Modified hyaluronic acid antibacterial gel and preparation method thereof

Abstract

The invention discloses antibacterial gel of modified hyaluronic acid and a preparation method of the antibacterial gel, and belongs to the technical field of antibacterial gel. The antibacterial gel is prepared from the following raw materials in parts by weight: 20-40 parts of polyvinyl alcohol, 10-15 parts of modified hyaluronic acid, 5-8 parts of modified chitosan, 1-10 parts of an emulsifier, 5-10 parts of a humectant and 30-50 parts of water. By accurately controlling the proportion of various functional components and the mass ratio of the antibacterial components, the optimal antibacterial performance of the antibacterial gel can be achieved, the antibacterial effect can be improved, it can be ensured that the gel has proper swelling performance and mechanical performance, and smearing and absorption are convenient.

Description

Technical Field

[0001] The invention belongs to the technical field of antibacterial gel, and specifically relates to an antibacterial gel of modified hyaluronic acid and a preparation method thereof. Background Art

[0002] When treating a patient's wound, choosing the right dressing is very important for promoting wound healing, preventing infection, and keeping the wound moist. Although traditional dressings such as gauze and cotton pads can provide a certain physical barrier to prevent external pollutants from entering the wound, they may cause dry wounds due to their lack of moisturizing and antibacterial properties. When changing the dressing, traditional dressings may adhere to the new tissue, causing pain and damaging the new skin, which is not conducive to healing and cannot effectively prevent bacterial infection.

[0003] In order to overcome the limitations of traditional dressings, new antibacterial gel dressings have been gradually developed and widely used in clinical practice. Antibacterial gel dressings are a three-dimensional mesh structure polymer colloid material with strong water absorption capacity and can promote wound healing. It is a new type of wound dressing with excellent performance. Antibacterial gel excipients have moisturizing and antibacterial properties, are easy to replace, and will not adhere to the wound surface, reducing secondary damage to the wound during replacement and alleviating the patient's pain.

[0004] There are many kinds of antibacterial ingredients in antibacterial gel excipients, which are divided into metal ions, synthetic antibacterial agents, natural antibacterial agents, polypeptide antibacterial agents, etc. according to their sources and chemical properties. Among them, metal ion antibacterial agents include silver ions and zinc ions, synthetic antibacterial agents include chlorhexidine, quaternary ammonium salts, polyhexamethylene biguanide, etc., natural antibacterial agents include tea tree oil, garlic extract, propolis, chitosan, etc., and polypeptide antibacterial agents include defensins, lactoferrin, etc.

[0005] Although there are many antibacterial agents available, the antibacterial properties of antibacterial gels are still limited, and their mechanical properties and swelling properties are poor, and they do not have high-efficiency and long-lasting antibacterial properties. Therefore, there is an urgent need for an antibacterial auxiliary material with good antibacterial properties, mechanical properties and swelling properties. Summary of the invention

[0006] The purpose of the present invention is to provide an antibacterial gel of modified hyaluronic acid and a preparation method thereof, so as to solve the problem that the antibacterial performance of the above-mentioned antibacterial gel still has certain limitations, and the mechanical properties and swelling properties are poor. The present invention can achieve the best antibacterial performance of the antibacterial gel by accurately controlling the ratio of various functional components and the mass ratio between the antibacterial components. The present invention can not only improve the antibacterial effect, but also ensure that the gel has suitable rheological properties, which is easy to apply and absorb.

[0007] To achieve the above object, the first aspect of the present invention provides an antibacterial gel of modified hyaluronic acid, the antibacterial gel comprising the following raw materials in parts by weight:

[0008] Polyvinyl alcohol 20-40 parts

[0009] 10-15 parts of modified hyaluronic acid

[0010] Modified chitosan 5-8 parts

[0011] Emulsifier 1-10 parts

[0012] 5-10 parts moisturizer

[0013] 30-50 parts of water;

[0014] The modified hyaluronic acid is prepared by the reaction of acrylated hyaluronic acid, inorganic guanidine salt, organic guanidine salt, polyamine small molecule and amino-terminated polymer; the mass ratio of acrylated hyaluronic acid, inorganic guanidine salt, organic guanidine salt, polyamine small molecule and amino-terminated polymer is (5-8): (1-3): (1-3): (2-4): 1;

[0015] The modified chitosan is prepared by reacting a protein composition, chitosan and a cross-linking agent; the mass ratio of the protein composition, chitosan and the cross-linking agent is (0.5-0.8):(1-1.5):1, and the protein composition includes collagen, silk fibroin and keratin in a mass ratio of (5-8):(2-4):1;

[0016] Preferably, the viscosity of the polyvinyl alcohol is 20 to 30 mPa·s, and the alcoholysis degree is 98.0 to 99.0 mol %.

[0017] Polyvinyl alcohol was purchased from Kuraray of Japan, model number R-1130.

[0018] As a new type of material that can replace traditional gauze accessories, the antibacterial performance of antibacterial gel has attracted much attention. There are many antibacterial ingredients that can be added to antibacterial gel, but the antibacterial performance of antibacterial gel is still not high. In view of this problem, the present invention adds hyaluronic acid, chitosan, inorganic guanidine salt, organic guanidine salt, polyamine small molecules and amino-terminated polymers to the antibacterial gel, and multiple antibacterials work together to significantly improve the antibacterial performance of the gel. Analysis of the reasons: Hyaluronic acid and chitosan, these two substances themselves have certain antibacterial activity, especially chitosan, whose positive charge can combine with the negatively charged bacterial cell wall, causing the cell wall to rupture. The modified hyaluronic acid also has antibacterial properties, and can construct an environment that is not conducive to bacterial survival by increasing the hygroscopicity of the gel. Different types of guanidine salts can play an antibacterial role against different types of bacteria. Inorganic guanidine salts and organic guanidine salts can penetrate the bacterial cell wall, interfere with its metabolic process, and even directly destroy the cell membrane structure, leading to bacterial death. The polyamine small molecules and the amino-terminated polymers can interact with the negatively charged groups on the surface of bacteria, destroying the integrity of the cell wall or membrane, thereby achieving an antibacterial effect. The present invention can produce a synergistic effect by mixing the above substances in an appropriate proportion, and multiple antibacterial components act together on different parts or stages of bacteria, thereby enhancing the antibacterial effect and reducing the possibility of bacteria developing drug resistance.

[0019] The present invention continues to study and finds that although adding multiple antibacterial ingredients can synergistically improve the antibacterial properties of the gel, the mechanical properties and swelling properties of the antibacterial gel are poor, which seriously affects the use of the antibacterial gel. To address this problem, the present invention specifically modifies hyaluronic acid and chitosan, significantly improving the mechanical properties and swelling properties of the antibacterial gel. Analysis of the reasons: In the preparation process of modified hyaluronic acid, a new functional group acrylate group is first introduced into the hyaluronic acid chain, which can increase the interaction between molecules and improve the mechanical properties of the gel. The modified hyaluronic acid can form a stable three-dimensional network structure at a lower concentration, which helps to improve the fluidity and thixotropy of the gel, making it easier to extend when applied, and can maintain its shape after application. The gel formed by unmodified hyaluronic acid may not be tough enough and is prone to deformation or rupture when external force is applied. Hyaluronic acid itself has strong water absorption, and its water absorption and swelling ability can be further enhanced after modification, so that the gel can expand rapidly when exposed to water to form a protective layer. Modified hyaluronic acid helps to maintain a moist environment inside the gel, which is very important for promoting wound healing and maintaining skin health. After chitosan is modified, its molecular chains can form stronger hydrogen bonds or other types of cross-links, which increases the strength and toughness of the gel, making it less likely to break when subjected to external forces. Modified chitosan can form a more uniform gel structure, which helps to improve the uniformity and stability of the gel, so that it can maintain consistent performance during long-term use. Although unmodified natural hyaluronic acid has good water absorption, its swelling properties may not be sufficient to meet the application requirements of antibacterial gel, especially when it needs to remain moist for a long time. Modified chitosan can adjust its swelling rate as needed, which is very important for controlling the drug release rate and the effect of the gel. Unmodified chitosan may easily lose structural stability during the swelling process, resulting in a decrease in gel performance. Modified chitosan can improve the water retention of the gel, prevent water from evaporating too quickly, and keep the gel moist.

[0020] Preferably, the preparation method of acrylated hyaluronic acid is:

[0021] A mixture is obtained by mixing hyaluronic acid with a molecular weight of 400,000 to 1,000,000, hyaluronic acid with a molecular weight of 1,000,000 to 1,800,000, and hyaluronic acid with a molecular weight of 1,800,000 to 2,200,000 in a mass ratio of 1:(3-5):1;

[0022] The mixture is dissolved in water to form a hyaluronic acid solution with a concentration of 3 to 5 mg / mL, adipic acid dihydrazide is added, and the mass ratio of adipic acid dihydrazide to the mixture is 1:11 to 14. After the solution is completely dissolved, the pH is adjusted to 5 to 7 with hydrochloric acid; carbodiimide hydrochloride is added, and the mass ratio of carbodiimide hydrochloride to the mixture is 1:1-5, and the pH is maintained at 5 to 7 until it is stable, reacting at room temperature for 10 to 12 hours, dialyzing with deionized water for 50 to 60 hours, and freeze-drying to obtain a hyaluronic acid-adipic acid dihydrazide derivative;

[0023] The hyaluronic acid-adipic acid dihydrazide derivative is dissolved in hydroxyethylpiperazine ethanesulfonic acid buffer to obtain a solution A with a concentration of 3 to 6 mg / mL, N-acryloyloxysuccinimide is dissolved in dimethyl sulfoxide to obtain a solution B with a concentration of 120 to 150 mg / mL, and then the solution B is added to the solution A, reacted at room temperature for 10 to 12 hours, dialyzed with deionized water for 65 to 85 hours, and freeze-dried to obtain acrylated hyaluronic acid.

[0024] The present invention limits the use of three kinds of molecular weight hyaluronic acid, including hyaluronic acid (low molecular weight HA) with a molecular weight of 400,000 to 1,000,000, hyaluronic acid (middle molecular weight HA) with a molecular weight of 1,000,000 to 1,800,000, and hyaluronic acid (high molecular weight HA) with a molecular weight of 1,800,000 to 2,200,000, wherein high molecular weight HA has a higher viscosity, can provide a stronger mechanical support effect, enhance the toughness and strength of the gel, and the network structure formed by high molecular weight HA is tighter, which helps to improve the stability of the gel. The middle molecular weight HA can provide better fluidity and permeability while enhancing mechanical properties. The HA of this molecular weight can maintain a certain viscosity and will not be too viscous, so that the gel is easy to apply. Low molecular weight HA can increase the fluidity of the gel, making it easier to apply and distribute, and low molecular weight HA can also more easily penetrate into the deep layer of the skin, exerting its moisturizing and repairing effects. In the present invention, three kinds of molecular weight hyaluronic acids are mixed at a specific ratio, which can not only improve the mechanical properties of the gel, but also improve swelling properties, enhance permeability and absorbency, improve moisturizing effects, promote wound healing, and achieve multifunctional synergy.

[0025] Preferably, the inorganic guanidine salt includes guanidine nitrate and guanidine hydrochloride in a mass ratio of 1:(6-8); the organic guanidine salt includes aminoguanidine hydrochloride, diaminoguanidine hydrochloride and triaminoguanidine hydrochloride in a mass ratio of (1-3):(4-6):1; the polyamine small molecule is diethylenetriamine; and the amino-terminated polymer is polyetheramine.

[0026] Hyaluronic acid was purchased from Shaanxi Zhenhe Biotechnology Co., Ltd.

[0027] Preferably, the amine value of the polyetheramine is 440 to 480 mgKOH / g.

[0028] Polyetheramine was purchased from Wuxi Akeli Technology Co., Ltd., model number MA-223.

[0029] Preferably, the preparation process of modified hyaluronic acid is:

[0030] Add acrylated hyaluronic acid, inorganic guanidine salt, organic guanidine salt, polyamine small molecule and amino-terminated polymer into a reactor, mix them evenly, and heat to 150-200° C. for reflux reaction for 5-10 hours under nitrogen protection to obtain modified hyaluronic acid.

[0031] Preferably, the preparation method of modified chitosan is:

[0032] At room temperature, chitosan was added to a reaction container, and a 0.3 mol / L acetic acid solution was added to fully swell the chitosan, the mass volume ratio of chitosan to the acetic acid solution was 1 g: (3-5) mL, and deionized water was added to fully dissolve the chitosan, the mass volume ratio of chitosan to deionized water was 1 g: (25-40) mL, to obtain a chitosan solution;

[0033] The cross-linking agent and the protein composition are added to the chitosan solution, the pH value is adjusted to 5.0, the solution is stirred evenly, and the solution is irradiated under ultraviolet light for 30 to 60 minutes. The solution is filtered, washed and dried to obtain the modified chitosan.

[0034] The present invention adds three proteins, including collagen, fibroin and keratin, during the chitosan modification process. Collagen and fibroin have certain mechanical strength, which can enhance the mechanical properties of the modified chitosan, making the gel tougher and less prone to breakage during use. The presence of keratin helps to improve the softness of the gel, making the user feel more comfortable. By combining these three proteins with chitosan, the synergistic effects of multiple functions such as antibacterial, moisturizing and repairing can be achieved, so that the final product has better comprehensive performance.

[0035] Preferably, the cross-linking agent is tea polyphenols.

[0036] Preferably, the ultraviolet light comes from an ultraviolet lamp, the power of the ultraviolet lamp is 500-800W, and the main wavelength is 365nm.

[0037] Preferably, the deacetylation degree of chitosan is greater than 90%, and the viscosity is 10 to 200 mPa·s.

[0038] Preferably, the emulsifier is at least one of lactic acid ester, betaine, polysorbate, gum arabic and soybean lecithin.

[0039] Preferably, the humectant is at least one of sorbitol, glycerol and 1,3-butylene glycol.

[0040] The second aspect of the present invention provides a method for preparing an antibacterial gel of modified hyaluronic acid, comprising the following steps:

[0041] S1: Weigh polyvinyl alcohol, modified hyaluronic acid, modified chitosan, emulsifier, moisturizer and water according to weight proportions;

[0042] S2: Mix the raw materials in step S1 and stir them evenly to obtain an antibacterial gel.

[0043] Therefore, the present invention adopts the above-mentioned antibacterial gel of modified hyaluronic acid and its preparation method, which has the following beneficial effects:

[0044] 1. In the present invention, modified hyaluronic acid and modified chitosan both have certain antibacterial properties, and the synergistic effect of the two can improve the antibacterial properties of the gel. At the same time, in the process of modified hyaluronic acid preparation, the present invention also adds inorganic guanidine salts, organic guanidine salts and other ingredients with broad-spectrum antibacterial activity, and these antibacterial ingredients can act on bacterial cell walls or membranes through different mechanisms, causing the physiological functions of bacteria to be impaired or directly killing bacteria. In particular, the selective combination of organic guanidine salts and inorganic guanidine salts can play a role for different types of pathogens, thereby improving the antibacterial breadth and depth of the entire gel system. In addition, the presence of polyamine small molecules and amino-terminated polymers can also adsorb negatively charged bacterial surfaces through electrostatic interactions, further enhancing the antibacterial effect.

[0045] 2. In the present invention, polyvinyl alcohol (PVA) is used as the main raw material. As a polymer material, polyvinyl alcohol plays an important skeleton role in the gel system due to its good film-forming property, adhesion and plasticity. The selection of PVA viscosity and alcoholysis degree in the present invention can ensure that the gel maintains sufficient toughness and strength during use and avoids breaking when subjected to external forces. At the same time, the addition of modified hyaluronic acid and modified chitosan can give the gel better elasticity, making it easier to extend and adhere to the skin surface when applied, and will not fall off easily.

[0046] 3. The hyaluronic acid used in the present invention is a naturally occurring polysaccharide with strong water absorption and water retention capabilities. After modification, the water absorption and swelling capabilities of hyaluronic acid are further enhanced. When the antibacterial gel contacts the skin surface, it can quickly absorb moisture from the skin and swell to form a moist protective layer, which helps to maintain the skin's hydration state, promote wound healing or relieve dry skin.

[0047] 4. The hyaluronic acid and chitosan used in the present invention are both naturally occurring biopolymers, which have good biocompatibility and are not likely to cause allergic reactions or other adverse reactions. The modified hyaluronic acid and chitosan of the present invention retain the original biocompatibility characteristics, while improving their functionality and stability through chemical modification. In addition, other ingredients used, such as polyetheramine, are also safe, so the entire gel system prepared by the present invention is relatively mild and safe for the human body.

[0048] 5. The present invention can achieve the best performance of the antibacterial gel by precisely controlling the ratio of various functional ingredients and the mass ratio between antibacterial ingredients, such as acrylated hyaluronic acid and other antibacterial ingredients. The present invention can not only improve the antibacterial effect, but also ensure that the gel has suitable rheological properties for easy application and absorption.

[0049] The technical solution of the present invention is further described in detail below through embodiments. DETAILED DESCRIPTION

[0050] The present invention will be further described below. It should be noted that this embodiment is based on the technical solution and provides a detailed implementation method and a specific operation process, but the present invention is not limited to this embodiment.

[0051] Example 1

[0052] This embodiment provides an antibacterial gel of modified hyaluronic acid, which includes the following raw materials in parts by weight:

[0053] 30 parts of polyvinyl alcohol, the viscosity of the polyvinyl alcohol is 20-30 mPa·s, the alcoholysis degree is 98.0-99.0 mol%, and the polyvinyl alcohol is purchased from Kuraray of Japan, model R-1130;

[0054] 12 parts of modified hyaluronic acid;

[0055] 6 parts of modified chitosan;

[0056] 5 parts of emulsifier, the emulsifier is polysorbate;

[0057] 8 parts of moisturizer, the moisturizer is sorbitol;

[0058] 40 parts of water.

[0059] The preparation process of modified hyaluronic acid is:

[0060] Add acrylated hyaluronic acid, inorganic guanidine salt, organic guanidine salt, polyamine small molecules and amino-terminated polymer into a reactor in a mass ratio of 6:2:1:3:1. Mix them evenly and heat to 180°C under nitrogen protection for reflux reaction for 7 hours to obtain modified hyaluronic acid.

[0061] The preparation method of acrylated hyaluronic acid is as follows:

[0062] A mixture is obtained by mixing hyaluronic acid with a molecular weight of 400,000 to 1,000,000, hyaluronic acid with a molecular weight of 1,000,000 to 1,800,000, and hyaluronic acid with a molecular weight of 1,800,000 to 2,200,000 according to a mass ratio of 1:4:1; hyaluronic acid is purchased from Shaanxi Zhenhe Biotechnology Co., Ltd.;

[0063] The mixture is dissolved in water to form a hyaluronic acid solution with a concentration of 4 mg / mL, adipic acid dihydrazide is added, and the mass ratio of adipic acid dihydrazide to the mixture is 1:12. After the solution is completely dissolved, the pH is adjusted to 6 with hydrochloric acid; carbodiimide hydrochloride is added, and the mass ratio of carbodiimide hydrochloride to the mixture is 1:3, and the pH is maintained at 6 until it is stable, reacting at room temperature for 11 hours, dialyzing with deionized water for 55 hours, and freeze-drying to obtain a hyaluronic acid-adipic acid dihydrazide derivative;

[0064] The hyaluronic acid-adipic acid dihydrazide derivative was dissolved in hydroxyethylpiperazine ethanesulfonic acid buffer to obtain a solution A with a concentration of 5 mg / mL, N-acryloyloxysuccinimide was dissolved in dimethyl sulfoxide to obtain a solution B with a concentration of 135 mg / mL, and then solution B was added to solution A, reacted at room temperature for 11 hours, dialyzed with deionized water for 70 hours, and freeze-dried to obtain acrylated hyaluronic acid. The hydroxyethylpiperazine ethanesulfonic acid buffer was purchased from Merck at a concentration of 0.5M.

[0065] Inorganic guanidine salts include guanidine nitrate (CAS: 506-93-4) and guanidine hydrochloride (CAS: 50-01-1) in a mass ratio of 1:7; organic guanidine salts include aminoguanidine hydrochloride (CAS: 1937-19-5), diaminoguanidine hydrochloride (CAS: 36062-19-8), and triaminoguanidine hydrochloride (CAS: 5329-29-3) in a mass ratio of 2:5:1; the small molecule polyamine is diethylenetriamine; the amino-terminated polymer is polyetheramine. The amine value of the polyetheramine is 440-480 mgKOH / g, purchased from Wuxi Akeli Technology Co., Ltd., model MA-223.

[0066] The preparation method of modified chitosan is:

[0067] At room temperature, chitosan was added into the reaction container. The chitosan had a deacetylation degree of >90% and a viscosity of 10-200 mPa·s, which was purchased from Shaanxi Yuanshengte Biotechnology Co., Ltd., and 0.3 mol / L acetic acid solution was added to fully swell the chitosan. The mass volume ratio of chitosan to acetic acid solution was 1 g:4 mL. Deionized water was then added to fully dissolve the chitosan. The mass volume ratio of chitosan to deionized water was 1 g:30 mL to obtain a chitosan solution.

[0068] A cross-linking agent and a protein composition are added to a chitosan solution, wherein the cross-linking agent is tea polyphenols. The pH is adjusted to 5.0, stirred evenly, and irradiated under ultraviolet light for 50 minutes. The ultraviolet light comes from an ultraviolet lamp with a power of 600W and a main wavelength of 365nm. After filtering, washing and drying, modified chitosan is obtained. The mass ratio of the protein composition, chitosan and the cross-linking agent is 0.6:1.2:1, and the protein composition includes collagen, silk fibroin and keratin in a mass ratio of 6:3:1.

[0069] A method for preparing an antibacterial gel of modified hyaluronic acid comprises the following steps:

[0070] S1: Weigh polyvinyl alcohol, modified hyaluronic acid, modified chitosan, emulsifier, moisturizer and water according to weight proportions;

[0071] S2: Mix the raw materials in step S1 and stir them evenly to obtain an antibacterial gel.

[0072] Example 2

[0073] The difference between this embodiment and embodiment 1 is that the formula of the antibacterial gel is different.

[0074] The antibacterial gel in this embodiment includes the following raw materials in parts by weight:

[0075]

[0076] Comparative Example 1

[0077] The difference between this comparative example and Example 1 is that the viscosity of the polyvinyl alcohol is 5.2-6.2 mPa·s, the alcoholysis degree is 74.0-80.0 mol%, and the polyvinyl alcohol is purchased from Kuraray of Japan, model KL-506.

[0078] Comparative Example 2

[0079] The difference between this comparative example and Example 1 is that during the preparation of the modified hyaluronic acid, the mixture is directly reacted with the inorganic guanidine salt, the organic guanidine salt, the polyamine small molecule, and the amino-terminated polymer without modification of the acrylate group.

[0080] Comparative Example 3

[0081] The difference between this comparative example and Example 1 is that during the preparation of the modified hyaluronic acid, hyaluronic acid with a molecular weight of 400,000 to 1,000,000 is not added to the mixture, and hyaluronic acid with a molecular weight of 1,000,000 to 1,800,000 is used to replace the hyaluronic acid with a molecular weight of 400,000 to 1,000,000.

[0082] Comparative Example 4

[0083] The difference between this comparative example and Example 1 is that during the preparation of the modified hyaluronic acid, the mass ratio of hyaluronic acid with a molecular weight of 400,000 to 1,000,000, hyaluronic acid with a molecular weight of 1,000,000 to 1,800,000 and hyaluronic acid with a molecular weight of 1,800,000 to 2,200,000 in the mixture is 1:4:5.

[0084] Comparative Example 5

[0085] The difference between this comparative example and Example 1 is that during the preparation of the modified hyaluronic acid, no inorganic guanidine salt is added, and an equal mass of organic guanidine salt is used to replace the inorganic guanidine salt.

[0086] Comparative Example 6

[0087] The difference between this comparative example and Example 1 is that during the preparation of the modified hyaluronic acid, the polyetheramine has an amine value of 220-280 mgKOH / g and is purchased from Wuxi Akeli Technology Co., Ltd. with a model number of MA-240.

[0088] Comparative Example 7

[0089] The difference between this comparative example and Example 1 is that a mixture of hyaluronic acid with a molecular weight of 400,000 to 1,000,000, hyaluronic acid with a molecular weight of 1,000,000 to 1,800,000 and hyaluronic acid with a molecular weight of 1,800,000 to 2,200,000 is directly added to the preparation process of the antibacterial gel, and the hyaluronic acid is not subjected to any modification treatment.

[0090] Comparative Example 8

[0091] The difference between this comparative example and Example 1 is that the deacetylation degree of chitosan is ≥85%, and the chitosan is purchased from Qingdao Hehai Biotechnology Co., Ltd.

[0092] Comparative Example 9

[0093] The difference between this comparative example and Example 1 is that during the preparation of the modified chitosan, no silk fibroin was added to the protein composition, and an equal mass of collagen was used to replace the silk fibroin.

[0094] Comparative Example 10

[0095] The difference between this comparative example and Example 1 is that during the preparation of the modified chitosan, the mass ratio of collagen, silk fibroin and keratin in the protein composition is 6:3:5.

[0096] Comparative Example 11

[0097] The difference between the comparative example and Example 1 is that chitosan is directly added into the preparation process of the antibacterial gel without any modification of the chitosan.

[0098] Performance Testing

[0099] (1) Antibacterial performance test

[0100] Medium preparation

[0101] ①LB liquid culture medium: Use a measuring cylinder to measure 100 mL of distilled water and pour it into a 250 mL reagent bottle. Use an analytical electronic balance to weigh 2.5 g of LB broth culture medium and add it to mix well. Sterilize it in a high-temperature and high-pressure steam sterilizer at 121°C for 15 minutes and set aside for use.

[0102] ②LB solid culture medium: Use a measuring cylinder to measure 100mL of distilled water and pour it into a 250mL reagent bottle. Use an analytical electronic balance to weigh 2.5g of LB broth culture medium and 1.5g of agar powder respectively. Add the above weighed reagents and mix well. Sterilize in a high-temperature and high-pressure steam sterilizer at 121℃ for 15min. When the culture medium cools to 45℃, use an electric pipette to draw 15mL of culture medium and pour it into a disposable sterile plate.

[0103] ③ Preparation of bacterial suspension: Take three 12mL bacterial culture tubes, add 3mL LB liquid culture medium to each, pick single colonies from the solid culture medium of Escherichia coli and Staphylococcus aureus and add them to the liquid culture medium. The other tube is used as a blank control and placed in a constant temperature oscillator (37℃, 200rpm) for shaking culture overnight (15h).

[0104] ④ Sample processing: Place the samples in disposable culture dishes according to the groups, sterilize them under ultraviolet light for 30 minutes on both sides, and set aside.

[0105] ⑤Inhibition zone test: Use sterile PBS solution to dilute Escherichia coli and Staphylococcus aureus to 10 6 CFU / mL, then draw 100 μL and evenly spread it on the LB solid culture medium. Use sterile tweezers to stick the sterilized gel prepared in Example 1 to the surface of the culture medium in sequence and gently press it with tweezers to make it fully contact, and place the culture dish in a constant temperature incubator at 37°C for 18 hours. After the culture is completed, take out and measure and record the size of the inhibition zone. The test results of the embodiment and the comparative example are shown in Table 1.

[0106] Table 1 Antibacterial performance test results

[0107]

[0108] (2) Swelling performance test of antibacterial gel

[0109] The cylindrical antibacterial gel samples of the embodiment and comparative example (made with a 48-well plate as a mold) were placed in pure water and PBS solution (0.1M, pH=7.4) in a 37°C incubator for testing. They were taken out of the pure water and PBS solution at regular intervals and the surface water was removed with filter paper. The initial weight (W0) and weight after swelling at different time periods (W t ), and calculate the swelling ratio. The test results are shown in Table 2.

[0110] Swelling ratio=(W t -W0) / W0

[0111] The equilibrium swelling ratio of hydrogel is an important factor in characterizing the structure of hydrogel. The reason why hydrogel has swelling characteristics is that it has a large number of cross-linking points inside, which can carry out molecular chain movement and allow water molecules to fill the interior of the hydrogel.

[0112] Table 2 Swelling performance test results

[0113]

[0114] (3) Mechanical properties test of antibacterial gel

[0115] In order to evaluate the tensile properties of the antibacterial gel, a universal tensile testing machine was used to carry out a tensile test on the gel. The tensile speed was fixed at 10%. The elongation at break of the hydrogel is shown in Table 3.

[0116] Table 3 Mechanical properties test results

[0117]

[0118]

[0119] (4) The stability of the antibacterial gels obtained in the above examples and comparative examples was tested with reference to Appendix C6 of GB 15979-2002 "Hygiene Standard for Disposable Hygiene Products". An accelerated test was performed in a constant temperature box at 54-57° C. for 14 days to observe the morphology of the gel. The test results are shown in Table 4.

[0120] Table 4 Stability test results

[0121]

[0122]

[0123] From the above performance test results, it can be seen that the antibacterial properties, mechanical properties, swelling properties and stability of the antibacterial gel prepared in Examples 1-2 are greatly improved, especially the comprehensive performance of Example 1 is the most outstanding, which is mainly because the present invention adds modified hyaluronic acid and modified chitosan to the antibacterial gel.

[0124] However, the comparative example is obviously inferior to the embodiment in the corresponding performance test because the necessary technical scheme is not adopted. In comparative example 1, the alcoholysis degree of the main ingredient polyvinyl alcohol is changed. It can be seen from the results that compared with embodiment 1, the various properties of the antibacterial gel prepared in comparative example 1 are reduced, indicating that the parameters of polyvinyl alcohol have a certain influence on the performance of the antibacterial gel. In comparative example 2, hyaluronic acid is not subjected to acrylate modification treatment. It can be seen from the results that the various properties of the antibacterial gel prepared therefrom are further reduced, indicating that the first step of acrylate modification of hyaluronic acid is very important for improving the comprehensive performance of the antibacterial gel. In comparative example 3, hyaluronic acid with a small molecular weight is not used. Compared with the embodiment, only hyaluronic acid with a medium molecular weight and a large molecular weight is used. It can be seen from the results that the comprehensive performance of the antibacterial gel prepared therefrom is also further reduced, indicating that the simultaneous use of hyaluronic acid with three molecular weights has an important influence on improving the various properties of the antibacterial gel. In Comparative Example 4, the mass ratio of three molecular weight hyaluronic acids was changed. It can be seen from the results that the mechanical properties, swelling properties, antibacterial properties and stability of the antibacterial gel prepared therefrom all decreased, indicating that the addition ratio of multiple hyaluronic acids will affect the performance of the antibacterial gel. In Comparative Example 5, no inorganic guanidine salt was added. It can be seen from the results that the comprehensive performance of the antibacterial gel prepared therefrom decreased, indicating that the simultaneous addition of various types of guanidine salts has a synergistic effect and can improve the performance of the antibacterial gel. In Comparative Example 6, the amine value of polyetheramine was changed. It can be seen from the results that the performance of the antibacterial gel prepared therefrom decreased, indicating that the amine value of polyetheramine has an impact on the various parameters of the antibacterial gel. In Comparative Example 7, hyaluronic acid was not modified. It can be seen from the results that the various properties of the antibacterial gel all decreased seriously, indicating that the modification of hyaluronic acid in the present invention is very necessary. In Comparative Example 8, the deacetylation degree of chitosan was changed, which decreased to a certain extent. It can be seen from the results that it has a certain impact on the performance of the antibacterial gel. In Comparative Example 9, no silk fibroin was added to the modification of chitosan. It can be seen from the results that the various properties of the antibacterial gel prepared therefrom decreased to varying degrees, indicating that the three proteins in the protein composition have a very important influence on synergistically improving the comprehensive performance of the antibacterial gel. In Comparative Example 10, the mass ratio of the three proteins was changed. It can be seen from the results that the comprehensive performance of the antibacterial gel prepared therefrom decreased, indicating that the addition ratio of the three proteins has a very important influence on improving the comprehensive performance of the antibacterial gel. In Comparative Example 11, no modification was made to chitosan. It can be seen from the results that the various properties of the antibacterial gel seriously decreased, indicating that the modification of chitosan in the present invention has an important influence on improving the antibacterial properties, mechanical properties, swelling properties and stability of the antibacterial gel. The above experimental results further prove the importance of the technical scheme defined in the present invention for its technical effect.

[0125] Therefore, the present invention adopts an antibacterial gel of modified hyaluronic acid with the above structure and a preparation method thereof. The present invention can achieve the best antibacterial performance of the antibacterial gel by accurately controlling the proportion of various functional components and the mass ratio between the antibacterial components. The present invention can not only improve the antibacterial effect, but also ensure that the gel has suitable rheological properties for easy application and absorption.

[0126] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. An antibacterial gel of modified hyaluronic acid, characterized in that: The antibacterial gel comprises the following raw materials in parts by weight: The modified hyaluronic acid is prepared by the reaction of acrylated hyaluronic acid, inorganic guanidine salt, organic guanidine salt, polyamine small molecule and amino-terminated polymer; the mass ratio of acrylated hyaluronic acid, inorganic guanidine salt, organic guanidine salt, polyamine small molecule and amino-terminated polymer is (5-8): (1-3): (1-3): (2-4): 1; The modified chitosan is prepared by reacting a protein composition, chitosan and a crosslinking agent; the mass ratio of the protein composition, chitosan and the crosslinking agent is (0.5-0.8):(1-1.5):1, and the protein composition includes collagen, silk fibroin and keratin in a mass ratio of (5-8):(2-4):

1.

2. The antibacterial gel of modified hyaluronic acid according to claim 1, characterized in that: The viscosity of polyvinyl alcohol is 20 to 30 mPa·s, and the alcoholysis degree is 98.0 to 99.0 mol%.

3. The antibacterial gel of modified hyaluronic acid according to claim 1, characterized in that: The preparation method of acrylated hyaluronic acid is as follows: A mixture is obtained by mixing hyaluronic acid with a molecular weight of 400,000 to 1,000,000, hyaluronic acid with a molecular weight of 1,000,000 to 1,800,000, and hyaluronic acid with a molecular weight of 1,800,000 to 2,200,000 in a mass ratio of 1:(3-5):1; The mixture is dissolved in water to form a hyaluronic acid solution with a concentration of 3 to 5 mg / mL, adipic acid dihydrazide is added, and the mass ratio of adipic acid dihydrazide to the mixture is 1:11 to 14. After the solution is completely dissolved, the pH is adjusted to 5 to 7 with hydrochloric acid; carbodiimide hydrochloride is added, and the mass ratio of carbodiimide hydrochloride to the mixture is 1:1-5, and the pH is maintained at 5 to 7 until it is stable, reacting at room temperature for 10 to 12 hours, dialyzing with deionized water for 50 to 60 hours, and freeze-drying to obtain a hyaluronic acid-adipic acid dihydrazide derivative; The hyaluronic acid-adipic acid dihydrazide derivative is dissolved in hydroxyethylpiperazine ethanesulfonic acid buffer to obtain a solution A with a concentration of 3 to 6 mg / mL, N-acryloyloxysuccinimide is dissolved in dimethyl sulfoxide to obtain a solution B with a concentration of 120 to 150 mg / mL, and then the solution B is added to the solution A, reacted at room temperature for 10 to 12 hours, dialyzed with deionized water for 65 to 85 hours, and freeze-dried to obtain acrylated hyaluronic acid.

4. The antibacterial gel of modified hyaluronic acid according to claim 1, characterized in that: The inorganic guanidine salts include guanidine nitrate and guanidine hydrochloride in a mass ratio of 1:(6-8); the organic guanidine salts include aminoguanidine hydrochloride, diaminoguanidine hydrochloride and triaminoguanidine hydrochloride in a mass ratio of (1-3):(4-6):1; the polyamine small molecule is diethylenetriamine; and the amino-terminated polymer is polyetheramine.

5. The antibacterial gel of modified hyaluronic acid according to claim 4, characterized in that: The amine value of the polyetheramine is 440 to 480 mgKOH / g.

6. The antibacterial gel of modified hyaluronic acid according to claim 1, characterized in that: The preparation process of modified hyaluronic acid is: Add acrylated hyaluronic acid, inorganic guanidine salt, organic guanidine salt, polyamine small molecule and amino-terminated polymer into a reactor, mix them evenly, and heat to 150-200° C. for reflux reaction for 5-10 hours under nitrogen protection to obtain modified hyaluronic acid.

7. The antibacterial gel of modified hyaluronic acid according to claim 1, characterized in that: The preparation method of modified chitosan is: At room temperature, chitosan was added to a reaction container, and a 0.3 mol / L acetic acid solution was added to fully swell the chitosan, the mass volume ratio of chitosan to the acetic acid solution was 1 g: (3-5) mL, and deionized water was added to fully dissolve the chitosan, the mass volume ratio of chitosan to deionized water was 1 g: (25-40) mL, to obtain a chitosan solution; The cross-linking agent and the protein composition are added to the chitosan solution, the pH value is adjusted to 5.0, the solution is stirred evenly, and the solution is irradiated under ultraviolet light for 30 to 60 minutes. The solution is filtered, washed and dried to obtain the modified chitosan.

8. The antibacterial gel of modified hyaluronic acid according to claim 1, characterized in that: The emulsifier is at least one of lactic acid ester, betaine, polysorbate, gum arabic and soybean lecithin.

9. The antibacterial gel of modified hyaluronic acid according to claim 1, characterized in that: The moisturizing agent is at least one of sorbitol, glycerol and 1,3-butylene glycol.

10. The method for preparing an antibacterial gel of modified hyaluronic acid according to any one of claims 1 to 9, characterized in that: The following steps are involved: S1: Weigh polyvinyl alcohol, modified hyaluronic acid, modified chitosan, emulsifier, moisturizer and water according to weight proportions; S2: Mix the raw materials in step S1 and stir them evenly to obtain an antibacterial gel.

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