阅读说明：本技术 一种复合水凝胶伤口敷料及其制备方法 (Composite hydrogel wound dressing and preparation method thereof ) 是由 郑允权 冯晓莲 石贤爱 杨建民 于 2020-05-13 设计创作，主要内容包括：本发明提供了一种复合水凝胶伤口敷料及其制备方法,其是以胺化胶原蛋白和氧化海藻酸钠为基材,并载入多粘菌素B硫酸盐与杆菌肽两种多肽类抗生素,通过席夫碱反应制备AC/OSA-PB复合水凝胶敷料,本发明制备方法操作简单,对反应条件要求也低,可在短时间内成胶,利于大规模生产,且所制得的伤口敷料不仅具有良好的杀菌作用,还能够最大程度的提高HSF细胞迁移率和HSF细胞增殖率,具有极好的促伤口愈合能力,同时还具有适用于皮肤伤口的力学性能与机械性能。(The invention provides a composite hydrogel wound dressing and a preparation method thereof, wherein aminated collagen and oxidized sodium alginate are used as base materials, two polypeptide antibiotics of polymyxin B sulfate and bacitracin are loaded, and the AC/OSA-PB composite hydrogel dressing is prepared through Schiff base reaction.)

1. A preparation method of the composite hydrogel wound dressing is characterized by comprising the following steps:

s1, dissolving aminated collagen, polymyxin B sulfate and bacitracin in a buffer solution to obtain a medicine-containing aminated collagen solution;

s2, dissolving oxidized sodium alginate in a buffer solution to obtain an oxidized sodium alginate solution;

s3, mixing the drug-containing aminated collagen solution obtained in the step S1 with the oxidized sodium alginate solution obtained in the step S2, immediately removing bubbles by ultrasonic waves, standing and curing to form gel, and obtaining the composite hydrogel wound dressing;

wherein the mass ratio of the polymyxin B sulfate to the bacitracin to the oxidized sodium alginate is 1:1 (75-150), and the mass ratio of the aminated collagen to the oxidized sodium alginate is (1-3: 3).

2. The preparation method of the composite hydrogel wound dressing according to claim 1, wherein the aminated collagen is prepared by dissolving collagen in a buffer solution, adding ethylenediamine according to the mass-to-volume ratio of 1 (1-3) g/mL after the collagen is dissolved, fully mixing uniformly, and adjusting the pH value to 5.0-6.0; then adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride according to the mass ratio of 1 to the collagen of (0.5-3), reacting for 4-8 hours to obtain reaction liquid, dialyzing the reaction liquid, and freeze-drying to obtain the collagen grafted and modified by the ethylenediamine; the buffer solution used was either 1mol/L, pH =6.0-7.2 sodium phosphate buffer solution or PBS.

3. The method for preparing a composite hydrogel wound dressing according to claim 1 or 2, wherein the aminated collagen is specifically aminated fish scale collagen, namely, the aminated fish scale collagen is prepared by taking fish scale collagen as a raw material, and the molecular weight of the fish scale collagen is more than 80 KDa.

4. The preparation method of the composite hydrogel wound dressing according to claim 1, wherein the preparation method of the oxidized sodium alginate comprises the following steps:

(1) dispersing 5-25 g of sodium alginate in 100 mL of absolute ethyl alcohol to prepare a sodium alginate solution, dissolving 5-20 g of sodium periodate in 100 mL of deionized water, slowly pouring the solution into the sodium alginate solution, reacting for 4-8 h at 20-40 ℃ in the dark, and adding ethylene glycol to terminate the reaction to obtain a reaction solution;

(2) and (2) adding absolute ethyl alcohol into the reaction liquid obtained in the step (1) to separate out oxidized sodium alginate, performing suction filtration, dissolving the oxidized sodium alginate obtained by suction filtration in deionized water, dialyzing, and freeze-drying to obtain the sodium alginate.

5. The method for preparing a composite hydrogel wound dressing according to claim 1, wherein the buffer solution is any one of 1mol/L, pH =6.0-7.2 sodium phosphate buffer solution and PBS.

6. The method for preparing a composite hydrogel wound dressing according to claim 1, wherein the steps S1-S3 are all performed at 10-50 ℃.

7. A composite hydrogel wound dressing prepared by the method of any one of claims 1 to 6.

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to a composite hydrogel wound dressing and a preparation method thereof.

Background

Skin wound repair is one of the major challenges facing the medical field at present, and according to the statistics of the world health organization, about millions of people suffer from skin injury every year worldwide, of which about 18 thousands die from burns, and non-fatal burns are the main causative factors. Large areas of non-lethal trauma destroy the normal physiological functions of the skin, and when timely and effective treatment is not obtained, the wounds are very likely to be infected by invasion of microorganisms, so that the healing of the wounds is delayed, and in severe cases, life risks are caused. Therefore, effective control of wound infection, and rapid promotion of wound healing, are important ways to reduce patient mortality. Full-thickness skin wound tissue undergoes a complex process of hemostasis, inflammation, proliferation, tissue remodeling and scarring during the healing process. Wound healing of damaged skin is slow without medication or other treatment, especially in the elderly and in patients with underlying diseases such as diabetes.

Autologous skin grafting is a commonly used method for treating skin injuries such as large-area burns and wounds, but the method has long operation time and high treatment cost, and can have many side effects, such as: blood circulation disorder, secondary hemorrhage or inflammatory infection, etc. After the operation is successful, the problems of large color difference between the transplanted skin area and the surrounding skin, influence on the appearance and the like can also occur. To date, wound dressings are one of the most practical methods of treating deep wounds of the skin. Hydrogel wound dressings have advantages over numerous wound dressings such as gauze, electrospun fibers, films, sponges, and the like in that the wound can be maintained in a moist environment, the wound is prevented from dehydrating, additional secretions are absorbed, gas exchange is permitted, and removal is easy without secondary damage to the wound.

In recent years, a large number of documents have reported different types of hydrogel wound dressings. The hydrogel wound dressings mainly take natural polymer materials and synthetic polymer materials as base materials, one of the two types of materials is used independently or the two types of materials are compounded, and antibacterial or hemostatic medicines are added according to the characteristics of the base materials to prepare hydrogels with different efficacies. The healing promoting effect of the fish skin collagen-chitosan composite alginate hydrogel dressing on the burn and scald wound surfaces (Song Wen mountain, and the like, Chinese ocean medicine, 2019) reports that sodium alginate is used as a raw material, chitosan and fish skin collagen are compounded, and Ca is used for Ca²⁺Crosslinking to prepare the alginate hydrogel dressing. The prepared fish skin collagen-chitosan composite alginate hydrogel dressing can effectively shorten the healing period, eliminate wound inflammation and inhibit scar formation, and has good prospects and application directions in the aspect of wound care, however, the method also has a plurality of defects, and concretely comprises the following steps: (1) preparing gel liquid by adopting sodium alginate, fish skin collagen, polyvinyl alcohol and glycerol, wherein the polyvinyl alcohol is an organic compound and is harmful to human bodies; (2) the hydrogel is required to be placed for 2 hours at the temperature of 4 ℃, then a cross-linking agent prepared from chitosan and anhydrous calcium chloride is added for cross-linking reaction, the whole cross-linking process usually needs several hours to realize gelling, the gelling time is long, the production efficiency is influenced, and the cross-linking step is complicated, so that uncertain factors are increased; (3) ca in contrast to other hydrogels²⁺The hydrogel prepared by crosslinking has the characteristics of unevenness, hardness, brittleness and the like, and is not favorable for the comfort of wounds. In addition, calcium ion impurities are doped, so that the healing of the wound is influenced to a certain extent; (4) chitosan is used as a natural antibacterial agent, compared with antibiotics, the antibacterial effect of the chitosan is poor, the antibacterial concentration and antibacterial performance of chitosan are closely related to the preparation process, molecular weight and molecular structure of the chitosan, and the chitosan is mainly obtained by an extraction method at present, so that the control requirements on raw materials and the production process are high, the production stability of chitosan is difficult to grasp, the antibacterial effect of chitosan products sold on the market is uneven, and the trouble of screening chitosan is increased.

Disclosure of Invention

In order to solve the defects in the prior art, the invention aims to provide a composite hydrogel wound dressing and a preparation method thereof, the operation is simple, the requirement on reaction conditions is low, the composite hydrogel wound dressing can be gelatinized in a short time, the mass production is facilitated, and the prepared wound dressing has good biocompatibility, excellent wound healing promoting capability and mechanical properties suitable for skin wounds.

In order to achieve the purpose, the invention adopts the following technical scheme:

one of the purposes of the invention is to provide a preparation method of a composite hydrogel wound dressing, which comprises the following steps:

s1, dissolving aminated collagen, polymyxin B sulfate and bacitracin in a buffer solution to obtain a medicine-containing aminated collagen solution;

s2, dissolving oxidized sodium alginate in a buffer solution to obtain an oxidized sodium alginate solution;

s3, mixing the drug-containing aminated collagen solution obtained in the step S1 with the oxidized sodium alginate solution obtained in the step S2, immediately removing bubbles by ultrasonic waves, standing and curing to form gel, and obtaining the composite hydrogel wound dressing.

Wherein the mass ratio of the polymyxin B sulfate, bacitracin and sodium alginate oxide is 1:1 (75-150), the mass ratio of the aminated collagen to the sodium alginate oxide is (1-3) to 3, and the mass ratio of the aminated collagen to the sodium alginate oxide is (1.5-2.5) to 3. According to the invention, by controlling the concentration of the aminated collagen, the problems that the aminated collagen content is too low and is difficult to solidify into gel and the aminated collagen content is too high, so that the investment cost of the aminated collagen is too high, and the time for solidifying into gel is too short, so that the materials before solidifying into gel are not uniformly mixed and the quality of the hydrogel wound dressing is influenced are avoided.

The aminated collagen is prepared by grafting amino on a collagen molecular chain by using ethylenediamine as a modifier, and specifically, the ethylenediamine and carboxyl in natural collagen are subjected to amidation reaction to form modified collagen. The grafting rate of the aminated collagen is 10-27%. The preparation method comprises the steps of dissolving collagen in a buffer solution, adding ethylenediamine according to the mass-volume ratio of 1 (1-3) g/mL after the collagen is dissolved, fully and uniformly mixing, and adjusting the pH value to 5.0-6.0; then, according to the mass ratio of the collagen to the collagen of 1: (0.5-3) adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, reacting for 4-8 h to activate carboxyl on the collagen, dialyzing the obtained reaction solution, and freeze-drying to obtain the ethylenediamine grafted and modified collagen.

The aminated collagen is preferably aminated fish scale collagen, namely, the fish scale collagen is prepared by taking the fish scale collagen as a raw material, and the fish scale collagen has a molecular weight of more than 80KDa and can have better gel property.

The preparation method of the oxidized sodium alginate comprises the following steps:

(1) dispersing 5-25 g of sodium alginate in 100 mL of absolute ethyl alcohol to prepare a sodium alginate solution, dissolving 5-20 g of sodium periodate in 100 mL of deionized water, slowly pouring the solution into the sodium alginate solution, reacting for 4-8 h at 20-40 ℃ in the dark, and adding ethylene glycol to terminate the reaction to obtain a reaction solution;

(2) and (2) adding absolute ethyl alcohol into the reaction liquid obtained in the step (1) to separate out oxidized sodium alginate, performing suction filtration on the separated oxidized sodium alginate, dissolving the oxidized sodium alginate obtained by suction filtration in deionized water, dialyzing, and freeze-drying to obtain the sodium alginate.

Each monomolecular structure of the sodium alginate contains cis-vicinal diol hydroxyl, and the cis-vicinal diol hydroxyl can react with a strong oxidant sodium periodate to break C-C bonds in the monomolecular structure of the sodium alginate and oxidize the C-C bonds into active aldehyde groups or ketones.

The buffer solution used in the above was either 1mol/L, pH =6.0-7.2 sodium phosphate buffer solution or PBS.

Preferably, the steps S1-S3 are all performed at 10-50 ℃ to avoid the problem that the amination collagen and the oxidized sodium alginate are difficult to completely dissolve due to too low temperature, the reaction speed is influenced, and the side reaction caused by too high temperature is avoided. More preferably, the steps S1-S3 are carried out at 30-40 ℃.

Preferably, the gel forming time of standing and curing in the step S3 is 40-210S, which not only can avoid too long gel forming time and reduction of production efficiency, but also can avoid too short gel forming time and short material mixing operation time, which bring product quality problems.

It is another object of the present invention to provide a composite hydrogel wound dressing prepared by the above method.

The invention has the following beneficial effects:

(1) the Schiff base reaction mainly refers to the reaction of aldehyde or ketone (active carbonyl) and ammonia or amine for condensation to generate a class of organic compounds containing imine or azomethine groups (-RC = N-). The oxidized sodium alginate used in the invention contains a large amount of active aldehyde groups, and the aminated collagen contains a large amount of amino groups, and the oxidized sodium alginate and the aminated collagen can rapidly carry out Schiff base reaction under mild conditions to generate amido bonds to form hydrogel.

(2) The two kinds of polypeptide antibiotics without drug resistance, namely polymyxin B sulfate and bacitracin, are loaded in the invention, compared with natural antibacterial agents, the two antibacterial agents have better antibacterial performance and stable performance, wherein the polymyxin B sulfate has a bactericidal effect on gram-negative bacteria, the bacitracin has a bactericidal effect on gram-positive bacteria, the two antibacterial agents are combined for use and have broad-spectrum bactericidal effect, and the two antibacterial agents have almost no drug resistance and do not generate cross drug resistance with other antibiotics.

(3) Since the cationic lipopeptide on the polymyxin B sulfate structure interacts with the bacterial cell membrane, resulting in a change in intracellular osmotic pressure and cell lysis and death, one of ordinary skill in the art would generally consider that polymyxin B sulfate concentration is inversely related to HSF (human epidermal fibroblast) cell proliferation rate. However, when the concentration of the antibacterial agent is examined, the inventor unexpectedly discovers that: with the continuous increase of the concentrations of the polymyxin B sulfate and bacitracin, the proliferation effect of the HSF cells on the hydrogel wound dressing has a trend of increasing first and then decreasing later, which shows that the polymyxin B sulfate with a certain concentration is used in combination with the bacitracin, so that the prepared hydrogel wound dressing has good sterilization effect, can avoid wound infection, and can also improve the migration rate and the proliferation rate of the HSF cells to the maximum extent, thereby promoting the wound healing to the maximum extent, and finally determining that the suitable concentration is that the mass ratio of the polymyxin B sulfate, the bacitracin and the sodium alginate oxide is 1:1: (75-150).

(4) The wound dressing is natural and nontoxic and has good biocompatibility.

(5) The collagen base material preferably adopts fish scale collagen with the molecular weight of more than 80KDa, and the macromolecular collagen has better gel property, so that the prepared wound dressing has better mechanical property;

(6) the invention also specifically controls the mass ratio of aminated collagen to oxidized sodium alginate in the range from (1-3) to (3) so as to further shorten the time for curing and gelling, and can provide enough time for uniform mixing of materials before curing and gelling, thereby avoiding the influence of uneven mixing of materials on the quality of hydrogel wound dressing.

Drawings

FIG. 1 is a comparison graph of the IR spectra of oxidized sodium alginate and sodium alginate prepared in the examples;

FIG. 2 is a comparison graph of infrared spectra of aminated fish scale collagen and fish scale collagen prepared in the examples;

FIG. 3 is an infrared spectrum of the AC/OSA hydrogel prepared in the example.

Detailed Description

In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.

The sources of the main chemicals used are shown in table 1.

TABLE 1