Chitosan-based composite sponge, preparation method and application of chitosan-based composite sponge in preparation of products for diabetic wound healing

Abstract

The invention provides a chitosan-based composite sponge, a preparation method and application of the chitosan-based composite sponge in preparation of a product for diabetic wound healing, and belongs to the technical field of biological medicines. The preparation method of the composite sponge comprises the following steps: preparing a chitosan / polyvinyl alcohol / ciprofloxacin composite sponge, preparing solid calcium peroxide / metformin, and preparing the chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge. The composite sponge can stably exist on the surface of a diabetic wound, and can continuously release oxygen for wound repair while absorbing wound exudate. In addition, an in-vitro drug release experiment shows that metformin can be slowly released from the composite sponge. The composite sponge is simple to prepare and high in biocompatibility, and can be widely applied to the field of biological medicines.

Description

Technical Field

[0001] The present invention belongs to the technical field of material synthesis and biomedicine, and particularly relates to a chitosan-based composite sponge, a preparation method and application thereof in preparing a product for diabetic wound healing. Technical Background

[0002] Typically, wounds may be caused by injuries, accidents, mechanical trauma, diabetes, burns, thermal damage or surgical operations. Wounds are generally divided into acute and chronic types. Among them, chronic wounds are usually related to underlying health conditions, such as diabetes or vascular diseases, which affect the body's normal recovery process. Since normal wound healing goes through four main stages: hemostasis, inflammation, proliferation and remodeling, chronic wounds usually remain in the inflammation stage, so healing is very slow or even impossible. More seriously, vascular damage caused by chronic inflammation and tissue hypoxia can directly lead to diabetic wounds and foot ulcers. About 15% of diabetic patients develop diabetic foot ulcers and chronic skin lesions. Currently, there is still no effective material that can be used for diabetic wound healing.

[0003] Ciprofloxacin is a broad-spectrum antibiotic belonging to the fluoroquinolone class. Fluoroquinolones are commonly used to treat microbial infections, resulting in bacterial death through inhibition of DNA spinase and topoisomerase IV. However, the hydrophobicity of ciprofloxacin reduces its bioavailability. Metformin hydrochloride is a traditional drug used to treat type 2 diabetes, offering advantages such as good biocompatibility and low cost. Recent studies have demonstrated that metformin can improve endothelial function and exhibits excellent anti-inflammatory activity, suggesting its potential application in inflammation-related tissue repair.

[0004] Chitosan, a deacetylated product of chitin, is the second most abundant biopolymer after cellulose. It is the only cationic polysaccharide among natural polysaccharides. The amino groups on chitosan can carry a positive charge in neutral and acidic media, interacting with the negative charges on bacterial cell membranes and causing microbial death. Polyvinyl alcohol (PVA), a hydrophilic, non-toxic, highly biocompatible, and biodegradable material, has a wide range of applications in biomedical fields such as drug delivery, tissue engineering, and wound healing. PVA can be blended with polymers to improve the mechanical properties of other polymeric materials, including ductility and flexibility. Calcium peroxide is a commonly used solid oxygen supply. Known for its slow and sustained release of oxygen, it can continuously provide oxygen for wound repair, enabling controlled, sustained release of oxygen and avoiding the cytotoxicity caused by localized accumulation of reactive oxygen species. Summary of the Invention

[0005] To address the shortcomings of the prior art, the present invention aims to provide a chitosan-based composite sponge, a preparation method, and its use in the manufacture of products for diabetic wound healing. In this method, chitosan and ciprofloxacin are dissolved in an acetic acid solution, mixed with a polyvinyl alcohol solution, and freeze-dried to produce a chitosan / polyvinyl alcohol / ciprofloxacin sponge. Calcium peroxide / metformin particles are then prepared by coprecipitation. Finally, the calcium peroxide / metformin particles are dispersed in ethanol and impregnated into a chitosan / polyvinyl alcohol / ciprofloxacin sponge, thereby loading the chitosan / polyvinyl alcohol / ciprofloxacin sponge with calcium peroxide / metformin, resulting in a chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge. This composite sponge can remain stable on the surface of diabetic wounds, absorbing wound exudate while continuously releasing oxygen for wound repair. Furthermore, in vitro drug release experiments demonstrate that metformin can be slowly released from this composite sponge. This composite sponge is simple to prepare and has high biocompatibility, making it suitable for widespread biomedical applications.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] In one aspect, the present invention provides a chitosan-based composite sponge, comprising chitosan / polyvinyl alcohol / ciprofloxacin sponge and calcium peroxide / metformin contained therein.

[0008] In a second aspect, the present invention provides a method for preparing a chitosan-based composite sponge, comprising the following steps:

[0009] (1) Weighing gelatin, glycerol, and heated polyvinyl alcohol solution, adding them to the chitosan solution, stirring evenly, cooling to room temperature, adding ciprofloxacin, mixing evenly, standing to form a hydrogel, and then freeze-drying to obtain a chitosan / polyvinyl alcohol / ciprofloxacin sponge;

[0010] (2) Weigh metformin and dissolve it in water, add calcium chloride, stir and dissolve, add sodium hydroxide solution, then dropwise add hydrogen peroxide under stirring, take the precipitate, and dry it to obtain solid calcium peroxide / metformin;

[0011] (3) Weigh the calcium peroxide / metformin obtained in step (2) above, dissolve it in ethanol, add the chitosan / polyvinyl alcohol / ciprofloxacin sponge obtained in step (1) above, stir evenly, and dry to obtain a chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge.

[0012] The preparation method, the preparation method of the polyvinyl alcohol solution in step (1) is: weighing polyvinyl alcohol and adding it to water;

[0013] Preferably, the mass volume ratio of the polyvinyl alcohol to water is 0.1-1.5 g:5-50 mL;

[0014] The chitosan solution is prepared by weighing chitosan and adding the chitosan into an acetic acid solution;

[0015] Preferably, the mass volume ratio of chitosan to acetic acid is 0.1-1.5 g:5-50 mL;

[0016] More preferably, the volume percentage concentration of acetic acid in the chitosan solution is 0.5-1.5%.

[0017] In the preparation method, the heating temperature in step (1) is 80-100° C.

[0018] The mass volume ratio of the gelatin, glycerol, polyvinyl alcohol solution, chitosan solution and ciprofloxacin is 0.1-1.0 g: 0.1-1.5 mL: 10-50 mL: 10-50 mL: 1-10 mg.

[0019] In the preparation method, the mass volume ratio of metformin, water, calcium chloride, sodium hydroxide solution and hydrogen peroxide in step (2) is 10-30 mg:100-300 mL:40-60 g:60-90 mL:80-120 mL.

[0020] 16. The preparation method according to claim 2, characterized in that the concentration of the sodium hydroxide solution in step (2) is 0.1 to 1.5 mol / L;

[0021] The dropping time is 20 to 40 minutes.

[0022] In the preparation method, the mass volume ratio of calcium peroxide / metformin, ethanol, chitosan / polyvinyl alcohol / ciprofloxacin sponge in step (3) is 0.1-1.5 g:80-120 mL:5-15 g.

[0023] In a third aspect, the present invention provides use of the chitosan-based composite sponge in preparing a product for diabetic wound healing.

[0024] In a fourth aspect, the present invention provides the use of the chitosan-based composite sponge in antibacterial and / or anti-inflammatory activities.

[0025] In a fifth aspect, the present invention provides a product for diabetic wound healing, comprising the chitosan-based composite sponge and pharmaceutically acceptable excipients.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The chitosan-based composite sponge prepared by this invention maintains stability in the skin environment of diabetic wounds, enabling controlled release of metformin and effectively inhibiting bacteria and inflammatory factors such as TNF-α. Calcium peroxide can slowly and persistently release oxygen, providing oxygen for wound repair and preventing vascular damage caused by hypoxia. This hydrogel is simple to prepare and has high biocompatibility, making it suitable for a wide range of biomedical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 IR spectra of calcium peroxide, metformin, and calcium peroxide / metformin in Example 1;

[0029] Figure 2 are scanning electron micrographs of the chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponges in Example 1, Comparative Example 1, and Comparative Example 2, wherein A is a scanning electron micrograph of the chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge in Comparative Example 1, B is a scanning electron micrograph of the chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge in Example 1, and C is a scanning electron micrograph of the chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge in Comparative Example 2;

[0030] Figure 3 IR spectra of the chitosan / polyvinyl alcohol, ciprofloxacin, chitosan / polyvinyl alcohol / ciprofloxacin, and chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponges in Example 1;

[0031] Figure 4 This is the oxygen release curve of the chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge in Example 1;

[0032] Figure 5 The release curves of metformin from the chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge under different pH conditions in Example 1 are shown;

[0033] Figure 6 The antibacterial effect diagram of the chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge in Example 1 and the antibacterial effect diagram of the chitosan / polyvinyl alcohol sponge in Comparative Example 3 are shown;

[0034] Figure 7 This is a bar graph showing the anti-inflammatory effect of the chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge in Example 1. DETAILED DESCRIPTION

[0035] The present invention will now be further described with reference to specific examples. The following examples are intended to illustrate the present invention rather than to further limit the present invention.

[0036] Example 1:

[0037] A method for preparing a chitosan-based composite sponge that can be used for diabetic wound healing comprises the following steps:

[0038] (1) Weigh 0.5 g of chitosan and dissolve it in 25 mL of 1% acetic acid solution to obtain a chitosan solution. Add 1 g of polyvinyl alcohol to 25 mL of deionized water and heat it to 90°C to obtain a polyvinyl alcohol solution. Then, add 0.5 g of gelatin, 1 mL of glycerol, and 25 mL of polyvinyl alcohol solution to the 25 mL chitosan solution and stir evenly. After the solution cools to room temperature, add 5 mg of ciprofloxacin to the above solution and mix evenly. Then, let the viscous mixed solution stand to form a hydrogel, and then freeze-dry it to obtain a composite sponge.

[0039] (2) 20 mg of metformin was dissolved in 200 mL of deionized water, 50 g of calcium chloride was added, and the mixture was stirred to uniformly dissolve. Then, 80 mL of 1 mol / L sodium hydroxide solution was added to the solution. While the mixture was stirred with a magnetic stirrer, 100 mL of hydrogen peroxide was added dropwise to the mixture over 30 min. White particles were observed to gradually precipitate, indicating the formation of calcium peroxide / metformin particles. After drying, the particles were ground to obtain a uniform powder.

[0040] Among them, the infrared spectra of calcium peroxide, metformin, and calcium peroxide / metformin are as follows: Figure 1 As shown, for calcium peroxide, 1420 cm –1 and 875cm –1 The peaks at 3375 cm-1 are attributed to the stretching vibration of O–Ca–O and O–O, respectively. –1 and 3293cm –1 Assigned to N–H stretching vibration, 3174 cm –1 Assigned to C–H stretching vibration. In calcium peroxide / metformin, 3375 cm –1 、3293cm –1 and 3174cm –1 The characteristic peak comes from metformin. 1420cm –1 and 875cm –1 Belongs to calcium peroxide.

[0041] (3) Weigh 1g of calcium peroxide / metformin particles and disperse them in 100mL of ethanol. Add 10g of chitosan / polyvinyl alcohol / ciprofloxacin composite sponge, stir evenly, and naturally dry to remove ethanol to obtain chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge. The field emission scanning electron microscopy image of the prepared chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge is shown in FIG. Figure 2 As shown in Figure B, it can be clearly seen that the chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge has an obvious three-dimensional network structure with an average pore size of 25.1 μm.

[0042] The infrared spectra of the chitosan / polyvinyl alcohol, ciprofloxacin, chitosan / polyvinyl alcohol / ciprofloxacin, chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponges prepared above are shown in FIG. Figure 3 For chitosan / polyvinyl alcohol, 3255 cm –1 The peak at 2930 cm is attributed to the superposition of O–H and N–H stretching vibrations. –1 The peak at 1400 cm is attributed to the stretching vibration of C–H. –1 and 1255cm –1 They are assigned to the stretching vibrations of C=O and C–O, respectively. For ciprofloxacin, 3445 cm –1 The peak at 1732cm is attributed to the superposition of O–H and N–H stretching vibrations. –1 The peak at 3436 cm is attributed to the stretching vibration of the carbonyl group. –1 2930 cm-1 is assigned to the stretching vibrations of O–H and N–H –1 and 1400cm –1 The characteristic peak comes from chitosan / polyvinyl alcohol, 1732 cm –1 The characteristic peak of 3375cm is from ciprofloxacin. For the chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge, –1 、1420cm –1 and 875cm –1 Belongs to calcium peroxide / metformin. 2930cm –1 Belongs to chitosan / polyvinyl alcohol / ciprofloxacin.

[0043] Example 2:

[0044] A method for preparing a chitosan-based composite sponge that can be used for diabetic wound healing comprises the following steps:

[0045] (1) Weigh 0.1 g of chitosan and dissolve it in 5 mL of 0.5% acetic acid solution to obtain a chitosan solution. Add 0.1 g of polyvinyl alcohol to 5 mL of deionized water and heat it to 80°C to obtain a polyvinyl alcohol solution. Then, add 0.1 g of gelatin, 0.1 mL of glycerol, and 10 mL of polyvinyl alcohol solution to 10 mL of the chitosan solution and stir evenly. After the solution cools to room temperature, add 1 mg of ciprofloxacin to the above solution and mix evenly. Then, let the viscous mixed solution stand to form a hydrogel, and then freeze-dry it to obtain a composite sponge.

[0046] (2) 10 mg of metformin was dissolved in 100 mL of deionized water, and 40 g of calcium chloride was added and stirred to uniformly dissolve. Then, 60 mL of 0.1 mol / L sodium hydroxide solution was added to the solution. While stirring the mixture with a magnetic stirrer, 80 mL of hydrogen peroxide was added dropwise to the mixture over 20 min. White particles were observed to gradually precipitate, indicating the formation of calcium peroxide / metformin particles. After drying, the particles were ground to obtain a uniform powder.

[0047] (3) Weigh 0.1 g of calcium peroxide / metformin particles and disperse them in 80 mL of ethanol. Add 5 g of chitosan / polyvinyl alcohol / ciprofloxacin composite sponge, stir evenly, and dry naturally to remove ethanol to obtain chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge.

[0048] Example 3:

[0049] A method for preparing a chitosan-based composite sponge that can be used for diabetic wound healing comprises the following steps:

[0050] (1) Weigh 1.5 g of chitosan and dissolve it in 50 mL of 1.5% acetic acid solution to obtain a chitosan solution. Add 1.5 g of polyvinyl alcohol to 50 mL of deionized water and heat it to 100°C to obtain a polyvinyl alcohol solution. Then, add 1.0 g of gelatin, 1.5 mL of glycerol, and 50 mL of polyvinyl alcohol solution to 50 mL of the chitosan solution and stir evenly. After the solution cools to room temperature, add 10 mg of ciprofloxacin to the above solution and mix evenly. Then, let the viscous mixed solution stand to form a hydrogel, and then freeze-dry it to obtain a composite sponge.

[0051] (2) 30 mg of metformin was dissolved in 300 mL of deionized water, and 60 g of calcium chloride was added and stirred to uniformly dissolve. Then, 90 mL of 1.5 mol / L sodium hydroxide solution was added to the solution. While stirring the mixture with a magnetic stirrer, 120 mL of hydrogen peroxide was added dropwise to the mixture over 40 min. White particles were observed to gradually precipitate, indicating the formation of calcium peroxide / metformin particles. After drying, the particles were ground to obtain a uniform powder.

[0052] (3) Weigh 1.5 g of calcium peroxide / metformin particles and disperse them in 120 mL of ethanol. Add 15 g of chitosan / polyvinyl alcohol / ciprofloxacin composite sponge, stir evenly, and dry naturally to remove ethanol to obtain a chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge.

[0053] Example 4: Oxygen production content test

[0054] 5 g of the chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge prepared in Example 1 was weighed and immersed in 20 mL of deionized water. An oxygen production test was performed at a constant temperature of 37° C. The dissolved oxygen content was measured at regular intervals and repeated three times.

[0055] The oxygen release curve of the chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge prepared in Example 1 is as follows: Figure 4 As shown in the figure, the composite sponge can continuously release oxygen within 200 hours. Within 60 hours, the oxygen release rate of the composite sponge is 27-32 mg L –1 Then, within 60 to 160 h, the oxygen release amount was between 11 and 22 mg L –1 Although the oxygen release of the chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge showed a decreasing trend over time, it still had a good oxygen release effect, thus avoiding vascular damage caused by tissue hypoxia.

[0056] Example 4: In vitro release effect test

[0057] Chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge was used to release metformin in vitro under different pH conditions:

[0058] 1 g of the chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge prepared in Example 1 was placed in a dialysis bag with a molecular cutoff of 3500, and the dialysis bag was placed in 50 mL of phosphate buffer solutions with pH values of 6.8, 7.4, and 9.0, respectively. The drug was released in vitro at a constant temperature of 37° C. with magnetic stirring. During the drug release process, 4 mL of the solution was removed every hour to determine the amount of metformin released, and 4 mL of fresh phosphate buffer solution was added. The characteristic absorption peak intensity of metformin at 233 nm was measured using a UV-visible spectrophotometer, and its concentration was calculated to calculate the cumulative percentage of metformin released at different pH values at different times.

[0059] The drug release curves of chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge at different pH values are shown in Figure 2. Figure 5As shown in the figure, metformin release at different pH values essentially reached equilibrium after 10 hours. At equilibrium, the cumulative release percentages in PBS at pH values of 6.8, 7.4, and 9.0 were 48.8%, 46.0%, and 41.7%, respectively, indicating that slightly acidic conditions are more conducive to metformin release.

[0060] Example 5: In vitro antibacterial effect test

[0061] In order to evaluate the in vitro antibacterial effect of the material, Escherichia coli grown to the logarithmic phase was diluted with sterile water to 1×10 8 CFU / mL, 1 mg of the chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge prepared in Example 1 was added to a 5 mL centrifuge tube and heated to 37°C, followed by the addition of 1 mL of E. coli bacterial suspension, mixed evenly, and incubated at 37°C for 40 min. 100 μL of bacterial suspension was diluted to 1×10 5 CFU / mL was plated on agar plates. Colonies were counted 24 hours later. The same antibacterial evaluation was performed against Staphylococcus aureus.

[0062] The in vitro antibacterial activity of the chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge prepared in Example 1 is as follows: Figure 6 As shown, the inhibition rate of chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge on Staphylococcus aureus and Escherichia coli can reach 100%.

[0063] Example 6: In vitro anti-inflammatory effect test

[0064] In order to evaluate the anti-inflammatory effect of the material in vitro, 5×10 4 Mouse macrophages were placed in a 12-well cell culture plate. 100 ng / mL lipopolysaccharide was co-cultured with mouse macrophages for 12 hours to induce mouse macrophage polarization to M1 type. Then 20 μL PBS (control group) or 20 μL chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge extract prepared in Example 1 was added to the wells. After incubation for 48 hours, total RNA was extracted. cDNA synthesis was performed using a reverse transcription kit. Quantitative PCR was performed. GAPDH was used as the internal reference gene and 2 -ΔΔCT Calculate the relative gene expression of the samples.

[0065] The in vitro anti-inflammatory activity of the chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge prepared in Example 1 is as follows: Figure 7As shown, TNF-α is a common pro-inflammatory cytokine that plays a key role in the development of inflammation. As can be seen in the figure, TNF-α expression increased in the LPS group 24 hours after LPS treatment, indicating successful inflammation induction. Compared to the LPS group, the LPS + chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge significantly reduced TNF-α expression, attributed to the enhanced inhibitory effect of metformin on inflammation.

[0066] Comparative Example 1:

[0067] (1) Weigh 0.1 g of chitosan and dissolve it in 25 mL of 1% acetic acid solution. Add 1 g of polyvinyl alcohol to 25 mL of deionized water and heat it to 90°C. Then add 0.5 g of gelatin, 1 mL of glycerol, and 25 mL of polyvinyl alcohol solution to the 25 mL chitosan solution and stir evenly. After the solution cools to room temperature, add 5 mg of ciprofloxacin to the above solution and mix evenly. Then, let the viscous mixed solution stand to form a hydrogel, and then freeze-dry it to obtain a composite sponge.

[0068] (2) 20 mg of metformin was dissolved in 200 mL of deionized water, 50 g of calcium chloride was added, and the mixture was stirred to uniformly dissolve. Then, 80 mL of 1 mol / L sodium hydroxide solution was added to the solution. While the mixture was stirred with a magnetic stirrer, 100 mL of hydrogen peroxide was added dropwise to the mixture over 30 min. White particles were observed to gradually precipitate, indicating the formation of calcium peroxide / metformin particles. After drying, the particles were ground to obtain a uniform powder.

[0069] (3) Weigh 1 g of calcium peroxide / metformin particles and disperse them in 100 mL of ethanol. Add 10 g of chitosan / polyvinyl alcohol / ciprofloxacin composite sponge, stir evenly, and dry naturally to remove ethanol to obtain a chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge.

[0070] (4) Scanning electron microscopy images of chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge Figure 2 As shown in Figure A, as the amount of chitosan added decreased, the composite sponge developed a looser three-dimensional network structure, with larger pores and an average pore size of 37.8 μm. This is likely due to a lower cross-linking density. Too low a cross-linking density would cause the composite sponge to decompose too quickly, preventing sustained drug release.

[0071] Comparative Example 2:

[0072] (1) Weigh 1.0 g of chitosan and dissolve it in 25 mL of 1% acetic acid solution. Add 1 g of polyvinyl alcohol to 25 mL of deionized water and heat it to 90°C. Then add 0.5 g of gelatin, 1 mL of glycerol, and 25 mL of polyvinyl alcohol solution to the 25 mL chitosan solution and stir evenly. After the solution cools to room temperature, add 5 mg of ciprofloxacin to the above solution and mix evenly. Then, let the viscous mixed solution stand to form a hydrogel, and then freeze-dry it to obtain a composite sponge.

[0073] (2) 20 mg of metformin was dissolved in 200 mL of deionized water, 50 g of calcium chloride was added, and the mixture was stirred to uniformly dissolve. Then, 80 mL of 1 mol / L sodium hydroxide solution was added to the solution. While the mixture was stirred with a magnetic stirrer, 100 mL of hydrogen peroxide was added dropwise to the mixture over 30 min. White particles were observed to gradually precipitate, indicating the formation of calcium peroxide / metformin particles. After drying, the particles were ground to obtain a uniform powder.

[0074] (3) Weigh 1 g of calcium peroxide / metformin particles and disperse them in 100 mL of ethanol. Add 10 g of chitosan / polyvinyl alcohol / ciprofloxacin composite sponge, stir evenly, and dry naturally to remove ethanol to obtain a chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge.

[0075] (4) Scanning electron microscopy images of chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge Figure 2 As shown in Figure C, increasing the amount of chitosan added further increases the crosslinking density, forming a denser three-dimensional network structure and shrinking the pore size to an average pore size of 12.6 μm. Because a porosity greater than 80% is required for dressings, a crosslinking density that is too high is not suitable for these applications.

[0076] Comparative Example 3:

[0077] (1) Weigh 0.5 g of chitosan and dissolve it in 25 mL of 1% acetic acid solution. Add 1 g of polyvinyl alcohol to 25 mL of deionized water and heat to 90°C. Then, add 0.5 g of gelatin, 1 mL of glycerol, and 25 mL of polyvinyl alcohol solution to the 25 mL chitosan solution and stir evenly. The viscous mixed solution is then allowed to stand to form a hydrogel, which is then freeze-dried to obtain a chitosan / polyvinyl alcohol sponge.

[0078] (2) The same in vitro antibacterial effect test as in Example 5 was used. The in vitro antibacterial effect of chitosan / polyvinyl alcohol sponge was as follows: Figure 6As shown, the inhibition rates against Staphylococcus aureus and Escherichia coli were 97.2% and 96.1%, respectively. This is due to the positive charge of chitosan, which attracts the negative charge on the bacterial cell membrane surface. This interaction enhances the permeability of the bacterial cell membrane, leading to the outflow of cellular contents and bacterial death. The results indicate that the chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge has higher antibacterial activity, which is due to the addition of ciprofloxacin.

Claims

1. A chitosan-based composite sponge, characterized in that: The composite sponge comprises chitosan / polyvinyl alcohol / ciprofloxacin sponge and calcium peroxide / metformin contained therein.

2. The method for preparing a chitosan-based composite sponge according to claim 1, wherein: The following steps are involved: (1) Weighing gelatin, glycerol, and heated polyvinyl alcohol solution, adding them to the chitosan solution, stirring evenly, cooling to room temperature, adding ciprofloxacin, mixing evenly, standing to form a hydrogel, and then freeze-drying to obtain a chitosan / polyvinyl alcohol / ciprofloxacin sponge; (2) Weigh metformin and dissolve it in water, add calcium chloride, stir and dissolve, add sodium hydroxide solution, then dropwise add hydrogen peroxide under stirring, take the precipitate, and dry it to obtain solid calcium peroxide / metformin; (3) Weigh the calcium peroxide / metformin obtained in step (2) above, dissolve it in ethanol, add the chitosan / polyvinyl alcohol / ciprofloxacin sponge obtained in step (1) above, stir evenly, and dry to obtain a chitosan / polyvinyl alcohol / ciprofloxacin / calcium peroxide / metformin composite sponge.

3. The preparation method according to claim 2, wherein The preparation method of the polyvinyl alcohol solution in step (1) is as follows: weighing polyvinyl alcohol and adding it to water; Preferably, the mass volume ratio of the polyvinyl alcohol to water is 0.1-1.5 g:5-50 mL; The chitosan solution is prepared by weighing chitosan and adding the chitosan into an acetic acid solution; Preferably, the mass volume ratio of chitosan to acetic acid is 0.1-1.5 g:5-50 mL; More preferably, the volume percentage concentration of acetic acid in the chitosan solution is 0.5-1.5%.

4. The preparation method according to claim 2, wherein The heating temperature in step (1) is 80-100° C.; The mass volume ratio of the gelatin, glycerol, polyvinyl alcohol solution, chitosan solution and ciprofloxacin is 0.1-1.0 g: 0.1-1.5 mL: 10-50 mL: 10-50 mL: 1-10 mg.

5. The preparation method according to claim 2, wherein The mass volume ratio of metformin, water, calcium chloride, sodium hydroxide solution and hydrogen peroxide in step (2) is 10-30 mg: 100-300 mL: 40-60 g: 60-90 mL: 80-120 mL.

6. The preparation method according to claim 2, wherein The concentration of the sodium hydroxide solution in step (2) is 0.1 to 1.5 mol / L; The dropping time is 20 to 40 minutes.

7. The preparation method according to claim 2, wherein The mass volume ratio of calcium peroxide / metformin, ethanol, chitosan / polyvinyl alcohol / ciprofloxacin sponge in step (3) is 0.1-1.5 g:80-120 mL:5-15 g.

8. Use of the chitosan-based composite sponge as claimed in claim 1 in preparing a product for diabetic wound healing.

9. Use of the chitosan-based composite sponge as claimed in claim 1 in antibacterial and / or anti-inflammatory activities.

10. A product for diabetic wound healing, characterized in that: The invention comprises the chitosan-based composite sponge as claimed in claim 1 and pharmaceutically acceptable excipients.

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