, 12.33±1.21 mg, 10.37±1.19 mg, 5.63±1 mg, and 2.53±1.55 mg ( Figure 5 c). The above experiments show that FTH-NO hydrogel exhibits good hemostatic properties with the assistance of NIR, which is a very important property in the application of wound dressing.

[0053] 2.5. In vivo activity study of hydrogels: 2.5.1. Decubitus wound repair in mice: By verifying the results of in vitro evaluation experiments, the present invention conducted in vivo experimental analysis on mice. The thermal imaging images of mice in the FTH-NO+NIR group before and after 10 minutes of irradiation are as follows: Figure 8 As shown in the figure, it can be seen that the FTH-NO+NIR responsive hydrogel can heat up to the temperature required by the local wound in a short time and exert its therapeutic effect without damaging the tissue around the wound. Figure 6 a) Observe the healing status of bedsore wounds on days 0, 3, 7, and 14. Meanwhile, the present invention uses Image J software to analyze the wound images ( Figure 6 b) On the 3rd day, compared with the control group, the scab area in the FTH-NO+NIR hydrogel group was relatively smaller, and the wound healing rates of the control group and the FTH-NO+NIR group were 8.94±2.55% and 32.65±4.8%, respectively. On the 7th day, the scab area in the FTH-NO+NIR group was significantly reduced, the scab gradually fell off, and new skin tissue grew around it. The wound healing rates were 23.62±8.2% and 67.38±1.51%, respectively. On the 14th day, when the control group was still completely scabbed, the scab in the FTH-NO+NIR group had completely fallen off, and the wound healing rate reached 82.95±4.42%. The regenerated epidermis had also completely covered the wound. The healing trend was clearly superior to that of the other groups ( Figure 6c) This indicates that wound healing was accelerated under NIR light irradiation. It can be seen that the temperature of the FTH-NO+NIR mouse group rose rapidly in a short period of time, reaching the required appropriate temperature, thereby improving the therapeutic effect without damaging the skin wound.

[0054] 2.5.2. Immunohistochemical analysis Angiogenesis is an important part of the skin tissue healing stage. Its more obvious feature is that some immature blood vessels gradually grow into mature vascular networks. Vascular growth factors CD31 and VEGF are important indicators for evaluating blood vessel growth and tissue wound healing. The inflammatory cytokine CD68 is a marker for identifying macrophages. When subjected to external trauma, it stimulates the macrophage matrix to release a large amount of pro-inflammatory cytokines, thereby hindering wound healing. From the experimental results, compared with the control group, the expression levels of CD31 and VEGF proteins in the NIR group increased significantly, indicating that under the irradiation of NIR light, FTH-NO hydrogel further promoted angiogenesis and accelerated wound healing. Compared with the control group, the expression level of CD68 protein in the NIR group was significantly reduced (see Figure 7 a to c), indicating that FTH-NO+NIR hydrogel can effectively reduce tissue inflammation and better promote the repair of tissue wounds.

[0055] The above description of the present invention using specific examples is intended only to facilitate understanding of the present invention and is not intended to limit the present invention. A person skilled in the art of the present invention may make several simple deductions, modifications, or substitutions based on the principles of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of protection of the claims.

Claims

1. A method for preparing a dihydroquercetin preparation, characterized in that: The method comprises the following steps: Step 1. Preparation of iron-dihydroquercetin nanoparticles: Step 1.

1. Dissolve ferric chloride hexahydrate in methanol, mix thoroughly, and sonicate. Then, add dropwise to a methanol solution of polyvinyl pyrrolidone and mix thoroughly to obtain a mixed solution. Step 1.

2. Dihydroquercetin was dissolved in methanol, sonicated, and then added dropwise to the mixed solution from step 1. The reaction was allowed to react at room temperature. The resulting solution was placed in a dialysis bag and dialyzed against ultrapure water overnight. The dialyzed solution was lyophilized to obtain iron-dihydroquercetin nanoparticles. Step 2. Preparation of hydrogel: The iron-dihydroquercetin nanoparticles prepared in step 1 were dissolved in an oxidized dextran aqueous solution, followed by addition of H2O2, and then mixed with an amino-treated gelatin aqueous solution to obtain FTH-NO hydrogel.

2. The method for preparing a dihydroquercetin preparation according to claim 1, wherein: In step 1.1, 20 mg of ferric chloride hexahydrate was dissolved in 1 mL of methanol. The concentration of the polyvinyl pyrrolidone methanol solution was 10 mg / mL. In step 1.2, 10 mg of dihydroquercetin was dissolved in 1 mL of methanol.

3. The method for preparing a dihydroquercetin preparation according to claim 1, wherein: The preparation method of amino gelatin is as follows: 5.0 g of gelatin is weighed and placed in 50 mL of phosphate buffer solution, heated at 50°C to dissolve, then 30 mL of ethylenediamine is added for modification, the pH is adjusted to 6.5 with hydrochloric acid, 2.0 g of DMTMM is added for dissolution, and the mixture is reacted at room temperature in the dark overnight. After the reaction is completed, the mixed solution is added to a dialysis bag, dialyzed in deionized water for three days, and freeze-dried to obtain amino gelatin.

4. The method for preparing a dihydroquercetin preparation according to claim 1, wherein: The preparation method of oxidized dextran is as follows: 8.0 g of dextran is weighed and dissolved in 200 mL of distilled water. After dissolution, 26 g of sodium periodate is added for oxidation, and the reaction is carried out at room temperature in the dark for 5 h. Subsequently, 16 mL of ethylene glycol is added to terminate the oxidation. The reaction mixture is placed in a dialysis bag and dialyzed for 72 h. The oxidized dextran is obtained after freeze-drying.

5. The method for preparing a dihydroquercetin preparation according to claim 1, wherein: In step 2, the concentration of the amino-gelatin aqueous solution is 10%, the concentration of the oxidized dextran aqueous solution is 5%, and the ratio of amino-gelatin to oxidized dextran is 2:1; the concentration of iron-dihydroquercetin nanoparticles in the prepared hydrogel is 250 μg / mL, and the final concentration of H2O2 is 1 mmol / mL.

6. Use of the FTH-NO hydrogel prepared by the method according to any one of claims 1 to 5 in the preparation of hemostatic materials.

7. Use of the FTH-NO hydrogel prepared by the method according to any one of claims 1 to 5 in preparing materials for promoting the healing of bedsores.

Images