Riboflavin photodynamic antibacterial hydrogel for diabetes wound dressing as well as preparation and application of riboflavin photodynamic antibacterial hydrogel

Abstract

The invention discloses riboflavin photodynamic antibacterial hydrogel for a diabetes wound dressing as well as preparation and application of the riboflavin photodynamic antibacterial hydrogel, and belongs to the technical field of biomedical materials. The hydrogel is prepared from carbomer 941, rhizoma bletillae polysaccharide and carboxymethyl chitosan through physical and chemical bi-crosslinking, glutaraldehyde and carboxymethyl chitosan are added to form dynamic imine bonds, and the stability and adaptability of the hydrogel are enhanced. Meanwhile, the photosensitizer riboflavin is introduced, so that the hydrogel is endowed with photodynamic sterilization characteristics, efficient antibiosis is realized, and rapid healing of diabetic wounds is promoted. The hydrogel has excellent wound environment adaptability and adhesion performance, and is suitable for complicated wound surfaces of diabetes mellitus. The successful research and development of the invention provide reference for the development of novel photodynamic wound dressings, have wide application prospects, provide better treatment choices for diabetics, and are helpful for accelerating wound healing, reducing infection risks and improving the life quality of patients.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and in particular relates to a riboflavin photodynamic antibacterial hydrogel for diabetic wound dressings and a preparation method and application thereof. Background Art

[0002] As a chronic metabolic disease, diabetes has become widely prevalent worldwide. Diabetic wound ulcers are the most common complications in diabetic patients, accounting for about 34% of diabetic patients. They are difficult to recover and have a high amputation rate, which seriously threatens the personal safety of patients. Bacterial infection and biofilm formation caused by abnormal wound metabolic environment (hyperglycemia) are considered to be key factors inhibiting diabetic wound healing. Therefore, effective treatment of diabetic wound infection is conducive to promoting diabetic wound healing. At present, antibiotics have become the most commonly used method to promote diabetic wound healing due to their high antibacterial activity and broad antibacterial spectrum. However, problems such as bacterial resistance, adverse reactions and environmental pollution caused by long-term use of antibiotics have restricted the application of antibiotics, forcing people to explore new methods for treating bacterial infections in diabetic wounds.

[0003] Photodynamic antimicrobial therapy (PDT) uses photosensitizers to generate reactive oxygen species (ROS) under the excitation of light of a specific wavelength. These reactive oxygen species can damage bacterial cell membranes, DNA and proteins, leading to bacterial death. PDT is considered to be an emerging and effective strategy for the treatment of bacterial infections due to its strong antibacterial ability, broad antibacterial spectrum, non-bacterial tolerance and non-invasiveness. The effect of photosensitizers is due to the generation of reactive oxygen species under the excitation of light of a specific wavelength, which leads to the destruction of bacterial cell membranes, damage to DNA and proteins, and leakage of contents. Photosensitive materials are the key to photodynamic therapy. Among most photosensitive materials, riboflavin has excellent water solubility, can promote cell metabolism and promote cell tissue proliferation. Riboflavin has three aromatic rings and multiple CN hybrid groups, has strong photosensitivity, and has significant bactericidal properties under light conditions. However, riboflavin has poor dispersibility. When used for wound sterilization, it will lead to excessive local concentration, and excessive reactive oxygen species will be generated under light, thereby damaging tissue cells. Therefore, in order to improve the dispersibility of riboflavin and improve its biocompatibility, a suitable and effective antibacterial carrier is needed.

[0004] Hydrogel is a three-dimensional cross-linked highly hydrophilic network structure with good biocompatibility, solubility and uniformity. It is an excellent antibacterial drug carrier and has been widely used. Among the many materials used to synthesize hydrogels, natural polysaccharides have attracted the attention of researchers due to their good biocompatibility, low toxicity and degradability. Among them, Bletilla striata polysaccharide is considered to be an excellent hydrogel wound dressing because of its significant anti-inflammatory, antibacterial and biocompatibility. Bletilla striata polysaccharide also has the ability to promote cell proliferation and collagen deposition during the proliferation and remodeling stages, which is beneficial to wound healing. In recent years, studies have used Bletilla striata polysaccharide to form physically cross-linked hydrogels through hydrogen bonds. The physically cross-linked hydrogels have excellent water retention, stability and good drug release, but the lack of mechanical properties, bioadhesion and shape adaptability make them unable to effectively adhere to irregular wound surfaces. For severe bacterial infection wounds, especially diabetic wounds, Bletilla striata polysaccharide hydrogels have weak inherent antibacterial properties, which limits their application in complex wound treatment. Therefore, there is an urgent need for photodynamic hydrogels with significant antibacterial properties, excellent mechanical properties, bioadhesion and shape adaptability for use in diabetic wound dressings. Summary of the invention

[0005] Bacterial infection and biofilm formation caused by abnormal metabolic environment (hyperglycemia) of diabetic wounds make it difficult to effectively treat bacterial infection on wounds. Riboflavin has poor biocompatibility and dispersibility. The existing Bletilla striata polysaccharide-based antibacterial hydrogel lacks mechanical properties, bioadhesion and shape adaptability, making it impossible for riboflavin to effectively adhere to irregular wound surfaces. The present invention aims to provide a riboflavin photodynamic antibacterial hydrogel for diabetic wound dressings, as well as its preparation and application.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: The invention provides a riboflavin photodynamic antibacterial hydrogel for diabetic wound dressing. The riboflavin photodynamic antibacterial hydrogel comprises, by weight, 10 to 90 parts of carbomer 941, 5 to 30 parts of carboxymethyl chitosan, 150 parts of triethanolamine, 50 to 450 parts of bletilla striata polysaccharide, 0 to 50 parts of riboflavin, 150 to 550 parts of glutaraldehyde and 10,000 to 13,000 parts of ultrapure water.

[0007] Furthermore, in parts by weight, the riboflavin photodynamic antibacterial hydrogel includes 50 parts of carbomer 941, 100 parts of carboxymethyl chitosan, 150 parts of triethanolamine, 2500 parts of bletilla striata polysaccharide, 50 parts of riboflavin, 350 parts of glutaraldehyde, and 12000 parts of ultrapure water.

[0008] The present invention provides a method for preparing the riboflavin photodynamic antibacterial hydrogel for diabetic wound dressing, which specifically comprises the following steps: (1) Adding carbomer 941 to the riboflavin aqueous solution to prepare a riboflavin-carbomer 941 dispersion system; (2) adding the carboxymethyl chitosan aqueous solution to the riboflavin-carbomer 941 dispersion system to obtain a carbomer-carboxymethyl chitosan mixed solution; (3) adjusting the pH value of the carbomer-carboxymethyl chitosan mixed solution, adding the aqueous solution of Bletilla striata polysaccharide to obtain a carbomer-carboxymethyl chitosan-Bletilla striata polysaccharide mixed solution; (4) Add ultrapure water to the carbomer-carboxymethyl chitosan-Bletilla striata polysaccharide mixed solution and stir evenly, add glutaraldehyde, and stir to obtain riboflavin photodynamic antibacterial hydrogel.

[0009] In step (1), the mass ratio of riboflavin to carbomer 941 is 1-5:5.

[0010] Furthermore, the concentration of the riboflavin aqueous solution is 1%, and the mass concentration of the Carbomer 941 is 1%.

[0011] In step (2), the mass ratio of the carboxymethyl chitosan aqueous solution to the riboflavin-carbomer 941 dispersion system is 1:50-60.

[0012] Furthermore, the mass concentration of the carboxymethyl chitosan is 10%.

[0013] The pH value is adjusted to 6-8.

[0014] Furthermore, the pH value is adjusted to 7.

[0015] Furthermore, triethanolamine is used to adjust the pH value.

[0016] In step (3), the mass ratio of the aqueous solution of Bletilla striata polysaccharide to the carbomer-carboxymethyl chitosan mixed solution is 1:2-3.

[0017] Furthermore, the mass concentration of the Bletilla striata polysaccharide is 10%.

[0018] In step (4), the mass ratio of the carbomer-carboxymethyl chitosan-Bletilla striata polysaccharide mixed solution to ultrapure water is 1-2:1.

[0019] The added amount of glutaraldehyde is 1% to 3% of the volume of the carbomer-carboxymethyl chitosan-Bletilla striata polysaccharide mixed solution.

[0020] The reaction conditions are room temperature and 200-400 rpm / min magnetic stirring.

[0021] The riboflavin photodynamic antibacterial hydrogel for diabetic wound dressings and its preparation and application in the preparation of diabetic wound healing dressings.

[0022] Compared with the prior art, the present invention has the following beneficial effects: The riboflavin photodynamic antibacterial hydrogel for diabetic wound dressing provided by the present invention combines the photodynamic antibacterial property of riboflavin and the excellent carrier performance of bletilla striata polysaccharide-based hydrogel, and has the advantages of strong antibacterial, excellent carrier performance, safety and non-invasiveness. The matrix is ​​composed of carbomer 941, carboxymethyl chitosan and bletilla striata polysaccharide, and has good biocompatibility, solubility and uniformity. Bletilla striata polysaccharide has significant anti-inflammatory, antibacterial and biocompatibility, and can promote cell proliferation and collagen deposition, which is beneficial to wound healing. The photodynamic antibacterial property of riboflavin is utilized to generate reactive oxygen species (ROS) under the excitation of light of a specific wavelength, effectively destroying bacterial cell membranes, DNA and proteins, leading to bacterial death, thereby achieving efficient antibacterial effect and avoiding the drug resistance problem that may be caused by traditional antibiotics. The novel hydrogel dressing provided by the present invention not only has strong antibacterial ability, but also can be used as a good carrier of drugs or other bioactive substances, providing a new choice for the treatment of diabetic wounds.

[0023] The preparation method provided by the present invention improves the mechanical properties of the natural polymer-based hydrogel through the combination of physical crosslinking and chemical crosslinking, enhances the adhesion strength and shape adaptability of the hydrogel, and enables the hydrogel to adhere to the irregular wound surface more effectively, providing lasting antibacterial protection; Carbomer 941 serves as the skeleton of the hydrogel, provides the necessary structure and stability for the riboflavin-Carbomer 941 dispersion system, contributes to the uniform distribution of riboflavin, and enhances the antibacterial effect of the hydrogel; Carboxymethyl chitosan further enhances the stability and adhesion of the system, and synergizes with riboflavin to improve the hydrogel. The antibacterial effect of the hydrogel; triethanolamine can adjust the pH value of the mixed solution to ensure that the subsequently added components can exist stably and play the best role; Bletilla striata polysaccharide further enhances the adhesion and stability of the hydrogel, and at the same time gives the hydrogel better moisturizing properties; glutaraldehyde as a cross-linking agent can react with carboxymethyl chitosan, carbomer 941 and other components in the mixed solution to form a cross-linked structure. The preparation method of the present invention is simple, and a hydrogel dressing with ideal properties can be prepared by adjusting the proportion of each component and the cross-linking conditions. The raw materials used in the preparation process are relatively easy to obtain, which is conducive to reducing production costs and promoting application.

[0024] The application provided by the present invention, the riboflavin photodynamic antibacterial gel in the present invention showed strong photodynamic antibacterial performance, excellent wound healing ability, good adhesion stability and moisturizing performance and suitable water absorption performance in the diabetic wound bacterial infection model, and has significant technical effects and clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1The single factor investigation diagram of the preparation of riboflavin photodynamic antibacterial hydrogel of the present invention, wherein (a) is the effect of different 10% BSP addition amounts on the gel surface adhesion, (b) is the effect of different 10% CMCS addition amounts on the gel surface adhesion, (c) is the effect of different GA volumes on the gel surface adhesion, and (d) is the effect of different Cbm941 addition amounts on the gel surface adhesion; Figure 2 The inversion test and Fourier transform infrared spectra of the riboflavin photodynamic antibacterial hydrogel of the present invention are shown, wherein (a) is a hydrogel inversion tilt test diagram, and (b) is a hydrogel FT-IR diagram; Figure 3 The SEM images of the riboflavin photodynamic antibacterial hydrogel of the present invention, wherein (a) is CCBG, (b) is CCBG-1, (c) is CCBG-2, (d) is CCBG-3, (e) is CCBG-4, and (f) is CCBG-5; Figure 4 The diagrams are the water retention rate and swelling test diagrams of the riboflavin photodynamic antibacterial hydrogel of the present invention, wherein (a) is the water retention diagram, and (b) is the swelling test result diagram; Figure 5 The adhesion test results of the riboflavin photodynamic antibacterial hydrogel of the present invention are shown in Figure 1, wherein (a) is a visual comparison diagram of the adhesion test, and (b) is a comparison of the adhesion test data; Figure 6 The figures are the evaluation diagrams of the antibacterial activity of the riboflavin photodynamic antibacterial hydrogel of the present invention; wherein (a) is the LB plate diagram of each group of hydrogels treating Staphylococcus aureus under visible light irradiation, (b) is the colony count of Staphylococcus aureus treated by each group of hydrogels under visible light irradiation, (c) is the LB plate diagram of each group of hydrogels treating Escherichia coli under visible light irradiation, and (d) is the colony count of Escherichia coli treated by each group of hydrogels under visible light irradiation; Figure 7 The in vivo evaluation diagram of the riboflavin photodynamic antibacterial hydrogel of the present invention includes (a) wound image of diabetic wound 48 hours after infection and real-time images of wound at 0, 3, 7, 10 and 14 days, (b) simulated image of wound healing area, (c) healing rate of diabetic wound after different treatments, (d) scab removal rate of diabetic wound after different treatments (n = 3), *p<0.05, **p<0.01, ***p<0.001. DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not 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 should fall within the scope of protection of the present invention.

[0027] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0028] Example 1 This embodiment provides a method for preparing a riboflavin photodynamic antibacterial hydrogel for diabetic wound dressings, which is obtained by the following steps: (1) Adding carbomer 941 to the riboflavin aqueous solution to prepare a riboflavin-carbomer 941 dispersion system; (2) adding the carboxymethyl chitosan aqueous solution to the riboflavin-carbomer 941 dispersion system to obtain a carbomer-carboxymethyl chitosan mixed solution; (3) adding triethanolamine to the carbomer-carboxymethyl chitosan mixed solution, adjusting the pH value, and continuing to add the Bletilla striata polysaccharide aqueous solution to obtain a carbomer-carboxymethyl chitosan-Bletilla striata polysaccharide mixed solution; (4) Add ultrapure water to the carbomer-carboxymethyl chitosan-Bletilla striata polysaccharide mixed solution and stir evenly, add glutaraldehyde, and stir to obtain riboflavin photodynamic antibacterial hydrogel.

[0029] See attached Figure 1 , Single factor experiment on the preparation of riboflavin photodynamic antibacterial hydrogel for diabetic wound dressing.

[0030] (1) Screening of 10% Bletilla striata polysaccharide BSP addition amount When 2.5 g of 10% BSP was added, the adhesion of the hydrogel was the greatest. This is because when the BSP content was low, the hydroxyl content in the hydrogel increased with the increase in BSP content, which promoted the formation of hydrogen bonds with the hydrophilic groups on the skin surface, thereby increasing the adhesion of the hydrogel. However, with the excessive increase in the BSP content in the hydrogel, the cross-linking degree of the hydrogel increased significantly, resulting in smaller pores, reducing the contact area with the skin, and not conducive to adhesion with the skin.

[0031] (2) Screening of 10% CMCS addition amount As the amount of carboxymethyl chitosan (CMCS) increases, the adhesion of the hydrogel increases first and then decreases. When 0.1g of 10% carboxymethyl chitosan is added, the adhesion of the hydrogel is the largest. This is because the content of free functional groups such as -OH, -NH2, and -COOH in the hydrogel increases with the increase in the amount of CMCS, which is conducive to the formation of hydrogen bonds on the skin surface, thereby increasing the adhesion to the skin. However, the CMCS network collapses at high concentrations due to the repulsion of the charged groups, resulting in bond breakage and reduced viscosity. In addition, the hydrogen bonds formed between the components give the hydrogel a higher physicochemical cross-linking density and a smaller pore size, thereby reducing the contact area with the skin and being unfavorable for adhesion to the skin.

[0032] (3) Glutaraldehyde GA volume screening As the volume of glutaraldehyde GA increases, the adhesion of the hydrogel increases first and then decreases. When the amount of GA added is 350 μL, the adhesion of the hydrogel reaches the maximum. This is because when the GA content is low, as the GA content increases, the reaction between the hydrogel and the protein amino group on the skin surface increases, thereby enhancing the adhesion of the hydrogel. However, as the volume of GA in the hydrogel continues to increase, the cross-linking degree of the hydrogel also increases, resulting in a smaller pore size, which is not conducive to adhesion to the skin. In addition, excessive GA will polymerize and also reduce the adhesion of the hydrogel.

[0033] (4) Screening of the amount of Cbm 941 added The effect of CBM941 dosage on hydrogel adhesion was studied. As the dosage of CBM941 increased, the adhesion of the hydrogel first increased and then decreased. When the dosage of CBM941 was 50 mg, the adhesion of the hydrogel was the largest. This is because the molecular chain repulsion of CBM941 was poor at low concentrations, and it could not swell, which was not conducive to cross-linking with CMCS and BSP, and the bonding performance was weak. However, high concentrations of CBM941 would lead to increased cross-linking of the hydrogel, resulting in a well-developed network and reduced pore size, thereby weakening the adhesion of the hydrogel.

[0034] Example 2 This embodiment provides a riboflavin photodynamic antibacterial hydrogel based on embodiment 1, and the specific preparation steps include: 50 mg of Carbomer 941 was weighed and placed in 5 mL of ultrapure water, and stirred evenly to disperse the Carbomer completely; 0.1 g of 10% carboxymethyl chitosan solution was added to the Carbomer dispersion system, stirred evenly, and triethanolamine was added to adjust the pH to neutral to obtain a Carbomer 941 / carboxymethyl chitosan solution; 2.50 g of 10% Bletilla striata polysaccharide was added to the Carbomer 941 / carboxymethyl chitosan solution, and stirred evenly to obtain a Carbomer 941 / carboxymethyl chitosan solution / Bletilla striata polysaccharide solution; 4.8 mL of ultrapure water was added to the Carbomer 941 / carboxymethyl chitosan solution / Bletilla striata polysaccharide solution, and stirred evenly to obtain an H-bond cross-linked hydrogel; 350 μL of 2.5% glutaraldehyde solution was added to the H-bond hydrogel, stirred, and allowed to stand to obtain a riboflavin photodynamic antibacterial hydrogel, named CCBG.

[0035] Example 3 This embodiment provides riboflavin photodynamic antibacterial hydrogel based on embodiment 1, and the specific preparation process is as follows: 10 mg of riboflavin was weighed and dispersed in 5 mL of ultrapure water, 50 mg of carbomer 941 was added, and the mixture was stirred evenly to completely disperse the carbomer to obtain a carbomer dispersion system; 0.1 g of 10% carboxymethyl chitosan solution was added to the carbomer dispersion system, the mixture was stirred evenly, and triethanolamine was added to adjust the pH to neutral to obtain a carbomer 941 / carboxymethyl chitosan solution; 2.50 g of 10% solution of Bletilla striata polysaccharide was added to the carbomer 941 / carboxymethyl chitosan solution, the mixture was stirred evenly to obtain a carbomer 941 / carboxymethyl chitosan solution / Bletilla striata polysaccharide solution; 4.8 mL of ultrapure water was added, the mixture was stirred evenly to obtain an H-bond cross-linked hydrogel; 350 μL of 2.5% glutaraldehyde solution was added to the H-bond hydrogel, the mixture was stirred, and the mixture was allowed to stand to obtain a riboflavin photodynamic antibacterial hydrogel, which was named CCBG-1.

[0036] Example 4 This embodiment provides a riboflavin photodynamic antibacterial hydrogel based on embodiment 1, and the specific preparation steps include: 20 mg of riboflavin was weighed and dispersed in 5 mL of ultrapure water, and 50 mg of carbomer 941 was added and stirred evenly to make the carbomer completely dispersed to obtain a carbomer dispersion system; 0.1 g of 10% carboxymethyl chitosan solution was added to the carbomer dispersion system, stirred evenly, and triethanolamine was added to adjust the pH to neutral to obtain a carbomer 941 / carboxymethyl chitosan solution; 2.50 g of 10% Bletilla striata polysaccharide was added to the carbomer 941 / carboxymethyl chitosan solution and stirred evenly to obtain a carbomer 941 / carboxymethyl chitosan solution / Bletilla striata polysaccharide solution; 4.8 mL of ultrapure water was added to the carbomer 941 / carboxymethyl chitosan solution / Bletilla striata polysaccharide solution and stirred evenly to obtain an H-bond cross-linked hydrogel; 350 μL of 2.5% glutaraldehyde solution was added to the H-bond hydrogel, stirred, and allowed to stand to obtain a riboflavin photodynamic antibacterial hydrogel, named CCBG-2.

[0037] Example 5 This embodiment provides a riboflavin photodynamic antibacterial hydrogel based on embodiment 1, and the specific preparation steps include: 30 mg of riboflavin was weighed and dispersed in 5 mL of ultrapure water, and 50 mg of carbomer 941 was added and stirred evenly to completely disperse the carbomer to obtain a carbomer dispersion system; 0.1 g of 10% carboxymethyl chitosan solution was added to the carbomer dispersion system, stirred evenly, and triethanolamine was added to adjust the pH to neutral to obtain a carbomer 941 / carboxymethyl chitosan solution; 2.50 g of 10% Bletilla striata polysaccharide was added to the carbomer 941 / carboxymethyl chitosan solution and stirred evenly to obtain a carbomer 941 / carboxymethyl chitosan solution / Bletilla striata polysaccharide solution; 4.8 mL of ultrapure water was added to the carbomer 941 / carboxymethyl chitosan solution / Bletilla striata polysaccharide solution and stirred evenly to obtain an H-bond cross-linked hydrogel; 350 μL of 2.5% glutaraldehyde solution was added to the H-bond hydrogel, stirred, and allowed to stand to obtain a riboflavin photodynamic antibacterial hydrogel, named CCBG-3.

[0038] Example 6 This embodiment provides a riboflavin photodynamic antibacterial hydrogel based on embodiment 1, and the specific preparation steps include: 40 mg of riboflavin was weighed and dispersed in 5 mL of ultrapure water, and 50 mg of carbomer 941 was added and stirred evenly to completely disperse the carbomer to obtain a carbomer dispersion system; 0.1 g of 10% carboxymethyl chitosan solution was added to the carbomer dispersion system, stirred evenly, and triethanolamine was added to adjust the pH to neutral to obtain a carbomer 941 / carboxymethyl chitosan solution; 2.50 g of 10% Bletilla striata polysaccharide was added to the carbomer 941 / carboxymethyl chitosan solution and stirred evenly to obtain a carbomer 941 / carboxymethyl chitosan solution / Bletilla striata polysaccharide solution; 4.8 mL of ultrapure water was added to the carbomer 941 / carboxymethyl chitosan solution / Bletilla striata polysaccharide solution and stirred evenly to obtain an H-bond cross-linked hydrogel; 350 μL of 2.5% glutaraldehyde solution was added to the H-bond hydrogel, stirred, and allowed to stand to obtain a riboflavin photodynamic antibacterial hydrogel, named CCBG-4.

[0039] Example 7 This embodiment provides a riboflavin photodynamic antibacterial hydrogel based on embodiment 1, and the specific preparation steps include: 50 mg of riboflavin was weighed and dispersed in 5 mL of ultrapure water, and 50 mg of carbomer 941 was added and stirred evenly to completely disperse the carbomer to obtain a carbomer dispersion system; 0.1 g of carboxymethyl chitosan solution was added to the carbomer dispersion system, stirred evenly, and triethanolamine was added to adjust the pH to neutral to obtain a carbomer 941 / carboxymethyl chitosan solution; 2.50 g of Bletilla striata polysaccharide was added to the carbomer 941 / carboxymethyl chitosan solution and stirred evenly to obtain a carbomer 941 / carboxymethyl chitosan solution / Bletilla striata polysaccharide solution; 4.8 mL of ultrapure water was added to the carbomer 941 / carboxymethyl chitosan solution / Bletilla striata polysaccharide solution and stirred evenly to obtain an H-bond cross-linked hydrogel; 350 μL of glutaraldehyde solution was added to the H-bond hydrogel, stirred, and allowed to stand to obtain a riboflavin photodynamic antibacterial hydrogel, named CCBG-4.

[0040] Example 8 This example performs a performance test on the riboflavin photodynamic antibacterial hydrogel provided in Examples 2-7.

[0041] (1) Tilt inversion and Fourier transform infrared spectroscopy test The hydrogel sample bottles prepared in Examples 2-7 were tilted and inverted to observe whether they maintained a gel state; CMCS (carboxymethyl chitosan), BSP (Bletilla striata polysaccharide), CBM941 (Carbomer 941) and the prepared hydrogel were subjected to spectral analysis using an FTIR spectrometer. The specific method was as follows: the samples were crushed, mixed with potassium bromide, pressed into tablets, and irradiated at 400 cm -1 -4000 cm -1 Infrared scanning inside.

[0042] By the attached Figure 2 The data show that the tilting and inversion of the sample bottle does not affect the transition of its gel state, which confirms the successful preparation of the hydrogel of the present invention. In order to clarify the interaction between the components and the cross-linking mechanism of the hydrogel, FTIR spectroscopy analysis was performed. In the CMCS spectrum, 3436 cm −1 The broad peak at 1620 cm is caused by the stretching vibration of the OH and NH bonds. −1 The absorption peak at 1315 cm is caused by the stretching vibration of C=O. −1 The absorption peak at 3407cm is caused by the stretching vibration of C-N. −1 and 2935 cm −1 The absorption peaks at 1150 cm-1 and 1160 cm-2 belong to the stretching vibrations of the OH bond and the CH bond, respectively. -1 and 1032 cm -1 The absorption peak at 2500 indicates the presence of pyranose, which is the characteristic peak of BSP; in the CBM941 spectrum, -1 ~ 3300 cm -1 The broad peak and 1715cm -1 The strong sharp absorption peaks belong to the stretching vibrations of the OH bond and C=O bond of COOH, which are the characteristic peaks of CBM941. Compared with BSP, CMCS and CBM941, the -OH absorption peak of the control hydrogel shifts to a lower wavelength, indicating that intermolecular hydrogen bonds are formed through physical cross-linking. -1 A new absorption peak appeared at 1315 cm -1 The NH absorption peak at 100 nm was weakened, indicating that -NH2 in CMCS and -C=O in glutaraldehyde underwent Schiff base condensation reaction to form C=N bonds. Therefore, the formation of the hydrogel can be attributed to the physicochemical double crosslinking of intermolecular H bonds and C=N bonds. In addition, compared with the control hydrogel, the riboflavin-loaded hydrogel has a peak belonging to the characteristic absorption peak of riboflavin, indicating that riboflavin is successfully loaded into the hydrogel.

[0043] In summary, the present invention successfully prepared a stable hydrogel, and the tilting and inverting tests of the sample bottle confirmed that the gel state of the hydrogel was stable and not easily affected by external influences. FTIR spectral analysis revealed the interactions between the components in the hydrogel, including intermolecular hydrogen bonds formed by physical crosslinking and C=N bonds formed by chemical crosslinking, and the Schiff base condensation reaction between CMCS and GA was one of the main mechanisms of chemical crosslinking. FTIR spectral analysis showed that riboflavin was successfully loaded into the hydrogel, which provided the possibility for the application of the hydrogel in the fields of drug loading, release, tissue engineering, etc.

[0044] (2) SEM test The hydrogels (CCBG, CCBG-1, CCBG-2, CCBG-3, CCBG-4 and CCBG-5) prepared in Examples 2-9 were observed using a scanning electron microscope (SEM). The test results are shown in the attached Figure 3 shown.

[0045] By the attached Figure 3 The data show that the hydrogel has a cross-linked microporous structure with a pore size of 100um-200um, which is conducive to the absorption of wound exudate, the interaction between active substances and bacteria, and gas exchange, thereby promoting wound healing. However, after adding photodynamic materials, the pore structure of the hydrogel becomes irregular, which may be due to the uneven cross-linking of the hydrogel caused by the addition of riboflavin.

[0046] (3) Water retention and water absorption test A moist wound environment plays a key role in promoting granulation formation and tissue growth. The good water absorption of the hydrogel plays a key role in the absorption of wound tissue exudates and the inhibition of bacterial proliferation. Therefore, the applicant evaluated the water retention properties of the hydrogel. The hydrogels (CCBG, CCBG-1, CCBG-2, CCBG-3, CCBG-4 and CCBG-5) prepared in Examples 2-9 were tested for water retention in a 37°C oven, and the concentration or percentage of water in the hydrogel at different time points was recorded. Specifically, a certain weight of hydrogel was weighed in a centrifuge tube, placed in an oven at 37°C for incubation, and weighed and recorded within a certain period of time; the lyophilized hydrogel sample was placed in a centrifuge tube, a certain amount of ultrapure water was added, and the sample was swelled by water absorption in a 37°C oven, and the concentration or percentage of water in the hydrogel at different time points was recorded. For details, see the attached Figure 4 shown.

[0047] By the attached Figure 4 The data show that all the tested hydrogel samples (CCBG, CCBG-1, CCBG-2, CCBG-3, CCBG-4 and CCBG-5) showed excellent water retention, among which CCBG-3 had better water retention, which was due to its internal structure and chemical composition, which enhanced the interaction between riboflavin and water and reduced the pore size of the hydrogel, thus effectively limiting the outward diffusion of water. Each hydrogel group showed excellent water absorption capacity, among which CCBG reached water absorption equilibrium after 3 hours, while the other groups reached water absorption equilibrium after 8 hours, which was due to the uneven pore structure of the hydrogel caused by the doping of riboflavin, thus hindering the water absorption process. CCBG-5 showed the highest water absorption rate, which was due to the strong interaction between riboflavin and water and the inhibition of water desorption by riboflavin loading.

[0048] In summary, this experiment tested the water retention and water absorption properties of different hydrogel samples and found that all samples showed excellent water retention and water absorption capacity. Among them, CCBG-3 performed best in water retention, while CCBG-5 performed best in water absorption. These results may be related to the internal structure, chemical composition, and riboflavin content and distribution of the hydrogel. In the future, the effects of these factors on the performance of the hydrogel can be further studied to optimize its performance in practical applications.

[0049] (4) Adhesion test The ability of hydrogel to effectively adhere to the wound surface is the basis for promoting wound healing. The ability of hydrogel to effectively adhere to the wound surface is the basis for promoting wound healing. The adhesion stability of pig skin and human finger joints was verified by the adhesion experiment. Fresh pig skin was used to simulate human skin and cut into rectangles of 60×25 mm². The hydrogels (CCBG, CCBG-1, CCBG-2, CCBG-3, CCBG-4 and CCBG-5) prepared in Examples 2-9 were placed on the surface of pig skin or human finger joints. Through the continuous bending of the finger joints and the stretching, curling and rotation changes of the pig skin, observe whether the hydrogel falls off. The hydrogel was applied to the pig skin, and another group of pig skin was attached to it. A 200 g weight was used for crimping for 30 minutes. The crimping area was 25×25 mm². The adhesion of the hydrogel was measured using an electronic tensiometer (WY-200A) at a tensile speed of 20 mm / min. For specific test results, see the attached Figure 5 shown.

[0050] After constant bending of the knuckles and stretching, curling, and rotation of the pig skin, all hydrogel samples still effectively adhered to the tissue surface without detachment, indicating that the hydrogel has adhesion stability.

[0051] The adhesion strength of hydrogel samples with the same riboflavin content to pig skin was different; CCBG had the highest adhesion strength of 2.15 kPa. As the riboflavin concentration increased, the adhesion strength gradually decreased. The adhesion strengths of CCBG-1, CCBG-2, CCBG-3, CCBG-4, and CCBG-5 were 1.73 kPa, 1.53 kPa, 1.44 kPa, 1.24 kPa, and 1.10 kPa, respectively. CCBG, as the control group, had the highest adhesion strength, indicating that the network structure of the hydrogel may be more complete when there is no riboflavin or when the concentration of riboflavin is low, which is conducive to adhesion. As the concentration of riboflavin increased, the adhesion strength gradually decreased, which may be due to the excessive riboflavin doping that destroyed the network structure of the hydrogel, resulting in a decrease in adhesion performance. CCBG-5 had the lowest adhesion strength, but it could still stably adhere to the tissue surface, indicating that even at high concentrations of riboflavin, the hydrogel still had a certain adhesion ability.

[0052] In summary, this experiment evaluated the effect of different riboflavin concentrations on the adhesion properties of hydrogels through adhesion stability verification and shear test. All hydrogel samples had good adhesion stability, but the adhesion strength decreased with the increase of riboflavin concentration. This was because excessive riboflavin destroyed the network structure of the hydrogel.

[0053] (5) In vitro antibacterial properties The plate method was used to evaluate the efficacy of riboflavin photodynamic antibacterial gel on Staphylococcus aureus ( S. aureus , ATCC25923) and Escherichia coli ( E. coli , ATCC 25922) in vitro to confirm the antibacterial activity of riboflavin photodynamic antibacterial gel against Gram-negative and Gram-positive bacteria. 1 g of hydrogel was applied on a 30 mm culture dish and then sterilized by ultraviolet for 30 min; then 1 mL of OD 600 =0.2 bacterial suspension, then irradiate with 400nm visible light for 7min, dilute the irradiated bacterial suspension 1000 times, take 100 μL diluted suspension and spread on standard agar medium, incubate the agar plate at 37℃ for 24h, count the colonies and calculate the antibacterial efficiency. , where N0 is the result of 1000-fold dilution of the untreated bacterial suspension, and N t is the number of colonies in the sample after irradiation with 400nm visible light. For specific test results, please refer to the attached Figure 6 shown.

[0054] By the attached Figure 6 The data show that compared with the control group, each group of hydrogels showed excellent antibacterial effects under VIS irradiation, especially the CCBG-5 group, which had a sterilization rate of 99.99% and 99.96% against Staphylococcus aureus and Escherichia coli, respectively. This is based on the synergistic antibacterial effect of photothermal, photodynamic and inherent antibacterial activity. Compared with VIS, CCBG+VIS has significant antibacterial activity (p<0.05), which is because the -NH3 + Binding to the bacterial cell wall changes the permeability of the bacterial cell membrane, which is an inherent antibacterial property of the hydrogel. In addition, the antibacterial ability of riboflavin photodynamic hydrogel increases with the increase of riboflavin content. This is because the production of ROS produced by riboflavin excitation under visible light irradiation increases with the increase of riboflavin content. However, compared with CCBG-50+VIS, CCBG-50 hydrogel exhibits weaker antibacterial activity, which is due to the inability of the hydrogel to effectively produce reactive oxygen under non-light conditions.

[0055] In summary, this experiment verified the excellent antibacterial properties of riboflavin photodynamic antibacterial gel against Staphylococcus aureus and Escherichia coli through the plate method. In particular, the CCBG-5 group had a sterilization rate of nearly 100% against both bacteria under visible light irradiation, which was related to the synergistic effects of photothermal, photodynamic and inherent antibacterial activity.

[0056] (5) In vivo wound healing assessment This experiment aims to establish a rat model of diabetic full-thickness wound defect to evaluate the effects of different treatment groups on wound healing in vivo. Sprague Dawley (SD) rats were selected for the experiment, and a series of operations were used to successfully establish a model of diabetes and diabetic wound infection. Subsequently, the rats were randomly divided into groups and given different treatments, including KB (possibly blank control or basic treatment), Model (model control group), VIS (visible light irradiation), CCBG (riboflavin photodynamic antibacterial gel basic group), and groups with different concentrations of CCBG combined with VIS, and AgNPs hydrogel (silver nanoparticle hydrogel group) as a comparison. A rat model of diabetic full-thickness wound defect was established to evaluate the wound healing effect in vivo. Sprague Dawley (SD) rats were purchased from Chengdu Dashuo Experimental Animal Co., Ltd. All animal experiments were approved by the Animal Ethics Committee of Shaanxi University of Chinese Medicine. SD rats were adaptively fed for 3 to 5 days under sterile conditions with suitable temperature, humidity and air. The rats were fasted for 12 hours and injected with 1% STZ once (65 mg / kg) according to their fasting body weight to establish a type I diabetes model. After 72 hours, blood was collected from the tail tip of the rats, and blood glucose was measured using blood glucose test strips. The model was considered successful when the blood glucose concentration was higher than 16.8mmol. Subsequently, all rats were fasted for 12 hours to establish the wound model, and then anesthetized with intraperitoneal injection of sodium pentobarbital (30mg / kg) according to the standard anesthesia procedure, and the back hair was removed. A circular full-thickness skin wound with a diameter of 1cm was created on the back of each rat using sterile scissors, and ~10 8 CFU, OD 600 = 0.5, 100 μL of Staphylococcus aureus was applied to the wound of the rat and the bacteria were allowed to grow for 48 hours. When the wound became red, swollen or even suppurated, it proved that the rat model of diabetic wound infection was successfully established.

[0057] Rats were randomly divided into 9 groups, 3 rats in each group, namely KB, Model, VIS, CCBG, CCBG-5, CCBG-2.5+VIS, CCBG-5+VIS, CCBG-7.5+VIS, AgNPs hydrogel. The dressing was changed every other day, and the wound images on the 0th, 3rd, 7th, 10th and 14th days were recorded. The wound area was measured using ImageJ software, and the wound healing rate and scab removal rate on the 14th day were calculated. Wound healing rate (%) = 1-(At / A0), Scab shedding rate (%) = 1-(S14 / A0). Where At and A0 are the wound area sizes on the tth day and the 0th day, respectively. S14 is the scab area remaining in the wound on the 14th day. The in vivo healing ability of riboflavin photodynamic antibacterial gel was studied in the diabetic wound bacterial infection model. Nanosilver hydrogel was used as the positive control group. The infected wound site was covered with hydrogel and irradiated with 400nm near-infrared light for 3min. Treatment was performed every other day, and the wound was photographed every day to observe the wound size to evaluate the therapeutic effect of the hydrogel. For specific results, see the attached Figure 7 shown.

[0058] By the attached Figure 7 The data show that the wound healing and scar healing speeds of the CCBG-5 photodynamic antibacterial gel of the present invention are the fastest. After 14 days of treatment, the wound healing rates of Model, VIS, CCBG, CCBG-5, CCBG-2.5+VIS, CCBG-5+VIS, CCBG-7.5+VIS, and AgNPs hydrogel are 51.85%, 59.70%, 72.39%, 73.72%, 65.92%, 84.46%, 84.41%, and 82.12%, respectively. The wound scab shedding rate of the CCBG-5+VIS group at 14 days is 99.75, which is significantly different (p < 0.05). This shows that CCBG-5 has the best therapeutic effect on diabetic wounds in rats, which is based on its powerful photodynamic bactericidal and anti-inflammatory properties, and can improve bacterial infection and inflammatory response at the wound. In addition, CCBG-5 photodynamic hydrogel can also promote cell proliferation and collagen deposition at the wound surface, thereby effectively promoting wound healing.

[0059] In summary, this experiment successfully evaluated the effects of different treatment groups on wound healing in vivo by constructing a rat diabetic full-thickness wound defect model. The treatment group of riboflavin photodynamic antibacterial gel combined with visible light (such as CCBG-5+VIS) showed a higher wound healing rate and scab removal rate, indicating that it has a significant effect in promoting wound healing.

[0060] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A riboflavin photodynamic antibacterial hydrogel for diabetic wound dressing, characterized in that: The riboflavin photodynamic antibacterial hydrogel comprises, by weight, 10 to 90 parts of carbomer 941, 50 to 300 parts of carboxymethyl chitosan, 150 parts of triethanolamine, 500 to 4500 parts of bletilla striata polysaccharide, 0 to 50 parts of riboflavin, 150 to 550 parts of glutaraldehyde and 10000 to 13000 parts of ultrapure water.

2. The riboflavin photodynamic antibacterial hydrogel for diabetic wound dressing according to claim 1, characterized in that: Calculated by weight, the riboflavin photodynamic antibacterial hydrogel comprises 50 parts of carbomer 941, 100 parts of carboxymethyl chitosan, 150 parts of triethanolamine, 2500 parts of bletilla striata polysaccharide, 50 parts of riboflavin, 350 parts of glutaraldehyde and 12000 parts of ultrapure water.

3. The method for preparing the riboflavin photodynamic antibacterial hydrogel for diabetic wound dressing according to any one of claims 1 to 2, characterized in that: The specific steps include: (1) Adding carbomer 941 to the riboflavin aqueous solution to prepare a riboflavin-carbomer 941 dispersion system; (2) adding the carboxymethyl chitosan aqueous solution to the riboflavin-carbomer 941 dispersion system to obtain a carbomer-carboxymethyl chitosan mixed solution; (3) adjusting the pH value of the carbomer-carboxymethyl chitosan mixed solution, adding the aqueous solution of Bletilla striata polysaccharide to obtain a carbomer-carboxymethyl chitosan-Bletilla striata polysaccharide mixed solution; (4) Add ultrapure water to the carbomer-carboxymethyl chitosan-Bletilla striata polysaccharide mixed solution and stir evenly, add glutaraldehyde, and stir to obtain riboflavin photodynamic antibacterial hydrogel.

4. The method for preparing the riboflavin photodynamic antibacterial hydrogel for diabetic wound dressing according to claim 3, characterized in that: In step (1), the mass ratio of riboflavin to carbomer 941 is 1-5:

5.

5. The method for preparing the riboflavin photodynamic antibacterial hydrogel for diabetic wound dressing according to claim 4, characterized in that: In step (2), the mass ratio of the carboxymethyl chitosan aqueous solution to the riboflavin-carbomer 941 dispersion system is 1:50-60.

6. The method for preparing the riboflavin photodynamic antibacterial hydrogel for diabetic wound dressing according to claim 3, characterized in that: In step (3), the pH value is adjusted to 6-8.

7. The method for preparing the riboflavin photodynamic antibacterial hydrogel for diabetic wound dressing according to claim 3, characterized in that: In step (3), the mass ratio of the aqueous solution of Bletilla striata polysaccharide to the carbomer-carboxymethyl chitosan mixed solution is 1:2-3.

8. The method for preparing the riboflavin photodynamic antibacterial hydrogel for diabetic wound dressing according to claim 3, characterized in that: In step (4), the mass ratio of the carbomer-carboxymethyl chitosan-Bletilla striata polysaccharide mixed solution to ultrapure water is 1-2:

1.

9. The method for preparing the riboflavin photodynamic antibacterial hydrogel for diabetic wound dressing according to claim 3, characterized in that: In step (4), the amount of glutaraldehyde added is 1% to 3% of the volume of the carbomer-carboxymethyl chitosan-Bletilla striata polysaccharide mixed solution.

10. Use of the riboflavin photodynamic antibacterial hydrogel for diabetic wound dressing according to any one of claims 1 to 2 in the preparation of diabetic wound healing dressing.

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