Double-layer hydrogel dressing with double functions as well as preparation method and application of double-layer hydrogel dressing

Abstract

The invention relates to a double-layer hydrogel dressing with double functions as well as a preparation method and application thereof, and belongs to the technical field of biomedical materials. The hydrogel dressing is composed of an inner layer and an outer layer, the inner layer is a treatment layer with a diabetes wound healing promoting effect, and the outer layer is a detection layer with a blood glucose monitoring effect; the aperture of the hydrogel of the inner treatment layer is more than 10 times that of the hydrogel of the outer detection layer, and the inner layer and the outer layer are attached in a physical and chemical mode. According to the hydrogel dressing, the outer layer is small in pore diameter and compact in structure, the inner layer is large in pore diameter and loose in structure, the two layers are tightly combined, extraction of percolate and colorimetric detection of blood sugar at a wound are achieved, and the hydrogel dressing has excellent biocompatibility, antibacterial property, self-healing performance and swelling performance.

Description

Technical Field

[0001] The invention relates to a double-layer hydrogel dressing with dual functions and a preparation method and application thereof, belonging to the technical field of biomedical materials. Background Art

[0002] Hyperglycemia is one of the main characteristics and high-risk factors of diabetes. Sustained hyperglycemia caused by insulin deficiency or insulin resistance leads to metabolic abnormalities and dysfunctions, and causes a variety of related complications. Diabetic wounds, as one of the complications of diabetes, are chronic wounds that do not heal normally. They can occur in any part of the body, and the environment around the wound is relatively complex. They are mainly manifested by hypoxia, excessive wound exudate, repeated bacterial infection, persistent inflammation, and impaired angiogenesis and tissue regeneration, which lead to overlapping wound healing processes and prolonged inflammation. The current common treatment measure is to lower blood sugar while adding antibacterial treatment to promote wound healing.

[0003] Nowadays, traditional dressings are increasingly unable to meet people's needs for high-quality wound care, and hydrogels with appropriate water absorption and swelling capabilities are being widely studied. Hydrogels can provide a moist environment, protect wounds from bacterial infection, and can be used as modern wound dressings in regenerative medicine. However, single-layer hydrogels prepared from natural polymers usually have poor mechanical properties and lack of sufficient barrier function against wound infection.

[0004] In diabetic patients, the wound healing process usually becomes slow and complicated, and the role of glucose (blood sugar) plays a key role in this process. By strictly controlling blood sugar levels, the healing speed and quality of wounds can be improved to a certain extent. Therefore, diabetic patients need to pay special attention to blood sugar management during wound care to reduce the risk of wound non-healing and infection. Glucose oxidase (GOx) is a class of natural enzymes with glucose as a substrate. It catalyzes glucose to produce gluconic acid and hydrogen peroxide. Hydrogen peroxide can generate hydroxyl radicals that oxidize the chromogenic substrate in the presence of horseradish peroxidase (HRP), resulting in oxidation and color development of the chromogenic substrate. The use of two enzymatic reactions makes it possible to detect glucose by color change. However, the natural glucose oxidase has poor stability, which will significantly affect the sensitivity, color development efficiency and color consistency of the color development reaction, thereby affecting the accuracy and reliability of the glucose detection results. Summary of the invention

[0005] In order to solve the shortcomings of the above-mentioned single-layer hydrogel dressing and natural enzyme in blood glucose detection, the present invention provides a double-layer hydrogel dressing with dual functions, a preparation method and application thereof. The present invention prepares a double-layer hydrogel by adding effective substances with different effects to the inner and outer layers, and applies it to promote diabetic wound healing, wherein the inner layer hydrogel is mainly a treatment layer composed of GOx@ZIF-8, oxidized hyaluronic acid (OHA) and carboxymethyl chitosan (CMCS), and the outer layer hydrogel is mainly a detection layer composed of polyvinyl alcohol (PVA), chitosan (CS), peroxidase and a chromogenic substrate, and the two are closely connected by physical and chemical means. The present invention utilizes the characteristics of different pore sizes and swelling differences of hydrogels to provide a double-layer hydrogel dressing with dual functions and a preparation method thereof. The hydrogel dressing has excellent biocompatibility, and the pores also meet the characteristics of small pore size of the detection layer and large pore size of the treatment layer. In addition to providing a moist environment, reducing blood glucose concentration and promoting wound healing, it can also simultaneously realize rapid colorimetric detection of blood glucose at the wound, realize the dual functions of treatment and detection, and accelerate the healing process of diabetic wounds.

[0006] One of the purposes of the present invention is to provide a double-layer hydrogel dressing with dual functions, which is composed of an inner layer and an outer layer. The inner layer is a therapeutic layer that promotes diabetic wound healing, and the outer layer is a detection layer that monitors blood glucose. The pore size of the hydrogel in the inner therapeutic layer is more than 10 times that of the hydrogel in the outer detection layer, and the inner and outer layers are bonded together by physical and chemical means.

[0007] The double-layer hydrogel dressing with dual functions of the present invention has an inner layer that contacts the skin and an outer layer that is away from the skin.

[0008] The double-layer hydrogel dressing with dual functions of the present invention, wherein the treatment layer contains GOx@ZIF-8 which can react with blood sugar at the wound and perform antibacterial treatment, has the effect of promoting the healing of diabetic wounds.

[0009] The double-layer hydrogel dressing with dual functions of the present invention, wherein the detection layer contains peroxidase and a chromogenic substrate capable of producing color changes and chitosan with antibacterial effects, can achieve the blood sugar monitoring function while ensuring that the hydrogel in the treatment layer is not infected by bacteria and maintains air permeability.

[0010] Furthermore, the pore size of the inner therapeutic layer hydrogel is 250-300 μm.

[0011] Furthermore, the pore size of the outer detection layer hydrogel is 15 to 25 μm.

[0012] The double-layer hydrogel dressing with dual functions of the present invention, the inner therapeutic layer hydrogel is made of the following raw materials: GOx@ZIF-8, oxidized hyaluronic acid solution and carboxymethyl chitosan solution.

[0013] The double-layer hydrogel dressing with dual functions of the present invention comprises an outer detection layer hydrogel made of the following raw materials: polyvinyl alcohol solution, chitosan solution, peroxidase solution and color developing substrate solution.

[0014] The double-layer hydrogel dressing with dual functions of the present invention is chemically bonded by the inner and outer layers of hydrogels through the imine bonds formed by chemical reaction between the carboxyl groups provided by oxidized hyaluronic acid in the inner layer of hydrogel and the amino groups provided by chitosan in the outer layer of hydrogel.

[0015] In the double-layer hydrogel dressing with dual functions of the present invention, the physical mode of bonding the inner and outer layers of hydrogel is hydrogen bonding force.

[0016] The double-layer hydrogel dressing with dual functions of the present invention, the preparation method of the GOx@ZIF-8 is to 2+ The methanol solution mixed with glucose oxidase was added dropwise to the 2-methylimidazole aqueous solution under stirring conditions of 500-960 rpm, stirred for 5-30 min, centrifuged, washed three times with water and freeze-dried to obtain GOx@ZIF-8.

[0017] Furthermore, the Zn 2+ Provided by either zinc nitrate hexahydrate or zinc acetate dihydrate.

[0018] In the above technical solution, the Zn 2+ The concentration is 0.1~1mol / L.

[0019] Preferably, the Zn 2+ The concentration is 0.1mol / L.

[0020] In the above technical solution, the concentration of glucose oxidase is 0.1-10 mg / mL.

[0021] Preferably, the concentration of glucose oxidase is 1 mg / mL.

[0022] In the above technical solution, the concentration of 2-methylimidazole is 0.4-20 mol / L.

[0023] Preferably, the concentration of 2-methylimidazole is 0.4 mol / L.

[0024] In the above technical solution, the Zn 2+ The molar ratio of 2-methylimidazole to 1:4-16.

[0025] Preferably, the Zn 2+ The molar ratio of 2-methylimidazole to 1:8.

[0026] In the above technical solution, the Zn 2+ The preparation method of the methanol solution mixed with glucose oxidase is to take a certain amount of Zn 2+ and glucose oxidase were dissolved in methanol solution.

[0027] Another object of the present invention is to provide a method for preparing the above-mentioned double-layer hydrogel dressing with dual functions, comprising the following steps:

[0028] Step 1: polyvinyl alcohol solution and chitosan solution are mixed according to a mass ratio, and then peroxidase solution and color substrate solution are added thereto, mixed evenly, and subjected to freeze-thaw cycles to obtain a detection layer hydrogel;

[0029] Step 2: After mixing GOx@ZIF-8 with the oxidized hyaluronic acid solution, add the carboxymethyl chitosan solution and mix well to obtain a mixed solution for the treatment layer;

[0030] Step 3: Pour the mixed solution of the treatment layer quickly onto the detection layer hydrogel, place it at room temperature and then demold it to obtain a double-layer hydrogel dressing with dual functions.

[0031] In the method for preparing the hydrogel of the present invention, in step 1, the final concentration of the peroxidase is 0.1-0.5 mg / L.

[0032] In the method for preparing the hydrogel of the present invention, in step 1, the final concentration of the chromogenic substrate is 0.8-2 mmol / L.

[0033] In the method for preparing the hydrogel of the present invention, in step 1, the mass ratio of the polyvinyl alcohol solution to the chitosan solution is 2:3.

[0034] Furthermore, the mass concentration of the polyvinyl alcohol solution is 4-10%, and the molecular weight is 27000-98000Da.

[0035] Furthermore, the chitosan solution has a mass concentration of 3-8% and a molecular weight of 500,000-1,000,000 Da.

[0036] Furthermore, the polyvinyl alcohol solution is obtained by dissolving polyvinyl alcohol in pure water and stirring the solution at 90 to 100° C. and 100 to 500 rpm.

[0037] Furthermore, the chitosan solution is obtained by dissolving chitosan in an acetic acid aqueous solution, wherein the mass concentration of acetic acid in the acetic acid aqueous solution is 1-3%.

[0038] In the method for preparing the hydrogel of the present invention, in step 2, the mass concentration of the GOx@ZIF-8 is 1-5%.

[0039] In the method for preparing the hydrogel of the present invention, in step 2, the volume ratio of the oxidized hyaluronic acid solution to the carboxymethyl chitosan solution is 2:3.

[0040] Furthermore, the mass concentration of the oxidized hyaluronic acid solution is 2-6%.

[0041] Furthermore, the mass concentration of the carboxymethyl chitosan solution is 4-8%.

[0042] Furthermore, the carboxymethyl chitosan solution is obtained by dissolving carboxymethyl chitosan in PBS buffer solution.

[0043] Furthermore, the preparation process of the oxidized hyaluronic acid solution is as follows: at room temperature, 5 mL of 0.5 mmol / L sodium periodate (NaIO4) aqueous solution is added dropwise to a hyaluronic acid (HA) aqueous solution with a mass concentration of 1%, protected from light, and stirred at 100-500 rpm for 2-6 hours. After the reaction, 1-3 mL of ethylene glycol solution is added, and the reaction is continued in the dark for 1 hour, followed by dialysis and freeze-drying to obtain oxidized hyaluronic acid, which is then dissolved with a PBS buffer solution to obtain an oxidized hyaluronic acid solution.

[0044] In the above technical solution, the molar ratio of hyaluronic acid to sodium periodate is 1:1.

[0045] In the above technical solution, the molecular weight of the hyaluronic acid is 200,000 to 400,000 Da.

[0046] In the above technical solution, the dialysis process is to transfer the reacted solution into a dialysis bag (with a diameter of 44 mm and a molecular weight cut-off of 14 KDa) and dialyze it in pure water for 3 to 7 days.

[0047] In the above technical solution, the pH of the PBS buffer solution is 6 to 7.4, and the concentration is 0.01 to 0.1M.

[0048] The mixing methods described in the present invention all adopt stirring mixing, and are all carried out in a stirring device disclosed in the prior art, preferably a magnetic stirring device.

[0049] In the preparation method of the hydrogel of the present invention, in the step 1, the number of freeze-thaw cycles is 2 to 3 times, the freezing condition is freezing at -18°C for 8 to 12 hours, and the melting condition is thawing at room temperature for 2 to 4 hours.

[0050] In the method for preparing the hydrogel of the present invention, in step 1, the peroxidase is horseradish peroxidase (HRP).

[0051] In the method for preparing the hydrogel of the present invention, the color developing substrate is any one of 3,3',5,5'-tetramethylbenzidine (TMB), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and o-phenylenediamine (OPD).

[0052] In the method for preparing the hydrogel of the present invention, in step 3, the room temperature placement time is 1 to 8 hours.

[0053] Another object of the present invention is to use the above-mentioned double-layer hydrogel dressing with dual functions or the double-layer hydrogel dressing with dual functions prepared by the above-mentioned method in the preparation of materials for promoting diabetic wound healing.

[0054] The oxidized hyaluronic acid contained in the hydrogel of the inner treatment layer of the hydrogel dressing provides carboxyl groups, and the chitosan contained in the hydrogel of the outer detection layer provides amino groups, and the two combine to form an imine bond. The detection layer hydrogel contains a chromogenic substrate that can react with glucose to produce color changes to achieve blood sugar detection. At the same time, the presence of chitosan can ensure that the treatment layer is not contaminated and maintains breathability. The GOx@ZIF-8 nanocomposite contained in the inner treatment layer hydrogel can react with blood sugar at the wound and has an antibacterial effect, thereby achieving dual functions of detection and treatment.

[0055] The present invention uses immobilized enzyme technology to fix GOx to ZIF-8, forming a GOx@ZIF-8 nanocomposite and then integrating it into the treatment layer hydrogel. When the treatment layer hydrogel contacts the wound, the strong swelling ability enables the hydrogel to absorb the exudate. Under the action of GOx, the glucose at the wound reacts to produce gluconic acid, the glucose concentration around the wound decreases, and the environment becomes an acidic environment, thereby causing ZIF-8 to cleave and release the antibacterial ion Zn 2+ , and cooperate with carboxymethyl chitosan for antibacterial treatment. Using cascade enzymatic reactions, the double-layer hydrogel produces specific and responsive changes to different glucose concentrations, promoting wound healing.

[0056] The double-layer hydrogel can realize the functions of absorbing exudate and retaining moisture for a long time and preventing bacterial infection. Absorbing exudate requires the hydrogel to have a larger pore size, while preventing bacteria and retaining moisture for a long time requires the hydrogel to have a smaller pore size. The pore size of the treatment layer hydrogel of the double-layer hydrogel dressing prepared by the present invention is more than ten times that of the detection layer hydrogel, and the pores meet the characteristics of small pore size of the detection layer and large pore size of the treatment layer. The water vapor permeability of the double-layer hydrogel dressing prepared by the present invention is relatively low, and it has better water retention capacity, so that the wound is in a moist environment, which can better promote wound healing.

[0057] The present invention at least has the following beneficial effects:

[0058] 1. The double-layer hydrogel dressing constructed by the present invention has dual functions of treatment and detection. While consuming glucose at the wound and exerting antibacterial effects, it can quickly absorb wound exudate. Combined with the characteristics of the double-layer hydrogel that produces specific and responsive changes to different glucose concentrations, it can achieve visual rapid blood sugar detection in the body, and the detection time is only 20 minutes. The color changes corresponding to normal blood sugar and high blood sugar are observable to the naked eye and can be quickly identified. At the same time, treating wounds from the two aspects of lowering blood sugar and releasing antibacterial components can promote wound healing. There will be no infection problems when treating wounds, which makes it easier to improve patient compliance.

[0059] 2. The gel matrix used in the present invention has good biocompatibility. Polyvinyl alcohol has the properties of strong adhesion and good film-forming properties; chitosan is a natural alkaline polysaccharide with antibacterial properties and high biocompatibility, which gives the detection layer hydrogel good mechanical properties and antibacterial properties. Carboxymethyl chitosan can interfere with the bacterial wall to exert stronger antibacterial activity, and can also overcome the problem of low solubility of chitosan, and prepare the therapeutic layer hydrogel through imine bond cross-linking with oxidized hyaluronic acid. GOx@ZIF-8 loaded on the therapeutic layer hydrogel converts glucose in a high sugar environment into hydrogen peroxide with antibacterial effect, and reacts with Zn 2+ Together with carboxymethyl chitosan, it exerts antibacterial and anti-inflammatory effects. It can alleviate the problem of difficult wound healing caused by the high-sugar environment of diabetic wounds without adding additional antibacterial agents, and overcome the problem of drug resistance of traditional antibacterial agents. As a dynamic covalent bond, the imine bond combines the reversibility of non-covalent bonds and the stability of covalent bonds, so that the therapeutic layer hydrogel has good self-healing properties, swelling properties and pH responsiveness. At the same time, the synthesis of the imine bond is simple and efficient, which can make the double layers tightly bonded, thereby extending the service life of the hydrogel dressing. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 These are optical images of the double-layer hydrogel in Example 1 before and after stretching (the purple part is the inner layer of the hydrogel dyed with crystal violet, and the white transparent part is the outer layer of the hydrogel).

[0061] Figure 2 This is a scanning electron micrograph of the therapeutic layer hydrogel and GOx@ZIF-8 in Example 1.

[0062] Figure 3 This is a scanning electron microscope image of the detection layer hydrogel in Example 1.

[0063] Figure 4 This is a scanning electron microscope image of the cross section of the bifunctional double-layer hydrogel in Example 1.

[0064] Figure 5 This is a diagram of the swelling performance of the detection layer hydrogel, the treatment layer hydrogel and the bifunctional double-layer hydrogel in Example 1.

[0065] Figure 6 This is a diagram showing the moisturizing performance of the detection layer hydrogel, treatment layer hydrogel and bifunctional double-layer hydrogel in Example 1.

[0066] Figure 7 This is a graph of the rheological properties of the detection layer hydrogel, the treatment layer hydrogel and the bifunctional double-layer hydrogel in Example 1.

[0067] Figure 8 This is a comparison chart of the antibacterial properties of the bifunctional double-layer hydrogel in Example 1.

[0068] Fig. 9 The concentration curve and color development diagram of different concentrations of glucose collected for the bifunctional double-layer hydrogel in Example 1.

[0069] Fig.10 This is a diagram showing the effect of the bifunctional double-layer hydrogel in Example 1 when detecting rats with different blood glucose concentrations.

[0070] Fig.11 The figures are diagrams showing the effects of the bifunctional bilayer hydrogel in Example 1 on the wounds of diabetic rats, wherein (a) is a flowchart showing the wound healing of rats, (b) is a diagram showing the effects of the wounds of diabetic rats on days 0, 3, 7, 10 and 13 after treatment with each hydrogel, (c) is the result of the wound healing rate, and (d) is a diagram showing the animal wound healing model on days 0, 3, 7, 10 and 13 after treatment with each hydrogel. DETAILED DESCRIPTION

[0071] The following non-limiting embodiments may enable a person skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0072] The test methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0073] Example 1

[0074] A preparation method of a double-layer hydrogel dressing with dual functions:

[0075] Step 1: Take a certain amount of PVA and add it to pure water, stir and dissolve it at 90°C and 400rpm to prepare a PVA solution with a mass concentration of 5%; take a certain amount of CS and dissolve it in 1% acetic acid aqueous solution to prepare a CS solution with a mass concentration of 3%; mix the above-obtained PVA solution and CS solution in a mass ratio of 2:3; then add horseradish peroxidase (HRP) solution and 3,3',5,5'-tetramethylbenzidine (TMB) solution to make their final concentrations of 0.1 mg / L and 0.8 mmol / L respectively; mix the above solutions evenly, freeze them at -18°C for 12 hours, thaw them at room temperature for 3 hours, and repeat 3 times to obtain a detection layer hydrogel.

[0076] Step 2: Add 1g HA to a single-necked flask, add 100mL water to completely dissolve, add 5mL NaIO4 aqueous solution (0.5mmol / L) at room temperature, protect from light, stir at 300rpm for 3h, add 1mL ethylene glycol to the reaction solution, continue to react for 1h in the dark, dialyze for 72h and freeze-dry to obtain OHA. Dissolve 0.1mol / L zinc acetate dihydrate and 2mg GOx in 2mL methanol, add it dropwise to 4mL 2-methylimidazole aqueous solution (concentration 0.4mol / L) under stirring at 960rpm, continue to stir vigorously at 960rpm for 5min, centrifuge and wash three times, and freeze-dry to obtain GOx@ZIF-8. OHA and CMCS were dissolved in PBS buffer solution (pH 7.4) to obtain 2% OHA solution and 4% CMCS solution; 5 mg GOx@ZIF-8 was mixed with 0.4 mL of 2% OHA solution, and then 0.6 mL of 4% CMCS solution was added and mixed evenly to obtain a therapeutic layer hydrogel.

[0077] Step 3: Pour the treatment layer hydrogel quickly onto the detection layer hydrogel, place at room temperature for 2 hours to gel, and demold to obtain a double-layer hydrogel dressing with dual functions.

[0078] Performance test of a double-layer hydrogel dressing with dual functions (taking the above-prepared double-layer hydrogel with dual functions as an example):

[0079] Figure 1 These are optical images of the double-layer hydrogel before and after stretching. It can be seen that under stretching conditions, the inner layer hydrogel stained with crystal violet (dyed for easy observation) and the transparent outer layer hydrogel show strong binding force at the interface, and the double-layer hydrogel interface is closely bonded. Figures 2 to 4 The scanning electron microscope images of the cross-sections of the treatment layer hydrogel and GOx@ZIF-8 (GZ), the detection layer hydrogel, and the bifunctional double-layer hydrogel show that: the detection layer hydrogel in the double-layer hydrogel dressing has a small pore size and a dense structure, while the treatment layer hydrogel has a large pore size and a loose structure. The two layers of hydrogel are tightly connected by an imine bond. The pores of the treatment layer hydrogel are about 250-300μm, and the particle size of GOx@ZIF-8 is about 500nm, which is evenly distributed on the treatment layer hydrogel; the pores of the detection layer hydrogel are about 15-25μm.

[0080] The swelling performance evaluation method includes: freeze-drying the hydrogel prepared above to obtain a freeze-dried hydrogel, weighing a certain amount of the freeze-dried hydrogel, and recording its weight (W0, g). Next, the hydrogel is immersed in a PBS buffer solution (pH 7.4, concentration 0.01M). At each set time point 0, 0.5, 1, 2, 4, 6, 8, 12, 24h, the hydrogel is taken out of the PBS buffer solution, the surface moisture is absorbed with filter paper, and the weight is immediately weighed (W t ,g), calculate the swelling ratio (SR), the calculation formula is as follows:

[0081]

[0082] The moisturizing performance evaluation method includes: weighing a sample bottle containing pure water (W1, g), and sealing the bottle mouth (diameter 12 mm, area S) with a sheet of hydrogel (about 4 mm thick). Then put the sealed sample bottle in an environment with a test temperature of 37°C for 24 hours (t), take it out immediately after the test, remove the hydrogel sample, weigh it (W2, g), and calculate the water vapor transmission rate (WVTR). The calculation formula is as follows:

[0083]

[0084] Figure 5 The swelling performance diagrams of the detection layer hydrogel (CP hydrogel), the treatment layer hydrogel (GZ-OC hydrogel) and the dual-functional double-layer hydrogel (GZ-OCP hydrogel) are shown in Figure 2. Figure 6 This is a diagram of the moisturizing performance of the detection layer hydrogel (CP hydrogel), the treatment layer hydrogel (GZ-OC hydrogel) and the dual-functional double-layer hydrogel (GZ-OCP hydrogel). It can be seen that the swelling performance of the treatment layer is significantly higher than that of the detection layer, which also allows the treatment layer hydrogel to absorb a large amount of exudate in a short time. The difference in swelling rate between the inner and outer layer hydrogels can enable the hydrogel to be quickly extracted and the outer layer hydrogel to be colored. Compared with the treatment layer, the water vapor permeability of the detection layer and the double-layer hydrogel dressing is relatively low, indicating that the double-layer hydrogel dressing has better water retention capacity, can provide a moist environment for the wound, and is conducive to wound healing.

[0085] The rheological property evaluation method includes: placing the hydrogel on a 20 mm parallel plate, and performing rheological analysis on the hydrogel using a rheometer. The analysis conditions are: at 32°C and a constant strain of 6%, the storage modulus (G') and loss modulus (G") of the hydrogel are evaluated by frequency scanning (scanning angular frequency is 0.1-100 rad / s). Figure 7The rheological properties of the detection layer hydrogel (CP hydrogel), the treatment layer hydrogel (GZ-OC hydrogel) and the dual-functional double-layer hydrogel (GZ-OCP hydrogel) are shown in Figure 2. It can be seen that G' always exceeds G" in the entire frequency range, which means that all hydrogels are stable and mainly exhibit elastic properties; the G' of all samples rises slowly, indicating that each hydrogel can withstand pressure.

[0086] The antibacterial performance evaluation method was a co-culture method, which included: the sample hydrogel was made into cylinders of equal volume (1 mL) in a 24-well plate, and after ultraviolet disinfection for 1 h, the prepared bacterial solution with a concentration of 1×10 6 200 μL of CFU / mL bacterial suspension of Escherichia coli and Staphylococcus aureus was added with 50 μL of glucose solution (20 mM) and co-cultured at 37°C and 100 rpm in a shaker. 1 mL of MH broth liquid culture medium was added to mix each of the above cultured bacterial suspensions, and 100 μL was spread on the plate. The plate was inverted and cultured in a 37°C constant temperature incubator for 18 hours, and the growth of the colonies on the plate was recorded. The antibacterial activity of the hydrogel was evaluated by comparing with the positive control (containing only bacterial solution and liquid culture medium). Figure 8 The figure is a comparison of the antibacterial properties of the bifunctional double-layer hydrogel. It can be seen that the hydrogel dressing has the ability to inhibit the growth of Escherichia coli and Staphylococcus aureus. Compared with the positive control, the blank inner layer hydrogel (OC hydrogel, the raw material does not contain GOx@ZIF-8) has a certain antibacterial effect, and the antibacterial effect of the blank double-layer hydrogel (OCP hydrogel, the raw material does not contain GOx@ZIF-8) is improved. It is speculated that the main reason is that the chitosan in the detection layer hydrogel and the carboxymethyl chitosan in the treatment layer are antibacterial together. After adding GOx@ZIF-8, the antibacterial properties of the treatment layer hydrogel (GZ-OC hydrogel) and the bifunctional double-layer hydrogel (GZ-OCP hydrogel) against Escherichia coli (E.Coil) and Staphylococcus aureus (S.aureus) are enhanced, showing stronger antibacterial properties.

[0087] Simulated application of a dual-functional double-layer hydrogel dressing on the skin:

[0088] The double-layer hydrogel dressing obtained above was placed on a plastic cover with glucose solution of concentrations of 0, 5, 8, 10, 15, and 20 mM in sequence, so that the treatment layer hydrogel was in contact with the surface of the glucose solution without overflowing the solution. It was left for a period of time. After different time points, the hydrogel was removed, and color photos were taken with a smartphone at the same position and light, and RGB analysis was performed. The results showed that the color distribution was uniform after 20 minutes of placement. Fig. 9 The concentration curve of different glucose concentrations and the color development diagram after 20 minutes of storage were collected for the above-obtained bifunctional double-layer hydrogel. It can be seen that the RGB chromaticity value is linearly related to the concentration of the glucose solution.

[0089] Application of a dual-functional double-layer hydrogel dressing in diabetic wounds:

[0090] The bifunctional double-layer hydrogel dressing obtained above was applied to the wound models of diabetic rats and normal rats. After 20 minutes, the hydrogel was removed, and color photos were taken under the same light using a smartphone, and RGB analysis was performed. Fig.10 This is the effect diagram of the dual-functional double-layer hydrogel dressing when testing rats with different blood sugar concentrations. It can be seen that the double-layer hydrogel dressing is also glucose responsive in the rat model. Under different blood sugar concentrations, the color development and RGB chromaticity values ​​are significantly different, and the experimental trends in in vivo and in vitro color development are consistent.

[0091] The obtained double-layer hydrogel is applied to treat diabetic wounds. Fig.11 (a) Flow chart of wound healing in rats. Fig.11 (b) is the effect diagram of the wound of diabetic rats on days 0, 3, 7, 10 and 13 after treatment with each hydrogel. Fig.11 (c) is the wound healing rate result, Fig.11 (d) is a diagram of the animal wound healing model at 0, 3, 7, 10 and 13 days after treatment with each hydrogel. It can be seen that compared with the control group without GOx@ZIF-8, all treatment groups showed the effect of promoting wound healing. It can be seen that the treatment layer hydrogel containing GOx@ZIF-8 and the bifunctional double-layer hydrogel group have better treatment effects and higher wound closure rates. On the 10th day, the wound closure area of ​​rats treated with bifunctional double-layer hydrogels reached 91.8%. The main reason is that the hydrogel prepared by this method can reduce the blood sugar level at the wound while performing antibacterial treatment, promoting wound healing from two aspects. At the same time, compared with the single-layer treatment layer hydrogel group, the double-layer hydrogel has better water retention and can better maintain the moisture level of the wound, making the wound heal faster and better.

Claims

1. A double-layer hydrogel dressing with dual functions, characterized in that: It consists of an inner layer and an outer layer, the inner layer is a therapeutic layer that promotes diabetic wound healing, and the outer layer is a detection layer that monitors blood sugar; The pore size of the inner treatment layer hydrogel is more than 10 times that of the outer detection layer hydrogel, and the inner and outer layers are bonded together by physical and chemical means.

2. The double-layer hydrogel dressing with dual functions according to claim 1, characterized in that: The pore size of the inner treatment layer hydrogel is 250-300 μm, and the pore size of the outer detection layer hydrogel is 15-25 μm.

3. The double-layer hydrogel dressing with dual functions according to claim 1, characterized in that: The inner therapeutic layer hydrogel is prepared from the following raw materials: GOx@ZIF-8, oxidized hyaluronic acid solution and carboxymethyl chitosan solution; The outer detection layer hydrogel is prepared from the following raw materials: polyvinyl alcohol solution, chitosan solution, peroxidase solution and color development substrate solution.

4. The double-layer hydrogel dressing with dual functions according to claim 1, characterized in that: The inner and outer hydrogel layers are adhered chemically by imine bonds formed by chemical reaction between carboxyl groups provided by oxidized hyaluronic acid in the inner hydrogel and amino groups provided by chitosan in the outer hydrogel, and are tightly adhered by hydrogen bonding.

5. The double-layer hydrogel dressing with dual functions according to claim 1 or 3, characterized in that: The preparation method of GOx@ZIF-8 is to add Zn 2+ and glucose oxidase mixed methanol solution is added dropwise to the 2-methylimidazole aqueous solution under stirring conditions of 500-960 rpm, and the stirring is continued for 5-30 minutes, then centrifuged, washed with water three times, and then freeze-dried to obtain, wherein, Zn 2+ Provided by either zinc nitrate hexahydrate or zinc acetate dihydrate, Zn 2+ The concentration is 0.1~1mol / L, the glucose oxidase concentration is 0.1~10mg / mL, the 2-methylimidazole concentration is 0.4~20mol / L, and the Zn 2+ The molar ratio of 2-methylimidazole to 1:4-16.

6. The method for preparing the double-layer hydrogel dressing with dual functions according to any one of claims 1 to 5, characterized in that: The steps include: Step 1: polyvinyl alcohol solution and chitosan solution are mixed according to a mass ratio, and then peroxidase solution and color substrate solution are added thereto, mixed evenly, and subjected to freeze-thaw cycles to obtain a detection layer hydrogel; Step 2: After mixing GOx@ZIF-8 with the oxidized hyaluronic acid solution, add the carboxymethyl chitosan solution and mix well to obtain a mixed solution for the treatment layer; Step 3: Pour the mixed solution of the treatment layer quickly onto the detection layer hydrogel, place it at room temperature and then demold it to obtain a double-layer hydrogel dressing with dual functions.

7. The method for preparing a double-layer hydrogel dressing with dual functions according to claim 6, characterized in that: In step 1, the final concentration of the peroxidase is 0.1-0.5 mg / L; the final concentration of the chromogenic substrate is 0.8-2 mmol / L; the mass ratio of the polyvinyl alcohol solution to the chitosan solution is 2:3, wherein, The mass concentration of the polyvinyl alcohol solution is 4-10%, the molecular weight is 27000-98000Da, the mass concentration of the chitosan solution is 3-8%, and the molecular weight is 500,000-1,000,000Da; In step 2, the mass concentration of GOx@ZIF-8 is 1-5%, and the volume ratio of oxidized hyaluronic acid solution to carboxymethyl chitosan solution is 2:3, wherein, The mass concentration of the oxidized hyaluronic acid is 2-6%, the molecular weight of the raw material hyaluronic acid is 200,000-400,000 Da, and the mass concentration of the carboxymethyl chitosan solution is 4-8%.

8. The method for preparing a double-layer hydrogel dressing with dual functions according to claim 6, characterized in that: In step 1, the number of freeze-thaw cycles is 2 to 3 times, the freezing condition is freezing at -18°C for 8 to 12 hours, and the melting condition is thawing at room temperature for 2 to 4 hours.

9. The method for preparing a double-layer hydrogel dressing with dual functions according to claim 6 or 7, characterized in that: In the step 1, the peroxidase is horseradish peroxidase, and the color developing substrate is any one of 3,3',5,5'-tetramethylbenzidine, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, and o-phenylenediamine.

10. Use of the double-layer hydrogel dressing with dual functions according to any one of claims 1 to 5 or the double-layer hydrogel dressing with dual functions prepared by the method according to any one of claims 6 to 9 in the preparation of materials for promoting diabetic wound healing.

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