Hydrogel for promoting wound healing and preparation method thereof

Abstract

The invention provides hydrogel for promoting wound healing and a preparation method thereof, and belongs to the field of hydrogel. The preparation method of the hydrogel for promoting wound healing comprises the following steps: preparing the composite synergist and preparing the dressing. According to the preparation method of the hydrogel for promoting wound healing, the biocompatibility of the hydrogel can be improved while the mechanical strength and long-term antibacterial performance of the hydrogel are improved, the problems that the hydrogel stimulates a wound and peripheral skin and hinders wound healing are avoided, and wound healing is effectively promoted; the stability of antibacterial components in the hydrogel is further improved, and the antibacterial components are prevented from losing and losing efficacy in the long-term storage or application process.

Description

Technical Field

[0001] The present invention relates to the field of hydrogels, and particularly to a hydrogel for promoting wound healing and a preparation method thereof. Background Art

[0002] Medical dressings are special wound treatment materials for medical use, mainly used for dressing surgical wounds or trauma wounds, etc. They have the characteristics of large usage amount and wide application range, and are widely used in the hemostasis and antibacterial treatment of various wounds.

[0003] Medical dressings play an indispensable role in protecting wounds. Whether it is a surgical incision or a trauma wound, existing medical dressings can effectively isolate harmful substances such as external bacteria and dust through physical or chemical means, reduce the risk of wound infection, and provide a relatively clean and suitable healing environment for the wound. Moreover, while being able to promote wound healing, medical dressings generally also have functions such as absorbing exudate, stopping bleeding, relieving pain, and preventing wound adhesion. They act directly on the wound surface, helping to relieve the pain of patients and accelerate the healing process. In addition, existing medical dressings can also have functions such as breathability and cooling, further promoting wound healing.

[0004] There are various existing medical dressings, including gauze, synthetic fibers, polyfilms, foamed polymeric materials, hydrogels, alginates, etc. Although gauze has low cost and is easy to use, its ability to absorb wound exudate is limited, the risk of wound infection is high, and the replacement frequency is high, which is likely to increase the pain of patients. Although synthetic fibers have a certain ability to absorb exudate and antibacterial properties, their breathability is poor, which is likely to cause the local temperature of the wound to be too high, affecting the wound healing speed; moreover, some synthetic fibers may also cause allergic reactions. Although polyfilms and foamed polymeric materials can provide a good protection barrier for the wound and effectively prevent the invasion of external bacteria, they are not breathable, which is likely to cause the local humidity of the wound to be too high, increasing the infection risk. Although alginates have high absorbency and good hemostatic function, they require secondary fixation with an outer dressing and have poor stability.

[0005] In recent years, hydrogels have been widely concerned and studied because they can provide a suitable moist healing environment for wounds, have good absorption performance, good biocompatibility, low irritation, and are easy to remove. However, the mechanical strength of hydrogels is relatively low, unable to meet the wound treatment requirements that need higher mechanical strength, and is prone to breakage or ineffective fixation during the application process; at the same time, hydrogels also cannot meet the wound treatment requirements that need strong antibacterial properties for a long period, and cannot effectively perform deep antibacterial and cleaning of wounds for a long period.

[0006] To solve the aforementioned technical deficiencies and improve the mechanical strength and long-term antibacterial performance of hydrogels, in the preparation of hydrogels, raw materials such as nanocellulose, titanium dioxide nanoparticles, silica nanoparticles, silver nanocomposite antibacterial agents, antibacterial plant extracts, and chemical antibacterial agents can generally be added to the hydrogels. However, the addition of the above raw materials will lead to a decrease in the biocompatibility of the hydrogels, cause greater irritation to the wound and the surrounding skin, and may also inhibit the normal proliferation of cells due to excessive deposition at the wound, thereby hindering wound healing. Moreover, the antibacterial components used have poor stability, are prone to rapid loss during the application process, and are prone to failure during long-term storage. Summary of the Invention

[0007] To solve the technical problems existing in the prior art, the present invention provides a hydrogel for promoting wound healing and a preparation method thereof, which can improve the mechanical strength and long-term antibacterial performance of the hydrogel while enhancing the biocompatibility of the hydrogel, avoiding the problems of the hydrogel irritating the wound and the surrounding skin and hindering wound healing, effectively promoting wound healing; and further improving the stability of the antibacterial components in the hydrogel to avoid their loss and failure during long-term storage or application.

[0008] To solve the above technical problems, the technical solutions adopted by the present invention are as follows: A preparation method of a hydrogel for promoting wound healing, comprising the following steps: preparing a composite synergist and preparing a dressing; The preparation of the composite synergist consists of the following steps: pretreatment and composite treatment; The method of the pretreatment is to treat nano-montmorillonite with a hydrochloric acid solution to obtain modified nano-montmorillonite; after uniformly ball-milling the modified nano-montmorillonite, titanium dioxide nanoparticles, and an ethanol solution, ball-milling modification is carried out using titanate coupling agent 101 to obtain a pretreated product; The method of the composite treatment is to disperse the pretreated product, zinc nitrate hexahydrate, and 2-methylimidazole in anhydrous methanol, then continue to add tea polyphenols and honeysuckle extract, and under room temperature conditions, after ultrasonic dispersion, carry out a stirring reaction; separate to obtain a solid, and the solid is washed with methanol and dried to obtain a composite synergist; The method of the dressing preparation is to uniformly mix gelatin, polyvinyl alcohol, modified chitosan, the composite synergist, alginic acid, hydroxypropyl methylcellulose, propylene glycol, and deionized water, and defoam to obtain a hydrogel for promoting wound healing.

[0009] Preferably, when treating nano-montmorillonite with a hydrochloric acid solution, the nano-montmorillonite is put into the hydrochloric acid solution, and under the temperature condition of 80-90 °C, reflux stirring is carried out, the solid is separated, washed with deionized water until neutral, and dried to obtain modified nano-montmorillonite; The concentration of the hydrochloric acid solution is 1-1.5 mol / L.

[0010] Preferably, in the pretreatment, the weight ratio of the modified nano-montmorillonite, nano-titanium dioxide, ethanol solution, and titanate coupling agent 101 is 20 - 25:4 - 5:15 - 20:2 - 2.5; The volume percentage concentration of the ethanol solution is 70 - 80%.

[0011] Preferably, in the composite treatment, the ultrasonic dispersion time after adding tea polyphenols and honeysuckle extract is 20 - 30 min, and the stirring reaction time is 20 - 24 h.

[0012] Preferably, in the composite treatment, the weight ratio of the pretreatment product, zinc nitrate hexahydrate, 2 - methylimidazole, anhydrous methanol, tea polyphenols, and honeysuckle extract is 30 - 35:30 - 32:68 - 70:160 - 170:8 - 9:10 - 12.

[0013] Preferably, in the preparation of the dressing, the weight ratio of gelatin, polyvinyl alcohol, modified chitosan, composite synergist, alginic acid, hydroxypropyl methylcellulose, propylene glycol, and deionized water is 10 - 12:8 - 9:3 - 3.5:2 - 2.5:6 - 7:8 - 10:3 - 4:90 - 100; The molecular weight of the gelatin is 20 - 30 KDa; the alcoholysis degree of the polyvinyl alcohol is 99%, and the polymerization degree is 1700; the molecular weight of the alginic acid is 50 - 60 KDa.

[0014] Furthermore, the preparation method further includes: preparing modified chitosan; The preparation of the modified chitosan consists of the following steps: primary modification and secondary modification; The method of the primary modification is to dissolve chitosan in an acetic acid aqueous solution, stir and dropwise add the first modification solution. After the addition of the first modification solution is completed, continue stirring for 1 - 2 h, then add sodium cyanoborohydride, stir for 10 - 12 h, adjust the pH value to neutral, precipitate with ethanol, separate the precipitate, wash the precipitate with absolute ethanol, and dry to obtain the primary modification product; The first modification solution is an ethanol solution dissolved with lauraldehyde.

[0015] Preferably, the mass concentration of lauraldehyde in the first modification solution is 15 - 18 wt%; In the primary modification, the weight ratio of lauraldehyde, chitosan, and sodium cyanoborohydride is 1.2 - 1.3:1:1.2 - 1.3.

[0016] Further, the method for secondary modification is as follows: dissolve the primary modification product in an acetic acid aqueous solution, adjust the pH value to 5.9 - 6.1, add the second modification solution, and stir evenly; then continue to add the genipin aqueous solution, stir evenly, and under the temperature condition of 32 - 37 °C, stand for reaction for 8 - 10 h, and then freeze-dry to obtain the modified chitosan; The second modification solution is a Tris-HCl buffer solution dissolved with sericin.

[0017] Preferably, the mass concentration of sericin in the second modification solution is 12 - 15 wt%; the concentration of the Tris-HCl buffer solution is 1 mol / L, and the pH value is 9.0; The mass concentration of the genipin aqueous solution is 0.9 - 1 wt%; In the secondary modification, the weight ratio of sericin, the primary modification product, and genipin is 1.5 - 1.8:1:0.2 - 0.25.

[0018] A hydrogel for promoting wound healing prepared by the foregoing method.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: (1)The preparation method of the hydrogel for promoting wound healing of the present invention. In the primary modification step of preparing modified chitosan, lauraldehyde is used for alkylation modification of chitosan. On the basis of improving the antibacterial property of chitosan, the mechanical property and adhesion ability of chitosan are improved, and the binding property with sericin is further enhanced. In the secondary modification step, in the presence of a crosslinking agent, sericin is crosslinked and compounded with the primary modification product (alkylated chitosan), further improving the mechanical property and adhesion ability of chitosan, while enhancing the biocompatibility of the hydrogel, reducing the irritation of the hydrogel to the wound surface, reducing the risk of rejection, and improving the ability of the hydrogel to promote wound healing. In the pretreatment step of preparing the composite synergist, first hydrochloric acid solution is used to treat nano-montmorillonite to prepare modified nano-montmorillonite, improving the adsorption property of nano-montmorillonite for its combination with subsequent active ingredients; then a titanate coupling agent is used to perform ball milling modification on the modified nano-montmorillonite and nano-titanium dioxide, further enhancing the binding property of the pretreatment product with subsequent active ingredients; in the composite treatment step, the pretreatment product is compounded with a metal-organic framework material and simultaneously loaded with plant active ingredients (tea polyphenols, honeysuckle extract) with antibacterial, anti-inflammatory and wound surface healing promoting effects. Through the combination of the porous nano-inorganic material (pretreatment product) and the metal-organic framework material, the mechanical property of the hydrogel is further improved, and the biocompatibility is enhanced at the same time; combined with the plant active ingredients, while reducing the irritation of the plant active ingredients to the wound surface, their stability and biocompatibility are improved, realizing the long-term slow release of the active ingredients, further enhancing the biocompatibility of the hydrogel, reducing the irritation of the hydrogel to the wound surface, reducing the risk of rejection, and improving the ability of the hydrogel to promote wound healing; in the dressing preparation step, specific raw materials are used to prepare the hydrogel; the combination of the above-mentioned technical means improves the mechanical strength and long-term antibacterial property of the hydrogel, while enhancing the biocompatibility of the hydrogel, avoiding the problem that the hydrogel stimulates the wound and the surrounding skin and hinders wound healing, and effectively promoting wound healing; and further improving the stability of the antibacterial components in the hydrogel, avoiding their loss and inactivation during long-term storage or application.

[0020] (2)Through experiments, for the preparation method of the hydrogel for promoting wound healing of the present invention, the tensile strength of the prepared hydrogel for promoting wound healing is 1.51 - 1.56 MPa, the elongation at break is 214 - 219%, and the water absorption swelling rate is 270 - 274%.

[0021] (3)Through experiments, for the preparation method of the hydrogel for promoting wound healing of the present invention, the antibacterial rate of the prepared hydrogel for promoting wound healing against Staphylococcus aureus is 99.7 - 99.8%, the antibacterial rate against Escherichia coli is 99.1 - 99.4%, and the antibacterial rate against Candida albicans is 97.7 - 98.0%; in the in vitro cytotoxicity test, the relative cell survival rate is 99.4 - 99.5%, and the cytotoxicity grade is grade 0.

[0022] (4)After testing, in the preparation method of the hydrogel for promoting wound healing of the present invention, the prepared hydrogel for promoting wound healing was applied to a circular wound surface with a diameter of 1 cm on the buttocks of mice, and the dressing was changed every 3 days. When the dressing was changed on the 6th day, the percentage of the remaining area of the unhealed wound surface was 5.8 - 6.0%, and the complete wound healing time was 7.5 - 7.9 d, which could effectively promote wound healing.

[0023] (5)After testing, in the preparation method of the hydrogel for promoting wound healing of the present invention, when the prepared hydrogel for promoting wound healing was stored statically for 60 days at a temperature of 25 °C and a relative humidity of 50%, the tensile strength was 1.43 - 1.49 MPa, the elongation at break was 204 - 210%, and the water absorption expansion rate was 260 - 266%; the antibacterial rate against Staphylococcus aureus was 99.0 - 99.3%, the antibacterial rate against Escherichia coli was 98.8 - 99.1%, and the antibacterial rate against Candida albicans was 97.2 - 97.5%. Detailed implementation manners

[0024] In order to have a clearer understanding of the technical features, objectives, and effects of the present invention, the detailed implementation manners of the present invention are described below.

[0025] Example 1 This example provides a preparation method of a hydrogel for promoting wound healing, specifically as follows: 1. Preparation of modified chitosan 1) Primary modification Chitosan was put into an acetic acid aqueous solution (acetic acid concentration: 1 wt%) with a weight 50 times that of chitosan. After stirring until completely dissolved, under stirring conditions, the first modification solution was dropped at a dropping rate of 0.8 mL / min. After the dropping of the first modification solution was completed, stirring was continued for 1 h, and then sodium cyanoborohydride was added. After stirring for 10 h, the pH value was adjusted to neutral with a sodium hydroxide solution with a concentration of 1.2 mol / L, and precipitation was carried out with ethanol. The precipitate was separated, washed twice with absolute ethanol, and then dried to obtain the primary modified product.

[0026] Among them, the first modification solution was an ethanol solution in which lauraldehyde was dissolved; the mass concentration of lauraldehyde in the first modification solution was 15 wt%.

[0027] In the primary modification, the weight ratio of lauraldehyde, chitosan, and sodium cyanoborohydride was 1.2:1:1.2.

[0028] 2) Secondary modification The primary modified product was put into an acetic acid aqueous solution with a concentration of 1 wt%, and the mass concentration of the primary modified product was controlled to be 1.1 wt%. After stirring until completely dissolved, the pH value was adjusted to 5.9 with a sodium hydroxide solution with a concentration of 1 mol / L. Then, the secondary modification solution was added with stirring. After stirring evenly, the genipin aqueous solution was added continuously. After stirring for another 20 min, the reaction was allowed to stand at 32 °C for 8 h, and then freeze-dried to obtain the modified chitosan.

[0029] Among them, the secondary modification solution was a tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer solution dissolved with sericin; the mass concentration of sericin in the secondary modification solution was 12 wt%; the concentration of the Tris-HCl buffer solution was 1 mol / L, and the pH value was 9.0.

[0030] The mass concentration of the genipin aqueous solution was 0.9 wt%.

[0031] In the secondary modification, the weight ratio of sericin, the primary modified product, and genipin was 1.5:1:0.2.

[0032] 2. Preparation of the composite synergist 1) Pretreatment The nano-montmorillonite was put into a hydrochloric acid solution with a volume 4 times that of the nano-montmorillonite. After stirring and heating to 80 °C, the mixture was kept warm and refluxed with stirring for 1 h. Then, the solid was separated, washed with deionized water until neutral, and dried to obtain the modified nano-montmorillonite. Then, the modified nano-montmorillonite, nano-titanium dioxide, and ethanol solution were put into a ball mill. The ball-to-material ratio was controlled to be 5:1, the ball milling speed was 100 rpm. After ball milling for 5 min, the titanate coupling agent 101 was sprayed in, and ball milling was continued for 40 min to obtain the ball-milled product, which was then dried to obtain the pretreated product.

[0033] Among them, the concentration of the hydrochloric acid solution was 1 mol / L.

[0034] The volume percentage concentration of the ethanol solution was 70%.

[0035] The weight ratio of the modified nano-montmorillonite, nano-titanium dioxide, ethanol solution, and titanate coupling agent 101 was 20:4:15:2.

[0036] 2) Composite treatment The pretreated product, zinc nitrate hexahydrate, and 2-methylimidazole were put into anhydrous methanol. After ultrasonic dispersion evenly, the mixture was stirred for 20 min. Then, tea polyphenols and honeysuckle extract were added continuously. Under room temperature conditions, after ultrasonic dispersion for 20 min, the mixture was stirred and reacted for 20 h. The solid was obtained by centrifugal separation. After the solid was washed twice with methanol, it was dried to obtain the composite synergist.

[0037] Among them, the weight ratio of the pretreatment product, zinc nitrate hexahydrate, 2-methylimidazole, anhydrous methanol, tea polyphenols, and honeysuckle extract is 30:30:68:160:8:10.

[0038] The tea polyphenols are obtained by purchasing through the commercial channel, and the purity is greater than 98 wt%.

[0039] The honeysuckle extract is obtained by purchasing through the commercial channel, and the extraction ratio is not less than 10:1.

[0040] 3. Dressing preparation Mix gelatin, polyvinyl alcohol, modified chitosan, composite synergist, alginic acid, hydroxypropyl methylcellulose, propylene glycol, and deionized water evenly, and prepare a hydrogel for promoting wound healing after standing and defoaming in a room temperature environment.

[0041] Among them, the weight ratio of gelatin, polyvinyl alcohol, modified chitosan, composite synergist, alginic acid, hydroxypropyl methylcellulose, propylene glycol, and deionized water is 10:8:3:2:6:8:3:90.

[0042] The molecular weight of the gelatin is 20 KDa.

[0043] The alcoholysis degree of the polyvinyl alcohol is 99%, and the degree of polymerization is 1700.

[0044] The molecular weight of the alginic acid is 50 KDa.

[0045] This example also provides a hydrogel for promoting wound healing prepared by the aforementioned method.

[0046] Example 2 This example provides a preparation method of a hydrogel for promoting wound healing, specifically: 1. Preparation of modified chitosan 1) Primary modification Put chitosan into an acetic acid aqueous solution (acetic acid concentration is 1.2 wt%) with a weight 52 times that of chitosan, stir until completely dissolved, and then, under stirring conditions, dropwise add the first modification solution at a dropping rate of 0.9 mL / min. After the addition of the first modification solution is completed, continue stirring for 1.5 h, then add sodium cyanoborohydride, stir for 11 h, adjust the pH value to neutral with a sodium hydroxide solution with a concentration of 1.3 mol / L, precipitate with ethanol, separate the precipitate, wash the precipitate 3 times with absolute ethanol, and then dry to obtain the primary modification product.

[0047] Among them, the first modification solution is an ethanol solution dissolved with lauraldehyde; the mass concentration of lauraldehyde in the first modification solution is 16.5 wt%.

[0048] In the primary modification, the weight ratio of lauraldehyde, chitosan, and sodium cyanoborohydride is 1.25:1:1.25.

[0049] 2) Secondary modification Put the primary modified product into an acetic acid aqueous solution with a concentration of 1.2 wt%, control the mass concentration of the primary modified product to be 1.2 wt%, stir until completely dissolved, then adjust the pH value to 6.0 with a sodium hydroxide solution with a concentration of 1.1 mol / L, stir and add the second modification solution, and stir evenly; continue to add the genipin aqueous solution, continue to stir for 30 min, then under the temperature condition of 35 °C, let it stand and react for 9 h, and freeze-dry to obtain the modified chitosan.

[0050] Among them, the second modification solution is a tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer solution dissolved with sericin; the mass concentration of sericin in the second modification solution is 14 wt%; the concentration of the Tris-HCl buffer solution is 1 mol / L, and the pH value is 9.0.

[0051] The mass concentration of the genipin aqueous solution is 0.95 wt%.

[0052] In the secondary modification, the weight ratio of sericin, the primary modified product, and genipin is 1.7:1:0.23.

[0053] 2. Preparation of the composite synergist 1) Pretreatment Put the nano-montmorillonite into a hydrochloric acid solution with a volume 4.5 times that of the nano-montmorillonite, stir and heat up to 85 °C, keep it warm and reflux and stir for 1.5 h, then separate the solid, wash it with deionized water until neutral, and dry it to obtain the modified nano-montmorillonite; then put the modified nano-montmorillonite, nano-titanium dioxide, and ethanol solution into a ball mill, control the ball-to-material ratio to be 5.5:1, the ball milling speed to be 180 rpm, ball mill for 8 min, then spray in the titanate coupling agent 101, and continue to ball mill for 50 min to obtain the ball milled product, and dry it to obtain the pretreatment product.

[0054] Among them, the concentration of the hydrochloric acid solution is 1.2 mol / L.

[0055] The volume percentage concentration of the ethanol solution is 75%.

[0056] The weight ratio of the modified nano-montmorillonite, nano-titanium dioxide, ethanol solution, and titanate coupling agent 101 is 23:4.5:18:2.3.

[0057] 2) Composite treatment Put the pretreatment product, zinc nitrate hexahydrate, and 2-methylimidazole into anhydrous methanol, ultrasonically disperse them evenly, and then stir for 25 min; continue to add tea polyphenols and honeysuckle extract, under the room temperature condition, ultrasonically disperse for 25 min, then stir and react for 22 h, centrifuge to separate the solid, wash the solid 3 times with methanol, and dry it to obtain the composite synergist.

[0058] Among them, the weight ratio of the pretreatment product, zinc nitrate hexahydrate, 2-methylimidazole, anhydrous methanol, tea polyphenols, and honeysuckle extract is 33:31:69:165:8.5:11.

[0059] The tea polyphenols are obtained by purchasing through the commercial channel, and the purity is greater than 98 wt%.

[0060] The honeysuckle extract is obtained by purchasing through the commercial channel, and the extraction ratio is not less than 10:1.

[0061] 3. Dressing preparation Mix gelatin, polyvinyl alcohol, modified chitosan, composite synergist, alginic acid, hydroxypropyl methylcellulose, propylene glycol, and deionized water evenly, and let it stand and defoam in a room temperature environment to obtain a hydrogel for promoting wound healing.

[0062] Among them, the weight ratio of gelatin, polyvinyl alcohol, modified chitosan, composite synergist, alginic acid, hydroxypropyl methylcellulose, propylene glycol, and deionized water is 11:8.5:3.3:2.4:6.5:9:3.7:95.

[0063] The molecular weight of the gelatin is 26 KDa.

[0064] The alcoholysis degree of the polyvinyl alcohol is 99%, and the degree of polymerization is 1700.

[0065] The molecular weight of the alginic acid is 55 KDa.

[0066] This example also provides a hydrogel for promoting wound healing prepared by the aforementioned method.

[0067] Example 3 This example provides a preparation method of a hydrogel for promoting wound healing, specifically as follows: 1. Preparation of modified chitosan 1) Primary modification Put chitosan into an acetic acid aqueous solution (acetic acid concentration is 1.5 wt%) with 55 times the weight, stir until completely dissolved, and then, under stirring conditions, dropwise add the first modification solution at a dropping rate of 1 mL / min. After the addition of the first modification solution is completed, continue stirring for 2 h, then add sodium cyanoborohydride, stir for 12 h, adjust the pH value to neutral with a sodium hydroxide solution with a concentration of 1.5 mol / L, precipitate with ethanol, separate the precipitate, wash the precipitate 3 times with absolute ethanol, and then dry to obtain the primary modification product.

[0068] Among them, the first modification solution is an ethanol solution dissolved with lauraldehyde; the mass concentration of lauraldehyde in the first modification solution is 18 wt%.

[0069] In the first modification, the weight ratio of lauraldehyde, chitosan, and sodium cyanoborohydride is 1.3:1:1.3.

[0070] 2) Second modification Put the first modified product into an acetic acid aqueous solution with a concentration of 1.5 wt%, control the mass concentration of the first modified product to be 1.3 wt%, stir until completely dissolved, then adjust the pH value to 6.1 using a sodium hydroxide solution with a concentration of 1.2 mol / L, stir and add the second modification solution, and stir evenly; continue to add the genipin aqueous solution, continue to stir for 40 min, then under the temperature condition of 37 °C, stand and react for 10 h, and freeze-dry to obtain the modified chitosan.

[0071] Among them, the second modification solution is a tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer solution dissolved with sericin; the mass concentration of sericin in the second modification solution is 15 wt%; the concentration of the Tris-HCl buffer solution is 1 mol / L, and the pH value is 9.0.

[0072] The mass concentration of the genipin aqueous solution is 1 wt%.

[0073] In the second modification, the weight ratio of sericin, the first modified product, and genipin is 1.8:1:0.25.

[0074] 2. Preparation of the composite synergist 1) Pretreatment Put the nano-montmorillonite into a hydrochloric acid solution with 5 times its volume, stir and heat up to 90 °C, keep it warm and reflux and stir for 2 h, then separate the solid matter, wash it with deionized water until neutral, and dry it to obtain the modified nano-montmorillonite; then put the modified nano-montmorillonite, nano-titanium dioxide, and ethanol solution into a ball mill, control the ball-to-material ratio to be 6:1, the ball milling speed to be 200 rpm, ball mill for 10 min, then spray in the titanate coupling agent 101, and continue to ball mill for 60 min to obtain the ball milled product, and dry it to obtain the pretreated product.

[0075] Among them, the concentration of the hydrochloric acid solution is 1.5 mol / L.

[0076] The volume percentage concentration of the ethanol solution is 80%.

[0077] The weight ratio of the modified nano-montmorillonite, nano-titanium dioxide, ethanol solution, and titanate coupling agent 101 is 25:5:20:2.5.

[0078] 2) Composite treatment The pretreated material, zinc nitrate hexahydrate and 2-methylimidazole were added to anhydrous methanol, ultrasonically dispersed and stirred for 30 minutes. Tea polyphenols and honeysuckle extract were then added, ultrasonically dispersed for 30 minutes at room temperature, stirred for reaction for 24 hours, and solid matter was obtained by centrifugation. The solid matter was washed 3 times with methanol and dried to obtain a composite synergist.

[0079] Among them, the weight ratio of the pretreated material, zinc nitrate hexahydrate, 2-methylimidazole, anhydrous methanol, tea polyphenols and honeysuckle extract is 35:32:70:170:9:12.

[0080] Tea polyphenols were purchased from commercial sources, with a purity of more than 98 wt%.

[0081] The honeysuckle extract is purchased from commercial sources, and the extraction ratio is not less than 10:1.

[0082] 3. Dressing preparation Gelatin, polyvinyl alcohol, modified chitosan, composite enhancer, alginate, hydroxypropyl methylcellulose, propylene glycol and deionized water are uniformly mixed, and the mixture is allowed to stand at room temperature for degassing to obtain a hydrogel that promotes wound healing.

[0083] Among them, the weight ratio of gelatin, polyvinyl alcohol, modified chitosan, composite enhancer, alginate, hydroxypropyl methylcellulose, propylene glycol and deionized water is 12:9:3.5:2.5:7:10:4:100.

[0084] The molecular weight of gelatin is 30KDa.

[0085] The alcoholysis degree of polyvinyl alcohol is 99% and the polymerization degree is 1700.

[0086] The molecular weight of alginic acid is 60KDa.

[0087] This embodiment also provides a hydrogel for promoting wound healing prepared by the aforementioned method.

[0088] Comparative Example 1 The technical solution of Example 2 is adopted, but the differences are as follows: 1) in the preparation of modified chitosan, a modification step is omitted; 2) in the preparation of the composite synergist, the pretreatment step is omitted, and nano-montmorillonite and nano-titanium dioxide are directly mixed in a weight ratio of 23:4.5 and used as a pretreatment product in the composite treatment step.

[0089] Comparative Example 2 The technical solution of Example 2 is adopted, with the differences being: 1) In the preparation of modified chitosan, the secondary modification step is omitted; 2) In the compound treatment step for preparing the compound synergist, the addition of zinc nitrate hexahydrate and 2-methylimidazole is omitted. The pretreatment agent, tea polyphenols, and honeysuckle extract are put into anhydrous methanol according to the original parts by weight, dispersed evenly, stirred for 22 h, and then centrifuged to obtain a solid, which is dried to obtain the compound synergist.

[0090] The tensile strength, elongation at break, and water absorption swelling ratio of the hydrogels of Examples 1-3 and Comparative Examples 1-2 were respectively detected. Specifically, each hydrogel was placed in a forced-air drying oven and dried to a constant weight at a temperature of 50 °C to obtain a dried hydrogel film, which was cut into specimens with a length of 30 mm and a width of 4 mm, and the thickness of the specimens was measured using a film thickness gauge; at room temperature, a tensile testing machine was used to measure and calculate the tensile strength and elongation at break of each specimen.

[0091] Among them, the calculation method of tensile strength (MPa) is the tension value N at the time of test fracture / (specimen width mm × specimen thickness mm). The calculation method of elongation at break (%) is [(elongation length mm at the time of specimen fracture / original length mm of the specimen)] × 100%.

[0092] The water absorption swelling ratio is obtained by completely immersing a dried hydrogel film with a thickness of 1.5 mm in deionized water, allowing it to stand and absorb water for 12 h, and then calculating using (weight after 12 h of water absorption / original weight of the dried hydrogel film) × 100%.

[0093] The specific test results are as follows:

[0094] Furthermore, the antibacterial properties and biocompatibility of the hydrogels of Examples 1-3 and Comparative Examples 1-2 were respectively detected. Specifically, Staphylococcus aureus, Escherichia coli, and Candida albicans were respectively inoculated into a medium containing 3 g / L beef extract, 5 g / L peptone, and 15 g / L agar, cultured for 12 h, the viable bacteria were counted, and then diluted to a viable bacteria concentration of 1000 cfu / ml (initial viable bacteria concentration) to obtain the test bacterial solution.

[0095] After sterilizing each hydrogel, 1 mL of each was taken and evenly smeared on an LB nutrient agar plate. After drying, 1 mL of the test bacterial solution with a viable bacteria concentration of 1000 cfu / ml (Staphylococcus aureus or Escherichia coli or Candida albicans) was evenly smeared on the aforementioned LB nutrient agar plate coated with the hydrogel, placed in a temperature environment of 37 °C, allowed to stand for 12 h, and then the viable bacteria concentration after 12 h was counted to calculate the antibacterial rate. The calculation method of the antibacterial rate is [(initial viable bacteria concentration - viable bacteria concentration after 12 h) / initial viable bacteria concentration] × 100%.

[0096] The detection method of biocompatibility refers to the relevant content of the MTT method in GB / T 16886.5-2017 "Biological evaluation of medical devices - Part 5: In vitro cytotoxicity test".

[0097] The specific detection results are as follows:

[0098] Furthermore, the hydrogels of Examples 1-3 and Comparative Examples 1-2 were respectively used for wound healing tests. Specifically, 25 healthy male mice with similar age and weight were selected and evenly divided into 5 groups; the buttocks of each mouse were depilated, and then an electric soldering iron at 75 °C was placed on the shaved bare skin for 10 s to form a circular wound surface with a diameter of 1 cm. The hydrogel was completely covered on the circular wound surface, and the dressing was changed every 3 days. When the dressing was changed on the 6th day, the remaining area of the unhealed wound surface was measured, and the percentage of the remaining area of the unhealed wound surface was calculated by (remaining area of the unhealed wound surface / area of the initial circular wound surface) × 100%, and the average value was taken. At the same time, the complete healing time of the wound surface was counted and the average value was taken.

[0099] The specific results are as follows:

[0100] Furthermore, the hydrogels of Examples 1-3 and Comparative Examples 1-2 were placed in an environment with a temperature of 25 °C and a relative humidity of 50% and stored statically for 60 days. Then, the tensile strength, elongation at break, water absorption expansion rate, and antibacterial performance of each hydrogel were detected by the aforementioned methods. The specific detection results are as follows:

[0101] It can be seen that in the preparation method of the hydrogel for promoting wound healing of the present invention, in the primary modification step of preparing the modified chitosan, lauric aldehyde is used to alkylate chitosan. On the basis of improving the antibacterial property of chitosan, the mechanical property and adhesion ability of chitosan are improved, and further the binding property with sericin is enhanced; in the secondary modification step, in the presence of a crosslinking agent, sericin is crosslinked and compounded with the primary modification product (alkylated chitosan), further improving the mechanical property and adhesion ability of chitosan, while enhancing the biocompatibility of the hydrogel, reducing the irritation of the hydrogel to the wound surface, reducing the risk of rejection, and improving the ability of the hydrogel to promote wound healing. In the pretreatment step of preparing the composite synergist, hydrochloric acid solution is first used to treat nano-montmorillonite to prepare modified nano-montmorillonite, improving the adsorption property of nano-montmorillonite for its combination with the subsequent active ingredients; then a titanate coupling agent is used to ball-mill and modify the modified nano-montmorillonite and nano-titanium dioxide, further enhancing the binding property of the pretreatment product with the subsequent active ingredients; in the composite treatment step, the pretreatment product is compounded with a metal-organic framework material and simultaneously loaded with plant active ingredients (tea polyphenols, honeysuckle extract) with antibacterial, anti-inflammatory and wound surface healing promoting effects. Through the combination of the porous nano-inorganic material (pretreatment product) and the metal-organic framework material, the mechanical property of the hydrogel is further improved, and at the same time the biocompatibility is enhanced; and in combination with the plant active ingredients, while reducing the irritation of the plant active ingredients to the wound surface, their stability and biocompatibility are improved, realizing the long-acting sustained release of the active ingredients, further enhancing the biocompatibility of the hydrogel, reducing the irritation of the hydrogel to the wound surface, reducing the risk of rejection, and improving the ability of the hydrogel to promote wound healing; in the dressing preparation step, specific raw materials are used to prepare the hydrogel; the combination of the foregoing technical means improves the mechanical strength and long-term antibacterial property of the hydrogel, while enhancing the biocompatibility of the hydrogel, avoiding the problem that the hydrogel irritates the wound and the surrounding skin and hinders wound healing, and effectively promoting wound healing; and further improving the stability of the antibacterial components in the hydrogel, avoiding their loss and inactivation during long-term storage or application.

[0102] It can be seen from Comparative Example 1 that after omitting the first modification step in the preparation of modified chitosan, the mechanical properties, antibacterial properties and the binding properties with sericin of chitosan decreased to a certain extent; in the pretreatment step of preparing the composite synergist, omitting the acid modification of nano-montmorillonite and the ball milling modification of nano-montmorillonite and nano-titanium dioxide led to a decrease in the binding properties between the porous inorganic material and the metal-organic antibacterial material and the plant active ingredient, and a decrease in the mechanical properties of the hydrogel; specifically, the tensile strength, elongation at break, antibacterial properties and biocompatibility of the hydrogel prepared in Comparative Example 1 all decreased to a certain extent; at the same time, the ability to promote wound healing was lower than that of Example 2, and the percentage of the residual area of the unhealed wound surface was 7.1% on the 6th day, and the complete healing time of the wound surface was 8.3 d; moreover, after the hydrogel was stored for a long time, the mechanical properties and antibacterial properties both decreased to a certain extent.

[0103] It can be seen from Comparative Example 2 that after omitting the second modification step in the preparation of modified chitosan, the mechanical properties of chitosan could not be further improved by the binding of sericin with the first modifier (alkylated chitosan), the biocompatibility of the hydrogel could not be further improved, and the ability to promote wound healing decreased; in the composite treatment step of preparing the composite synergist, omitting the composite with the metal-organic framework material and directly loading the plant active ingredient with the porous inorganic material could not further improve the mechanical properties and biocompatibility of the hydrogel; it was impossible to effectively avoid the irritation of the plant active ingredient to the wound surface, and the stability of the active ingredient deteriorated significantly; specifically, the tensile strength, elongation at break, antibacterial properties and biocompatibility of the hydrogel prepared in Comparative Example 2 all decreased more significantly; at the same time, the ability to promote wound healing was significantly lower than that of Example 2, and the percentage of the residual area of the unhealed wound surface was 11.4% on the 6th day, and the complete healing time of the wound surface was 9.4 d; moreover, after the hydrogel was stored for a long time, the mechanical properties and antibacterial properties deteriorated significantly.

[0104] Unless otherwise specified, the percentages used in the present invention are all mass percentages.

[0105] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a hydrogel for promoting wound healing, characterized in that: The method comprises the following steps: preparing a composite enhancer and preparing a dressing; The preparation of the composite synergist comprises the following steps: pretreatment, composite treatment; The pretreatment method comprises: treating nano-montmorillonite with a hydrochloric acid solution to obtain a modified nano-montmorillonite; ball-milling the modified nano-montmorillonite, nano-titanium dioxide and an ethanol solution to obtain a pretreated product; and then ball-milling the modified nano-montmorillonite, nano-titanium dioxide and an ethanol solution to obtain a pretreated product. The composite treatment method comprises dispersing the pretreated material, zinc nitrate hexahydrate and 2-methylimidazole in anhydrous methanol, adding tea polyphenols and honeysuckle extract, stirring at room temperature for reaction, separating and obtaining a solid, washing and drying the solid to obtain a composite synergist; The method for preparing the dressing comprises the following steps: uniformly mixing gelatin, polyvinyl alcohol, modified chitosan, composite enhancer, alginic acid, hydroxypropyl methylcellulose, propylene glycol and deionized water, and degassing to obtain a hydrogel for promoting wound healing.

2. The method for preparing a hydrogel for promoting wound healing according to claim 1, characterized in that: The method of treating the nano-montmorillonite with a hydrochloric acid solution comprises placing the nano-montmorillonite in a hydrochloric acid solution, refluxing and stirring at a temperature of 80-90° C., separating out solid matter, and washing and drying the solid matter to obtain the modified nano-montmorillonite. The concentration of the hydrochloric acid solution is 1-1.5 mol / L.

3. The method for preparing a hydrogel for promoting wound healing according to claim 1, characterized in that: In the pretreatment, the weight ratio of modified nano-montmorillonite, nano-titanium dioxide, ethanol solution, and titanate coupling agent 101 is 20-25:4-5:15-20:2-2.5; The volume percentage concentration of the ethanol solution is 70-80%.

4. The method for preparing a hydrogel for promoting wound healing according to claim 1, characterized in that: In the composite treatment, the stirring reaction time after adding tea polyphenols and honeysuckle extract is 20-24 hours; The weight ratio of the pretreated material, zinc nitrate hexahydrate, 2-methylimidazole, anhydrous methanol, tea polyphenols and honeysuckle extract is 30-35:30-32:68-70:160-170:8-9:10-12.

5. The method for preparing a hydrogel for promoting wound healing according to claim 1, characterized in that: In the preparation of the dressing, the weight ratio of gelatin, polyvinyl alcohol, modified chitosan, composite enhancer, alginic acid, hydroxypropyl methylcellulose, propylene glycol and deionized water is 10-12:8-9:3-3.5:2-2.5:6-7:8-10:3-4:90-100.

6. The method for preparing a hydrogel for promoting wound healing according to claim 1, characterized in that: The preparation method also includes: preparing modified chitosan; The preparation of modified chitosan comprises the following steps: primary modification, secondary modification; The primary modification method comprises dissolving chitosan in an acetic acid aqueous solution, dripping a first modification liquid, stirring after the first modification liquid is dripped, then adding sodium cyanoborohydride, adjusting the pH value to neutral after stirring, using ethanol for precipitation, separating the precipitate, washing and drying the precipitate to obtain a primary modified product; The first modifying liquid is an ethanol solution in which lauryl aldehyde is dissolved.

7. The method for preparing a hydrogel for promoting wound healing according to claim 6, characterized in that: The mass concentration of lauryl aldehyde in the first modified liquid is 15-18wt%; The weight ratio of lauryl aldehyde, chitosan and sodium cyanoborohydride is 1.2-1.3:1:1.2-1.

3.

8. The method for preparing a hydrogel for promoting wound healing according to claim 6, characterized in that: The secondary modification method comprises dissolving the primary modified substance in an acetic acid aqueous solution, adjusting the pH value to 5.9-6.1, adding a second modified solution, and mixing evenly; continuing to add a genipin aqueous solution, mixing evenly, standing for reaction at a temperature of 32-37° C., and freeze-drying to obtain modified chitosan; The second modifying solution is a Tris-HCl buffer solution in which sericin is dissolved.

9. The method for preparing a hydrogel for promoting wound healing according to claim 8, characterized in that: The mass concentration of sericin in the second modified solution is 12-15wt%; The concentration of Tris-HCl buffer was 1 mol / L and the pH value was 9.0; The mass concentration of the genipin aqueous solution is 0.9-1wt%; In the secondary modification, the weight ratio of sericin, primary modified product, and genipin is 1.5-1.8:1:0.2-0.

25.

10. A hydrogel for promoting wound healing, characterized in that: The method is prepared by the method according to any one of claims 1 to 9.

Images