A collagen dressing, a wound dressing and a preparation method thereof

Abstract

This application relates to the technical field of medical dressings, and provides a collagen dressing, a dressing and a preparation method thereof. The collagen dressing includes a essence liquid. By weight, the essence liquid totals 100 parts of the weight, and includes the following components: 0.02 - 2 parts of recombinant collagen, 0.01 - 1 part of biomimetic collagen, 0.001 - 0.5 part of a humectant, 0.02 - 4 parts of a stabilizing agent, and 2 - 25 parts of a thickening agent, with the balance being water or phosphate buffer solution. The stabilizing agent is one or more of trehalose, gallic acid, resveratrol and sinapic acid. This application uses biomimetic collagen with good structural stability, including good thermal stability and irradiation stability, to prepare a sterile collagen dressing and dressing. At the same time, a stabilizing agent is added to synergistically stabilize the recombinant and biomimetic collagen, simplifying the sterilization process of the dressing, reducing the production sterility requirements, and also reducing the impact of the dressing processing and sterilization process on the self-activity of the collagen.

Description

Technical Field

[0001] The present application relates to the technical field of medical dressings, and in particular to a collagen dressing, a dressing and a preparation method thereof. Background Art

[0002] When the skin is diseased or suffers from burns, scalds, or mechanical damage, the skin barrier function will be damaged, causing water and protein loss, metabolism, and immune system-related problems. It usually takes three or four weeks for the skin to heal, and scars often form during the automatic healing process, seriously affecting the appearance. The theory of moist wound healing emphasizes keeping the wound surface moist during the healing process to promote cell migration, proliferation, and regeneration, thereby accelerating the healing process.

[0003] Collagen dressings can physically isolate the wound from the outside world by covering and caring for the wound surface, provide a suitable microenvironment for wound healing, and prevent infection by pathogenic microorganisms. At the same time, they can help improve or prevent the formation of pathological scars. In the process of wound healing, such as hemostasis, inflammation, proliferation and remodeling, collagen can participate in them.

[0004] However, in the preparation process of collagen dressings, especially for recombinant collagen, its poor stability has always been a problem that has troubled technicians. In the preparation process of dressings, terminal sterilization will be carried out. Common terminal sterilization methods, such as high temperature sterilization and irradiation sterilization, will cause the decomposition of the recombinant collagen structure, leading to the denaturation of collagen and the loss of its natural inherent biological properties. Therefore, strict aseptic control is required in the raw materials and processing process, which directly increases the difficulty and cost of using recombinant collagen in products such as medical dressings. For this reason, there are related technologies that avoid the influence of irradiation sterilization on collagen and other ingredients by adding irradiation improvers, but this is not a good way to solve the structural stability problem of natural or recombinant collagen from the root.

[0005] Therefore, it is urgent to find a method that can fundamentally solve the problem of poor stability of recombinant collagen dressings and reduce the difficulty and cost of applying recombinant collagen in products such as medical dressings. Summary of the invention

[0006] Based on this, the present application prepares a sterile collagen dressing by using a bionic collagen with a specially designed additive structure and thermal stability to match with recombinant collagen. At the same time, it is necessary to add a collagen thermal stabilizing agent to coordinate and stabilize the recombinant collagen to solve the problem of poor structural stability of the recombinant collagen, thereby ensuring the biological activity and efficacy of collagen in wound repair.

[0007] The specific technical solution of this application is:

[0008] In a first aspect, the present application provides a collagen dressing. The collagen dressing includes a essence solution. In terms of parts by weight, the total amount of the essence solution is 100 parts of the weight, and includes the following components: 0.02 - 2 parts of recombinant collagen, 0.01 - 1 part of biomimetic collagen, 0.001 - 0.5 part of a humectant, 0.02 - 4 parts of a stabilizing agent, and 2 - 25 parts of a thickening agent, with the balance being water or phosphate buffer solution. The stabilizing agent is one or more of trehalose, gallic acid, resveratrol, and sinapic acid.

[0009] In some embodiments, the stabilizing agent is preferably 0.02 - 2 parts of gallic acid and / or 0.02 - 2 parts of sinapic acid.

[0010] In some embodiments, the parts by weight of the recombinant collagen are 0.02 - 1 part.

[0011] In some embodiments, the parts by weight of the biomimetic collagen are 0.01 - 0.5 part.

[0012] In some embodiments, the parts by weight of the stabilizing agent are 0.04 - 4 parts.

[0013] Among them, preferably, the humectant is 0.02 - 0.5 part.

[0014] Among them, preferably, the humectant is hyaluronic acid and / or sodium hyaluronate.

[0015] Among them, preferably, the thickening agent includes 1 - 20 parts of glycerol, 0.2 - 2 parts of butanediol, and 0.1 - 1 part of carbomer.

[0016] In some embodiments, in terms of parts by weight, the essence solution includes the following components: 0.02 - 1 part of recombinant collagen, 0.01 - 0.5 part of biomimetic collagen, 0.001 - 0.3 part of hyaluronic acid, 1 - 6 parts of glycerol, 0.2 - 2 parts of butanediol, 0.1 - 0.6 part of carbomer, 0.02 - 0.8 part of gallic acid, and 0.02 - 0.8 part of sinapic acid, with the balance being water or phosphate buffer solution.

[0017] In some embodiments, in terms of parts by weight, the essence solution includes the following components: 0.4 part of recombinant collagen, 0.2 part of biomimetic collagen, 0.1 part of hyaluronic acid, 3 parts of glycerol, 0.2 - 1 part of butanediol, 0.3 part of carbomer, 0.4 part of gallic acid, and 0.4 part of sinapic acid, with the balance being water or phosphate buffer solution.

[0018] In some embodiments, when the balance is water, the essence solution further includes 0.01 - 5 parts of a pH regulator and 0.2 - 0.5 part of a preservative.

[0019] Among them, the pH regulator is generally triethanolamine and citric acid. Preferably, the mass ratio of triethanolamine to citric acid is 1:10 - 10:1.

[0020] In some embodiments, when the balance is phosphate buffer solution, the essence further comprises 0.2 - 0.5 parts of preservative. In this application, the preservative is generally octanoyl hydroxamic acid and glycerol caprylate. Preferably, the mass ratio of octanoyl hydroxamic acid to glycerol caprylate is 9:1 - 1:9.

[0021] In this application, the concentration of the phosphate buffer solution is 0.01 - 0.1M, and the pH is 4 - 7.4.

[0022] In a second aspect, the present application also provides a preparation method of the aforementioned collagen dressing, comprising the following steps:

[0023] S1. Dissolve the recombinant collagen, the bionic collagen and the humectant with a solvent to obtain solution A;

[0024] S2. Dissolve the thickener with a solvent to obtain solution B;

[0025] S3. Dissolve the stabilizing aid with a solvent to obtain solution C;

[0026] S4. Mix solution A and solution B evenly, add solution C, and then continue to add solvent to make up to 100 parts by weight of the essence, and mix evenly.

[0027] Among them, the parts by weight of the substances in steps S1 - S4 adopt the parts by weight of the foregoing components.

[0028] In some embodiments, the solvent in steps S1 - S4 is water or phosphate buffer solution. When the solvent is water, in step S4, after adding solution C and before continuing to add the solvent to make up, add the preservative and the pH regulator; when the solvent is phosphate buffer solution, in step S4, after adding solution C and before continuing to add the solvent to make up, add the preservative.

[0029] In some embodiments, after step S4, it further comprises the steps of standing, bagging, and sterilizing.

[0030] Among them, the sterilization method is generally moist heat sterilization or irradiation sterilization. When moist heat sterilization is adopted, the temperature of the moist heat sterilization is 121°C, and the time of the moist heat sterilization is 15 - 30 min; when irradiation sterilization is adopted, the dose of the irradiation sterilization is 10 - 25 KGy.

[0031] The present application also provides a method for preparing a collagen dressing, which is the same as the steps of the method for preparing the aforementioned collagen patch. Preferably, the addition amounts of the components are 2-5 times the addition amounts of the components in the method for preparing the collagen patch, and 0.05-5 parts by weight of sodium carboxymethylcellulose is additionally dissolved in step S1.

[0032] The present application also provides a collagen dressing prepared by the method for preparing the aforementioned collagen dressing, which includes a collagen patch and a hydrogel.

[0033] Compared with the prior art, the present application has the following advantages:

[0034] (1) The present application prepares a sterile collagen patch and dressing by using a biomimetic collagen with good structural stability, including good thermal stability and irradiation stability. At the same time, a stabilizing aid is added to synergistically stabilize the recombinant collagen, which simplifies the sterilization process of the dressing, reduces the aseptic requirements for production, and also reduces the impact of the dressing processing and sterilization processes on the self-activity of collagen.

[0035] (2) By further screening the specific ratios and dosages within the components of the stabilizing aid, and screening the specific ratios between different components, the obtained patches and dressings have better thermal stability. At the same time, the system formula comprehensively balances the adhesiveness and fluidity, and the product has good conformability and water permeability, and a high usage experience score. Description of the Drawings

[0036] In order to more clearly illustrate the technical solutions of the present application, the drawings required for use in the description of the present application will be briefly introduced below.

[0037] Figure 1 It is a temperature-variable circular dichroism spectrum and a collagen thermal denaturation temperature analysis chart, where Figure 1 (a) is the temperature-variable circular dichroism spectrum chart of the biomimetic collagen; Figure 1 (b) is the temperature-variable circular dichroism spectrum of the recombinant collagen; Figure 1 (c) is the temperature-variable circular dichroism spectrum chart of bovine type I collagen; Figure 1 (d) is the curve of the CD value of the above three collagens changing with temperature at the positive peak wavelength, that is, the thermal denaturation analysis curve.

[0038] Figure 2 It is the circular dichroism spectrum chart of collagen after moist heat sterilization in Comparative Examples 1-3.

[0039] Figure 3 It is a data diagram showing the usage experience scores of the patches obtained in different examples.

[0040] Figure 4 Data trend chart of the thermal decomposition temperature of collagen. Detailed Embodiments

[0041] In the description of the embodiments of the present application, it should be noted that all ranges disclosed in the present application are understood to cover any and all sub-ranges subsumed therein. For example, the stated range "0.005 - 2" should be considered to include any and all of the following sub-ranges: starting with a minimum value of 0.005 or greater and ending with a maximum value of 2 or less, for example, 0.005 to 0.5 parts, or 0.5 to 1 part, or 1 to 2 parts. At the same time, all ranges disclosed in the present application are also considered to include the endpoints of the stated range, unless otherwise clearly stated. For example, a range "between 1 and 20" or "1 to 20" or "1 - 20" should generally be considered to include the endpoints 1 and 20.

[0042] Similarly, it can be understood that the phrases and terms used in the present application are for descriptive purposes and should not be considered restrictive. The use of "including", "comprising", or "having" and their variants in the present application is intended to open - endedly include the items listed thereafter, their equivalents, and additional items.

[0043] In this article, for the sake of brevity of description, not all possible combinations of all technical features in each embodiment or example are described. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each embodiment or example can be combined arbitrarily, and all possible combinations should be considered to be within the scope described in this specification.

[0044] Since sterile products of collagen dressings often have a risk of denaturation of recombinant collagen after terminal sterilization due to the relatively low structural stability of collagen, especially for recombinant collagen with poor stability, it is necessary to create a sterile environment throughout the preparation process of the dressing, which increases the difficulty and cost of applying recombinant collagen in dressing products. The present application aims to solve this problem by specifically using biomimetic collagen with good structural stability to prepare sterile collagen patches and dressings, and at the same time adding stabilizing aids to cooperate with recombinant and biomimetic collagen synergistically, so as to simplify the sterilization process of the dressing and reduce the requirements for sterile production.

[0045] On the one hand, the present application provides a collagen patch, the collagen patch includes essence. By weight, the essence totals 100 parts of the weight, and includes the following components: 0.02 - 2 parts of recombinant collagen, 0.01 - 1 part of biomimetic collagen, 0.001 - 0.5 part of humectant, 0.02 - 4 parts of stabilizing aid, and 2 - 25 parts of thickener, with the balance being water or phosphate buffer solution. The stabilizing aid is one or several of trehalose, gallic acid, resveratrol, and sinapic acid.

[0046] In this application, the collagen dressing generally consists of essence liquid and non-woven fabric, and is packaged in an aluminum foil bag.

[0047] In this application, the recombinant collagen has a smaller molecular weight compared to the bionic collagen. The recombinant collagen has a certain degree of transdermal absorption ability. The purpose of mixing the recombinant collagen and the bionic collagen is, on the one hand, to increase the amount of collagen absorbed through the skin, and on the other hand, to add collagen with thermal stability. Since the thermal stability of the recombinant collagen is poor, it is difficult to change its thermal stability only by adding bionic collagen, and a thermal stability aid needs to be added to improve its thermal stability.

[0048] In this application, the recombinant collagen is selected from human DNA gene fragments, then introduced into fermenting bacteria such as Escherichia coli or Pichia pastoris for fermentation, and obtained after centrifugal filtration, sterilization ultrafiltration, and chromatography purification. Its molecular weight distribution is 2KDa - 50KDa.

[0049] In this application, the bionic collagen is a bionic biomaterial that mimics the composition, structure, and function of natural collagen. Its basic structure is the same as the repeating amino acid sequence of natural collagen (Gly-X-Y)n, mainly composed of glycine, proline, and hydroxyproline, and is obtained through liquid-phase synthesis technology, that is, through hydrolysis, polymerization, extraction, rotary evaporation, and purification means. Its molecular weight distribution is 100KDa - 1000KDa. For the specific preparation method, reference can be made to the prior art patent CN202410309839.7.

[0050] The above-mentioned bionic collagen is composed of the following structural units (tripeptides) repeating and polymerizing to form a single α-chain, and then self-assembling through hydrogen bonding to form a triple helix structure:

[0051]

[0052] The above structural unit compound first synthesizes a dipeptide from proline and hydroxyproline, and then synthesizes a tripeptide from the dipeptide and glycine. This tripeptide is the repeating structural unit.

[0053] In some embodiments, the stability aid is preferably gallic acid and / or sinapic acid.

[0054] Among them, trehalose can, on the one hand, accelerate the self-assembly kinetics of collagen, improve the degree of self-assembly of collagen and the mechanical properties of the resulting hydrogel, manifested as higher turbidity, but has no effect on the microstructure of the resulting collagen fibers. On the other hand, it can improve the thermal stability of collagen.

[0055] Gallic acid can change the thermal denaturation temperature of the protein, increase the compactness of the protein structure, and improve the thermal stability of the protein.

[0056] Resveratrol can slow down the conformational change of the non-α-helical domain of α-lactalbumin. An increase in the content of resveratrol helps to improve the stability of collagen, but too high an incubation temperature will have the opposite effect.

[0057] An increase in the concentration of sinapic acid will cause the protein denaturation temperature to shift to a higher temperature. In the presence of sinapic acid, during the unfolding process of the protein, the hydrophobic core of the protein is exposed to solvent molecules, the protein structure becomes more compact, stabilizing the protein structure and preventing thermal denaturation. The stabilization of the protein structure can be generated by the electrostatic attraction between negatively charged sinapic acid and positively charged amino acid residues.

[0058] In some embodiments, the addition amount of the stabilizing agent is 0.5 - 10 times the addition amount of collagen.

[0059] In some embodiments, the stabilizing agent is 0.02 - 2 parts of gallic acid and / or 0.02 - 2 parts of sinapic acid.

[0060] Among them, the humectant can generally be hyaluronic acid and / or sodium hyaluronate.

[0061] Among them, the thickener generally includes glycerol, butanediol, and carbomer. Preferably, the weight parts of glycerol are 1 - 20 parts, the weight parts of butanediol are 0.2 - 2 parts, and the weight parts of carbomer are 0.1 - 1 part.

[0062] In this application, when the balance is water, the essence also includes 0.01 - 5 parts of pH regulator and 0.2 - 0.5 parts of preservative. Preferably, the pH regulator is triethanolamine and citric acid, and the mass ratio of triethanolamine to citric acid is 1:10 - 10:1; the preservative is octanoyl hydroxamic acid and glycerol caprylate, and the mass ratio of octanoyl hydroxamic acid to glycerol caprylate is 9:1 - 1:9. In this application, when the balance is phosphate buffer solution, the essence also includes 0.2 - 0.5 parts of preservative. Preferably, the concentration of the phosphate buffer solution is 0.01 - 0.1M, the pH is 4 - 7.4, and the preservative is the same as the foregoing.

[0063] On the other hand, this application provides a preparation method of the aforementioned collagen dressing, including the following steps:

[0064] S1. Dissolve the recombinant collagen, the bionic collagen, and the humectant with a solvent to obtain solution A;

[0065] S2. Dissolve the thickener with a solvent to obtain solution B;

[0066] S3. Dissolve the stabilizing agent with a solvent to obtain solution C;

[0067] S4. Mix the solution A and the solution B evenly, add the solution C, and continue to add the solvent to make up to 100 parts by weight of the essence, and mix evenly.

[0068] Among them, the parts by weight of the substances in steps S1 - S4 adopt the parts by weight of the foregoing components.

[0069] In this application, the solvent in steps S1 - S4 is water or phosphate buffer solution.

[0070] When the solvent is water, in step S4, after adding the solution C and before continuing to add the solvent to make up, add a preservative and a pH regulator.

[0071] When the solvent is phosphate buffer solution, in step S4, after adding the solution C and before continuing to add the solvent to make up, add a preservative.

[0072] In this application, after step S4, it also includes the steps of standing, bagging, and sterilization.

[0073] Among them, the sterilization method is generally moist heat sterilization or irradiation sterilization.

[0074] When using moist heat sterilization, the temperature of the moist heat sterilization is 121 °C, and the time of the moist heat sterilization is 15 - 30 min.

[0075] When using irradiation sterilization, the dose of the irradiation sterilization is 10 - 25 KGy.

[0076] This application also provides a preparation method of a collagen dressing. The steps of the preparation method are the same as those of the foregoing preparation method of the collagen patch. Among them, preferably, the addition amounts of the components are 2 - 5 times the addition amounts of the components in the preparation method of the collagen patch, and in step S1, it also includes dissolving sodium carboxymethylcellulose, and the parts by weight of the sodium carboxymethylcellulose are 0.05 - 5 parts.

[0077] This application also provides a collagen dressing prepared by the foregoing preparation method, including a collagen patch and a hydrogel.

[0078] The following details the examples of this embodiment. The examples are only used to explain this embodiment and cannot be understood as a limitation to this embodiment.

[0079] The molecular weight distribution of the bionic collagen is 100 KDa - 1000 KDa, and the specific preparation method can refer to the prior art patent CN202410309839.7.

[0080] The molecular weight distribution of the recombinant collagen is 2KDa - 50KDa. It is purchased from Zhejiang Zhuji Juyuan Biotechnology Co., Ltd., and is recombinant type I collagen with the model number SSP01 and the batch number YFPB20240929.

[0081] Bovine type I collagen is purchased from the National Institutes for Food and Drug Control.

[0082] Raw materials, reagents, etc. without indicating the manufacturer are all conventional products that can be obtained through commercial purchase.

[0083] Example 1

[0084] A preparation method of a collagen dressing, comprising the following steps:

[0085] S1: Respectively take 0.2 parts of recombinant collagen, 0.1 part of bionic collagen and 0.5 part of hyaluronic acid, and dissolve them with phosphate buffer solution to obtain solution A;

[0086] S2: Respectively take 3 parts of glycerol, 1 part of butanediol, 0.3 part of carbomer, and dissolve them with phosphate buffer solution to obtain solution B;

[0087] S3: Take 0.4 part of gallic acid and 0.4 part of sinapic acid, and dissolve them with phosphate buffer solution to obtain solution C;

[0088] S4: After stirring and mixing solution A and solution B evenly, add solution C and continue to stir and mix evenly. Then add 0.3 part of the preservative octanoyl hydroxamic acid and glycerol caprylate (mass ratio 5:9). After making up the phosphate buffer solution to 100 parts, stir and mix evenly. After standing to remove bubbles, fill it into an aluminum foil bag containing non-woven fabric, and obtain the dressing after moist heat sterilization. The above phosphate buffer solution has a pH of 6 and a concentration of 0.1M.

[0089] Examples 2 - 9

[0090] The difference between Examples 2 - 9 and Example 1 is only that the component amounts in solution A and solution B in steps S1 and S2 are changed. See the following table for details:

[0091] Table 1

[0092] Number Recombinant collagen Bionic collagen Hyaluronic acid Glycerol Butanediol Carbomer Example 1 0.2 0.1 0.5 3 1 0.3 Example 2 0.02 0.01 0.01 1 0.5 0.1 Example 3 0.4 0.2 0.01 3 1 0.3 Example 4 1 0.5 0.01 3 1 0.3 Example 5 0.4 0.2 0.1 3 1 0.3 Example 6 0.4 0.2 0.1 3 0.2 0.3 Example 7 0.4 0.2 0.1 5 1 0.2 Example 8 0.4 0.2 0.1 10 1 0.5 Example 9 0.4 0.2 0.1 20 1 0.1

[0093] Example 10

[0094] A preparation method of a collagen dressing, comprising the following steps:

[0095] S1: Respectively take 0.4 parts of recombinant collagen, 0.2 part of bionic collagen and 0.1 part of hyaluronic acid, and dissolve them with pure water to obtain solution A;

[0096] S2: Take 3 parts of glycerol, 1 part of butanediol, and 0.3 part of carbomer, and dissolve them in pure water to obtain Solution B;

[0097] S3: Take 0.4 part of gallic acid and 0.4 part of sinapic acid, and dissolve them in pure water to obtain Solution C;

[0098] S4: After stirring and mixing Solution A and Solution B evenly, add Solution C and continue to stir and mix evenly. Then add 0.3 part of the preservative octanoyl hydroxamic acid and glycerol caprylate (mass ratio 5:9), and add 2 parts of pH regulator, where the mass ratio of triethanolamine to citric acid is 3:1. Make up the balance with pure water to 100 parts, stir evenly, let stand to remove bubbles, then fill into an aluminum foil bag containing non-woven fabric, and obtain the dressing after moist heat sterilization.

[0099] Example 11

[0100] A preparation method of a collagen hydrogel dressing, comprising the following steps:

[0101] S1: Take 0.8 part of recombinant collagen, 0.4 part of biomimetic collagen, 0.15 part of hyaluronic acid, and 1 part of sodium carboxymethylcellulose, and dissolve them in phosphate buffer solution to obtain Solution A;

[0102] S2: Take 6 parts of glycerol, 2 parts of butanediol, and 0.6 part of carbomer, and dissolve them in phosphate buffer solution to obtain Solution B;

[0103] S3: Take 0.8 part of gallic acid and 0.8 part of sinapic acid, and dissolve them in phosphate buffer solution to obtain Solution C;

[0104] S4: After stirring and mixing Solution A and Solution B evenly, add Solution C and continue to stir and mix evenly. Then add 0.4 part of the preservative octanoyl hydroxamic acid and glycerol caprylate (mass ratio 2:3), make up 0.1M pH = 7.4 phosphate buffer solution to 100 parts, stir and mix evenly, let stand to remove bubbles, and obtain the hydrogel dressing after filling and sterilization.

[0105] Example 12

[0106] The difference from Example 11 is that in step S4, after adding 0.4 part of the preservative, then add 3.5 parts of pH regulator, where the mass ratio of triethanolamine to citric acid is 4:1, make up with pure water to 100 parts, and the steps are the same as those in Example 11.

[0107] Example 13

[0108] A preparation method of a collagen hydrogel dressing, comprising the following steps:

[0109] S1: Take 2 parts of recombinant collagen, 1 part of bionic collagen, 0.2 part of hyaluronic acid and 0.2 part of sodium carboxymethylcellulose, and dissolve them with phosphate buffer to obtain solution A;

[0110] S2: Take 10 parts of glycerol, 5 parts of butanediol, and 1.5 parts of carbomer, and dissolve them with phosphate buffer to obtain solution B;

[0111] S3: Take 1.2 parts of gallic acid and 1.2 parts of sinapic acid, and dissolve them with phosphate buffer to obtain solution C;

[0112] S4: After stirring and mixing solution A and solution B evenly, add solution C and continue to stir and mix evenly. Then add 0.5 part of preservatives octanoyl hydroxamic acid and glycerol caprylate (mass ratio 2:3), make up to 100 parts with 0.1M pH = 7.4 phosphate buffer, stir and mix evenly, let stand to remove bubbles, and obtain hydrogel dressing after filling and sterilization.

[0113] Comparative Example 1

[0114] The difference from Example 1 is only that in step S1, only 0.4 part of recombinant collagen is dissolved with phosphate buffer to obtain solution A.

[0115] Comparative Example 2

[0116] The difference from Example 1 is only that in step S1, only 0.4 part of bovine type I collagen is dissolved with phosphate buffer to obtain solution A.

[0117] Comparative Example 3

[0118] The difference from Example 1 is only that in step S1, only 0.4 part of bionic collagen is dissolved with phosphate buffer to obtain solution A.

[0119] Comparative Example 4

[0120] A preparation method of a collagen dressing, comprising the following steps:

[0121] S1: Take 0.2 part of recombinant collagen and dissolve it with phosphate buffer to obtain solution A;

[0122] S2: Take 3 parts of glycerol, 1 part of butanediol, and 0.3 part of carbomer, and dissolve them with phosphate buffer to obtain solution B;

[0123] S3: Take 0.4 part of trehalose and dissolve it with phosphate buffer to obtain solution C;

[0124] S4: After stirring and mixing solution A and solution B evenly, add solution C and continue to stir and mix evenly. Then add 0.3 parts of preservatives, octanoyl hydroxamic acid and glyceryl caprylate (mass ratio is 5:9). After making up the phosphate buffer solution to 100 parts, stir and mix evenly. After standing to remove air bubbles, fill it into an aluminum foil bag containing non-woven fabric, and obtain the dressing after moist heat sterilization. The pH of the above phosphate buffer solution is 6 and the concentration is 0.1M.

[0125] Comparative Examples 5 - 12

[0126] The differences between Comparative Examples 5 - 12 and Comparative Example 4 are only that the types and ratios of the stabilizers added to solution C in step S3 are changed. For details, see the following table:

[0127] Table 2

[0128] Number Trehalose Gallic acid Resveratrol Sinapic acid Comparative Example 4 0.4 / / / Comparative Example 5 / 0.8 / / Comparative Example 6 / / 0.8 / Comparative Example 7 / / / 0.8 Comparative Example 8 / 0.4 / 0.4 Comparative Example 9 0.3 0.3 / 0.3 Comparative Example 10 / 0.3 0.3 0.3 Comparative Example 11 0.2 0.2 0.2 0.2 Comparative Example 12 / / / /

[0129] Comparative Example 13

[0130] The difference from Comparative Example 5 is that in step S3, only gallic acid is added, and the addition amount is 0.01 part.

[0131] Comparative Example 14

[0132] The difference from Comparative Example 5 is that in step S3, only gallic acid is added, and the addition amount is 8 parts.

[0133] Comparative Example 15

[0134] The difference from Comparative Example 5 is that in step S3, only gallic acid is added, and the addition amount is 16 parts.

[0135] Comparative Example 16

[0136] The difference from Comparative Example 7 is that in step S3, only sinapic acid is added, and the addition amount is 8 parts.

[0137] Effect Examples

[0138] I. Results of variable temperature circular dichroism spectrum and collagen thermal denaturation temperature analysis

[0139] Figure 1 They are the variable temperature circular dichroism spectra and collagen thermal denaturation temperature analysis diagrams of bionic collagen, bovine type I collagen and recombinant collagen. Figure 1 It not only shows that the bionic collagen has better thermal stability, but also shows that the way to obtain the collagen thermal stability, that is, the thermal decomposition temperature, is the first derivative of the thermal denaturation analysis curve.

[0140] Among them, Figure 1(a) shows the temperature-variable circular dichroism spectrum of bionic collagen. It can be seen that as the temperature increases from 20 °C to 60 °C, bionic collagen has a positive peak in the range of 222 - 225 nm and a negative peak in the range of 195 - 197 nm. At 20 °C, 40 °C, and 60 °C, the absolute values of the positive and negative peak ratios are 0.117, 0.112, and 0.083 respectively (the lower the ratio, the lower the degree of collagen triple helix). As the temperature increases to 60 °C, the triple helix structure of bionic collagen does not disappear. When the temperature increases to 80 °C, the triple helix structure disappears.

[0141] Figure 1 (b) is the temperature-variable circular dichroism spectrum of recombinant collagen. Similarly, the absolute values of the positive and negative peak ratios at 4 °C, 35 °C, and 45 °C are 0.103, 0.059, and 0 respectively. The positive peak of recombinant collagen disappears at 45 °C, the triple helix structure disappears, and the collagen is inactivated.

[0142] Figure 1 (c) is the temperature-variable circular dichroism spectrum of bovine type I collagen. Similarly, the absolute values of the positive and negative peak ratios at 15 °C, 40 °C, and 60 °C are 0.107, 0.055, and 0 respectively. The positive peak of bovine type I collagen disappears at 60 °C, the triple helix structure disappears, and the collagen is inactivated.

[0143] Figure 1 (d) is the curve of the CD value of the three collagens changing with temperature at the positive peak wavelength, that is, the thermal denaturation analysis curve, and its first derivative is calculated. It can be seen that the thermal denaturation temperatures of bionic collagen, bovine type I collagen, and recombinant collagen are 61 °C, 50 °C, and 38 °C respectively. Among them, the positive peaks of the circular dichroism spectra of bovine type I collagen and recombinant collagen disappear after the thermal denaturation temperature, and both undergo denaturation. However, the positive peak of the circular dichroism spectrum of bionic collagen does not disappear above 60 °C, indicating that its triple helix structure has excellent heat resistance. This is mainly because bionic collagen is mainly composed of proline and hydroxyproline with rigid pyrrole rings, and it is more inclined to form a helical coiled structure, thus having better thermal stability.

[0144] Figure 2It is the circular dichroism spectra of collagen corresponding to Comparative Examples 1-3 after moist heat sterilization. As shown in the figure, it can be seen that recombinant collagen, bovine type I collagen, and bionic collagen all have positive and negative absorption peaks in the wavelength range of 190-260 nm. The positive peak is in the range of 222-225 nm, and the negative peak is in the range of 195-197 nm. The CD zero absorption value (MRE≈0) is near 218 nm. This indicates that the three types of collagen still possess a certain triple helix structure after high-temperature sterilization, which shows that adding heat-stable additives can improve the thermal stability of recombinant collagen and bovine type I collagen (still having a triple helix structure after moist heat sterilization). The ratio of the absolute values of the peak values of the positive and negative peaks of the circular dichroism spectra of bionic collagen, bovine type I collagen, and recombinant collagen gradually decreases, being 0.100, 0.041, and 0.032 in sequence, indicating that their triple helix degrees gradually decrease in sequence: (1) Moist heat sterilization does cause the unwinding of collagen and a decrease in the triple helix degree; (2) Bionic collagen has better thermal stability compared to recombinant collagen and bovine type I collagen; (3) Recombinant collagen still has a triple helix structure after moist heat sterilization. On the one hand, it is because the heat-stable additives improve its stability, and on the other hand, the unwound collagen still has the ability to self-assemble through hydrogen bonds to restore the triple helix structure after the temperature returns to room temperature.

[0145] II. Usage experience effect

[0146] Table 3

[0147] Number pH Viscosity mPa.s <![CDATA[Water vapor transmission rate in 24 hours g / m 2 > Use experience (1 - 10 points) Example 1 4.33 / / 6.2 Example 2 4.42 42.15 48543 5.4 Example 3 5.32 / / 7.5 Example 4 5.64 / / 7.6 Example 5 5.10 151.43 41715 8.7 Example 6 4.60 / / 8.4 Example 7 5.24 / / 7.1 Example 8 5.32 / / 5.8 Example 9 5.31 331.12 33739 4.6

[0148] Table 3 evaluates the performance of the patch through pH, viscosity, water vapor transmission rate, and usage experience parameters. Among them, the usage experience includes the overall evaluation of the appearance of the patch before use and the situation of liquid dripping, as well as the subjective feelings of freshness and irritation during use.

[0149] Figure 3 It is the data display chart corresponding to the usage experience column in the above table. It can be seen from the figure that the patch experience score corresponding to Example 5 is relatively high because its formulation comprehensively balances adhesiveness and fluidity, and it has good conformability and water permeability; the experience scores of Example 2 and Example 9 are relatively low. The former has too strong fluidity and lacks conformability, while the latter has a relatively high viscosity and poor air permeability. By adjusting the thickening and moisturizing agents to a reasonable ratio, the corresponding patch experience score can be improved, and its conformability and water permeability can be greatly improved, better meeting the usage experience.

[0150] III. Stability test of the patch or dressing

[0151] Test method: Adopt the variable-temperature circular dichroism test method.

[0152] Test results: In the examples, since the decomposition temperature of recombinant collagen is relatively low, the stability of the tested product patch or dressing is reflected by the thermal decomposition temperature of bionic collagen, and the stability of the comparative product is reflected by the thermal decomposition temperature of recombinant collagen.

[0153] The following Table 4 shows the thermal decomposition temperature data of bionic collagen corresponding to different examples:

[0154] Table 4

[0155] Number Thermal decomposition temperature Tm of bionic collagen (℃) Example 1 64 Example 5 64 Example 11 66

[0156] The following Table 5 shows the thermal decomposition temperature data of recombinant collagen corresponding to different comparative examples:

[0157] Table 5

[0158] Number Thermal decomposition temperature Tm of recombinant collagen (℃) Comparative Example 4 41 Comparative Example 5 45 Comparative Example 6 41 Comparative Example 7 46 Comparative Example 8 53 Comparative Example 9 51 Comparative Example 10 50 Comparative Example 11 47 Comparative Example 12 40 Comparative Example 13 38 Comparative Example 14 32 Comparative Example 15 35 Comparative Example 16 33

[0159] Figure 4 is the diagram corresponding to the data in Tables 4 - 5. Figure 4 The abscissa represents the groups of different examples and comparative examples, and the ordinate represents the thermal decomposition temperature Tm℃ of collagen. It can be seen from Figure 4 that Comparative Example 8 compared with Comparative Example 12 shows that adding 0.4 parts of gallic acid and 0.4 parts of sinapic acid as stabilizing aids is more conducive to improving the stability of recombinant collagen than other combinations of stabilizing aids. Comparative Example 13 shows that when the addition amount of gallic acid stabilizer is very small, it has no positive or negative effect on the thermal stability of recombinant collagen; while in Comparative Examples 14 - 16, the usage amount of gallic acid or sinapic acid is greatly increased, which does not improve the thermal stability of collagen, but instead destroys its thermal stability. It is analyzed that excessive acidic stabilizers will cause slow acidolysis of collagen, and the increase in negative charge ions also increases the repulsion between collagen molecules, inhibiting the aggregation of collagen molecules.

[0160] The thermal stability data of Examples 1, 5, and 11 show that adding bionic collagen to the collagen patch or dressing, the thermal stability tested by variable temperature circular dichroism spectroscopy is the thermal stability of bionic collagen, and its thermal stability is further improved on the basis of pure bionic collagen (61℃). Compared with recombinant collagen added with thermal stability aids, the improvement amplitude is smaller.

[0161] In summary, adding only stabilizing aids to the patch or dressing has limited improvement in the stability of recombinant collagen. Adding stabilizing aids and bionic collagen together and cooperating synergistically can greatly improve the stability of collagen.

[0162] Although the embodiments of the present embodiment have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present embodiment, and the scope of the present embodiment is defined by the claims and their equivalents.

Claims

1. A collagen patch, comprising an essence, characterized in that: According to the weight percentage, the essence contains 100 parts by weight, including the following components: 0.02-2 parts of recombinant collagen, 0.01-1 parts of biomimetic collagen, 0.001-0.5 parts of moisturizer, 0.02-4 parts of stabilizing agent and 2-25 parts of thickener, and the balance is water or phosphate buffer. The stabilizing agent is one or more of trehalose, gallic acid, resveratrol and sinapinic acid. The bionic collagen is composed of glycine, proline and hydroxyproline, and is obtained by a liquid phase synthesis method, i.e., by hydrolysis, polymerization, extraction, rotary evaporation and purification steps. Its molecular weight distribution is 100KDa~1000KDa, and its thermal denaturation temperature is 61°C. Its structure is a triple helix structure formed by repeated arrangement and polymerization of the following tripeptide structural units to form a single α chain, and then self-assembled through hydrogen bonding: ， The structural unit compound is firstly synthesized into a dipeptide from the proline and the hydroxyproline, and then synthesized into a tripeptide from the dipeptide and the glycine, and the tripeptide is a repeating structural unit.

2. The collagen patch according to claim 1, characterized in that: The stabilizing agent is 0.02 to 2 parts of gallic acid and / or 0.02 to 2 parts of sinapic acid.

3. The collagen patch according to any one of claims 1 to 2, characterized in that: The moisturizing agent is hyaluronic acid and / or sodium hyaluronate, and the thickener includes 1 to 20 parts of glycerin, 0.2 to 2 parts of butylene glycol and 0.1 to 1 part of carbomer.

4. The collagen patch according to claim 3, characterized in that: In terms of weight, the essence comprises the following components: 0.02 to 1 parts of the recombinant collagen, 0.01 to 0.5 parts of the biomimetic collagen, 0.001 to 0.3 parts of the hyaluronic acid, 1 to 6 parts of the glycerol, 0.2 to 2 parts of the butylene glycol, 0.1 to 0.6 parts of the carbomer, 0.02 to 0.8 parts of the gallic acid and 0.02 to 0.8 parts of the sinapinic acid, and the balance is water or phosphate buffer.

5. The collagen patch according to claim 4, characterized in that: When the balance is water, the essence further comprises 0.01 to 5 parts of a pH adjuster and 0.2 to 0.5 parts of a preservative; When the balance is phosphate buffer, the essence further comprises 0.2-0.5 parts of preservative.

6. The collagen patch according to claim 5, characterized in that: The following conditions must be met: a. The pH regulator is triethanolamine and citric acid, and the mass ratio of the triethanolamine to the citric acid is 1:10 to 10:1; b. The preservatives are caprylhydroxamic acid and glyceryl caprylate, and the mass ratio of caprylhydroxamic acid to glyceryl caprylate is 9:1 to 1:9; c. The concentration of the phosphate buffer is 0.01-0.1 M, and the pH is 4-7.

4.

7. A method for preparing the collagen patch according to any one of claims 1 to 6, characterized in that: The steps include: S1, dissolving the recombinant collagen, the biomimetic collagen and the moisturizing agent with a solvent to obtain a solution A; S2, dissolving the thickener in the solvent to obtain solution B; S3, dissolving the stabilizing agent with the solvent to obtain a solution C; S4. Mix the solution A and the solution B evenly, add the solution C, continue to add the solvent until the weight portion of the essence is 100 parts, and mix evenly.

8. The preparation method according to claim 7, characterized in that: The following conditions must be met: a. In steps S1 to S4, the solvent is water or phosphate buffer. When the solvent is water, in step S4, after adding the solution C, before continuing to add the solvent to make up, a preservative and a pH adjuster are added; When the solvent is a phosphate buffer, in step S4, after adding the solution C, before continuing to add the solvent to make up, the preservative is added; b. After step S4, the method further includes the steps of standing, bagging, and sterilization. The sterilization method is moist heat sterilization or radiation sterilization. When the moist heat sterilization is adopted, the temperature of the moist heat sterilization is 121° C., and the time of the moist heat sterilization is 15 to 30 minutes; when the radiation sterilization is adopted, the dose of the radiation sterilization is 10 to 25 KGy.

9. A method for preparing a collagen dressing, characterized in that: The steps are the same as the steps of the preparation method of the collagen patch according to any one of claims 7 to 8, wherein the amount of each component added is 2 to 5 times the amount of each component added in the preparation method of the collagen patch, and 0.05 to 5 parts by weight of sodium carboxymethyl cellulose is additionally dissolved in step S1.

10. A collagen dressing, comprising a collagen dressing and a hydrogel, characterized in that: The method according to claim 9 is used to prepare the product.

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