Preparation method of recombinant double-protein medical application and dressing

Abstract

The invention relates to the field of dressings, and particularly discloses a preparation method of a recombinant double-protein medical application and dressing. The application and the dressing provided by the invention are low in irritation, virus hidden dangers and rejection reaction are isolated by compounding the recombinant mussel mucin and the recombinant III type humanized collagen, rapid repair of wounded skin is realized, the activation ability of beta-glucan on macrophages is enhanced by treating beta-glucan, secretion of growth factors and collagen is promoted, and the application has a good application prospect. When being combined with the recombinant III-type humanized collagen for use, the recombinant III-type humanized collagen can accelerate the proliferation of fibroblasts.

Description

Technical Field

[0001] The present invention belongs to the field of dressings, and particularly relates to a recombinant double-protein medical dressing and a preparation method thereof. Background Art

[0002] For patients with allergies, eczema, acne, and those after photoelectric and hydrodermabrasion aesthetic medical procedures, the skin barrier function is damaged to varying degrees. Therefore, it is necessary to establish a new skin barrier to prevent the continued infection of the damaged part by external bacteria and other microorganisms. When skin injury occurs, the body initiates endogenous tissue regeneration and repair, that is, wound healing, which is a highly coordinated complex physiological process. Wound healing involves 4 different and overlapping stages, namely hemostasis, inflammation, proliferation, and remodeling (repair). Dressings can replace the damaged skin during the wound healing and treatment process to provide temporary protection, avoid or control wound infection, and provide a suitable healing environment for the traumatized surface.

[0003] The wound healing process is a continuous dynamic process, which is a process of interaction between cells, between cells and the cell matrix, and with soluble mediators. As the main structural component of the extracellular matrix, collagen has good biocompatibility, biodegradability, and biological activity, and is widely used in the fields of food, medicine, tissue engineering, cosmetics, etc. Currently, there are various dressing products for collagen-based skin barrier repair on the market. For example, patent CN112076340B provides a skin barrier repair dressing mask and its preparation method, and its raw materials include trehalose, sodium hyaluronate, collagen, glycerol, and some plant extracts: Saxifraga stolonifera extract, Peucedanum praeruptorum extract, and Platycodon grandiflorum root extract. Although this dressing has the effect of promoting wound healing and the growth of epithelial cells, for some skin-sensitive people, the active substances in the plant extracts are likely to cause allergic reactions. Summary of the Invention

[0004] In order to overcome the deficiencies of the above-mentioned prior art, the present invention provides a low-irritation collagen-based dressing, which reduces the virus risk and rejection reaction by compounding recombinant mussel adhesive protein and recombinant type III humanized collagen, realizes the rapid repair of traumatized skin, and exposes more glycosidic bonds by treating β-glucan, enhancing the activation ability of macrophages, promoting the secretion of growth factors and collagen. When used in combination with recombinant type III humanized collagen, it can accelerate the proliferation of fibroblasts; and on the basis of the high adhesion provided by recombinant mussel adhesive protein, the micelle structure of the glucan provided in this application can serve as a microscopic scaffold to guide cell directional migration and shorten the wound healing cycle.

[0005] To achieve the above object, the technical solution adopted by the present invention is: The present invention provides a method for preparing a recombinant double-protein medical dressing, which comprises the following steps: S1: Vacuum stir the recombinant double-protein ingredients at 10-90 °C until completely mixed to form a stock solution; S2: Fill the stock solution and the non-woven dressing into an aluminum foil bag through a filling machine, or fill the stock solution into a loading bottle through a filling machine; S3: Heat-seal the aluminum foil bag filled in step S2, or seal the loading bottle; S4: Irradiate and sterilize the sealed aluminum foil bag or loading bottle obtained in step S3 to obtain the recombinant double-protein medical dressing and dressing.

[0006] In some embodiments, in step S1, based on 100% of the mass percentage of the stock solution, it comprises the following components: 0.001-8% of recombinant double-protein, 0.1-6% of β-glucan, 0.1-5% of emulsifier, 0.1-15% of humectant, 0.1-3% of thickener, 0.1-1% of preservative, 0.1-2% of pH regulator, and the balance is purified water.

[0007] In some embodiments, the recombinant double-protein comprises recombinant mussel adhesive protein and recombinant type III humanized collagen.

[0008] In some embodiments, the mass ratio of the recombinant mussel adhesive protein to the recombinant type III humanized collagen is (0.00001-3):(0.001-7).

[0009] This application realizes the rapid repair of damaged skin by adding recombinant mussel adhesive protein and recombinant type III humanized collagen to the dressing. Among them, the recombinant type III humanized collagen is a collagen generated by genetic engineering technology, which is 100% homologous to human collagen, has no virus risk and rejection reaction, and can be used as the attachment and support for the growth of epithelial cells, inducing the proliferation, differentiation and migration of epithelial cells to promote wound healing.

[0010] As a polyphenol protein secreted by the foot silk gland of marine mussels, mussel adhesive protein is the only known protein with a high content of multi-benzyl groups. Recombinant mussel adhesive protein is composed of multiple repeating polypeptide fragments, which contains 20% lysine and 10% multi-benzyl groups. Lysine can attract negatively charged epidermal cells, fibroblasts, vascular endothelial cells and nerve cells in the human body to adhere and crawl through electrostatic interaction; the multi-benzyl groups can combine with oxygen in the air to form dopaquinone, and dopaquinone and unoxidized dopa can cross-link to form a polymer, thereby promoting the healing of the wound. Compared with traditional mussel adhesive protein, recombinant mussel adhesive protein has no animal-derived components, low virus contamination risk, low endotoxin content, and also has the elasticity and viscosity of natural mussel foot silk protein, which can promote wound healing.

[0011] In some embodiments, the preparation steps of the dextran are as follows: Prepare a casein solution with a concentration of 1-5 wt% using purified water, let it stand at 1-5 °C for 5-15 h, then add β-dextran, adjust the pH of the system to 3-8 with triethylamine, heat it to 80-100 °C and stir for 1-3 h. After the stirring is completed, immediately place it in an ice bath to terminate the reaction, and separate and dry to obtain dextran.

[0012] In some embodiments, the mass ratio of casein to β-dextran is 1:(0.8-1.2).

[0013] In this application, the treatment effect of the dressing is improved by adding β-dextran. β-Dextran is soluble in water and has the ability to improve the recognition and clearance of pathogens by immune cells, is non-irritating to the skin and can repair skin allergy symptoms. At the same time, in this application, casein is used to treat β-dextran to form a micelle structure with casein as the center and β-dextran as the outer surface, exposing more glycosidic bonds of β-dextran, enhancing the activation ability of macrophages, promoting the secretion of growth factors and collagen. When used in combination with recombinant type III humanized collagen, it can accelerate the proliferation of fibroblasts; and on the basis of the high adhesion provided by recombinant mussel adhesive protein, the micelle structure of β-dextran provided in this application can serve as a microscopic scaffold to guide cell directional migration and shorten the wound healing cycle.

[0014] In some embodiments, the emulsifier is at least one of glycerol polyether-26, polyoxypropylene ether, and sodium dodecyl sulfate.

[0015] Preferably, the emulsifier is glycerol polyether-26.

[0016] The semi-rigid chain conformation of β-dextran provided in this application forms an interpenetrating network with the flexible chain of glycerol polyether-26, enhancing the ductility and adhesion of the dressing.

[0017] In some embodiments, the humectant is at least one of glycerol, propylene glycol, and sodium hyaluronate.

[0018] In some embodiments, the thickener is at least one of carbomer, xanthan gum, and cellulose.

[0019] In some embodiments, the preservative is at least one of methyl paraben, sodium benzoate, and potassium sorbate.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a low-irritation collagen dressing, which reduces the virus risk and rejection reaction by compounding recombinant mussel adhesive protein and recombinant type III humanized collagen, and realizes the rapid repair of traumatic skin. On this basis, the therapeutic effect of the dressing is improved by adding β-glucan. β-glucan has the ability to improve the recognition and clearance of pathogens by immune cells, is non-irritating to the skin and can repair skin allergy symptoms. At the same time, by treating yeast β-glucan, more glycosidic bonds are exposed, enhancing the activation ability of macrophages, promoting the secretion of growth factors and collagen. When used in combination with recombinant type III humanized collagen, it can accelerate the proliferation of fibroblasts; and on the basis of the high adhesion provided by recombinant mussel adhesive protein, the micelle structure of the treated glucan provided in this application can serve as a microscopic scaffold to guide the directional migration of cells and shorten the wound healing cycle. In addition, this application uses glycerol polyether-26 as an emulsifier, and its flexible chain and the semi-rigid chain conformation of β-glucan form an interpenetrating network to enhance the ductility and adhesiveness of the dressing. Detailed implementation mode

[0021] The following will illustrate the present invention in combination with specific implementation examples. It should be noted that the following examples are examples of the present invention, only used to illustrate the present invention, and not used to limit the present invention. Other combinations and various improvements within the concept of the present invention can be made without departing from the main idea or scope of the present invention.

[0022] Unless otherwise specified, the raw materials used in the following preparation examples and examples can be from any commercially available manufacturer: Recombinant mussel adhesive protein is purchased from Shenzhen Baiyin Biotechnology or Xi'an Denohis Medical Technology Co., Ltd.; Recombinant type III humanized collagen is purchased from Bloomage Biotechnology Co., Ltd. or Jiangsu Jiangshan Juyuan Biotechnology Co., Ltd.

[0023] Preparation Example 1 The preparation steps of glucan A are as follows: Prepare a 3 wt% casein solution with 10 g of casein and purified water, let it stand at 2 ± 2 °C for 10 h, then add 10 g of β-glucan, adjust the pH of the system to 8 with triethylamine, heat up to 90 °C and stir for 2 h. After the stirring is completed, immediately place it in an ice bath to terminate the reaction, separate and dry to obtain glucan A.

[0024] Preparation Example 2 The difference between the preparation steps of glucan B and Preparation Example 1 is that the dosage of β-glucan is 7.5 g.

[0025] Preparation Example 3 The difference between the preparation steps of glucan C and Preparation Example 1 is that the dosage of β-glucan is 12.5 g.

[0026] Example 1 A preparation method of a recombinant double-protein medical dressing and dressing, comprising the following steps: S1: Vacuum stir the recombinant double-protein ingredients at 10 - 90 °C until completely mixed to form a stock solution; S2: Fill the stock solution and the non-woven dressing into an aluminum foil bag through a filling machine, or fill the stock solution into a loading bottle through a filling machine; S3: Heat-seal the aluminum foil bag filled in step S2, or seal the loading bottle; S4: Irradiate and sterilize the sealed aluminum foil bag or loading bottle obtained in step S3 to obtain the recombinant double-protein medical dressing and dressing.

[0027] In step S1 of this example, based on 100% of the mass percentage of the stock solution, it contains the following components: 5% of recombinant double-protein, 4% of dextran A, 3% of glycerol polyether-26, 10% of humectant, 2% of carbomer, 0.2% of methylparaben, 0.4% of triethanolamine, and the balance is purified water; The recombinant double-protein contains 1% of recombinant mussel adhesive protein and 4% of recombinant type III humanized collagen; the humectant contains 7% of glycerol and 3% of propylene glycol.

[0028] Example 2 In this example, the preparation method of the recombinant double-protein medical dressing and dressing is the same as that in Example 1, the difference is that: in step S1, based on 100% of the mass percentage of the stock solution, it contains the following components: 2% of recombinant double-protein, 2% of dextran A, 1% of glycerol polyether-26, 7% of humectant, 1% of xanthan gum, 0.1% of sodium benzoate, 0.1% of triethanolamine, and the balance is purified water; The recombinant double-protein contains 1% of recombinant mussel adhesive protein and 1% of recombinant type III humanized collagen; the humectant contains 5% of glycerol and 2% of propylene glycol.

[0029] Example 3 In this example, the preparation method of the recombinant double-protein medical dressing and dressing is the same as that in Example 1, the difference is that: in step S1, based on 100% of the mass percentage of the stock solution, it contains the following components: 8% of recombinant double-protein, 6% of dextran A, 5% of glycerol polyether-26, 15% of humectant, 3% of carbomer, 1% of potassium sorbate, 0.6% of triethanolamine, and the balance is purified water; The recombinant double-protein contains 3% of recombinant mussel adhesive protein and 5% of recombinant type III humanized collagen; the humectant contains 10% of glycerol and 5% of propylene glycol.

[0030] Example 4 This example provides a preparation method of a recombinant double-protein medical dressing and dressing. The specific implementation method is the same as that in Example 1, the difference is that: dextran A is replaced by an equal amount of dextran B.

[0031] Example 5 This example provides a method for preparing a recombinant double-protein medical dressing, and the specific implementation manner is the same as that of Example 1, except that: dextran A is replaced by an equal amount of dextran C.

[0032] Example 6 This example provides a method for preparing a recombinant double-protein medical dressing, and the specific implementation manner is the same as that of Example 1, except that: dextran A is replaced by an equal amount of β-dextran.

[0033] Example 7 This example provides a method for preparing a recombinant double-protein medical dressing, and the specific implementation manner is the same as that of Example 1, except that: glycerol polyether-26 is replaced by an equal amount of sodium dodecyl sulfate.

[0034] Performance Test Experimental group: the stock solutions provided in Examples 1 to 7; Blank group: normal saline; Control group: calculated by 100% mass percentage, containing the following components: recombinant human epidermal growth factor 9%, glycerol polyether-26 3%, glycerol 7%, propylene glycol 3%, carbomer 2%, methylparaben 0.2%, triethanolamine 0.4%, and the balance is purified water.

[0035] 1. Wound repair experiment: Test animals: Randomly select transgenic vascular green fluorescent zebrafish Fli-1 strain at 2 days after fertilization, and use a microinjector to inject 5 μL of 10% glacial acetic acid solution at the base of the zebrafish caudal fin to establish a local injury model; Test method: Place the caudal fin-transected zebrafish in the culture solutions of the experimental group, blank group and control group respectively, incubate in a 28°C incubator for 2 days, and use a confocal microscope to observe the incidence rate of new blood vessels (%), and the specific operation steps refer to the 1.2 experimental method in "In Vivo and In Vitro Studies on Cabozantinib Inhibiting Zebrafish Angiogenesis and Choroidal Neovascularization - Zhang Xiaoli".

[0036] 2. Tissue regeneration test: Test animals: Randomly select wild-type AB strain zebrafish at 3 days after fertilization, anesthetize the zebrafish with 0.016% tricaine methanesulfonate, and use surgery to cut off to establish a zebrafish tissue regeneration promotion model at the proximal 1 / 3 of the caudal fin; Test method: Place the caudal fin-transected zebrafish in the culture solutions of the experimental group, blank group and control group respectively, incubate in a 28°C incubator for 3 days, change the culture solution every 24h, and observe the growth length of the caudal fin (pixels).

[0037] 3. Anti-inflammatory test: Test animals: Transgenic vascular green fluorescent zebrafish at 3 days after fertilization were randomly selected, and a 5 μL 10% copper sulfate solution was injected into the base of the zebrafish caudal fin with a micro syringe to induce an inflammation model in zebrafish. Test method: The inflammatory fin zebrafish were respectively placed in the culture media of the experimental group, blank group and control group, and incubated in an incubator at 28 °C for 2 h to obtain the inflammation remission rate (%). The specific experimental operation steps refer to item 2.4 of "Study on the Antithrombotic and Anti - inflammatory Activities of Water - soluble Curcumin Preparations Based on the Zebrafish Model - Li Honglong -".

[0038] The results are shown in Table 1.

[0039] Table 1 Performance test results

[0040] It can be seen from the data in Table 1 that compared with the blank group, Examples 1 - 7 and the control group can all promote wound repair, tissue growth and inflammation remission. Among them, the effects of Examples 1 - 3 are significantly higher than those of the control group, specifically manifested in the increase in the incidence of new blood vessels, the growth of the caudal fin and the speed of inflammation remission.

[0041] Compared with Example 1, when casein was used to treat β - glucan in Examples 4 and 5, the dosage ratio of the two was changed, resulting in a partial decline in the effects of wound repair, tissue growth and inflammation remission. The possible reason is that it affects the micelle structure formed by casein and β - glucan, which is not conducive to the directional migration of cells, resulting in an extended wound healing cycle.

[0042] Compared with Example 1, in Example 6, β - glucan was directly used without casein treatment, which further decreased the effect of the ingredient solution, especially in terms of wound repair and tissue growth. The possible reason is that the exposure amount of the glycosidic bond of β - glucan is reduced; in Example 7, glycerol polyether - 26 was changed to sodium dodecyl sulfate, which affected the synergy between the emulsifier and β - glucan, resulting in a decrease in the effect.

[0043] The above - mentioned embodiments do not impose any formal restrictions on the present invention. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the relevant art, without departing from the scope of the technical solution of the present invention, can make some changes or modifications to the above - disclosed technical content to obtain equivalent embodiments with equivalent changes. However, as long as it does not depart from the content of the technical solution of the present invention, any simple modification, equivalent change and modification made to the above - mentioned embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A preparation method of a recombinant double-protein medical dressing, characterized in that, It includes the following steps: S1: Vacuum stir the recombinant double protein ingredient at 10 - 90 °C until completely mixed to form a stock solution; S2: Fill the stock solution and the non-woven dressing into an aluminum foil bag through a filling machine, or fill the stock solution into a loading bottle through a filling machine; S3: Heat-seal the aluminum foil bag filled in step S2, or seal the loading bottle; S4: Irradiate and sterilize the sealed aluminum foil bag or loading bottle obtained in step S3 to obtain the recombinant double protein medical dressing and the dressing; In step S1, based on 100% of the mass percentage of the stock solution, it includes the following components: recombinant double protein 0.001 - 8%, dextran 0.1 - 6%, emulsifier 0.1 - 5%, humectant 0.1 - 15%, thickener 0.1 - 3%, preservative 0.1 - 1%, pH regulator 0.1 - 2%, and the balance is purified water; The preparation steps of the dextran are as follows: Prepare a casein solution with a concentration of 1 - 5 wt% with purified water, stand at 1 - 5 °C for 5 - 15 h, then add β-dextran, adjust the pH of the system to 7.5 - 10 with triethylamine, heat up to 80 - 100 °C and stir for 1 - 3 h, immediately place it in an ice bath to terminate the reaction after stirring, and separate and dry to obtain dextran.

2. The preparation method of the recombinant double-protein medical dressing according to claim 1, characterized in that, The recombinant double protein includes recombinant mussel adhesive protein and recombinant type III humanized collagen.

3. The preparation method of the recombinant double-protein medical dressing and wound dressing according to claim 2, characterized in that, The mass ratio of the recombinant mussel adhesive protein to the recombinant type III humanized collagen is (0.000001 - 3):(0.001 - 7).

4. The preparation method of the recombinant double-protein medical dressing according to claim 1, characterized in that, The mass ratio of the casein to the β-dextran is 1:(0.8 - 1.2).

5. The preparation method of the recombinant double-protein medical dressing and dressing according to claim 1, characterized in that, The emulsifier is at least one of glycerol polyether - 26, polyoxypropylene ether, and sodium dodecyl sulfate.

6. The preparation method of the recombinant double-protein medical dressing and wound dressing according to claim 1, characterized in that, The humectant is at least one of glycerol, propylene glycol, and sodium hyaluronate.

7. The preparation method of the recombinant double-protein medical dressing and dressing according to claim 1, characterized in that, The thickener is at least one of carbomer, xanthan gum, and cellulose.

8. The preparation method of the recombinant double-protein medical dressing according to claim 1, characterized in that, The preservative is at least one of methyl paraben, sodium benzoate, and potassium sorbate.

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