Collagen patch, dressing and preparation method thereof

Abstract

The invention relates to the technical field of medical dressings, and provides a collagen patch, a dressing and a preparation method thereof. The collagen patch comprises essence, and the essence comprises the following components in parts by weight: 0.02 to 2 parts of recombinant collagen, 0.01 to 1 part of bionic collagen, 0.001 to 0.5 part of a humectant, 0.02 to 4 parts of a stabilizing auxiliary agent, 2 to 25 parts of a thickening agent and the balance of water or a phosphate buffer solution, and the total amount of the essence is 100 parts by weight, and the essence comprises the following components in parts by weight: 0.01 to 2 parts of recombinant collagen, 0.01 to 1 part of bionic collagen, 0.001 to 0.5 part of a humectant, 0.02 to 4 parts of a stabilizing auxiliary agent, 2 to 25 parts of a thickening agent and the balance of water or a phosphate buffer solution. The stabilizing aid is one or more of trehalose, gallic acid, resveratrol and sinapic acid. The sterile collagen patch and dressing are prepared by using the bionic collagen with good structural stability, including good thermal stability and irradiation stability, and meanwhile, the stabilizing additive is added to synergistically stabilize recombination and the bionic collagen, so that the sterilization process of the dressing is simplified, the production sterility requirement is reduced, and the production cost is reduced. And the influence of dressing processing and sterilizing processes on the activity of the collagen is also reduced.

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 the first aspect, the present application provides a collagen patch, which includes an essence. The essence, calculated by weight, has a total of 100 parts by weight and includes 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 remainder is water or phosphate buffer. The stabilizing agent is one or more of trehalose, gallic acid, resveratrol and sinapinic acid.

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

[0010] In some embodiments, the weight portion of the recombinant collagen is 0.02-1 part.

[0011] In some embodiments, the weight portion of the biomimetic collagen is 0.01-0.5 parts.

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

[0013] Wherein, preferably, the moisturizing agent is 0.02-0.5 parts.

[0014] Wherein, preferably, the moisturizing agent is hyaluronic acid and / or sodium hyaluronate.

[0015] Wherein, preferably, the thickener comprises 1-20 parts of glycerin, 0.2-2 parts of butylene glycol and 0.1-1 part of carbomer.

[0016] In some embodiments, the essence comprises the following components, measured in parts by weight: 0.02-1 parts of recombinant collagen, 0.01-0.5 parts of biomimetic collagen, 0.001-0.3 parts of hyaluronic acid, 1-6 parts of glycerol, 0.2-2 parts of butylene glycol, 0.1-0.6 parts of carbomer, 0.02-0.8 parts of gallic acid and 0.02-0.8 parts of sinapic acid, and the balance is water or phosphate buffer.

[0017] In some embodiments, the essence comprises the following components, measured by weight: 0.4 parts of recombinant collagen, 0.2 parts of biomimetic collagen, 0.1 parts of hyaluronic acid, 3 parts of glycerol, 0.2-1 parts of butylene glycol, 0.3 parts of carbomer, 0.4 parts of gallic acid and 0.4 parts of sinapic acid, and the balance is water or phosphate buffer.

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

[0019] 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, the essence further comprises 0.2-0.5 parts of preservatives. In the present application, the preservatives are generally caprylhydroxamic acid and glyceryl caprylate, preferably, the mass ratio of caprylhydroxamic acid to glyceryl caprylate is 9:1-1:9.

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

[0022] In a second aspect, the present application also provides a method for preparing the aforementioned collagen patch, comprising the following steps:

[0023] S1, dissolving the recombinant collagen, the biomimetic collagen and the moisturizing agent with a solvent to obtain a solution A;

[0024] S2, dissolving the thickener with a solvent to obtain a solution B;

[0025] S3, dissolving the stabilizing agent with a solvent to obtain a solution C;

[0026] S4. Mix the solution A and the solution B evenly, add the solution C, continue to add solvent until the weight portion of the essence is 100 parts, and mix evenly.

[0027] The weight percentages of the substances in steps S1-S4 are the weight percentages of the aforementioned components.

[0028] In some embodiments, the solvent in steps S1-S4 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 phosphate buffer, in step S4, after adding the solution C, before continuing to add the solvent to make up, a preservative is added.

[0029] In some embodiments, after step S4, the steps of standing, bagging, and sterilization are also included.

[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 minutes; when irradiation sterilization is adopted, the dose of the irradiation sterilization is 10-25KGy.

[0031] The present application also provides a method for preparing a collagen dressing, which has the same steps as the aforementioned method for preparing a collagen dressing, wherein, preferably, the amount of each component added is 2-5 times the amount of each component added in the method for preparing the collagen dressing, and 0.05-5 parts by weight of sodium carboxymethyl cellulose is additionally dissolved in step S1.

[0032] The present application also provides a collagen dressing prepared by the preparation method of the aforementioned collagen dressing, including a collagen dressing and a hydrogel.

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

[0034] (1) The present application prepares sterile collagen patches and dressings by using biomimetic collagen with good structural stability, including good thermal stability and radiation stability, and at the same time adds stabilizing additives to synergistically stabilize the recombinant collagen, thereby simplifying the sterilization process of the dressing, reducing the production sterility requirements, and reducing the impact of the dressing processing and sterilization process on the activity of the collagen itself.

[0035] (2) This application further screens the specific ratios and amounts of the components of the stabilizing auxiliary agents, as well as the specific ratios between different components. The resulting patches and dressings have better thermal stability. The system formula comprehensively balances adhesion and fluidity. The product has good conformability and water permeability, and the user experience score is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solution 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 the variable temperature circular dichroism spectrum and collagen thermal denaturation temperature analysis diagram, among which, Figure 1 (a) is the variable temperature circular dichroism spectrum of biomimetic collagen; Figure 1 (b) is the variable temperature circular dichroism spectrum of recombinant collagen; Figure 1 (c) is the temperature-dependent circular dichroism spectrum of bovine type I collagen; Figure 1 (d) is the curve of CD value variation with temperature at the positive peak wavelength of the above three collagens, that is, the thermal denaturation analysis curve.

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

[0039] Figure 3 It is a graphical representation of the usage experience score data of the patch obtained from different embodiments.

[0040] Figure 4 Thermal decomposition temperature data trend chart of collagen. DETAILED DESCRIPTION

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

[0042] Likewise, it is to be understood that the phrases and terms used in this application are for descriptive purposes and should not be considered as limiting. The use of "include", "comprise" or "have" and variations thereof in this application is intended to openly include the items listed thereafter and their equivalents and additional items.

[0043] In this document, in order to make the description concise, not all possible combinations of various technical features in various embodiments or examples are described. Therefore, as long as there is no contradiction in the combination of these technical features, the various technical features in various embodiments or examples can be combined arbitrarily, and all possible combinations should be considered to be within the scope of this specification.

[0044] Since the sterile products of collagen dressings often have the risk of denaturation of recombinant collagen after terminal sterilization due to the low structural stability of collagen, especially for recombinant collagen with poor stability, it is necessary to create a sterile environment during the entire preparation process of the dressing, which increases the difficulty and cost of using recombinant collagen in dressing products. This application is to solve this problem, and specially uses bionic collagen with good structural stability to prepare sterile collagen dressings and dressings, and adds stabilizing additives to cooperate with recombinant and bionic collagen, so that the sterilization process of the dressing is simplified and the production sterility requirements are reduced.

[0045] On the one hand, the present application provides a collagen patch, which includes an essence. Calculated by weight, the essence contains 100 parts by weight and includes 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.

[0046] In the present application, the collagen patch is generally composed of essence and non-woven fabric, and is packaged in an aluminum foil bag.

[0047] In the present application, the molecular weight of recombinant collagen is smaller than that of biomimetic collagen, and recombinant collagen has a certain degree of transdermal absorption ability. The purpose of matching recombinant collagen and biomimetic collagen is, on the one hand, to increase the amount of collagen absorbed through the skin, and on the other hand, to add thermally stable collagen. Since the thermal stability of recombinant collagen is poor, it is difficult to change its thermal stability by only adding biomimetic collagen, and it is necessary to add a thermal stabilizing agent to improve its thermal stability.

[0048] In the present application, the recombinant collagen is selected from human DNA gene fragments, which are then introduced into fermentation bacteria, such as Escherichia coli or Pichia pastoris, and then centrifuged, sterilized ultrafiltration, and chromatographically purified to obtain the recombinant collagen, whose molecular weight distribution is 2KDa-50KDa.

[0049] In the present application, the bionic collagen is a bionic biomaterial that imitates the composition, structure and function of natural collagen. Its basic structure is the same as the natural collagen (Gly-XY)n repeating amino acid sequence, and it is mainly composed of glycine, proline and hydroxyproline. It is obtained by liquid phase synthesis technology, that is, by hydrolysis, polymerization, extraction, rotary evaporation and purification. Its molecular weight distribution is 100KDa-1000KDa. The specific preparation method can be found in the prior art patent CN202410309839.7.

[0050] The biomimetic collagen described above is composed of the following structural units (tripeptides) repeatedly arranged and polymerized into a single α chain, which then self-assembles into a triple helix structure through hydrogen bonding:

[0051]

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

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

[0054] Among them, trehalose can accelerate the self-assembly kinetics of collagen, improve the self-assembly degree of collagen and the mechanical properties of the resulting hydrogel, which is 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 protein, increase the density of protein structure, and improve the thermal stability of protein.

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

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

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

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

[0060] Wherein, the moisturizing agent can generally be hyaluronic acid and / or sodium hyaluronate.

[0061] Wherein, the thickener generally includes glycerol, butylene glycol and carbomer. Preferably, the weight portion of the glycerol is 1-20 parts, the weight portion of the butylene glycol is 0.2-2 parts, and the weight portion of the carbomer is 0.1-1 part.

[0062] In the present application, when the balance is water, the essence further 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, the mass ratio of triethanolamine and citric acid is 1:10-10:1; the preservative is caprylhydroxamic acid and glyceryl caprylate, the mass ratio of caprylhydroxamic acid and glyceryl caprylate is 9:1-1:9. In the present application, when the balance is phosphate buffer, the essence further includes 0.2-0.5 parts of preservative, preferably, the concentration of the phosphate buffer is 0.01-0.1M, the pH is 4-7.4, and the preservative is the same as above.

[0063] On the other hand, the present application provides a method for preparing the aforementioned collagen patch, comprising the following steps:

[0064] S1, dissolving the recombinant collagen, the biomimetic collagen and the moisturizing agent with a solvent to obtain a solution A;

[0065] S2, dissolving the thickener with a solvent to obtain a solution B;

[0066] S3, dissolving the stabilizing agent with a solvent to obtain a solution C;

[0067] 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.

[0068] The weight percentages of the substances in steps S1-S4 are the weight percentages of the aforementioned components.

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

[0070] 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.

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

[0072] In the present application, after step S4, the steps of standing, bagging and sterilization are also included.

[0073] The sterilization method is generally moist heat sterilization or irradiation sterilization.

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

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

[0076] The present application also provides a method for preparing a collagen dressing, wherein the steps of the preparation method are the same as the steps of the preparation method of the aforementioned collagen dressing, wherein, preferably, the amount of each component added is 2-5 times the amount of each component added in the preparation method of the collagen dressing, and step S1 also includes dissolving sodium carboxymethyl cellulose, and the weight portion of the sodium carboxymethyl cellulose is 0.05-5 parts.

[0077] The present application also provides collagen dressings prepared by the aforementioned preparation method, including collagen dressings and hydrogels.

[0078] Examples of the present embodiment are described in detail below. The examples are only used to explain the present embodiment and are not to be construed as limiting the present embodiment.

[0079] The molecular weight distribution of bionic collagen is 100KDa-1000KDa. The specific preparation method can be found in the prior art patent CN202410309839.7.

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

[0081] Bovine type I collagen was purchased from China Food and Drug Administration.

[0082] All raw materials, reagents, etc. used without specifying the manufacturer are conventional products that can be purchased from the market.

[0083] Example 1

[0084] A method for preparing a collagen patch comprises the following steps:

[0085] S1: Dissolve 0.2 parts of recombinant collagen, 0.1 parts of biomimetic collagen and 0.5 parts of hyaluronic acid in phosphate buffer to obtain solution A;

[0086] S2: Dissolve 3 parts of glycerol, 1 part of butylene glycol, and 0.3 parts of carbomer in phosphate buffer to obtain solution B;

[0087] S3: Dissolve 0.4 parts of gallic acid and 0.4 parts of sinapinic acid in phosphate buffer to obtain solution C;

[0088] S4: After stirring and mixing solution A and solution B evenly, add solution C and continue stirring and mixing evenly, add 0.3 parts of preservatives caprylhydroxamic acid and glyceryl caprylate (mass ratio is 5:9), make up the phosphate buffer to 100 parts, stir and mix evenly, let it stand to remove bubbles, fill it into an aluminum foil bag filled with non-woven fabric, and obtain a patch after wet heat sterilization. The above phosphate buffer has a pH of 6 and a concentration of 0.1M.

[0089] Embodiment 2-9

[0090] The difference between Example 2-9 and Example 1 is that the amounts of components in solution A and solution B in steps S1 and S2 are changed. For details, see the following table:

[0091] Table 1

[0092] serial number Recombinant collagen Bionic collagen Hyaluronic acid glycerin 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 method for preparing a collagen patch comprises the following steps:

[0095] S1: 0.4 parts of recombinant collagen, 0.2 parts of biomimetic collagen and 0.1 parts of hyaluronic acid were respectively hydrolyzed with pure water to obtain solution A;

[0096] S2: Dissolve 3 parts of glycerol, 1 part of butylene glycol, and 0.3 parts of carbomer in pure water to obtain solution B;

[0097] S3: Dissolve 0.4 parts of gallic acid and 0.4 parts of sinapinic acid in pure water to obtain solution C;

[0098] S4: After stirring solution A and solution B to mix evenly, add solution C and continue stirring to mix evenly, add 0.3 parts of preservatives caprylhydroxamic acid and glyceryl caprylate (mass ratio is 5:9), add 2 parts of pH adjuster, wherein the mass ratio of triethanolamine: citric acid is 3:1, add the balance of pure water to 100 parts and stir evenly, let it stand to remove bubbles, fill it into an aluminum foil bag filled with non-woven fabric, and obtain an application after wet heat sterilization.

[0099] Embodiment 11

[0100] A method for preparing a collagen hydrogel dressing comprises the following steps:

[0101] S1: Dissolve 0.8 parts of recombinant collagen, 0.4 parts of biomimetic collagen, 0.15 parts of hyaluronic acid and 1 part of sodium carboxymethyl cellulose in phosphate buffer to obtain solution A;

[0102] S2: Dissolve 6 parts of glycerol, 2 parts of butylene glycol, and 0.6 parts of carbomer in phosphate buffer to obtain solution B;

[0103] S3: Dissolve 0.8 parts of gallic acid and 0.8 parts of sinapinic acid in phosphate buffer to obtain solution C;

[0104] S4: After stirring solution A and solution B to mix evenly, add solution C and continue stirring to mix evenly, add 0.4 parts of preservatives caprylhydroxamic acid and glyceryl caprylate (mass ratio is 2:3), add 0.1M pH=7.4 phosphate buffer to 100 parts, stir and mix evenly, let stand to remove bubbles, and obtain a hydrogel dressing after filling and sterilization.

[0105] Example 12

[0106] The difference from Example 11 is that in step S4, after adding 0.4 parts of preservatives, 3.5 parts of pH adjusters are added, wherein the mass ratio of triethanolamine: citric acid is 4:1, and pure water is added to 100 parts, and the steps are the same as Example 11.

[0107] Example 13

[0108] A method for preparing a collagen hydrogel dressing comprises the following steps:

[0109] S1: Dissolve 2 parts of recombinant collagen, 1 part of biomimetic collagen, 0.2 parts of hyaluronic acid and 0.2 parts of sodium carboxymethyl cellulose in phosphate buffer to obtain solution A;

[0110] S2: Dissolve 10 parts of glycerol, 5 parts of butylene glycol, and 1.5 parts of carbomer in phosphate buffer to obtain solution B;

[0111] S3: Dissolve 1.2 parts of gallic acid and 1.2 parts of sinapinic acid in phosphate buffer to obtain solution C;

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

[0113] Comparative Example 1

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

[0115] Comparative Example 2

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

[0117] Comparative Example 3

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

[0119] Comparative Example 4

[0120] A method for preparing a collagen patch comprises the following steps:

[0121] S1: Dissolve 0.2 parts of recombinant collagen in phosphate buffer to obtain solution A;

[0122] S2: Dissolve 3 parts of glycerol, 1 part of butylene glycol, and 0.3 parts of carbomer in phosphate buffer to obtain solution B;

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

[0124] S4: After stirring solution A and solution B to mix evenly, add solution C and continue stirring to mix evenly, add 0.3 parts of preservatives, caprylhydroxamic acid and glyceryl caprylate (mass ratio is 5:9), add phosphate buffer to 100 parts, stir and mix evenly, let stand to remove bubbles, fill into an aluminum foil bag filled with non-woven fabric, and obtain a patch after wet heat sterilization. The above phosphate buffer has a pH of 6 and a concentration of 0.1M.

[0125] Comparative Examples 5-12

[0126] The difference between Comparative Examples 5 to 12 and Comparative Example 4 is that the type and ratio of the stabilizer added to the solution C in step S3 are changed. For details, see the following table:

[0127] Table 2

[0128] serial 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 in an amount of 0.01 parts.

[0131] Comparative Example 14

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

[0133] Comparative Example 15

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

[0135] Comparative Example 16

[0136] The difference from Comparative Example 7 is that in step S3, only sinapinic acid is added in an amount of 8 parts.

[0137] Effect Example

[0138] 1. Results of variable temperature circular dichroism and thermal denaturation temperature analysis of collagen

[0139] Figure 1 These are the variable temperature circular dichroism spectra of bionic collagen, bovine type I collagen and recombinant collagen, as well as the thermal denaturation temperature analysis diagram of collagen. Figure 1 It not only shows that biomimetic collagen has better thermal stability, but also explains the thermal stability of collagen, that is, the thermal decomposition temperature is obtained by the first derivative of the thermal denaturation analysis curve.

[0140] in, Figure 1(a) shows the variable temperature circular dichroism spectrum of bionic collagen. It can be seen that as the temperature increases from 20℃ to 60℃, the bionic collagen has a positive peak in the range of 222-225nm and a negative peak in the range of 195-197nm. The absolute values ​​of the positive and negative peak ratios at 20℃, 40℃ and 60℃ 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℃, the triple helix structure of bionic collagen does not disappear, and the triple helix structure disappears when the temperature increases to 80℃.

[0141] Figure 1 (b) is the variable temperature circular dichroism spectrum of recombinant collagen. Similarly, it can be obtained that 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 variable temperature circular dichroism spectrum of bovine type I collagen. Similarly, the absolute values ​​of the positive and negative peak ratios at 15℃, 40℃, and 60℃ are 0.107, 0.055, and 0, respectively. The positive peak of bovine type I collagen disappears at 60℃, the triple helix structure disappears, and the collagen is inactivated.

[0143] Figure 1 (d) is the curve of CD value variation with temperature at the positive peak wavelength of the three collagens, i.e., the thermal denaturation analysis curve, and the first-order 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 is higher than that, and both undergo denaturation. However, the positive peak of the circular dichroism spectra of bionic collagen does not disappear at a temperature higher than 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, which tend to form a helical coiled structure and thus have better thermal stability.

[0144] Figure 2This is the circular dichroism spectrum corresponding to the collagen of Examples 1-3 after moist heat sterilization. As shown in the figure, it can be seen that the recombinant collagen, bovine type I collagen and biomimetic collagen have positive and negative absorption peaks in the wavelength range of 190-260nm, and the positive peak is in the range of 222-225nm, the negative peak is in the range of 195-197nm, and the CD zero absorption value (MRE≈0) is near 218nm, which indicates that the three collagens still have a certain triple helix structure after high temperature sterilization, which means that adding heat stabilizing additives can improve the thermal stability of recombinant collagen and bovine type I collagen (still have a triple helix structure after moist heat sterilization). The ratios of the absolute values ​​of the positive and negative peaks of the circular dichroism spectra of biomimetic collagen, bovine type I collagen and recombinant collagen gradually decreased, which were 0.100, 0.041 and 0.032, respectively, indicating that their triple helix degree gradually decreased: (1) Moist heat sterilization does cause collagen unwinding and the triple helix degree decreases; (2) Biomimetic collagen has better thermal stability than recombinant collagen and bovine type I collagen; (3) Recombinant collagen still has a triple helix structure after moist heat sterilization. On the one hand, this is because the thermal stabilizing agent improves its stability. On the other hand, the unwinding collagen is still able to restore the triple helix structure through hydrogen bond self-assembly after the temperature returns to room temperature.

[0145] 2. User experience effect

[0146] Table 3

[0147] serial number pH Viscosity mPa.s <![CDATA[Water vapor transmission rate in 24 hours g / m 2 > User 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 by pH, viscosity, water vapor permeability and usage experience parameters, where the usage experience includes a comprehensive evaluation of the appearance of the patch before use, the dripping of liquid, and the subjective feeling of freshness and irritation during use.

[0149] Figure 3 It is a data display chart corresponding to the use experience column in the above table. It can be seen from the figure that the application experience score corresponding to Example 5 is higher, because its formula comprehensively balances adhesion and fluidity, and its conformability is good and its water permeability is good; the experience scores of Example 2 and Example 9 are lower, the former has too strong fluidity and lacks conformability, and the latter has high viscosity and poor air permeability. By adjusting the thickener and moisturizer to a reasonable ratio, the corresponding application experience score can be improved, and its conformability and water permeability can be greatly improved, which better meets the use experience.

[0150] 3. Stability test of dressings or dressings

[0151] Testing method: variable temperature circular dichroism testing method is adopted.

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

[0153] Table 4 below shows the thermal decomposition temperature data of biomimetic collagen corresponding to different embodiments:

[0154] Table 4

[0155] serial number Thermal decomposition temperature of biomimetic collagen Tm ℃ Example 1 64 Example 5 64 Embodiment 11 66

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

[0157] Table 5

[0158] serial number Recombinant collagen thermal decomposition temperature Tm ℃ 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 This is the diagram corresponding to the data in Table 4-5. Figure 4 The horizontal axis represents the groups of different embodiments and comparative examples, and the vertical axis represents the thermal decomposition temperature Tm°C of collagen. Figure 4 It can be seen that, compared with Comparative Example 12, Comparative Example 8 shows that adding 0.4 parts of gallic acid and 0.4 parts of sinapinic acid as stabilizing agents is more conducive to improving the stability of recombinant collagen than the combination of other stabilizing agents. Comparative Example 13 shows that when the amount of gallic acid stabilizer added is very small, it has no positive or negative effect on the thermal stability of recombinant collagen; while Comparative Examples 14-16 greatly increase the amount of gallic acid or sinapinic acid used, which does not improve the thermal stability of collagen, but destroys its thermal stability. It is analyzed that excessive acidic stabilizers will cause slow acid hydrolysis of collagen, and the increase in negatively charged 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 indicate that the thermal stability of bionic collagen added to collagen patches or dressings as 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°C). Compared with recombinant collagen with added thermal stabilizing agents, the improvement is smaller.

[0161] In summary, adding only stabilizing agents to patches or dressings can only improve the stability of recombinant collagen to a limited extent. Adding stabilizing agents and biomimetic collagen at the same time can synergistically improve the stability of collagen to a large extent.

[0162] Although examples of the present embodiment have been shown and described, those skilled in the art will appreciate that various changes, modifications, substitutions and variations may be made to the 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: Calculated by weight, 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.

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

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

4. The collagen patch according to claim 3, characterized in that: In terms of weight, the essence comprises the following components: 0.02-1 parts of the recombinant collagen, 0.01-0.5 parts of the biomimetic collagen, 0.001-0.3 parts of the hyaluronic acid, 1-6 parts of the glycerol, 0.2-2 parts of the butylene glycol, 0.1-0.6 parts of the carbomer, 0.02-0.8 parts of the gallic acid and 0.02-0.8 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 0.01-5 parts of a pH adjuster and 0.2-0.5 parts of a preservative; When the balance is phosphate buffer, the essence further comprises 0.2-0.5 parts of preservatives.

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 1:10-10:1; b. The preservatives are caprylhydroxamic acid and glyceryl caprylate, and the mass ratio of caprylhydroxamic acid to glyceryl caprylate is 9:1-1:9; 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-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, the preservative and the 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 irradiation 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 15-30 minutes; when the irradiation sterilization is adopted, the dose of the irradiation sterilization is 10-25KGy.

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 7-8, wherein the amount of each component added is 2-5 times the amount of each component added in the preparation method of the collagen patch, and 0.05-5 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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