Preparation method of recombinant dual-protein medical dressing and dressing

Abstract

The present invention relates to the field of dressings and specifically discloses a method for preparing a recombinant dual-protein medical dressing. The dressing provided herein has low irritation. By compounding recombinant mussel mucin and recombinant humanized type III collagen, the dressing isolates viral hazards and rejection reactions, achieving rapid repair of traumatic skin. Furthermore, by treating β-glucan, the dressing enhances its ability to activate macrophages, promoting the secretion of growth factors and collagen. When used in combination with recombinant humanized type III collagen, the dressing accelerates fibroblast proliferation.

Description

Technical Field

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

[0002] The skin barrier function of patients suffering from allergies, eczema, acne, and after photoelectric and mesotherapy procedures will be damaged to varying degrees, so a new skin barrier needs to be established to prevent external bacteria and other microorganisms from continuing to infect the damaged part. When skin damage occurs, the body initiates endogenous tissue regeneration and repair, namely wound healing, which is a highly coordinated and complex physiological process. Wound healing involves four different and overlapping stages: hemostasis, inflammation, proliferation, and remodeling (repair). Dressings can replace damaged skin during wound healing and treatment to provide temporary protection, avoid or control wound infection, and provide a suitable healing environment for the injured surface.

[0003] The wound healing process is a continuous dynamic process, which is a process of interaction between cells, cells and cell matrix, and soluble media. Collagen, as the main structural component of the extracellular matrix, has good biocompatibility, biodegradability and biological activity, and is widely used in food, medicine, tissue engineering, cosmetics and other fields. At present, there are many collagen-based skin barrier repair dressing products on the market. For example, patent CN112076340B provides a skin barrier repair dressing mask and its preparation method. Its raw materials include trehalose, sodium hyaluronate, collagen, glycerol and some plant extracts: strawberry saxifrage extract, yellow flower peucedanum extract and platycodon root extract. Although this dressing has the effect of promoting wound healing and epithelial cell growth, for some people with sensitive skin, the active substances in the plant extracts are prone to cause allergies. 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 dressing, which reduces viral risks and rejection reactions by compounding recombinant mussel mucin and recombinant type III humanized collagen, thereby achieving rapid repair of traumatic skin. Beta-glucan is processed to expose more glycosidic bonds, thereby enhancing the ability to activate macrophages and promoting the secretion of growth factors and collagen. When used in combination with recombinant type III humanized collagen, it can accelerate fibroblast proliferation. Furthermore, on the basis of the high adhesion provided by recombinant mussel mucin, the micellar structure of the glucan provided in this application can serve as a microscopic scaffold to guide the directional migration of cells and shorten the wound healing cycle.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] The present invention provides a method for preparing a recombinant dual-protein medical dressing, comprising the following steps:

[0007] S1: Stir the recombinant double protein ingredients under vacuum at 10-90°C until completely mixed to form a stock solution;

[0008] S2: Fill the stock solution and non-woven fabric patch into the aluminum foil bag through a filling machine, or fill the stock solution into the loading bottle through a filling machine;

[0009] S3: heat-sealing the aluminum foil bag filled in step S2, or sealing the loading bottle;

[0010] S4: sterilize the sealed aluminum foil bag or loading bottle obtained in step S3 by irradiation to obtain the recombinant dual-protein medical dressing.

[0011] In some embodiments, in step S1, based on 100% by mass of the stock solution, the following components are included: 0.001-8% of recombinant biprotein, 0.1-6% of β-glucan, 0.1-5% of emulsifier, 0.1-15% of moisturizer, 0.1-3% of thickener, 0.1-1% of preservative, 0.1-2% of pH regulator, and the balance is purified water.

[0012] In some embodiments, the recombinant double protein comprises recombinant mussel mucin and recombinant humanized type III collagen.

[0013] In some embodiments, the mass ratio of the recombinant mussel mucin to the recombinant humanized type III collagen is (0.00001-3): (0.001-7).

[0014] This application achieves rapid repair of traumatic skin by adding recombinant mussel mucin and recombinant type III humanized collagen into the dressing. The recombinant type III humanized collagen is a collagen produced by genetic engineering technology. It is 100% homologous to human collagen, has no viral risks and rejection reactions, and can serve as an attachment and support for epithelial cell growth, inducing epithelial cell proliferation, differentiation and migration to promote wound healing.

[0015] Mussel mucin, a polyphenol protein secreted by the byssus gland of the marine organism mussel, is the only known protein with a high content of dopa groups. Recombinant mussel mucin is composed of multiple repeating polypeptide fragments, containing 20% ​​lysine and 10% dopa groups. Lysine can attract the negatively charged epidermal cells, fibroblasts, vascular endothelial cells, and nerve cells of the human body to adhere to the wall and crawl through electrostatic effects; dopa groups can combine with oxygen in the air to form dopaquinone. Dopaquinone and unoxidized dopa can be cross-linked to form polymers, thereby promoting wound healing. Compared with traditional mussel mucin, recombinant mussel mucin does not contain any animal-derived ingredients, has a low risk of viral contamination, and a low endotoxin content. It also has the elasticity and viscosity of natural mussel byssus protein, which can promote wound healing.

[0016] In some embodiments, the preparation steps of the glucan are as follows:

[0017] Prepare a casein solution with a concentration of 1-5wt% using purified water, let it stand at 1-5°C for 5-15 hours, then add β-glucan, adjust the pH of the system to 3-8 with triethylamine, heat to 80-100°C and stir for 1-3 hours. After stirring, immediately place it in an ice bath to terminate the reaction, separate and dry to obtain glucan.

[0018] In some embodiments, the mass ratio of casein to β-glucan is 1:(0.8-1.2).

[0019] This application enhances the therapeutic efficacy of the dressing by adding β-glucan. β-glucan is water-soluble and has the ability to enhance immune cells' ability to recognize and eliminate pathogens. It is non-irritating to the skin and can repair skin allergy symptoms. Furthermore, this application treats β-glucan with casein to form a micellar structure with casein as the core and β-glucan as the outer surface. This exposes more glycosidic bonds in the β-glucan, enhancing its ability to activate macrophages and promote the secretion of growth factors and collagen. When combined with recombinant humanized type III collagen, it can accelerate fibroblast proliferation. Furthermore, based on the high adhesion provided by recombinant mussel mucin, the micellar structure of β-glucan provided in this application can serve as a microscopic scaffold to guide directional cell migration and shorten the wound healing period.

[0020] In some embodiments, the emulsifier is at least one of glyceryl polyether-26, polyoxypropylene ether, and sodium lauryl sulfate.

[0021] Preferably, the emulsifier is glycereth-26.

[0022] The semi-rigid chain conformation of β-glucan provided in the present application forms an interpenetrating network with the flexible chain of glycerol polyether-26, thereby enhancing the ductility and adhesion of the dressing.

[0023] In some embodiments, the moisturizing agent is at least one of glycerin, propylene glycol, and sodium hyaluronate.

[0024] In some embodiments, the thickener is at least one of carbomer, xanthan gum, and cellulose.

[0025] In some embodiments, the preservative is at least one of methylparaben, sodium benzoate, and potassium sorbate.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention provides a low-irritation collagen dressing. By combining recombinant mussel mucin and recombinant humanized type III collagen, it reduces viral risks and rejection reactions, achieving rapid repair of wounded skin. Furthermore, the dressing's therapeutic efficacy is enhanced by the addition of β-glucan. β-glucan enhances the immune cell's ability to recognize and eliminate pathogens, is non-irritating to the skin, and can repair skin allergy symptoms. Furthermore, yeast β-glucan is treated to expose more glycosidic bonds, enhancing macrophage activation and promoting the secretion of growth factors and collagen. When combined with recombinant humanized type III collagen, it can accelerate fibroblast proliferation. Furthermore, based on the high adhesion provided by recombinant mussel mucin, the micellar structure of the treated glucan provided in this application can serve as a microscopic scaffold, guiding directional cell migration and shortening the wound healing period. Furthermore, this application uses glycerol polyether-26 as an emulsifier, leveraging its flexible chains to form an interpenetrating network with the semi-rigid chains of β-glucan, enhancing the dressing's ductility and adhesion. DETAILED DESCRIPTION

[0028] The present invention will be described below in conjunction with specific embodiments. It should be noted that the following examples are illustrative of the present invention and are intended only to illustrate the present invention and are not intended to limit the present invention. Other combinations and various modifications within the scope of the present invention may be made without departing from the spirit or scope of the present invention.

[0029] Unless otherwise specified, the raw materials used in the following preparations and examples can be obtained from any commercially available manufacturer:

[0030] Recombinant mussel mucin was purchased from Shenzhen Boyin Biotechnology or Xi'an Denuohaisi Medical Technology Co., Ltd.;

[0031] Recombinant humanized type III collagen was purchased from Bloomage Biotech Co., Ltd. or Jiangsu Jiangshan Juyuan Biotechnology Co., Ltd.

[0032] Preparation Example 1

[0033] The preparation steps of glucan A are as follows:

[0034] A 3 wt% casein solution was prepared by mixing 10 g of casein with purified water. The solution was allowed to stand at 2 ± 2 °C for 10 h. Then, 10 g of β-glucan was added. The pH of the system was adjusted to 8 with triethylamine. The solution was heated to 90 °C and stirred for 2 h. After stirring, the solution was immediately placed in an ice bath to terminate the reaction. Glucan A was obtained by separation and drying.

[0035] Preparation Example 2

[0036] The preparation steps of glucan B differ from those of Preparation Example 1 in that the amount of β-glucan used is 7.5 g.

[0037] Preparation Example 3

[0038] The preparation steps of Glucan C differ from those of Preparation Example 1 in that the amount of β-glucan used is 12.5 g.

[0039] Example 1

[0040] A method for preparing a recombinant dual-protein medical dressing comprises the following steps:

[0041] S1: Stir the recombinant double protein ingredients under vacuum at 10-90°C until completely mixed to form a stock solution;

[0042] S2: Fill the stock solution and non-woven fabric patch into the aluminum foil bag through a filling machine, or fill the stock solution into the loading bottle through a filling machine;

[0043] S3: heat-sealing the aluminum foil bag filled in step S2, or sealing the loading bottle;

[0044] S4: sterilize the sealed aluminum foil bag or loading bottle obtained in step S3 by irradiation to obtain the recombinant dual-protein medical dressing.

[0045] In step S1 of this embodiment, the following components are included based on the mass percentage of the stock solution as 100%: 5% recombinant biprotein, 4% dextran A, 3% glycerol polyether-26, 10% moisturizer, 2% carbomer, 0.2% methylparaben, 0.4% triethanolamine, and the balance is purified water;

[0046] The recombinant double protein contains 1% recombinant mussel mucin and 4% recombinant type III humanized collagen; the moisturizer contains 7% glycerol and 3% propylene glycol.

[0047] Example 2

[0048] The preparation method of the recombinant dual-protein medical dressing and dressing in this embodiment is the same as that in Example 1, except that: in step S1, the following components are included, based on the mass percentage of the stock solution as 100%, the following components are included: 2% recombinant dual-protein, 2% dextran A, 1% glycerol polyether-26, 7% moisturizer, 1% xanthan gum, 0.1% sodium benzoate, 0.1% triethanolamine, and the balance is purified water;

[0049] The recombinant dual protein contains 1% recombinant mussel mucin and 1% recombinant type III humanized collagen; the moisturizer contains 5% glycerol and 2% propylene glycol.

[0050] Example 3

[0051] The preparation method of the recombinant dual-protein medical dressing and dressing in this embodiment is the same as that in Example 1, except that: in step S1, based on the mass percentage of the stock solution as 100%, the following components are included: 8% recombinant dual-protein, 6% dextran A, 5% glycerol polyether-26, 15% moisturizer, 3% carbomer, 1% potassium sorbate, 0.6% triethanolamine, and the balance is purified water;

[0052] The recombinant double protein contains 3% recombinant mussel mucin and 5% recombinant type III humanized collagen; the moisturizer contains 10% glycerol and 5% propylene glycol.

[0053] Example 4

[0054] This example provides a method for preparing a recombinant dual-protein medical patch and dressing. The specific implementation method is the same as that of Example 1, except that glucan A is replaced by an equal amount of glucan B.

[0055] Example 5

[0056] This example provides a method for preparing a recombinant dual-protein medical patch and dressing. The specific implementation method is the same as that of Example 1, except that glucan A is replaced by an equal amount of glucan C.

[0057] Example 6

[0058] This example provides a method for preparing a recombinant dual-protein medical patch and dressing. The specific implementation method is the same as that of Example 1, except that glucan A is replaced by an equal amount of β-glucan.

[0059] Example 7

[0060] This embodiment provides a method for preparing a recombinant dual-protein medical patch and dressing. The specific implementation method is the same as that of Example 1, except that glycerol polyether-26 is replaced by an equal amount of sodium lauryl sulfate.

[0061] Performance Testing

[0062] Experimental group: stock solution provided in Examples 1 to 7;

[0063] Blank group: normal saline;

[0064] Control group: The following components were included by mass percentage: 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 was purified water.

[0065] 1. Wound repair experiment:

[0066] Experimental animals: Transgenic zebrafish (Fli-1) expressing green fluorescent blood vessels were randomly selected 2 days after fertilization. 5 μL of 10% glacial acetic acid solution was injected into the base of the caudal fin using a microsyringe to establish a local injury model.

[0067] Experimental method: The zebrafish with severed tail fins were placed in the culture medium of the experimental group, blank group, and control group, respectively, and incubated in a 28°C incubator for 2 days. The incidence rate (%) of neovascularization was observed using a confocal microscope. The specific operation steps refer to the experimental method 1.2 in "In vitro and in vivo study of cabozantinib inhibiting angiogenesis and choroidal neovascularization in zebrafish - Zhang Xiaoli".

[0068] 2. Tissue regeneration test:

[0069] Experimental animals: Wild-type AB zebrafish 3 days post-fertilization were randomly selected and anesthetized with 0.016% tricaine methanesulfonic acid. The proximal 1 / 3 of the caudal fin was surgically amputated to establish the zebrafish tissue regeneration model.

[0070] Experimental method: The zebrafish with severed tail fins were placed in the culture medium of the experimental group, blank group and control group respectively, and incubated in a 28℃ incubator for 3 days. The culture medium was replaced every 24 hours, and the growth length (pixels) of the tail fin was observed.

[0071] 3. Anti-inflammatory test:

[0072] Experimental animals: Transgenic zebrafish expressing green fluorescent blood vessels were randomly selected 3 days after fertilization. 5 μL of 10% copper sulfate solution was injected into the base of the zebrafish tail fin using a microsyringe to induce the zebrafish inflammation model.

[0073] Experimental method: The inflamed fin zebrafish were placed in the culture medium of the experimental group, blank group and control group respectively, and incubated in an incubator at 28℃ for 2 hours to obtain the inflammation resolution rate (%). The specific experimental steps refer to Item 2.4 of "Study on the Antithrombotic and Anti-inflammatory Activity of Water-soluble Curcumin Preparations Based on Zebrafish Model - Li Honglong -".

[0074] The results are shown in Table 1.

[0075] Table 1 Performance test results

[0076]

[0077] As can be seen from the data in Table 1, compared with the blank group, Examples 1 to 7 and the control group can promote wound repair, tissue growth and inflammation resolution, among which the effects of Examples 1 to 3 are significantly higher than those of the control group, which are specifically manifested in the increase in the incidence of neovascularization, the growth of the tail fin and the speed of inflammation resolution.

[0078] Compared with Example 1, when treating β-glucan with casein, Examples 4 and 5 changed the dosage ratio of the two, resulting in a partial decrease in the effects of wound repair, tissue growth, and inflammation resolution. The possible reason is that the micelle structure formed by casein and β-glucan was affected, which was not conducive to the directional migration of cells and led to a prolonged wound healing period.

[0079] Compared with Example 1, Example 6 directly used β-glucan without casein treatment, which further reduced the effect of the formulation, especially in wound repair and tissue growth. The possible reason is that the exposure of the glycosidic bonds of β-glucan was reduced. In Example 7, glycerol polyether-26 was replaced with sodium lauryl sulfate, which affected the synergy between the emulsifier and β-glucan and reduced the effect.

[0080] The embodiments described above do not impose any form of limitation on the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any technician familiar with the present profession can make some changes or modifications to equivalent embodiments using the technical content disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing a recombinant dual-protein medical dressing or dressing, characterized in that: The following steps are involved: S1: Stir the recombinant double protein ingredients under vacuum at 10-90°C until completely mixed to form a stock solution; S2: Filling the stock solution and non-woven fabric dressing into the aluminum foil bag through a filling machine, or filling the stock solution into the loading bottle through a filling machine; S3: heat-sealing the aluminum foil bag filled in step S2, or sealing the loading bottle; S4: sterilizing the sealed aluminum foil bag or loading bottle obtained in step S3 by irradiation to obtain a recombinant dual-protein medical dressing; In step S1, based on 100% by mass of the stock solution, the following components are included: 0.001-8% of recombinant biprotein, 0.1-6% of dextran, 0.1-5% of emulsifier, 0.1-15% of moisturizer, 0.1-3% of thickener, 0.1-1% of preservative, 0.1-2% of pH regulator, and the balance is purified water; The preparation steps of the glucan are as follows: preparing a casein solution with a concentration of 1 to 5 wt% with purified water, standing at 1 to 5° C. for 5 to 15 hours, then adding β-glucan, adjusting the pH of the system to 7.5 to 10 with triethylamine, heating to 80 to 100° C. and stirring for 1 to 3 hours, immediately placing in an ice bath after stirring to terminate the reaction, separating and drying to obtain the glucan; The recombinant double protein comprises recombinant mussel mucin and recombinant type III humanized collagen; The mass ratio of casein to β-glucan is 1:(0.8-1.2); The emulsifier is glyceryl polyether-26.

2. The method for preparing the recombinant dual-protein medical dressing according to claim 1, characterized in that: The mass ratio of the recombinant mussel mucin to the recombinant type III humanized collagen is (0.000001-3): (0.001-7).

3. The method for preparing the recombinant dual-protein medical dressing according to claim 1, characterized in that: The moisturizing agent is at least one of glycerin, propylene glycol, and sodium hyaluronate.

4. The method for preparing the recombinant dual-protein medical dressing according to claim 1, characterized in that: The thickener is at least one of carbomer, xanthan gum and cellulose.

5. The method for preparing the recombinant dual-protein medical dressing according to claim 1, characterized in that: The preservative is at least one of methylparaben, sodium benzoate and potassium sorbate.

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