Antibacterial hydrogel dressing for diabetic wounds and preparation method thereof

Abstract

The present invention relates to an antibacterial hydrogel dressing for diabetic wounds and a preparation method thereof, and relates to the field of hydrogel dressings. Sodium alginate is used as a carrier material, and attapulgite is used to improve the mechanical properties of the gel through the principle of fiber toughening, and the loading amount of glucose oxidase can be increased. The glucose oxidase can consume glucose at the wound in situ and generate hydrogen peroxide. The prepared hydrogel dressing can not only effectively consume glucose and alleviate the high-sugar environment of diabetic wounds, but also generate hydrogen peroxide. It can overcome the problem of difficult wound healing caused by the high-sugar environment of diabetes without adding additional antibacterial agents, and also overcome the problems of drug resistance and cytotoxicity of traditional antibacterial gels; the combination of calcium carbonate and GDL described in the present invention can effectively control Ca 2+ The formation rate of sodium alginate-based composite hydrogel is enhanced, which enhances the mechanical properties, drug loading properties and sustained release properties.

Description

Technical Field

[0001] The invention relates to the field of hydrogel dressings, and in particular to an antibacterial hydrogel dressing for diabetic wounds and a preparation method thereof. Background Art

[0002] Diabetic wounds refer to wounds that are difficult to heal or slowly heal on the body of diabetic patients. They are one of the complications of diabetes. Excessive local blood sugar concentration at the wound site can lead to blood vessel blockage, obstruction of oxygen and nutrient circulation, damage to the patient's immune system, low immune function, inhibition of immune cell function, and difficulty in resisting external bacterial infection. At the same time, due to peripheral nerve damage, patients cannot perceive wound pain and infection, and cannot deal with it in time, aggravating wound infection. The difficulty of diabetic wounds to heal will lead to long-term exposure of the wound, persistent bacterial infection, and further deterioration of the wound.

[0003] Alginate (SA) can form a three-dimensional network structure with metal ions through ion cross-linking. This network structure makes alginate gel have good mechanical properties and stability, and also gives it unique physical and chemical properties. In addition, the surface of alginate molecules contains a large number of hydrophilic groups, which have excellent properties such as high hygroscopicity, oxygen permeability, cell adhesion and biocompatibility. It is the most commonly used hydrogel material and also a good carrier material. Chinese patent CN 106668870 A uses the electrostatic attraction between the carboxyl group in the molecular structure of sodium alginate and the amino group in the pyrrole monomer, and uses the ion exchange between sodium alginate and calcium ions to form a gel, load the drug into the sodium alginate gel, and achieve sustained release of the drug.

[0004] Attapulgite (APT) is a crystalline hydrated magnesium aluminum silicate mineral, whose chemical components mainly include MgO, Al2O3, and SiO2. These components form a unique layered chain structure of attapulgite in a certain proportion in a specific environment. Because attapulgite has a special fibrous structure and contains active groups and bonding points on the surface, it can be used to improve the mechanical properties of mechanical gels. Chinese patent CN 112315976 A prepared an injectable antibacterial composite hydrogel cross-linked with silver-loaded attapulgite, in which the attapulgite can not only increase the loading amount of nanosilver, but also effectively enhance the mechanical properties of the gel.

[0005] Glucose oxidase (GOD) is an enzyme protein, and its molecular structure is usually composed of four identical protein subunits, each of which contains an auxiliary pigment molecule FAD and a tetraphosphate molecule. Glucose oxidase can catalyze the oxidation reaction of glucose, consume oxygen, and obtain hydrogen peroxide and gluconolactone. The generated gluconolactone is harmless to the human body, while the generated hydrogen peroxide has a sterilizing effect. Chinese patent CN 115212316 A once loaded glucose oxidase into a thermosensitive hydrogel to reduce blood sugar levels in the body.

[0006] At present, the research on diabetic wounds is mainly focused on alleviating various complications such as excessive inflammation, oxidative stress, and vascular occlusion caused by high-sugar environments. Almost all of the research attaches great importance to preventing wound infection. There are two main ways to achieve antibacterial function, loading antibacterial substances and selecting materials that have antibacterial functions themselves. Loading antibacterial substances is a more common antibacterial method. In recent years, due to the abuse of antibiotics, bacteria have developed drug resistance, and other substances with antibacterial functions have become the current research hotspot. However, for diabetic patients, the high-sugar environment at the wound is the root cause of the difficulty in wound healing. Current research is limited to dealing with diabetic wound complications, and has not solved the problem of difficulty in wound healing caused by high-sugar environments. Therefore, it is urgent to develop a gel that can alleviate the high-sugar environment of diabetic wounds and has antibacterial functions. Summary of the invention

[0007] The present invention aims to provide an antibacterial hydrogel dressing for diabetic wounds and a preparation method thereof, so as to solve the above problems.

[0008] The invention also provides an antibacterial hydrogel dressing for diabetic wounds.

[0009] The present invention is achieved through the following technical solutions:

[0010] A method for preparing an antibacterial hydrogel dressing for diabetic wounds comprises the following steps:

[0011] (1) adding sodium alginate powder into a container containing deionized water under stirring conditions until the sodium alginate is completely dispersed and dissolved to obtain a uniform and transparent sodium alginate solution;

[0012] (2) adding gluconolactone GDL to the sodium alginate solution under stirring to completely dissolve it, and continuing stirring for 5 minutes;

[0013] (3) dissolving glucose oxidase in a phosphate buffer solution to obtain a GOD-PBS solution, and adding the GOD-PBS solution into the container under stirring conditions, and continuing to stir for 5 minutes;

[0014] (4) weighing attapulgite powder, adding the attapulgite powder to deionized water under stirring and ultrasonic conditions to obtain an attapulgite suspension, and adding the attapulgite suspension to the container under stirring conditions;

[0015] (5) Weighing CaCO3, and dispersing the CaCO3 in deionized water under stirring and ultrasonic conditions to obtain a CaCO3 suspension;

[0016] (6) adding the CaCO3 suspension dropwise into the container under stirring conditions, and after the addition is completed, stirring is continued for 40 seconds and then stopped;

[0017] (7) The container is sealed and allowed to stand at room temperature to allow the system to gradually gel, thereby preparing an antibacterial hydrogel dressing for diabetic wounds.

[0018] The GDL solution can slowly release calcium ions from calcium carbonate, thereby initiating gelation and obtaining the desired alginate hydrogel. The addition of GDL leads to the formation of gluconic acid, which reacts with water to form H3O + , H3O + The increase in calcium carbonate leads to the slow decomposition of CO2 and Ca 2+ , thus Ca 2+ The combination of calcium carbonate and GDL can effectively control the Ca release compared with other divalent cation releasing compounds. 2+ The formation rate of calcium carbonate is slowly released in the gluconolactone solution. 2+ Sodium alginate hydrogel is formed in situ, and the formed gel structure is uniform and stable, which enhances the mechanical properties, drug loading properties and sustained release properties of the sodium alginate-based composite hydrogel.

[0019] Furthermore, the mass fraction of sodium alginate in the sodium alginate solution in step (1) is 1 wt% to 5 wt%, preferably 2 wt%; and the container is a beaker, a flask or a reactor.

[0020] Furthermore, in order to maintain the pH value of the system in step (2), the mass ratio of sodium alginate to GDL is (2-3):1, preferably sodium alginate:GDL=2.5:1.

[0021] Furthermore, the content of glucose oxidase in step (3) is 50 to 400 activity units per 40 mL of sodium alginate solution, preferably 300 activity units.

[0022] It should be noted that glucose oxidase, as an aerobic dehydrogenase, cannot be secreted in the digestive tract of animals. It can specifically oxidize and decompose β-D-glucose while consuming a large amount of oxygen. The definition of its activity unit helps to accurately measure and control the activity of glucose oxidase, thereby ensuring its effective application. The activity unit of glucose oxidase is defined as: the amount of enzyme required to form 1 μmol of hydrogen peroxide per minute at 37°C and pH 5.7. This unit is often used to quantify the rate and efficiency of glucose oxidase in oxidizing glucose into gluconic acid and hydrogen peroxide.

[0023] Furthermore, the mass of the attapulgite powder added in step (4) is 1 wt% to 20 wt% of the mass of the sodium alginate powder, preferably 5 wt% to 10 wt%.

[0024] Furthermore, after the CaCO3 suspension is added in step (6), the Ca 2+ The final concentration is maintained at 0.01M to 0.03M, preferably 0.0158M.

[0025] Furthermore, in the method for preparing the antibacterial hydrogel dressing, the temperature of the reaction system in steps (2) to (6) is always maintained at 35° C. to 40° C., preferably 37° C.

[0026] Furthermore, the CaCO3 described in the present invention is one or more of calcium carbonate powder, microporous calcium carbonate or hollow calcium carbonate; the porous structure of microporous calcium carbonate can increase the specific surface area in contact with the GDL solution, thereby increasing the CaCO3 content. 2+ The release rate of calcium alginate gel can be accelerated; the cavities inside the hollow calcium carbonate can also increase its specific surface area in contact with GDL to a certain extent. At the same time, due to its unique hollow structure, the specific surface area of ​​calcium carbonate will not change significantly with the consumption of the reaction, which can ensure that Ca 2+ The release is maintained at a constant rate, which is more conducive to obtaining a gel with uniform and stable structure, thereby enhancing the mechanical properties, drug loading properties and sustained-release stability of the sodium alginate-based composite hydrogel.

[0027] The preparation method of the microporous calcium carbonate is:

[0028] Sa1. Add sodium polystyrene sulfonate to the Na2CO3 solution and mix well;

[0029] Sa2, quickly add CaCl2 solution to Na2CO3 solution, stir and precipitate;

[0030] Sa3, centrifuge, wash with acetone and distilled water to remove sodium polystyrene sulfonate, filter and dry to obtain microporous calcium carbonate.

[0031] Furthermore, the concentration of Na2CO3 in the Na2CO3 solution in step Sa1 is 0.02M to 0.2M, and the amount of sodium polystyrene sulfonate added is such that the concentration of sodium polystyrene sulfonate in the Na2CO3 solution is 1g / L to 10g / L;

[0032] Further, the CaCl2 concentration in the CaCl2 solution in step Sa2 is 0.02M-0.2M; the volume ratio of the Na2CO3 solution to the CaCl2 solution is (0.2-5):1; the stirring rate is 100rpm-500rpm, the stirring time is 0.5h-2h, and the stirring temperature is 5°C-10°C;

[0033] Furthermore, the centrifugal speed in step Sa3 is 3500-4500 r / min; and the centrifugal time is 5-20 min.

[0034] The preparation method of the hollow calcium carbonate is:

[0035] Sb1, respectively adding polyacrylic acid (PAA) solution and sodium dodecyl sulfate (SDS) solution into Na2CO3 solution, and mixing them evenly to obtain solution A;

[0036] Sb2, add the PAA solution into the CaCl2 solution, mix well to obtain solution B;

[0037] Sb3. Quickly pour solution B into solution A, and carry out precipitation reaction under stirring. After the reaction is completed, filter and rinse with ethanol and distilled water to remove PAA, and dry to obtain hollow calcium carbonate.

[0038] Furthermore, in the solution A in step Sb1, the final Na2CO3 concentration is 0.02M to 0.2M, the PAA concentration is 0.2g / L to 2.0g / L, and the SDS concentration is 1g / L to 10g / L;

[0039] Further, in step Sb2, the final CaCl2 concentration in solution B is 0.02M to 0.2M, and the PAA concentration is 0.2g / L to 2.0g / L;

[0040] Furthermore, the stirring rate in step Sb3 is 100 rpm to 500 rpm, the precipitation reaction time is 0.5 h to 2 h; the precipitation reaction temperature is 5° C. to 10° C.; and the volume ratio of solution A to solution B is (0.2 to 5):1.

[0041] The present invention also provides an antibacterial hydrogel dressing prepared by the method for preparing the antibacterial hydrogel dressing for diabetic wounds.

[0042] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0043] 1. The present invention uses sodium alginate as a carrier material. Since attapulgite has a special fiber structure and lamellar structure, the mechanical properties of the gel can be improved through the principle of fiber toughening. At the same time, the attapulgite contains elements such as Si, Mg, and Al, and most of them exist in the form of ions, which can assist in the formation of the gel network structure and enhance the mechanical properties of the gel to a certain extent. It has the advantages of simple preparation process and low cost.

[0044] 2. The attapulgite used in the present invention has a hollow fiber structure and contains a large number of active groups on the surface, which can load glucose oxidase and make it uniformly dispersed in the gel. Glucose oxidase can consume glucose at the wound in situ and generate hydrogen peroxide. The active groups in the attapulgite can load and fix glucose oxidase through interactions such as hydrogen bonds and chemical bonds, thereby increasing the loading amount of glucose oxidase while preventing it from entering the blood in large quantities. A small amount of free glucose oxidase entering the blood can appropriately reduce the blood sugar content of diabetics, which will not only not harm human health, but is more beneficial. The prepared hydrogel dressing can not only effectively consume glucose and alleviate the high-sugar environment of diabetic wounds, but also generate hydrogen peroxide with excellent antibacterial properties. It does not need to add additional antibacterial agents to overcome the problem of difficult wound healing caused by the high-sugar environment of diabetes, but also can play an anti-inflammatory and antibacterial role through hydrogen peroxide, and also overcomes the problems of drug resistance and cytotoxicity of traditional antibacterial gels.

[0045] 3. Compared with other divalent cation releasing compounds, the combination of calcium carbonate and GDL described in the present invention can effectively control Ca 2+ The formation rate of alginate hydrogel can be regulated to enhance the mechanical properties, drug loading properties and sustained release properties of sodium alginate-based composite hydrogel, and the formed gel is uniform, thus expanding the application range of sodium alginate as a drug carrier, dressing and tissue engineering material.

[0046] 4. The present invention further provides a microporous calcium carbonate and a hollow calcium carbonate. Compared with the hydrogel dressing made from ordinary calcium carbonate powder, the antibacterial hydrogel dressing prepared with the microporous calcium carbonate and the hollow calcium carbonate has a more uniform and stable structure and better mechanical properties, drug loading properties, sustained-release properties and antibacterial effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, constitute a part of this application, and do not constitute a limitation of the embodiments of the present invention. In the drawings:

[0048] Figure 1 This is a scanning electron microscope image of the hydrogel dressing prepared in Example 1;

[0049] Figure 2 The mechanical properties test diagram of the hydrogel dressings prepared in Examples 1-3 and Comparative Example 1;

[0050] Figure 3 The figures are the results of in vitro antibacterial experiments of the hydrogel dressings prepared in Example 1 and Examples 4-5. DETAILED DESCRIPTION

[0051] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments and drawings. The schematic implementation mode of the present invention and its description are only used to explain the present invention and are not intended to limit the present invention. The embodiments described below are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0052] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0053] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0054] The terms "include", "comprising", "having", "containing", etc. used herein are all open-ended terms, which mean including but not limited to. The CaCO3 powders described in the following examples are all common commercially available calcium carbonate powders (brand number JS0033).

[0055] Example 1

[0056] (1) Weigh 0.8 g of sodium alginate powder and add it to a beaker containing 40 ml of deionized water. Stir in a 37°C water bath until the sodium alginate is completely dissolved to obtain a uniform and transparent sodium alginate solution.

[0057] (2) Add 0.35 g of GDL to the sodium alginate solution, dissolve it completely, and continue stirring for 5 minutes;

[0058] (3) Dissolve glucose oxidase in PBS solution to obtain GOD-PBS solution, and add the GOD-PBS solution containing 300 units of glucose oxidase to the sodium alginate solution, and continue stirring for 5 minutes;

[0059] (4) adding 0.4 g of attapulgite powder to 20 mL of deionized water under ultrasonic conditions to obtain an attapulgite suspension, and adding 2 mL of the attapulgite suspension to the beaker under stirring conditions, and continuing to stir for 5 minutes;

[0060] (5) Weigh 2.75 g of CaCO3 powder and disperse it in 100 mL of deionized water under stirring and ultrasonic conditions to obtain a CaCO3 suspension;

[0061] (6) Add 2.5 mL of CaCO suspension to the beaker under stirring. After the addition is completed, continue stirring for 40 seconds and then stop;

[0062] (7) The beaker is sealed and allowed to stand at room temperature to allow the system to gradually gel, thereby preparing an antibacterial hydrogel dressing for diabetic wounds.

[0063] The antibacterial hydrogel material prepared in this example was observed under a scanning electron microscope. Figure 1 As shown, it can be seen that the antibacterial hydrogel dressing prepared in this example has a porous structure and can load glucose oxidase.

[0064] Example 2

[0065] (1) Weigh 0.8 g of sodium alginate powder and add it to a beaker containing 40 ml of deionized water. Stir in a 37°C water bath until the sodium alginate is completely dissolved to obtain a uniform and transparent sodium alginate solution.

[0066] (2) Add 0.35 g of GDL to the sodium alginate solution, dissolve it completely, and continue stirring for 5 minutes;

[0067] (3) Dissolve glucose oxidase in PBS solution to obtain GOD-PBS solution, and add the GOD-PBS solution containing 300 units of glucose oxidase to the sodium alginate solution, and continue stirring for 5 minutes;

[0068] (4) adding 0.4 g of attapulgite powder to 20 mL of deionized water under ultrasonic conditions to obtain an attapulgite suspension, and adding 4 mL of the attapulgite suspension to the beaker under stirring conditions, and continuing to stir for 5 minutes;

[0069] (5) Weigh 2.75 g of CaCO3 powder and disperse it in 100 mL of deionized water under stirring and ultrasonic conditions to obtain a CaCO3 suspension;

[0070] (6) Add 2.5 mL of CaCO3 suspension to the beaker under stirring conditions. After the addition is completed, continue stirring for 40 seconds and then stop;

[0071] (7) The beaker is sealed and allowed to stand at room temperature to allow the system to gradually gel, thereby preparing an antibacterial hydrogel dressing for diabetic wounds.

[0072] Example 3

[0073] (1) Weigh 0.8 g of sodium alginate powder and add it to a beaker containing 40 ml of deionized water. Stir in a 37°C water bath until the sodium alginate is completely dissolved to obtain a uniform and transparent sodium alginate solution.

[0074] (2) Add 0.35 g of GDL to the sodium alginate solution, dissolve it completely, and continue stirring for 5 minutes;

[0075] (3) Dissolve glucose oxidase in PBS solution to obtain GOD-PBS solution, and add the GOD-PBS solution containing 300 units of glucose oxidase to the sodium alginate solution, and continue stirring for 5 minutes;

[0076] (4) adding 0.4 g of attapulgite powder to 20 mL of deionized water under ultrasonic conditions to obtain an attapulgite suspension, and adding 8 mL of the attapulgite suspension to the beaker under stirring conditions, and continuing to stir for 5 minutes;

[0077] (5) Weigh 2.75 g of CaCO3 powder and disperse it in 100 mL of deionized water under stirring and ultrasonic conditions to obtain a CaCO3 suspension;

[0078] (6) Add 2.5 mL of CaCO3 suspension to the beaker under stirring conditions. After the addition is completed, continue stirring for 40 seconds and then stop;

[0079] (7) The beaker is sealed and allowed to stand at room temperature to allow the system to gradually gel, thereby preparing an antibacterial hydrogel dressing for diabetic wounds.

[0080] Example 4

[0081] (1) Weigh 0.8 g of sodium alginate powder and add it to a beaker containing 40 ml of deionized water. Stir in a 37°C water bath until the sodium alginate is completely dissolved to obtain a uniform and transparent sodium alginate solution.

[0082] (2) Add 0.35 g of GDL to the sodium alginate solution, dissolve it completely, and continue stirring for 5 minutes;

[0083] (3) Dissolve glucose oxidase in PBS solution to obtain GOD-PBS solution, and add the GOD-PBS solution containing 300 units of glucose oxidase to the sodium alginate solution, and continue stirring for 5 minutes;

[0084] (4) adding 0.4 g of attapulgite powder to 20 mL of deionized water under ultrasonic conditions to obtain an attapulgite suspension, and adding 2 mL of the attapulgite suspension to the beaker under stirring conditions, and continuing to stir for 5 minutes;

[0085] (5) Weigh 2.75 g of microporous calcium carbonate and disperse it in 100 mL of deionized water under stirring and ultrasonic conditions to obtain a CaCO3 suspension;

[0086] (6) Add 2.5 mL of CaCO3 suspension to the beaker under stirring conditions. After the addition is completed, continue stirring for 40 seconds and then stop;

[0087] (7) The beaker is sealed and allowed to stand at room temperature to allow the system to gradually gel, thereby preparing an antibacterial hydrogel dressing for diabetic wounds.

[0088] The preparation method of the microporous calcium carbonate is:

[0089] Sa1. Add 0.25g sodium polystyrene sulfonate to 100ml 0.1M Na2CO3 solution and mix well.

[0090] Sa2. Quickly add 100 ml of 0.1 M CaCl2 solution to the above Na2CO3 solution, and stir and precipitate at 300 rpm at 8°C for 1 hour;

[0091] Sa3, centrifuge at 4000r / min for 10min, wash with acetone and distilled water to remove sodium polystyrene sulfonate, filter and dry to obtain microporous calcium carbonate.

[0092] Example 5

[0093] (1) Weigh 0.8 g of sodium alginate powder and add it to a beaker containing 40 ml of deionized water. Stir in a 37°C water bath until the sodium alginate is completely dissolved to obtain a uniform and transparent sodium alginate solution.

[0094] (2) Add 0.35 g of GDL to the sodium alginate solution, dissolve it completely, and continue stirring for 5 minutes;

[0095] (3) Dissolve glucose oxidase in PBS solution to obtain GOD-PBS solution, and add the GOD-PBS solution containing 300 units of glucose oxidase to the sodium alginate solution, and continue stirring for 5 minutes;

[0096] (4) adding 0.4 g of attapulgite powder to 20 mL of deionized water under ultrasonic conditions to obtain an attapulgite suspension, and adding 2 mL of the attapulgite suspension to the beaker under stirring conditions, and continuing to stir for 5 minutes;

[0097] (5) Weigh 2.75 g of hollow calcium carbonate and disperse it in 100 mL of deionized water under stirring and ultrasonic conditions to obtain a CaCO3 suspension;

[0098] (6) Add 2.5 mL of CaCO3 suspension to the beaker under stirring conditions. After the addition is completed, continue stirring for 40 seconds and then stop;

[0099] (7) The beaker is sealed and allowed to stand at room temperature to allow the system to gradually gel, thereby preparing an antibacterial hydrogel dressing for diabetic wounds.

[0100] The preparation method of the hollow calcium carbonate is:

[0101] Sb1, respectively add polyacrylic acid (PAA) solution and sodium dodecyl sulfate (SDS) solution to Na2CO3 solution, mix well to obtain solution A, the added amount makes the final Na2CO3 concentration in solution A be 0.01M, PAA concentration be 1g / L, SDS concentration be 2.5g / L;

[0102] Sb2, adding the PAA solution to the CaCl2 solution, mixing evenly to obtain solution B, the amount added is such that the final CaCl2 concentration in solution B is 0.1M, and the PAA concentration is 1g / L;

[0103] Sb3. Quickly pour 100 ml of solution B into 100 ml of solution A, stir at 300 rpm at 8°C for precipitation reaction for 1 hour. After the reaction is completed, filter and rinse with ethanol and distilled water to remove PAA, and dry to obtain hollow calcium carbonate.

[0104] Example 6

[0105] (1) Weigh 0.8 g of sodium alginate powder and add it to a beaker containing 40 ml of deionized water. Stir in a 37°C water bath until the sodium alginate is completely dissolved to obtain a uniform and transparent sodium alginate solution.

[0106] (2) Add 0.35 g of GDL to the sodium alginate solution, dissolve it completely, and continue stirring for 5 minutes;

[0107] (3) Dissolve glucose oxidase in PBS solution to obtain GOD-PBS solution, and add the GOD-PBS solution containing 200 units of glucose oxidase to the sodium alginate solution, and continue stirring for 5 minutes;

[0108] (4) adding 0.4 g of attapulgite powder to 20 mL of deionized water under ultrasonic conditions to obtain an attapulgite suspension, and adding 2 mL of the attapulgite suspension to the beaker under stirring conditions, and continuing to stir for 5 minutes;

[0109] (5) Weigh 2.75 g of CaCO3 powder and disperse it in 100 mL of deionized water under stirring and ultrasonic conditions to obtain a CaCO3 suspension;

[0110] (6) Add 2.5 mL of CaCO3 suspension to the beaker under stirring conditions. After the addition is completed, continue stirring for 40 seconds and then stop;

[0111] (7) The beaker is sealed and allowed to stand at room temperature to allow the system to gradually gel, thereby preparing an antibacterial hydrogel dressing for diabetic wounds.

[0112] Example 7

[0113] (1) Weigh 0.8 g of sodium alginate powder and add it to a beaker containing 40 ml of deionized water. Stir in a 37°C water bath until the sodium alginate is completely dissolved to obtain a uniform and transparent sodium alginate solution.

[0114] (2) Add 0.35 g of GDL to the sodium alginate solution, dissolve it completely, and continue stirring for 5 minutes;

[0115] (3) Dissolve glucose oxidase in PBS solution to obtain GOD-PBS solution, and add GOD-PBS solution containing 400 units of glucose oxidase to the sodium alginate solution, and continue stirring for 5 minutes;

[0116] (4) adding 0.4 g of attapulgite powder to 20 mL of deionized water under ultrasonic conditions to obtain an attapulgite suspension, and adding 2 mL of the attapulgite suspension to the beaker under stirring conditions, and continuing to stir for 5 minutes;

[0117] (5) Weigh 2.75 g of CaCO3 powder and disperse it in 100 mL of deionized water under stirring and ultrasonic conditions to obtain a CaCO3 suspension;

[0118] (6) Add 2.5 mL of CaCO suspension to the beaker under stirring. After the addition is completed, continue stirring for 40 seconds and then stop;

[0119] (7) The beaker is sealed and allowed to stand at room temperature to allow the system to gradually gel, thereby preparing an antibacterial hydrogel dressing for diabetic wounds.

[0120] Comparative Example 1

[0121] (1) Weigh 0.8 g of sodium alginate powder and add it to a beaker containing 40 ml of deionized water. Stir in a 37°C water bath until the sodium alginate is completely dissolved to obtain a uniform and transparent sodium alginate solution.

[0122] (2) Add 0.35 g of GDL to the sodium alginate solution, dissolve it completely, and continue stirring for 5 minutes;

[0123] (3) Dissolve glucose oxidase in PBS solution to obtain GOD-PBS solution, and add the GOD-PBS solution containing 300 units of glucose oxidase to the sodium alginate solution, and continue stirring for 5 minutes;

[0124] (4) Weigh 2.75 g of CaCO3 powder and disperse it in 100 mL of deionized water under stirring and ultrasonic conditions to obtain a CaCO3 suspension;

[0125] (5) Add 2.5 mL of CaCO suspension to the beaker under stirring. After the addition is completed, continue stirring for 40 seconds and then stop;

[0126] (6) Seal the beaker and let it stand at room temperature to wait for the system to gradually gel, thereby preparing an antibacterial hydrogel dressing.

[0127] Comparative Example 2

[0128] (1) Weigh 0.8 g of sodium alginate powder and add it to a beaker containing 40 ml of deionized water. Stir in a 37°C water bath until the sodium alginate is completely dissolved to obtain a uniform and transparent sodium alginate solution.

[0129] (2) Add 0.35 g of GDL to the sodium alginate solution, dissolve it completely, and continue stirring for 5 minutes;

[0130] (3) adding 0.4 g of attapulgite powder to 20 mL of deionized water under ultrasonic conditions to obtain an attapulgite suspension, and adding 2 mL of the attapulgite suspension to the beaker under stirring conditions, and continuing to stir for 5 minutes;

[0131] (4) Weigh 2.75 g of CaCO3 powder and disperse it in 100 mL of deionized water under stirring and ultrasonic conditions to obtain a CaCO3 suspension;

[0132] (5) Add 2.5 mL of CaCO3 suspension to the beaker under stirring. After the addition is completed, continue stirring for 40 seconds and then stop;

[0133] (6) Seal the beaker and let it stand at room temperature to wait for the system to gradually gel, thereby preparing a hydrogel dressing.

[0134] Comparative Example 3

[0135] (1) Weigh 0.8 g of sodium alginate powder and add it to a beaker containing 40 ml of deionized water. Stir in a 37°C water bath until the sodium alginate is completely dissolved to obtain a uniform and transparent sodium alginate solution.

[0136] (2) Add 0.35 g of GDL to the sodium alginate solution, dissolve it completely, and continue stirring for 5 minutes;

[0137] (3) adding 0.4 g of attapulgite powder to 20 mL of deionized water under ultrasonic conditions to obtain an attapulgite suspension, and adding 2 mL of the attapulgite suspension to the beaker under stirring conditions, and continuing to stir for 5 minutes;

[0138] (4) Weigh 2.75 g of CaCO3 powder and disperse it in 100 mL of deionized water under stirring and ultrasonic conditions to obtain a CaCO3 suspension;

[0139] (5) Add 3 mL of CaCO suspension to the beaker under stirring. After the addition is completed, continue stirring for 40 seconds and then stop;

[0140] (6) Seal the beaker and let it stand at room temperature to wait for the system to gradually gel, thereby preparing a hydrogel dressing.

[0141] Dynamic thermal mechanical analysis (DMA) was used to test the mechanical properties of the hydrogel dressings prepared in Examples 1-5 and Comparative Examples 1-3, respectively. The test mode was selected as the compression mode, the sample shape was cylindrical, and the compression speed was 1.5N / min to 4.5N. The diameter of each group of samples was measured in advance, and the sample height was automatically measured by the instrument before the formal test (the prepared sample height was preferably 2mm~3mm). The hydrogels used for testing were controlled to have the same size, and the test results are shown in Table 1.

[0142] Table 1 Mechanical properties test results of hydrogel dressings

[0143]

[0144] It can be seen from Table 1 that the mechanical strength and elastic modulus of the hydrogel dressings prepared in Examples 1-5 are significantly improved compared with those in Comparative Example 1, which proves that the antibacterial hydrogel dressings described in the present invention have significantly improved mechanical strength and elastic deformation capacity compared with the gel dressing without adding attapulgite; in particular, the mechanical strength of the gel dressing prepared in Example 1 is increased by 22.5%, and the elastic deformation is increased by 66.0% compared with Comparative Example 1, which shows that attapulgite has a significant enhancing effect on the mechanical strength of the antibacterial hydrogel dressing.

[0145] The corresponding APT addition amounts of Examples 1-3 are 5%, 10%, and 20%, respectively, and the corresponding APT addition amount of Comparative Example 1 is 0%. It can be seen from Table 1 that the mechanical strength and elastic deformation of Example 1 are the highest among the three, indicating that the mechanical properties of the antibacterial hydrogel dressing obtained when the APT addition amount is 5% are the best. The above results can be more clearly obtained from the stress-strain curve, as shown in the attached figure. Figure 2 As shown, the slope of the curve corresponding to the 5% APT addition in Example 1 is very close to the slope of the curve corresponding to the 20% APT addition in Example 3, but the final mechanical strength and elastic deformation of Example 1 are significantly higher than those of Example 3. The final deformation degrees are from large to small, namely, Example 1, Example 3, Example 2, and Comparative Example 1, that is, the elastic deformation degrees of the hydrogels prepared with 5%, 20%, 10%, and 0% APT additions decrease in sequence.

[0146] The mechanical strengths of Examples 4-5 are higher than those of Example 1, proving that microporous calcium carbonate and hollow calcium carbonate can effectively improve the mechanical strength of the antibacterial hydrogel dressing of the present invention compared with ordinary calcium carbonate powder, but the elastic deformation of the hydrogel dressing prepared in Example 4 is slightly lower than that in Example 1, indicating that microporous calcium carbonate may have a certain adverse effect on the elastic deformation of the hydrogel dressing; and the mechanical strength and elastic deformation of Example 5 are significantly higher than those of Example 1 and Example 4, proving that the hollow calcium carbonate prepared by a specific method of the present invention can significantly improve the mechanical strength and elastic deformation capacity of the hydrogel dressing compared with microporous calcium carbonate and ordinary calcium carbonate powder.

[0147] Compared with Example 1, Comparative Example 2-3 did not add glucose oxidase, and the mechanical strength and elastic deformation were significantly reduced, which confirmed that glucose oxidase had a significant improvement effect on the mechanical properties of the hydrogel dressing. In the prior art, glucose oxidase was only used to oxidize and consume substances such as glucose, and was not used to enhance the gel strength while consuming glucose. It can be seen that the present invention has achieved unexpected technical effects; among them, Comparative Example 3 increased the amount of CaCO3 powder compared to Comparative Example 2, and its mechanical strength and elastic deformation were both improved to a certain extent, which confirmed that the increase in the content of CaCO3 powder is beneficial to improving the mechanical properties of the hydrogel dressing.

[0148] Cultivate Staphylococcus aureus in liquid culture medium. First, use a sterile inoculation loop to pick up an appropriate amount of Staphylococcus aureus colonies and shake them in a centrifuge tube containing an appropriate amount of liquid culture medium to make a bacterial suspension, which is used as the experimental bacterial liquid and cultured at 37°C for 24 hours. Dilute the strain 10 3 The experimental environment was sterile. The hydrogel dressings prepared in Example 1 and Example 6-7 were mixed with bacterial solution, and glucose was added to the bacterial solution to make the concentration in the bacterial solution 15mmol / L. The bacterial solution without hydrogel dressing was used as the blank group, and the control group was replaced with an equal amount of PBS solution instead of hydrogel dressing. Co-culture was performed at 37°C. After 24 hours, 100μL of bacterial solution was taken from each group and diluted to 10 6 Take 200 μL of the bacterial dilution and spread it on the agar plate. Spread 3 plates for each group. Place the spread plate in a 37℃ constant temperature incubator for 24 hours and take photos to record the number of colonies formed (see Figure 3 ).

[0149] like Figure 3 As shown, a large number of colonies can be clearly observed on the agar plate in both the blank group and the control group, while no colonies can be observed with the naked eye in the groups of embedding 300unit GOD, embedding 200unit GOD and embedding 400unit GOD corresponding to Example 1 and Example 6-7, respectively. It can be seen that the antibacterial hydrogel dressing of the present invention can almost reach 100% inhibition effect on Staphylococcus aureus when embedding 200unit-400unit GOD at high glucose concentration, which reflects that the antibacterial hydrogel dressing of the present invention can still play a significant antibacterial effect for the high sugar environment of diabetic wounds. At the same time, the significant antibacterial effect also shows that the antibacterial hydrogel dressing of the present invention successfully converts glucose in the high sugar environment into hydrogen peroxide with antibacterial effect, thereby reducing the sugar concentration of the wound, thereby alleviating the problem of difficult wound healing caused by the high sugar environment of diabetic wounds.

[0150] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing an antibacterial hydrogel dressing for diabetic wounds, The following steps are involved: (1) adding sodium alginate powder into a container containing deionized water under stirring conditions until the sodium alginate is completely dispersed and dissolved to obtain a uniform and transparent sodium alginate solution; (2) adding gluconolactone GDL to the sodium alginate solution under stirring to completely dissolve it, and continuing stirring for 5 minutes; (3) dissolving glucose oxidase in a phosphate buffer solution to obtain a GOD-PBS solution, and adding the GOD-PBS solution into the container under stirring conditions, and continuing to stir for 5 minutes; (4) weighing attapulgite powder, adding the attapulgite powder to deionized water under stirring and ultrasonic conditions to obtain an attapulgite suspension, and adding the attapulgite suspension to the container under stirring conditions; (5) Weigh CaCO 3 , and the CaCO 3 Dispersed in deionized water under stirring and ultrasonic conditions, CaCO 3 Suspension; (6) Add the CaCO dropwise into the container under stirring conditions. 3 After the suspension was added dropwise, stirring was continued for 40 seconds and then stopped; (7) The container is sealed and allowed to stand at room temperature to allow the system to gradually gel, thereby preparing an antibacterial hydrogel dressing for diabetic wounds.

2. The method for preparing the antibacterial hydrogel dressing for diabetic wounds according to claim 1, Features: The mass fraction of sodium alginate in the sodium alginate solution in step (1) is 1 wt % to 5 wt %.

3. The method for preparing the antibacterial hydrogel dressing for diabetic wounds according to claim 2, Features: The mass ratio of sodium alginate to GDL in step (2) is 2 to 3:

1.

4. The method for preparing the antibacterial hydrogel dressing for diabetic wounds according to claim 2, Features: The content of glucose oxidase in step (3) is 50 to 400 activity units of glucose oxidase per 40 mL of sodium alginate solution.

5. The method for preparing the antibacterial hydrogel dressing for diabetic wounds according to claim 3, Features: The mass of the attapulgite powder added in step (4) is 1wt% to 20wt% of the mass of the sodium alginate powder.

6. The method for preparing the antibacterial hydrogel dressing for diabetic wounds according to claim 5, Features: In step (6), the CaCO 3 After suspension, the Ca of the whole system 2+ The final concentration was maintained at 0.01M~0.03M.

7. The method for preparing the antibacterial hydrogel dressing for diabetic wounds according to claim 5, Features: In the method for preparing the antibacterial hydrogel dressing, the temperature of the reaction system from step (2) to step (6) is always maintained at 35° C. to 40° C.

8. The method for preparing the antibacterial hydrogel dressing for diabetic wounds according to any one of claims 1 to 7, Features: The CaCO 3 It is one or more of calcium carbonate powder, microporous calcium carbonate or hollow calcium carbonate.

9. The method for preparing the antibacterial hydrogel dressing for diabetic wounds according to claim 8, Features: The preparation method of the hollow calcium carbonate is: Sb1, respectively add polyacrylic acid PAA solution and sodium dodecyl sulfate SDS solution into Na 2 CO 3 solution, and mixed uniformly to obtain solution A; Sb2, add PAA solution to CaCl 2 solution, and mixing well to obtain solution B; Sb3. Quickly pour solution B into solution A, and carry out precipitation reaction under stirring. After the reaction is completed, filter and rinse with ethanol and distilled water to remove PAA, and dry to obtain hollow calcium carbonate.

10. An antibacterial hydrogel dressing for diabetic wounds prepared by the method for preparing an antibacterial hydrogel dressing for diabetic wounds according to any one of claims 1 to 9.

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