Natural hydrogel dressing for promoting wound healing and preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to a natural hydrogel dressing for promoting wound healing and a preparation method and application thereof. The natural hydrogel dressing for promoting wound healing is prepared from 10wt% of an oxidized bletilla striata polysaccharide aqueous solution, 10wt%-20wt% of a dopamine grafted epsilon-polylysine aqueous solution and 1wt%-2wt% of a vitamin B1-loaded phenylboronic acid grafted chitosan micelle aqueous solution through a Schiff base dynamic cross-linking reaction. The natural hydrogel dressing for promoting wound healing has the multifunctional characteristics of injectability, self-healing ability, biocompatibility, tissue adhesion, mechanical strength and pH / glucose dual responsiveness. In addition, the compound also has anti-sugar, anti-inflammatory and antibacterial biological activities, and can improve the diabetes wound inflammation microenvironment and promote rapid healing and regeneration of infectious diabetes wounds by inhibiting AGEs formation, regulating macrophage M2 phenotypic polarization and preventing / treating bacterial infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and specifically relates to a natural hydrogel dressing for promoting wound healing, its preparation method and application. Background Art

[0002] Diabetic wounds are one of the most common chronic complications in diabetic patients. Due to the hyperglycemic microenvironment at the wound surface, diabetic wounds often accumulate a large amount of advanced glycation end products (AGEs) and are infected by bacteria, which will trigger an excessive amount of reactive oxygen species (ROS) and inflammatory responses, damaging cell proliferation, angiogenesis, tissue granulation, and wound healing and regeneration, ultimately resulting in the inability of the wound to heal itself. If the treatment is improper or untimely, diabetic wounds may lead to amputation and even endanger life. Therefore, developing a new strategy to inhibit the formation of AGEs, regulate macrophage phenotype polarization, and effectively treat bacterial infections is crucial for achieving the healing of infectious diabetic wounds.

[0003] Currently, many anti-diabetic drugs (such as insulin, Tirzepatide, and Semagutide) can relieve the inflammatory response of diabetic wounds by inhibiting AGEs, but they cannot fundamentally solve the problem of excessive inflammatory response. In addition, these drugs are usually costly, have unsustainable effects, and even have side effects. With the rapid development of materials science and nanomedicine, some inorganic nanoparticles (such as CeO2 and AuNP) have also been used as hypoglycemic agents to inhibit skin glycation. However, their poor biodegradability in vivo remains a problem that cannot be ignored. On the other hand, a large number of antibacterial drugs or antibacterial agents, including antibiotics, silver nanoparticles, metal oxide nanoparticles, and 2D nanosheets, have been reported to solve bacterial infections, but their antibacterial resistance and cytotoxic side effects on cells usually limit their therapeutic effects on wound healing.

[0004] As an important product in modern wound care, dressings can not only protect the wound surface from further damage but also isolate microbial infections to a certain extent. Among numerous dressings, hydrogel dressings have become the most promising wound dressings due to their excellent biocompatibility, biodegradability, ability to absorb tissue exudate, and the property of providing a moist microenvironment for the wound. However, most traditional hydrogels often have a single function, and their therapeutic effects and application prospects are not ideal. Summary of the Invention

[0005] Aiming at the deficiencies of the above-mentioned existing technologies, the purpose of the present invention is to provide a natural hydrogel dressing for promoting wound healing, its preparation method and application. The present invention dynamically cross-links phenylboronic acid grafted chitosan micelles loaded with vitamin B1 and dopamine grafted ε-polylysine with oxidized bletilla striata polysaccharide through Schiff base to prepare a natural multifunctional hydrogel dressing, that is, a natural hydrogel dressing for promoting wound healing. The natural hydrogel dressing for promoting wound healing prepared by the method of the present invention has physical multifunctionalities such as injectability, self-healing ability, biocompatibility, tissue adhesiveness, mechanical strength, and pH / glucose dual responsiveness, and prevents local stress damage by fully filling and long-term adhering to the damaged site. At the same time, the natural hydrogel dressing has bioactivities of anti-glycation, anti-inflammation and anti-bacterial. By effectively inhibiting the formation of AGEs, regulating the polarization of macrophage M2 phenotype and preventing / treating bacterial infection, it improves the inflammatory microenvironment of diabetic wounds, accelerates the rapid healing and regeneration of infectious diabetic wounds, and overcomes the technical defect of single function of existing technology dressings.

[0006] The present invention is realized by adopting the following technical solutions:

[0007] The present invention protects a preparation method of a natural hydrogel dressing for promoting wound healing, comprising the following steps:

[0008] After mixing 3-carboxyphenylboronic acid and a carboxyl activating reagent N-hydroxysuccinimide (NHS), adding a carboxyl activating reagent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) to obtain a phenylboronic acid solution; mixing the phenylboronic acid solution with a chitosan solution, and performing an amidation reaction between the carboxyl group of 3-carboxyphenylboronic acid and the amino group of chitosan. The combination of EDC and NHS promotes the amidation reaction between the carboxyl group and the amino group. The reaction conditions are: stirring at room temperature for 12 h to 24 h to obtain phenylboronic acid grafted chitosan; dissolving the phenylboronic acid grafted chitosan in a vitamin B1 solution and performing self-assembly. The self-assembly conditions are: stirring at room temperature for 12 h to 24 h. At this time, vitamin B1 is physically coated inside the phenylboronic acid grafted chitosan to obtain phenylboronic acid grafted chitosan micelles loaded with vitamin B1.

[0009] Dissolve sodium borate and sodium bicarbonate in water together, add dopamine hydrochloride under anaerobic conditions, and then dropwise add a tetrahydrofuran solution containing acryloyl chloride to perform an amidation reaction. The acyl chloride group of acryloyl chloride reacts with the amino group of dopamine hydrochloride. Sodium borate and sodium bicarbonate promote the amidation reaction between dopamine hydrochloride and acryloyl chloride; the reaction conditions are: stirring at room temperature for 12 h to 24 h to obtain double bond modified dopamine; mixing the double bond modified dopamine aqueous solution with an ε-polylysine dimethyl sulfoxide solution, and performing a Michael addition reaction between the double bond of the double bond modified dopamine and the amino group in ε-polylysine. The Michael addition reaction conditions are: stirring and reacting at 45 °C to 60 °C for 48 h to 72 h to obtain dopamine grafted ε-polylysine.

[0010] Mix a 10 wt% aqueous solution of oxidized Bletilla striata polysaccharide, a 10 wt% - 20 wt% aqueous solution of dopamine-grafted ε-polylysine, and a 1 wt% - 2 wt% aqueous solution of phenylboronic acid-grafted chitosan micelles loaded with vitamin B1. Then, Schiff base reactions occur between the amino groups in dopamine-grafted ε-polylysine and phenylboronic acid-grafted chitosan micelles loaded with vitamin B1 and the aldehyde groups in oxidized Bletilla striata polysaccharide, resulting in a natural hydrogel dressing that promotes wound healing.

[0011] Among them, the volume ratio of the 10 wt% aqueous solution of oxidized Bletilla striata polysaccharide, the 10 wt% - 20 wt% aqueous solution of dopamine-grafted ε-polylysine, and the 1 wt% - 2 wt% aqueous solution of phenylboronic acid-grafted chitosan micelles loaded with vitamin B1 is 4.5 - 5:1 - 2:4.5 - 2.

[0012] Preferably, the mass ratio of 3-carboxyphenylboronic acid to chitosan is 1:1.1 - 1.8. Outside this ratio range, the grafting rate will be too low to form micelles.

[0013] Preferably, the mass ratio of vitamin B1 to phenylboronic acid-grafted chitosan is 1:10 - 20. Outside this ratio range, the anti-glycation property of the natural hydrogel dressing that promotes wound healing is weakened.

[0014] Preferably, the molar ratio of dopamine hydrochloride to acryloyl chloride is 1:2 - 5. Outside this ratio range, it cannot be ensured that the double bond of acryloyl chloride can completely modify dopamine hydrochloride.

[0015] Preferably, the molar ratio of double bond-modified dopamine to ε-polylysine is 1:2 - 4. Outside this ratio range, the adhesion and antibacterial properties of the natural hydrogel dressing that promotes wound healing are affected.

[0016] Preferably, the conditions for the Schiff base reaction are: standing at room temperature for 0.5 h - 1 h.

[0017] Preferably, oxidized Bletilla striata polysaccharide is prepared according to the following steps: Mix sodium periodate with Bletilla striata polysaccharide, stir and oxidize in the dark at room temperature for 24 h, and use sodium periodate to oxidize the hydroxyl groups in Bletilla striata polysaccharide molecules to obtain oxidized Bletilla striata polysaccharide.

[0018] The present invention also protects the natural hydrogel dressing that promotes wound healing prepared by the above preparation method.

[0019] The present invention also protects the application of the natural hydrogel dressing that promotes wound healing in the preparation of drugs for diabetic infectious skin injuries, drugs for treating inflammatory reaction diseases, or drugs for treating bacterial infectious diseases.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] 1. In order to obtain a natural hydrogel dressing with anti-glycation, anti-inflammatory and antibacterial bioactivities for promoting wound healing, starting from the raw materials, the present invention first separately prepares a phenylboronic acid-grafted chitosan micelle loaded with vitamin B1, dopamine-grafted ε-polylysine, and oxidized bletilla striata polysaccharide, and then performs a Schiff base reaction on the three of them. Among them, the phenylboronic acid-grafted chitosan micelle loaded with vitamin B1 is prepared by first performing an amidation reaction between 3-carboxyphenylboronic acid and chitosan to obtain phenylboronic acid-grafted chitosan, and then coating vitamin B1 with phenylboronic acid-grafted chitosan; dopamine-grafted ε-polylysine is prepared by first performing an amidation reaction between acryloyl chloride and dopamine hydrochloride to obtain double bond-modified dopamine, and then performing a Michael addition reaction between double bond-modified dopamine and ε-polylysine; oxidized bletilla striata polysaccharide is prepared by oxidizing the hydroxyl groups in the bletilla striata polysaccharide molecule with sodium periodate.

[0022] Among them, vitamin B1 has anti-glycation properties. The phenylboronic acid-grafted chitosan micelle loaded with vitamin B1 not only solves the problems of low bioavailability and fast metabolism of vitamin B1, but also endows the natural hydrogel dressing for promoting wound healing with anti-glycation properties and inhibits the generation of AGEs; oxidized bletilla striata polysaccharide has an immunomodulatory function. Therefore, oxidized bletilla striata polysaccharide endows the natural hydrogel dressing for promoting wound healing with anti-inflammatory properties and regulates macrophage phenotype polarization; dopamine-grafted ε-polylysine has tissue adhesion and antibacterial properties, and endows the natural hydrogel dressing for promoting wound healing with adhesion properties and antibacterial properties.

[0023] The present invention combines vitamin B1 with an ε-polylysine-based hydrogel material with controllable drug release characteristics to prepare a natural anti-glycation, anti-inflammatory and antibacterial multifunctional hydrogel dressing, which can be used to achieve the rapid healing and regeneration of infectious diabetic wounds.

[0024] 2. The present invention provides a natural hydrogel dressing for promoting wound healing based on natural products and with a green and environmentally friendly preparation process. It has excellent injectability, self-healing ability, biocompatibility, tissue adhesion, appropriate mechanical strength, and pH / glucose dual-responsive physical multifunctional characteristics. The injectability can fully fill the wound. Due to the presence of imine dynamic chemical bonds in the natural hydrogel dressing for promoting wound healing, it has self-healing ability, and the pH / glucose dual-responsive property realizes the controllable release of vitamin B1.

[0025] 3. The natural hydrogel dressing for promoting wound healing prepared by the present invention has anti-glycation, anti-inflammatory and antibacterial biological activities, and can improve the inflammatory microenvironment of diabetic wounds by effectively inhibiting the formation of AGEs, regulating the polarization of macrophage M2 phenotype and preventing / treating bacterial infections, so as to promote the rapid healing and regeneration of infectious diabetic wounds. The mechanism of anti-glycation using vitamin B1 in the present invention is as follows: in the hyperglycemic microenvironment, the main site of non-enzymatic glycosylation of proteins is the free amino group in lysine, which can react with the carbonyl groups of various sugars to form Amadori products, and finally form isomeric AGEs. In the present invention, vitamin B1 acts as an inhibitor to inhibit the generation of AGEs by masking the lysine residues in proteins, thereby achieving the anti-glycation effect.

[0026] 4. The natural hydrogel dressing for promoting wound healing prepared by the present invention is used for the repair of infectious diabetic skin wounds, and obvious therapeutic effects have been achieved in both in vitro and in vivo experiments. Therefore, the natural hydrogel dressing for promoting wound healing has potential application value in the treatment of infectious diabetic wounds. Brief Description of the Drawings

[0027] Figure 1 In it, A is the 1H NMR spectrum of phenylboronic acid grafted chitosan and dopamine grafted ε-polylysine in Example 1; B is the infrared spectrum of Bletilla striata polysaccharide, oxidized Bletilla striata polysaccharide and multifunctional hydrogel 1 in Example 1; C is the transmission scanning electron micrograph of phenylboronic acid grafted chitosan in Example 1. 1 H NMR spectrum; B is the infrared spectrum of Bletilla striata polysaccharide, oxidized Bletilla striata polysaccharide and multifunctional hydrogel 1 in Example 1; C is the transmission scanning electron micrograph of phenylboronic acid grafted chitosan in Example 1.

[0028] Figure 2 are the scanning electron micrograph and element distribution map of multifunctional hydrogel 1 in Example 1.

[0029] Figure 3 In it, A is the injectability test diagram of multifunctional hydrogel 1 in Example 1; B is the self-healing ability diagram of multifunctional hydrogel 1 in Example 1; C is the tissue adhesion diagram of multifunctional hydrogel 1 in Example 1; D is the rheological property diagram of multifunctional hydrogel 1 in Example 1.

[0030] Figure 4 In it, A is the cytotoxicity test result diagram of multifunctional hydrogel 1, dopamine grafted ε-polylysine, oxidized Bletilla striata polysaccharide, phenylboronic acid grafted chitosan and blank group on NIH3T3 cells; B is the proliferation result diagram of multifunctional hydrogel 1, dopamine grafted ε-polylysine, oxidized Bletilla striata polysaccharide, phenylboronic acid grafted chitosan and blank group on NIH3T3 cells.

[0031] Figure 5Anti-glycation performance diagram of the multifunctional hydrogel 1 in Example 1. Among them, A is the ultraviolet-visible spectrum diagram of human serum albumin (HSA) after 28 days of treatment in the multifunctional hydrogel group and the control group; B is the fluorescence intensity diagram of AGEs-specific fluorescence after 28 days of treatment in the multifunctional hydrogel group and the control group.

[0032] Figure 6 Anti-inflammatory performance diagram of the multifunctional hydrogel 1 in Example 1. Among them, A is the statistical analysis diagram of the CD206 ratio of macrophages RAW264.7 in the multifunctional hydrogel group and the control group under AGEs stimulation; B is the statistical analysis diagram of the CD86 ratio of macrophages RAW264.7 in the multifunctional hydrogel group and the control group under AGEs stimulation.

[0033] Figure 7 Antibacterial performance diagram of the multifunctional hydrogel 1 in Example 1. Among them, A is the in vitro antibacterial photos of E. coli, S. aureus and MRSA under the treatment of the multifunctional hydrogel group and the control group; B is the bacterial activity diagram of E. coli, S. aureus and MRSA under the treatment of the multifunctional hydrogel group and the control group.

[0034] Figure 8 Among them, A is the repair result diagram of the infectious skin injury of diabetic mice infected with the multifunctional hydrogel 1 in Example 1, the multifunctional hydrogel 4 in Example 4 and the control group; B is the in vivo antibacterial performance diagram of the multifunctional hydrogel 1, the multifunctional hydrogel 4 in Example 4 and the control group and the control group. Detailed implementation mode

[0035] The following is a detailed description of the specific implementation mode of the present invention, but it should be understood that the protection scope of the present invention is not limited by the specific implementation mode. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work belong to the protection scope of the present invention. The experimental methods described in each embodiment of the present invention are conventional methods unless otherwise specified.

[0036] Considering the technical defect of the single function of traditional hydrogel dressings, for the first time, the present invention uses oxidized Bletilla striata polysaccharide, dopamine-grafted ε-polylysine, and phenylboronic acid-grafted chitosan micelles loaded with vitamin B1 as raw materials to prepare a natural multifunctional hydrogel dressing through Schiff base reaction, and uses it for the repair of infectious diabetic skin injuries. This method is based on natural products and the preparation process is green and environmentally friendly. The prepared multifunctional hydrogel dressing has excellent injectability, self-healing ability, biocompatibility, tissue adhesiveness, appropriate mechanical strength, and pH / glucose dual-responsive physical multifunctionality. In addition, the multifunctional hydrogel of the present invention also has biological activities of anti-glycation, anti-inflammatory, and antibacterial, and can improve the inflammatory microenvironment of diabetic wounds by effectively inhibiting the formation of AGEs and preventing / treating bacterial infections, and promote the rapid healing and regeneration of infectious diabetic wounds. Therefore, the natural hydrogel dressing that promotes wound healing is expected to become a new multifunctional biomaterial that can simultaneously achieve hemostasis, anti-glycation, anti-inflammatory, antibacterial, and promote wound surface healing, and has good application prospects in the clinical treatment of chronic infectious skin wounds.

[0037] The following uses examples to study the technical solutions of the present invention, and the specific research methods and results are as follows:

[0038] Example 1

[0039] A preparation method of a natural hydrogel dressing that promotes wound healing, comprising the following steps:

[0040] S1. Preparation of oxidized Bletilla striata polysaccharide: Dissolve 1 g of Bletilla striata polysaccharide in 500 mL of deionized water, then add 4.28 g of sodium periodate, and stir at room temperature in the dark for 24 h; then add 2 mL of ethylene glycol to terminate the oxidation reaction, and continue to stir for 2 h. After the reaction is completed, load it into a dialysis bag (MWCO = 3500), dialyze with water for 3 days for purification, and finally freeze-dry to obtain oxidized Bletilla striata polysaccharide.

[0041] S2. Preparation of phenylboronic acid-grafted chitosan: Dissolve 0.5 g of chitosan in 60 mL of acetic acid solution and stir for 1 h to obtain a chitosan solution; dissolve 0.45 g of 3-carboxyphenylboronic acid and 286.6 mg of N-hydroxysuccinimide in 40 mL of methanol and stir for 30 min, then add 353.8 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and continue to stir to obtain a phenylboronic acid solution; add the phenylboronic acid solution to the chitosan solution and stir for 24 h. After the reaction is completed, load it into a dialysis bag (MWCO = 3000), dialyze with water for 3 days for purification, and finally freeze-dry to obtain phenylboronic acid-grafted chitosan.

[0042] Preparation of vitamin B1-loaded phenylboronic acid-grafted chitosan micelles: Dissolve 200 mg of phenylboronic acid-grafted chitosan in 800 μL of a solution containing 10 mg of vitamin B1, stir at room temperature for 18 h, and finally perform freeze-drying to obtain vitamin B1-loaded phenylboronic acid-grafted chitosan micelles.

[0043] S3. Preparation of double bond-modified dopamine: Dissolve 10 g of sodium borate and 4 g of sodium bicarbonate in 100 mL of deionized water. After bubbling the solution with nitrogen for 20 min, add 5 g of hydrochloric acid dopamine, then dropwise add a THF solution containing 4.7 mL of acryloyl chloride, and stir at room temperature for 18 h. After the reaction, wash with 50 mL of ethyl acetate, then adjust the pH to 2 with 4 mol / L hydrochloric acid solution, extract 3 times from the aqueous solution with 50 mL of ethyl acetate, dry the combined organic layer with anhydrous magnesium sulfate and concentrate to 25 mL. Subsequently, slowly add 250 mL of n-hexane with stirring, and keep the suspension in a refrigerator at 4 °C overnight. Filter the precipitate and dry it in vacuo for 1 day to obtain double bond-modified dopamine.

[0044] Preparation of dopamine-grafted ε-polylysine: Dissolve 4.2 g of ε-polylysine and 0.2 g of double bond-modified dopamine in 20 mL of deionized water and 60 mL of dimethyl sulfoxide respectively. Then add the double bond-modified dopamine solution to the ε-polylysine solution, react at 60 °C for 72 h. After the reaction, load it into a dialysis bag (MWCO = 3000), dialyze with water for 3 days for purification, and finally perform freeze-drying to obtain dopamine-grafted ε-polylysine.

[0045] S4. Preparation of a natural hydrogel dressing for promoting wound healing: Prepare 10 wt% oxidized bletilla striata polysaccharide aqueous solution, 10 wt% dopamine-grafted ε-polylysine aqueous solution, and 1 wt% vitamin B1-loaded phenylboronic acid-grafted chitosan micelle aqueous solution respectively. Then add 20 μL of dopamine-grafted ε-polylysine aqueous solution and 50 μL of vitamin B1-loaded phenylboronic acid-grafted chitosan micelle aqueous solution to 50 μL of oxidized bletilla striata polysaccharide aqueous solution in sequence at room temperature. After vortex oscillation, obtain a natural hydrogel dressing for promoting wound healing, denoted as multifunctional hydrogel 1.

[0046] Example 2

[0047] A preparation method of a natural hydrogel dressing for promoting wound healing, comprising the following steps:

[0048] Prepare oxidized bletilla striata polysaccharide, dopamine-grafted ε-polylysine, and vitamin B1-loaded phenylboronic acid-grafted chitosan micelles by the same method as in Example 1.

[0049] Preparation of a natural hydrogel dressing for promoting wound healing: Prepare a 10 wt% aqueous solution of oxidized Bletilla striata polysaccharide, a 15 wt% aqueous solution of dopamine-grafted ε-polylysine, and a 1.5 wt% aqueous solution of phenylboronic acid-grafted chitosan micelles loaded with vitamin B1, respectively. Then, at room temperature, add 20 μL of the aqueous solution of dopamine-grafted ε-polylysine and 50 μL of the aqueous solution of phenylboronic acid-grafted chitosan micelles loaded with vitamin B1 to 50 μL of the aqueous solution of oxidized Bletilla striata polysaccharide in sequence. After vortexing, a natural hydrogel dressing for promoting wound healing is obtained, denoted as multifunctional hydrogel 2.

[0050] Example 3

[0051] A method for preparing a natural hydrogel dressing for promoting wound healing, comprising the following steps:

[0052] Prepare oxidized Bletilla striata polysaccharide, dopamine-grafted ε-polylysine, and phenylboronic acid-grafted chitosan micelles loaded with vitamin B1 by the same method as in Example 1.

[0053] Preparation of a natural hydrogel dressing for promoting wound healing: Prepare a 10% aqueous solution of oxidized Bletilla striata polysaccharide, a 20 wt% aqueous solution of dopamine-grafted ε-polylysine, and a 2 wt% aqueous solution of phenylboronic acid-grafted chitosan micelles loaded with vitamin B1, respectively. Then, at room temperature, add 20 μL of the aqueous solution of dopamine-grafted ε-polylysine and 50 μL of the aqueous solution of phenylboronic acid-grafted chitosan micelles loaded with vitamin B1 to 50 μL of the aqueous solution of oxidized Bletilla striata polysaccharide in sequence. After vortexing, a natural hydrogel dressing for promoting wound healing is obtained, denoted as multifunctional hydrogel 3.

[0054] Example 4

[0055] A method for preparing a natural hydrogel dressing for promoting wound healing, comprising the following steps:

[0056] Prepare oxidized Bletilla striata polysaccharide, dopamine-grafted ε-polylysine, and phenylboronic acid-grafted chitosan micelles by the same method as in Example 1.

[0057] Preparation of a natural hydrogel dressing for promoting wound healing: Prepare a 10 wt% aqueous solution of oxidized Bletilla striata polysaccharide, a 20 wt% aqueous solution of dopamine-grafted ε-polylysine, and a 2 wt% aqueous solution of phenylboronic acid-grafted chitosan micelles, respectively. Then, at room temperature, add 20 μL of the aqueous solution of dopamine-grafted ε-polylysine and 50 μL of the aqueous solution of phenylboronic acid-grafted chitosan micelles loaded with vitamin B1 to 50 μL of the aqueous solution of oxidized Bletilla striata polysaccharide in sequence. After vortexing, a natural hydrogel dressing for promoting wound healing is obtained, denoted as multifunctional hydrogel 4.

[0058] Example 5

[0059] A method for preparing a natural hydrogel dressing for promoting wound healing, comprising the following steps:

[0060] S1. Preparation of oxidized Bletilla striata polysaccharide: Dissolve 1 g of Bletilla striata polysaccharide in 500 mL of deionized water, then add 4.28 g of sodium periodate, and stir in the dark at room temperature for 24 h; then add 2 mL of ethylene glycol to terminate the oxidation reaction, and continue stirring for 2 h. After the reaction is completed, transfer it into a dialysis bag (MWCO = 3500), and dialyze with water for 3 days for purification. Finally, freeze-dry to obtain oxidized Bletilla striata polysaccharide.

[0061] S2. Preparation of phenylboronic acid-grafted chitosan: Dissolve 0.675 g of chitosan in 60 mL of acetic acid solution and stir for 1 h to obtain a chitosan solution; dissolve 0.45 g of 3-carboxyphenylboronic acid and 286.6 mg of N-hydroxysuccinimide in 40 mL of methanol and stir for 30 min, then add 353.8 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and continue stirring to obtain a phenylboronic acid solution; add the phenylboronic acid solution to the chitosan solution and stir for 12 h. After the reaction is completed, transfer it into a dialysis bag (MWCO = 3000), and dialyze with water for 3 days for purification. Finally, freeze-dry to obtain phenylboronic acid-grafted chitosan.

[0062] Preparation of phenylboronic acid-grafted chitosan micelles loaded with vitamin B1: Dissolve 150 mg of phenylboronic acid-grafted chitosan in 800 μL of a solution containing 10 mg of vitamin B1, stir at room temperature for 12 h, and finally freeze-dry to obtain phenylboronic acid-grafted chitosan micelles loaded with vitamin B1.

[0063] S3. Preparation of double-bond modified dopamine: Dissolve 10 g of sodium borate and 4 g of sodium bicarbonate in 100 mL of deionized water, bubble the solution with nitrogen for 20 min, then add 5 g of dopamine hydrochloride, and then dropwise add a THF solution containing 9 mL of acryloyl chloride, and stir at room temperature for 24 h. After the reaction is completed, wash with 50 mL of ethyl acetate, then adjust the pH to 2 with 4 mol / L hydrochloric acid solution, extract 3 times from the aqueous solution with 50 mL of ethyl acetate, dry the combined organic layer with anhydrous magnesium sulfate and concentrate to 25 mL. Subsequently, slowly add 250 mL of n-hexane with stirring, and keep the suspension in a refrigerator at 4 °C overnight. Filter the precipitate and dry it in vacuo for 1 day to obtain double-bond modified dopamine.

[0064] Preparation of dopamine-grafted ε-polylysine: Dissolve 4.2 g of ε-polylysine and 0.2 g of double-bond modified dopamine in 20 mL of deionized water and 60 mL of dimethyl sulfoxide respectively, then add the double-bond modified dopamine solution to the ε-polylysine solution, and react at 45 °C for 60 h. After the reaction is completed, transfer it into a dialysis bag (MWCO = 3000), and dialyze with water for 3 days for purification. Finally, freeze-dry to obtain dopamine-grafted ε-polylysine.

[0065] S4. Preparation of a natural hydrogel dressing for promoting wound healing: Prepare a 10 wt% aqueous solution of oxidized Bletilla striata polysaccharide, a 10 wt% aqueous solution of dopamine-grafted ε-polylysine, and a 1 wt% aqueous solution of phenylboronic acid-grafted chitosan micelles loaded with vitamin B1 respectively. Then, add 20 μL of the dopamine-grafted ε-polylysine aqueous solution and 50 μL of the phenylboronic acid-grafted chitosan micelles aqueous solution loaded with vitamin B1 to 50 μL of the oxidized Bletilla striata polysaccharide aqueous solution in sequence at room temperature. After vortex oscillation, a natural hydrogel dressing for promoting wound healing is obtained.

[0066] Example 6

[0067] A preparation method of a natural hydrogel dressing for promoting wound healing, comprising the following steps:

[0068] S1. Preparation of oxidized Bletilla striata polysaccharide: Dissolve 1 g of Bletilla striata polysaccharide in 500 mL of deionized water, then add 4.28 g of sodium periodate, and stir in the dark at room temperature for 24 h; add 2 mL of ethylene glycol to terminate the oxidation reaction, and continue stirring for 2 h. After the reaction is completed, put it into a dialysis bag (MWCO = 3500), and dialyze with water for 3 days for purification. Finally, freeze-dry to obtain oxidized Bletilla striata polysaccharide.

[0069] S2. Preparation of phenylboronic acid-grafted chitosan: Dissolve 0.81 g of chitosan in 60 mL of acetic acid solution and stir for 1 h to obtain a chitosan solution; dissolve 0.45 g of 3-carboxyphenylboronic acid and 286.6 mg of N-hydroxysuccinimide in 40 mL of methanol and stir for 30 min, then add 353.8 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and continue stirring to obtain a phenylboronic acid solution; add the phenylboronic acid solution to the chitosan solution and stir for 18 h. After the reaction is completed, put it into a dialysis bag (MWCO = 3000), and dialyze with water for 3 days for purification. Finally, freeze-dry to obtain phenylboronic acid-grafted chitosan.

[0070] Preparation of phenylboronic acid-grafted chitosan micelles loaded with vitamin B1: Dissolve 100 mg of phenylboronic acid-grafted chitosan in 800 μL of a solution containing 10 mg of vitamin B1, stir at room temperature for 24 h, and finally freeze-dry to obtain phenylboronic acid-grafted chitosan micelles loaded with vitamin B1.

[0071] S3. Preparation of double bond-modified dopamine: Dissolve 10 g of sodium borate and 4 g of sodium bicarbonate in 100 mL of deionized water. After bubbling the solution with nitrogen for 20 min, add 5 g of hydrochloric acid dopamine, then dropwise add a THF solution containing 11.7 mL of acryloyl chloride, and stir at room temperature for 12 h. After the reaction is completed, wash with 50 mL of ethyl acetate, then adjust the pH to 2 with 4 mol / L hydrochloric acid solution, extract 3 times from the aqueous solution with 50 mL of ethyl acetate, dry the combined organic layer with anhydrous magnesium sulfate and concentrate to 25 mL. Subsequently, slowly add 250 mL of n-hexane with stirring, and keep the suspension in a refrigerator at 4 °C overnight. Filter the precipitate and dry it in vacuo for 1 day to obtain double bond-modified dopamine.

[0072] Preparation of dopamine-grafted ε-polylysine: Dissolve 4.2 g of ε-polylysine and 0.2 g of double bond-modified dopamine in 20 mL of deionized water and 60 mL of dimethyl sulfoxide respectively. Then add the double bond-modified dopamine solution to the ε-polylysine solution, and react at 50 °C for 48 h. After the reaction is completed, load it into a dialysis bag (MWCO = 3000) and dialyze with water for 3 days for purification. Finally, perform freeze-drying to obtain dopamine-grafted ε-polylysine.

[0073] S4. Preparation of a natural hydrogel dressing for promoting wound healing: Prepare 10 wt% aqueous solution of oxidized bletilla striata polysaccharide, 10 wt% aqueous solution of dopamine-grafted ε-polylysine, and 1 wt% aqueous solution of phenylboronic acid-grafted chitosan micelles loaded with vitamin B1 respectively. Then, at room temperature, sequentially add 20 μL of the aqueous solution of dopamine-grafted ε-polylysine and 50 μL of the aqueous solution of phenylboronic acid-grafted chitosan micelles loaded with vitamin B1 to 50 μL of the aqueous solution of oxidized bletilla striata polysaccharide. After vortex oscillation, a natural hydrogel dressing for promoting wound healing is obtained.

[0074] The present invention investigated the biocompatibility, anti-glycation, anti-inflammatory, and in vitro antibacterial properties of the natural hydrogel dressing for promoting wound healing. The specific methods are as follows:

[0075] The experimental steps for investigating the biocompatibility are as follows:

[0076] Take NIH3T3 cells in good growth state and inoculate them in a 96-well plate at a density of 1.0×10 4 cells / well. When the cells adhere to the wall and reach 80%, aspirate the old culture medium, and add 100 μL of culture medium containing different concentrations (0 μg mL -1 、10 μg mL -1 、20 μg mL -1 、30 μg mL -1 、50 μg mL -1 and 100 μg mL -1) Oxidized Bletilla striata polysaccharide culture medium, dopamine grafted ε-polylysine culture medium, phenylboronic acid grafted chitosan culture medium and multifunctional hydrogel culture medium, and incubate for 24 hours, then remove the old culture medium, add 100 μL of DMEM solution containing 5% CCK-8 to each well, and then place in a cell culture incubator for further incubation for 1 hour. The absorbance of the well plate at 450nm was detected by a multifunctional microplate reader to quantitatively calculate the cell survival rate.

[0077] NIH3T3 cells that have grown well were taken and 1×10 3 After the cells adhered to the wall, the old culture medium was aspirated and 200 μL of 100 μg mL -1 Multifunctional hydrogel culture medium and 200 μL control culture medium (200 μL fresh culture medium, 200 μL containing 100 μg mL -1 Oxidized Bletilla striata polysaccharide culture medium, 100 μL containing 100 μg mL -1 Dopamine grafted ε-polylysine culture medium and 100 μL containing 100 μg mL -1 After adding phenylboronic acid grafted chitosan culture medium), the well plate was placed in a cell culture incubator for incubation. The cells in the 48-well plate were stained with a live cell / dead cell staining kit on the 1st day, 3rd day and 5th day, and the fluorescence intensity was recorded and calculated using a fluorescence microscope.

[0078] The experimental steps for investigating the anti-glycation performance are as follows:

[0079] At 37℃, human serum albumin (300mmol / L) and glucose (165mmol / L) were incubated in 10mmol / L PBS solution (pH 7.4) for 30 days as blank control; aminoguanidine (10mmol / L) and HSA (300mmol / L) were incubated in the presence of glucose (165mmol / L) for 30 days as positive control; vitamin B1 (500mmol / L) or multifunctional hydrogel were incubated with HSA (300mmol / L) in the presence of glucose (165mmol / L) for 30 days as experimental group; after incubation, all samples were dialyzed with PBS solution overnight to remove excess glucose. All samples were analyzed by UV-visible spectrophotometer and fluorescence photometer to investigate the effects of vitamin B1 and multifunctional hydrogel on the glycation process of human serum albumin.

[0080] UV-Vis spectroscopy studies:

[0081] The UV spectra of all samples in the range of 200 nm to 600 nm were measured and recorded using a UV-visible spectrophotometer.

[0082] AGEs fluorescence measurement:

[0083] Advanced glycation end products (AGEs) show strong fluorescence emission when excited between 300 nm and 400 nm. Therefore, all samples were excited at 370 nm by a fluorescence spectrophotometer to detect the formation of AGEs.

[0084] The experimental steps for investigating the anti-inflammatory properties are as follows:

[0085] Flow cytometry was used to detect the expression of CD86 and CD206 in Raw 264.7 cells cultured under different conditions. Raw 264.7 cells that grew well were taken and cultured at 2×10 5 / well in a 24-well plate. After the cells adhered to the wall, the old culture medium was aspirated and 400 μL of 20 ng mL -1 Interleukin-4, 400 μL containing 100 ng mL -1 LPS, 400 μL blank culture medium and 400 μL containing 10 μg mL -1 Multifunctional hydrogel and incubated for 24 hours. Collect cells from each group and incubate with 100 μL of PBS solution containing CD86 / APC (CD86 / APC antibody incubated at 4°C for 0.5 hours, CD206 / FITC antibody incubated at room temperature for 1 hour) for flow cytometry determination. Finally, analyze by Flowjo analysis software.

[0086] The experimental steps for investigating the antibacterial properties are as follows:

[0087] The multifunctional hydrogel and 990 μL phosphate buffer were placed in a 24-well plate, and 10 μL and 10 μL were taken respectively. 6 Gram-positive bacteria (Staphylococcus aureus and methicillin-resistant Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli) were dropped into the above-mentioned well plate, cultured at 37°C for 2 hours, and then phosphate buffer was added to 1 mL. 10 μL of the above bacterial solution was added to LB medium and cultured at 37°C for 18 hours. The in vitro antibacterial properties of the multifunctional hydrogel were investigated by counting the number of colonies.

[0088] The experimental steps for investigating the repair effect and antibacterial effect on infectious diabetic skin damage are as follows:

[0089] Female Kunming mice (17-22 g, 4-5 weeks old) were selected to establish an infectious diabetic mouse model. After anesthetizing the diabetic mice, the hair on the back was removed, and a full-thickness skin injury wound with a diameter of 8 mm was made. 10 μL, 10 8CFU / mL methicillin-resistant Staphylococcus aureus solution was used to establish a skin injury model of diabetic mice infected with drug-resistant bacteria. The mice were randomly divided into 4 groups, with 15 mice in each group. The multifunctional hydrogel was coated on the wound surface, and 4 groups of controls were set: (1) 10 μL of normal saline (blank control); (2) Tegaderm 3 mol / L film dressing; (3) hydrogel without vitamin B1. On the 3rd day, 7th day, 14th day, and 21st day, the mice were sacrificed, and the wound diameter was measured with a vernier caliper to prove that the multifunctional hydrogel accelerated the repair of infectious diabetic skin injuries. After cutting and chopping the wound, it was placed in a shaking tube containing 1 mL of PBS, shaken 4 times at 200 revolutions per minute at 37 °C, and after diluting the supernatant by 100 times, 10 μL was taken and added to the LB medium, and cultured at 37 °C for 18 h. The antibacterial performance of the multifunctional hydrogel was proved by calculating the number of colonies.

[0090] The multifunctional hydrogel prepared by the present invention has excellent injectability, self-healing ability, biocompatibility, tissue adhesiveness, appropriate mechanical strength, and pH / glucose dual-responsive multifunctional characteristics. In addition, the multifunctional hydrogel has anti-glycation, anti-inflammatory, and antibacterial biological activities, can improve the inflammatory microenvironment of diabetic wounds by effectively inhibiting the formation of AGEs and preventing / treating bacterial infections, promote cell proliferation and angiogenesis, and ultimately effectively accelerate the healing and regeneration of infectious diabetic wounds. The following is a detailed analysis in combination with experimental data.

[0091] Figure 1 Above A is the 1 1H-NMR nuclear magnetic resonance spectrum. It can be seen from the figure that δ = 8.11 ppm, 7.88 ppm, 7.78 ppm, and 7.42 ppm belong to the protons of the benzene ring on 3-carboxyphenylboronic acid, indicating that phenylboronic acid was successfully grafted onto the chitosan backbone; Figure 1 Below A is the 1 1H-NMR nuclear magnetic resonance spectrum of dopamine-grafted ε-polylysine. It can be seen from the figure that δ = 8.30 ppm - 8.40 ppm belongs to the protons of the benzene ring on dopamine, and δ = 3.82 ppm, 3.14 ppm, 1.75 ppm, 1.46 ppm, and 1.28 ppm belong to the methylene and methylene groups of ε-polylysine, confirming the successful preparation of dopamine-grafted ε-polylysine.

[0092] Figure 1 Above and in the middle of B are the infrared spectra of Bletilla striata polysaccharide and oxidized Bletilla striata polysaccharide respectively. Compared with the infrared spectrum of Bletilla striata polysaccharide, in the infrared spectrum of oxidized Bletilla striata polysaccharide, an absorption of aldehyde group (-CHO) appears at 1734 cm -1 This indicates the successful preparation of oxidized Bletilla striata polysaccharide. Compared with oxidized Bletilla striata polysaccharide, the multifunctional hydrogel shows an absorption at 1734 cm -1The absorption peak corresponding to the aldehyde group disappeared, and a stretching vibration attributed to the imine bond (-C=N) appeared at 1668 cm -1 This confirmed that a multifunctional hydrogel was constructed through a Schiff base reaction between phenylboronic acid grafted chitosan, dopamine grafted ε-polylysine, and oxidized Bletilla striata polysaccharide (as shown in Figure 1 Figure B below). Figure 1 Figure C is the transmission electron microscope image of self-assembled phenylboronic acid grafted chitosan. It can be seen from the image that the particle size is about 50.3 nm.

[0093] Figure 2 Figure D is the scanning electron microscope image and elemental distribution map of multifunctional hydrogel 1. It can be seen from the figure that the multifunctional hydrogel presents a 3D porous morphology with an average pore size of about 15.2 μm. Through the surface elemental analysis of the multifunctional hydrogel by energy dispersive spectrometer, it was found that carbon, oxygen, and boron elements were evenly distributed on the surface of the multifunctional hydrogel.

[0094] Figure 3 Figure A is the injectability diagram of multifunctional hydrogel 1. It can be extruded from an 18G needle and write the word "NPU". Figure 3 Figure B is the self-healing ability diagram of multifunctional hydrogel 1. As time goes by, the multifunctional hydrogel cut in half can repair itself. Figure 3 Figure C is the tissue adhesion performance diagram of multifunctional hydrogel 1. When the multifunctional hydrogel is applied to the knuckle and the finger joint is bent at different angles, the multifunctional hydrogel can adhere well to the finger skin. Figure 3 Figure D is the rheological property diagram of multifunctional hydrogel 1. Multifunctional hydrogel 1 was placed between the plates of a TA rheometer, with a shear frequency of 1 Hz, a strain of 1%, and a temperature of 37 °C. The storage modulus (G′) and loss modulus (G″) of the sample were measured. The results showed that multifunctional hydrogel 1 had a certain mechanical strength.

[0095] Figure 4 Figure A is the cytotoxicity results of multifunctional hydrogel 1 and its components after co-incubation with NIH3T3 cells for 24 h at different concentrations. It can be seen from the figure that multifunctional hydrogel 1 and its components have little toxicity to NIH3T3 cells. Even at a concentration of 100 μg / mL, their cell viability is above 90%. In addition, Figure 4 Figure B is the cell proliferation of multifunctional hydrogel 1 and its components after co-incubation with NIH3T3 cells for 5 days. It can be seen from the figure that the cells in all groups showed a continuous increase, indicating that the multifunctional hydrogel prepared in this invention has excellent biocompatibility.

[0096] Figure 5Figure A shows the UV-visible spectrum of human serum albumin after adding vitamin B1 and multifunctional hydrogel 1. After adding vitamin B1 or multifunctional hydrogel 1, the absorption of the human serum albumin sample at λmax decreased significantly, comparable to that of the positive control (aminoguanidine), indicating that both vitamin B1 and multifunctional hydrogel 1 can protect human serum albumin from structural changes. Figure 5 Figure B shows the statistical chart of the fluorescence intensity of advanced glycation end products generated after adding vitamin B1 or multifunctional hydrogel 1. After adding vitamin B1 and multifunctional hydrogel 1, the fluorescence intensity of advanced glycation end products decreased significantly, indicating that multifunctional hydrogel 1 has excellent anti-glycation performance.

[0097] Figure 6 Figure shows the statistical analysis chart of the proportions of CD206 ( Figure 6 A) and CD86 ( Figure 6 B) in macrophages RAW264.7 in the multifunctional hydrogel 1 group and the control group under AGEs stimulation. As can be seen from the figure, compared with the blank group, the expression level of CD206 (anti-inflammatory M2) in the multifunctional hydrogel 1 group increased, and the expression level of CD86 (pro-inflammatory M1) decreased, demonstrating that multifunctional hydrogel 1 has excellent anti-inflammatory performance.

[0098] Figure 7 Figure shows the antibacterial results of multifunctional hydrogel 1 against Escherichia coli, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus. As can be seen from the figure, the multifunctional hydrogel showed high antibacterial performance against the above three bacteria, and the bacterial survival rate was 0%, indicating that the multifunctional hydrogel 1 prepared in the present invention has high antibacterial ability.

[0099] Figure 8 Figure shows the repair results of the bacterial-infected skin wounds of diabetic mice by multifunctional hydrogel 1, normal saline, 3M, and multifunctional hydrogel 4. As can be seen from the figure, as time extended, the skin defect area of each group gradually decreased. Multifunctional hydrogel 1 showed the best wound repair effect and in vivo antibacterial effect, indicating that the multifunctional hydrogel 1 prepared in the present invention can promote the healing of infected diabetic wounds.

[0100] It is understood that many detailed changes are possible, but this does not violate the scope and spirit of the present invention. Any appropriate changes made by those of ordinary skill in the art to the present invention should be regarded as not departing from the scope of the present invention patent.

Claims

1. A method for preparing a natural hydrogel dressing for promoting wound healing, characterized in that: The steps include: After mixing 3-carboxyphenylboronic acid and N-hydroxysuccinimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is added to obtain a phenylboronic acid solution; the phenylboronic acid solution is mixed with a chitosan solution, and the carboxyl group of the 3-carboxyphenylboronic acid and the amino group of the chitosan are subjected to an amidation reaction to obtain phenylboronic acid grafted chitosan; the phenylboronic acid grafted chitosan is dissolved in a vitamin B1 solution and self-assembled, and the vitamin B1 is physically coated in the phenylboronic acid grafted chitosan to obtain phenylboronic acid grafted chitosan micelles loaded with vitamin B1; Dissolve sodium borate and sodium bicarbonate in water, add dopamine hydrochloride under anaerobic conditions, then drop a tetrahydrofuran solution containing acryloyl chloride, and the acyl chloride group of acryloyl chloride reacts with the amino group of dopamine hydrochloride to obtain double-bond modified dopamine; mix the double-bond modified dopamine solution with the ε-polylysine solution, and the double bond of the double-bond modified dopamine reacts with the amino group of the ε-polylysine to obtain dopamine grafted ε-polylysine; A 10wt% aqueous solution of oxidized bletilla striata polysaccharide, a 10wt%-20wt% aqueous solution of dopamine grafted ε-polylysine and a 1wt%-2wt% aqueous solution of phenylboronic acid grafted chitosan micelles loaded with vitamin B1 are mixed, and the amino groups of the dopamine grafted ε-polylysine and the phenylboronic acid grafted chitosan micelles loaded with vitamin B1 undergo Schiff base reaction with the aldehyde groups of the oxidized bletilla striata polysaccharide, respectively, to obtain a natural hydrogel dressing that promotes wound healing.

2. The method for preparing the natural hydrogel dressing for promoting wound healing according to claim 1, characterized in that: The volume ratio of 10wt% oxidized bletilla striata polysaccharide aqueous solution, 10wt%-20wt% dopamine grafted ε-polylysine aqueous solution and 1wt%-2wt% vitamin B1 loaded phenylboronic acid grafted chitosan micelle aqueous solution is 4.5-5:1-2:4.5-2.

3. The method for preparing the natural hydrogel dressing for promoting wound healing according to claim 1, characterized in that: The mass ratio of 3-carboxyphenylboronic acid to chitosan is 1:1.1-1.

8.

4. The method for preparing the natural hydrogel dressing for promoting wound healing according to claim 1, characterized in that: During the preparation of phenylboronic acid grafted chitosan, the conditions for the amidation reaction are: stirring at room temperature for 12 h to 24 h; During the preparation of double-bond modified dopamine, the conditions for the amidation reaction are: stirring at room temperature for 12 h to 24 h.

5. The method for preparing the natural hydrogel dressing for promoting wound healing according to claim 1, characterized in that: The mass ratio of vitamin B1 to phenylboronic acid grafted chitosan is 1:10-20.

6. The method for preparing the natural hydrogel dressing for promoting wound healing according to claim 1, characterized in that: The molar ratio of dopamine hydrochloride to acryloyl chloride is 1:2-5.

7. The method for preparing the natural hydrogel dressing for promoting wound healing according to claim 1, characterized in that: The molar ratio of double-bond modified dopamine to ε-polylysine is 1:2-4.

8. The method for preparing the natural hydrogel dressing for promoting wound healing according to claim 1, characterized in that: The conditions for the Schiff base reaction are: standing at room temperature for 0.5h to 1h.

9. A natural hydrogel dressing for promoting wound healing, prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the natural hydrogel dressing for promoting wound healing according to claim 9 in the preparation of a drug for treating diabetic infectious skin lesions, a drug for treating inflammatory response diseases, or a drug for treating bacterial infection diseases.

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