A transparent, highly thermostable biomimetic collagen, cosmetic, drug, tissue engineering material or medical device, and a preparation method and use thereof

Abstract

The present invention belongs to the technical field of medical and cosmetic raw materials, and particularly relates to a transparent and highly thermostable bionic collagen, cosmetics, drugs, tissue engineering materials or medical devices, and a preparation method and uses thereof. The structural formula of the bionic collagen of the present invention is H2N-(P-O-G) n -COOH; wherein, P-O-G is a peptide chain composed of proline, hydroxyproline and glycine residues. The present invention has prepared a bionic collagen with a transparent texture through multi-stage temperature control and improvement of the material ratio. After high-temperature sterilization, the bionic collagen still has a stable triple helix structure. The bionic collagen promotes wound healing and the expression of cellular collagen; it maintains the triple helix structure and fiber network structure within a large temperature range, has good thermal stability, and can be better applied to the fields of cosmetics, drugs, medical devices, biomaterials, etc., and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical fields of medical and cosmetic raw materials, and particularly relates to a transparent and highly thermostable bionic collagen, cosmetics, drugs, tissue engineering materials or medical devices, and a preparation method and use thereof. Background Art

[0002] Collagen is a protein widely present in animal connective tissues. Due to its good biocompatibility, excellent degradability, moisturizing property, repair and regeneration, and anti-aging properties, it is widely used in fields such as beauty skin care, cosmetic surgery, tissue engineering, wound repair, and health products.

[0003] However, since natural collagen is mostly extracted from animal tissues, there may be risks of immune rejection reaction or virus transmission; moreover, the molecular weight and quality of natural collagen are difficult to control, its mechanical properties are poor, and its degradation rate is difficult to regulate, which limits the clinical practical application of natural collagen. Therefore, researchers have developed bionic collagen, which overcomes the limitations of natural collagen by regulating the composition, structure, and preparation process of collagen, and provides safer, more effective, and more controllable collagen products.

[0004] From the perspective of existing literature and molecular design principles, the basic repeating unit of natural collagen is the tripeptide "Gly–X–Y", where the X and Y positions are often replaced by proline and hydroxyproline respectively (i.e., forming "Gly–Pro–Hyp"), and this region is the key "code" for maintaining the stability of the triple helix fold. Existing studies have shown that the homology between bovine collagen and the full-length sequence of human type I collagen is about 97.47%, and the homology with type III collagen is about 90.18%. The design goal of the bionic collagen Poly(POG)n is to simulate the triple helix fold structure of natural collagen by continuously repeating the "POG" unit (i.e., corresponding to the core "Gly–Pro–Hyp" in the actual sequence expression) during chemical synthesis. From the perspective of the core repeating unit, its sequence is exactly the same as that of "Gly–Pro–Hyp" which constitutes the key region of the triple helix in natural collagen, that is, the homology reaches 100% in this part.

[0005] The color of the bionic collagen depends on its purity and quality. There are various colors of bionic collagen, generally divided into white, transparent, yellow, gray, etc. Generally, it is considered that the color of a collagen solution with good quality should be close to colorless and in a clear and transparent state; the color of high-quality bionic collagen in a granular state should be white or off-white; the color of collagen peptides of general or low quality in a granular state may be yellow, gray, yellowish-gray or yellowish-brown mixed with a slightly reddish tint, and the color is dull; the darker the color, the worse the quality. Moreover, compared with milky bionic collagen, transparent bionic collagen has unique advantages. For example, it can better simulate natural tissues such as the cornea; it avoids the Tyndall effect and provides better visual effects and aesthetics. Therefore, researching and preparing a bionic collagen with a transparent texture is the common goal of experts in this field.

[0006] Most of the bionic collagens provided in the prior art are difficult to achieve a transparent texture. In transparent bionic collagen products, their thermal stability is difficult to meet the requirements. After being treated by steps such as high temperature or sterilization, their structures will be damaged and their biological activities will also be greatly affected. Therefore, constructing a bionic collagen with good thermal stability and a transparent texture is an urgent problem to be solved in this field. Summary of the Invention

[0007] In view of the problems of the prior art, the present invention provides a transparent and highly thermostable bionic collagen, cosmetics, drugs, tissue engineering materials or medical devices, and their preparation methods and uses.

[0008] The present invention provides a transparent and highly thermostable bionic collagen, whose structural formula is shown in Formula I:

[0009] Formula I: H2N-(P-O-G) n -COOH;

[0010] Wherein, P is a proline residue, O is a hydroxyproline residue, G is a glycine residue, and n≧4;

[0011] The bionic collagen is prepared by the following steps:

[0012] A condensation reaction is carried out using a condensation aid, a tripeptide and a dehydrating condensing agent to obtain a crude product solution; the reaction procedure of the condensation reaction is to raise the temperature to 30-50°C and then lower the temperature to 10-25°C;

[0013] The crude product solution is subjected to homogenization treatment; separation is carried out to obtain the product.

[0014] The feeding weight ratio of the condensation aid, tripeptide and dehydrating condensing agent is 0.7-2.3:7.2-24.6:27.4-36.2; the homogenization treatment is carried out in a homogenizer, and the rotation speed of the homogenizer is 5000-10000.

[0015] Preferably, the feeding weight ratio of the condensation aid, tripeptide and dehydrating condensing agent is 1-2:10-20:30-34.

[0016] Preferably, the feeding weight ratio of the condensation aid, tripeptide and dehydrating condensing agent is 2:10-15:30-34.

[0017] Preferably, the steps include the following:

[0018] Step 1, prepare a condensation aid solution;

[0019] Step 2, add the tripeptide with the structural formula of H2N-P-O-G-COOH to the condensation aid solution to obtain a tripeptide solution;

[0020] Step 3, prepare a dehydrating condensing agent solution;

[0021] Step 4, mix the tripeptide solution and the dehydrating condensing agent solution to carry out a condensation reaction to obtain a crude product solution;

[0022] Step 5, carry out homogenization treatment on the crude product solution;

[0023] Step 6, separate and purify to obtain.

[0024] Preferably, the temperature of the mixing in Step 4 is 0-10°C.

[0025] Preferably, the temperature of the temperature rise is raised to 40-50°C.

[0026] Preferably, the temperature of the re-cooling is re-cooled to 25°C.

[0027] Preferably, after re-cooling to 10-25°C, keep warm for 34-72 h.

[0028] Preferably, the rotation speed of the homogenizer is 9000.

[0029] Preferably, the dehydrating condensing agent is selected from at least one of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide or 1,3-dicyclohexylcarbodiimide or N,N'-diisopropylcarbodiimide.

[0030] Preferably, the dehydrating condensing agent is 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide.

[0031] Preferably, in step 1, after the condensation aid solution is prepared, it is stirred at 0-10°C for 1-4 h;

[0032] And / or, in step 2, the tripeptide solution is stirred at 15-30°C for 1-4 h, and then cooled to 0-10°C and maintained for 1-4 h;

[0033] And / or, in step 4, the mixing is that the dehydrating condensing agent solution is dropped into the tripeptide solution and dropped within 0.2-1.6 h;

[0034] And / or, in step 4, it is maintained at 30-50°C for 1.5-6 h;

[0035] And / or, the homogenization treatment in step 5 is carried out at ≤40°C for 0.25-1.2 h and stopped for 0.25-1.2 h; this is repeated 2-3 cycles;

[0036] And / or, the specific steps of the separation and purification in step 6 are: adding water with a volume 2.5-6 times that of the reaction solution, and performing membrane filtration using a hollow fiber membrane with a molecular weight of 3-5W; the membrane filtration is carried out 2-3 times.

[0037] Preferably, in step 1, the solvent of the condensation aid solution is phosphate buffer; in step 3, the solvent of the dehydrating condensing agent solution is phosphate buffer; the phosphate buffer is prepared from the following raw materials in molar parts:

[0038] Potassium chloride 3.2-4.3 mM,

[0039] Disodium hydrogen phosphate 8.8-13.1 mM,

[0040] Potassium dihydrogen phosphate 1.2-2.5 mM,

[0041] Sodium chloride 124-151 mM;

[0042] The pH of the phosphate buffer is 6.9-7.6;

[0043] And / or, the condensation aid is selected from at least one of 1-hydroxybenzotriazole or 1-hydroxy-7-azabenzotriazole or N-hydroxysuccinimide.

[0044] The present invention provides a method for preparing the above-mentioned transparent and high thermal stability bionic collagen, which is characterized by including the following steps:

[0045] A condensation reaction is carried out using a condensation aid, a tripeptide and a dehydrating condensing agent to obtain a crude product solution; the reaction procedure of the condensation reaction is to heat to 30-50°C and then cool to 10-25°C;

[0046] The crude product solution is subjected to homogenization treatment; separation is carried out to obtain the product.

[0047] The feeding weight ratio of the condensation aid, tripeptide and dehydrating condensing agent is 0.7 - 2.3:7.2 - 24.6:27.4 - 36.2;

[0048] The homogenization treatment is carried out in a homogenizer, and the rotation speed of the homogenizer is 5000 - 10000.

[0049] The present invention provides the use of the transparent and high thermal stability bionic collagen described in any one of the above in the preparation of cosmetics, drugs or tissue engineering materials.

[0050] Preferably, the cosmetic is a functional skin care product; the tissue engineering materials include tissue engineering materials for drug carriers, cosmetic plastic surgery or medical devices, and bio-based packaging materials; the medical devices include dressings, filling materials or engineering scaffolds for skin, bones, ligaments, peripheral nerves, and blood vessels.

[0051] Preferably, the functional skin care product is a facial mask, essence or facial cleanser.

[0052] The present invention provides a cosmetic or a drug, which is characterized in that it is made by using the transparent and high thermal stability bionic collagen described in any one of the above as an active ingredient and adding cosmetically or pharmaceutically acceptable excipients.

[0053] The present invention provides a tissue engineering material, which is characterized in that it is made by using the high thermal stability bionic collagen described in any one of the above as an active ingredient and adding medically acceptable excipients.

[0054] The present invention provides a medical device, which is characterized in that it is made by using the high thermal stability bionic collagen described in any one of the above as an active ingredient and adding medically acceptable excipients.

[0055] Preferably, the concentration of the high thermal stability bionic collagen is

[0056] The present invention provides a transparent and high thermal stability bionic collagen, its preparation method and use. The bionic collagen of the present invention has a structural formula of H2N-(P-O-G) n-COOH; wherein, P-O-G is a peptide chain composed of proline, hydroxyproline and glycine residues. Through multi-stage temperature control and improvement of the material feeding ratio and homogenization conditions, the present invention prepares a bionic collagen with a transparent texture. After high-temperature sterilization, the bionic collagen still has a stable triple helix structure. The bionic collagen promotes wound healing and the expression of cellular collagen, and its effect is similar to that of human-derived collagen; it maintains the triple helix structure and fiber network structure within the temperature range of 20°C - 95°C, has good thermal stability, and can be better applied to the fields of cosmetics, pharmaceuticals, medical devices, biomaterials, etc., with good application prospects.

[0057] Bionic collagen is an artificially synthesized collagen material designed and constructed through a bionic technology that crosses multiple disciplines. Its core feature lies in precisely simulating the stable triple helix structure of natural collagen and the collagen network fiber structure formed by self-assembly. The biological material of the present invention can be called "collagen" and has the same multi-level structure, function and quality controllability as natural collagen.

[0058] It is reasonable to call the bionic collagen material Poly(POG)n of the present invention "collagen". First of all, the multilevel structure of Poly(POG)n is consistent with that of natural collagen, and the multilevel structure includes the primary structure, secondary structure, tertiary structure and quaternary structure. In the primary structure, one of the main characteristics of natural collagen is that its amino acid sequence follows the "Gly-X-Y" repeating tripeptide unit (X / Y are mostly proline and hydroxyproline), which is also the basis for collagen to form a triple helix structure; Poly(POG)n constructs molecular chains through repeated P-O-G sequences. It should be noted that "POG" and "GPO" are equivalent in cyclic arrangement because the key to the function of collagen tripeptide is that one of every three amino acids must be glycine (Gly) to ensure the tight stacking and stability of the triple helix. Structurally speaking, Poly(POG)n does simulate the basic tripeptide repeating unit of natural collagen and is consistent with the primary structure of natural collagen; in the secondary structure, natural collagen forms a stable triple helix structure based on the tripeptide repeating unit through hydrogen bonds and hydrophobic interactions, while through precise chemical design and strict control of the preparation process, Poly(POG)n has successfully achieved a triple helix secondary structure similar to that of natural collagen; in the tertiary structure, natural collagen forms a tight three-dimensional folded structure through internal and external molecular interactions to maintain its water solubility and multifunctionality; while Poly(POG)n will also exhibit the molecular aggregation effect of natural collagen and form a similar triple helix network in an appropriate hydrated environment; in the quaternary structure, the Poly(POG)n material can be prepared into a composite structure similar to natural collagen fibers through process regulation and is widely used in tissue repair and scaffold materials. The bionic collagen molecules can form a highly cooperative collagen fiber structure through non-covalent bonds (such as hydrogen bonds and van der Waals forces), simulating the supramolecular properties of natural collagen.

[0059] In addition, the functions of Poly(POG)n are consistent with those of natural collagen. Natural collagen is the core component of the extracellular matrix (ECM), providing cell adhesion sites and promoting the proliferation and migration of fibroblasts, keratinocytes, endothelial cells, etc. Poly(POG)n has been experimentally verified to stably bind to cell surface receptors (such as integrins) and play the same biological role as natural collagen; after natural collagen degrades, it mainly generates small molecules such as glycine, proline and hydroxyproline, and these degradation products can be absorbed and utilized by the body, while Poly(POG)n also releases the above non-toxic products after degradation under various physiological conditions (pH, enzyme environment), showing degradability and harmlessness of the products; natural collagen is often used as a filling material and structural scaffold in medical and cosmetic applications. By adjusting parameters such as molecular weight and crosslinking density, Poly(POG)n has controllable mechanical properties and performs the same as natural collagen when applied to soft tissue repair, skin support and other scenarios.

[0060] The China National Medical Products Administration (NMPA) has clear regulations on the naming and application of collagen products in accordance with relevant regulations on drugs, medical devices, and cosmetics. The "Technical Specifications for Cosmetics Safety" (2021 edition) defines "collagen" as "proteins or oligopeptides containing a collagen triple helix structure". Poly(POG)n meets this definition, is functionally consistent with natural collagen, and has no source risks. China's "Medical Device Classification Rules and Classification Catalogue" (2021 edition) clearly classifies "collagen-based materials" as high-risk class III medical devices (such as implantable filling materials) and class II dressings. According to the "Regulations on the Supervision and Administration of Cosmetics" and the "Measures for the Registration and Filing of New Cosmetic Raw Materials", bionic collagen can be filed as a cosmetic raw material, and the safety of the raw material and its skin repair-promoting effect can be supported by existing functional verification methods. Among class III medical devices, Poly(POG)n can be applied to soft tissue filling materials, nerve regeneration catheters, bone repair scaffolds, etc. Among class II dressings, Poly(POG)n can be applied to burn and wound dressings, etc. In cosmetics, Poly(POG)n can be applied to anti-aging skin care products, and in cosmetic raw materials, Poly(POG)n can be applied to high-grade functional cosmetic raw materials.

[0061] The bionic collagen Poly(POG)n and its related materials have achieved a multi-level structure, function, and application value highly consistent with natural collagen through molecular design and synthesis technology. At the same time, they are superior to naturally extracted collagen in terms of quality safety and production controllability. These characteristics meet the NMPA and relevant regulations' definition of "collagen" and have high adaptability in the declaration in the fields of medical devices, cosmetics, and raw materials, and can normally enter the fields of class III medical devices, class II dressings, and cosmetics.

[0062] The bionic collagen of the present invention is highly consistent with human-derived type I and type III collagen in terms of sequence and function. However, due to the chemical synthesis method, it is not a humanized collagen in the traditional sense and can be regarded as bionic type I / III collagen. As a new generation of bionic biomaterials that break through the bottlenecks of traditional animal-extracted collagen and recombinant collagen, the bionic collagen of the present invention completely gets rid of the dependence on animal-derived components and eliminates risks such as pathogen carriage, virus contamination, and poor batch stability of materials from the source. By optimizing molecular design, it solves the performance bottlenecks of animal collagen such as easy degradation, poor thermal stability, and difficulty in chemical modification, as well as the problem of insufficient triple helix structure stability of recombinant collagen, and has unique advantages in biocompatibility, structural stability, industrial production, and sustainable development.

[0063] The bionic collagen of the present invention has the following advantages: (1) It has no risk of animal origin and is completely independent of animal extraction, avoiding the potential risks of animal-derived collagen; (2) It has a high degree of intelligent controllability. According to specific application requirements, it can accurately regulate the molecular weight, molecular structure and amino acid sequence, degradation rate, crosslinking density and pore structure, functional modification, etc. It can design artificial blood vessel patches with high tensile toughness and stability to meet the physiological conditions of blood flow impact and stress load. Without secondary crosslinking treatment, sufficient mechanical strength can be obtained, and the flexibility of the material is close to that of natural tissues, facilitating doctors to operate and use during surgery. By controlling the density of the triple helix collagen sequence, its adhesion to fibroblasts, smooth muscle cells and endothelial cells can be regulated, providing an ideal scaffold environment for blood vessel regeneration; (3) It has excellent biocompatibility and can be used in artificial collagen blood vessel patches. It gradually degrades with tissue regeneration while promoting the repair of endothelial cells and surrounding vascular tissues; (4) It can be mass-produced efficiently; (5) It has flexible self-assembly ability and can meet short-term and long-term medical needs. Short-term uses include wound dressings and functional skin care repair materials, and long-term uses include filling injections, artificial scaffolds and transplantation materials, providing long-term support for tissue reconstruction. The bionic collagen of the present invention is widely applicable to various tissue engineering and medical fields, and particularly shows significant value and application potential in high-end functional skin care, wound healing, soft tissue repair, regenerative medicine, tissue engineering scaffolds and blood vessel repair, etc.

[0064] Obviously, based on the above content of the present invention, according to the common general knowledge and customary means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can also be made.

[0065] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be understood as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. Brief Description of the Drawings

[0066] Figure 1 The circular dichroism spectrum of the bionic collagen prepared in Example 1 after heat treatment at 20 - 95 °C, where A is the circular dichroism spectrum of the bionic collagen before high-temperature sterilization after heat treatment at 20 - 95 °C, and B is the circular dichroism spectrum of the bionic collagen after high-temperature sterilization after heat treatment at 20 - 95 °C;

[0067] Figure 2 The circular dichroism spectrum of the collagen in the comparative example after moist heat sterilization after heat treatment at 20 - 95 °C;

[0068] Figure 3 The molecular weight result diagram of the bionic collagen prepared in Example 1;

[0069] Figure 4 Infrared spectrum result graph of the bionic collagen prepared in Example 1;

[0070] Figure 5 Circular dichroism spectrum graph of the bionic collagen prepared in Example 2 after heat treatment at 20 - 95°C;

[0071] Figure 6 Circular dichroism spectrum graph of the bionic collagen prepared in Example 3 after heat treatment at 20 - 95°C;

[0072] Figure 7 Circular dichroism spectrum graph of the bionic collagen prepared in Example 4 after heat treatment at 20 - 95°C;

[0073] Figure 8 Circular dichroism spectrum graph of the bionic collagen prepared in Example 5 after heat treatment at 20 - 95°C;

[0074] Figure 9 Circular dichroism spectrum graph of the bionic collagen prepared in Example 6 after heat treatment at 20 - 95°C;

[0075] Figure 10 Circular dichroism spectrum graph of the bionic collagen prepared in Example 7 after heat treatment at 20 - 95°C;

[0076] Figure 11 Circular dichroism spectrum graph of the bionic collagen prepared in Example 8 after heat treatment at 20 - 95°C;

[0077] Figure 12 Circular dichroism spectrum graph of the bionic collagen prepared in Example 9 after heat treatment at 20 - 95°C;

[0078] Figure 13 Circular dichroism spectrum graph of the bionic collagen prepared in Example 10 after heat treatment at 20 - 95°C;

[0079] Figure 14 Cell viability result graph of the bionic collagen incubated with cells for 1 day, where A is the cell viability result graph of the bionic collagen prepared in Example 1 incubated with cells for 1 day, and B is the cell viability result graph of the bionic collagen prepared in Comparative Example 1 incubated with cells for 1 day;

[0080] Figure 15 Cell viability result graph of the bionic collagen incubated with cells for 4 days, where A is the cell viability result graph of the bionic collagen prepared in Example 1 incubated with cells for 4 days, and B is the cell viability result graph of the bionic collagen prepared in Comparative Example 1 incubated with cells for 4 days;

[0081] Figure 16 It is a graph of cell viability results when the bionic collagen is incubated with cells for 7 days. Among them, A is the graph of cell viability results when the bionic collagen prepared in Example 1 is incubated with cells for 7 days, and B is the graph of cell viability results when the bionic collagen prepared in Comparative Example 1 is incubated with cells for 7 days;

[0082] Figure 17 It is a graph of the scratch test results of cells treated with bionic collagen in Experimental Example 3. Among them, the pictures in the first row are the scratch test results of the control group, and the pictures in the second row are the scratch test results of the experimental group;

[0083] Figure 18 It is a graph of the scratch test results of cells treated with the comparative collagen in Experimental Example 3. Among them, the pictures in the first row are the scratch test results of the control group, and the pictures in the second row are the scratch test results of the experimental group;

[0084] Figure 19 It is a graph of the experimental results of the collagen gene expression levels in HaCat cells treated with bionic collagen in Experimental Example 3. Among them, A is the graph of the relative expression level of the CollagenⅠ gene, and B is the graph of the relative expression level of the CollagenⅢ gene;

[0085] Figure 20 It is a graph of the experimental results of the collagen gene expression levels in HSF cells treated with bionic collagen in Experimental Example 3. Among them, A is the graph of the relative expression level of the CollagenⅠ gene, and B is the graph of the relative expression level of the CollagenⅢ gene;

[0086] Figure 21 It is a graph of the experimental results of the collagen gene expression levels in HaCat cells treated with the comparative collagen in Experimental Example 3. Among them, A is the graph of the relative expression level of the CollagenⅠ gene, and B is the graph of the relative expression level of the CollagenⅢ gene;

[0087] Figure 22 It is a graph of the experimental results of the collagen gene expression levels in HSF cells treated with the comparative collagen in Experimental Example 3. Among them, A is the graph of the relative expression level of the CollagenⅠ gene, and B is the graph of the relative expression level of the CollagenⅢ gene;

[0088] Figure 23 It is a graph of the state of collagen in aqueous solution after moist heat sterilization; among them, the solution graph in the left test tube shows the state of the comparative collagen in aqueous solution, and the solution graph in the right test tube shows the state of the bionic collagen prepared in Example 1 in aqueous solution;

[0089] Figure 24 It is a state diagram of collagen in aqueous solution after PBS treatment; among them, the solution diagram in the left test tube shows the state diagram of collagen in the comparative example in aqueous solution, and the solution diagram in the left test tube shows the state diagram of the biomimetic collagen prepared in Example 1 in aqueous solution;

[0090] Figure 25 It is a result diagram of the cell scratch experiment of the biomimetic collagen prepared in Example 1 after moist heat sterilization or filtration sterilization. Among them, the pictures in the first row are the results of the control group, the pictures in the second row are the results of the biomimetic collagen after moist heat sterilization, and the pictures in the third row are the results of the biomimetic collagen after filtration sterilization;

[0091] Figure 26 It is an experimental result diagram of the collagen gene expression level after the biomimetic collagen prepared in Example 1 is treated with HaCat cells after moist heat sterilization. Among them, A is the experimental result diagram of the relative expression level of the CollagenⅠ gene, and B is the experimental result diagram of the relative expression level of the CollagenⅢ gene;

[0092] Figure 27 It is an experimental result diagram of the collagen gene expression level after the biomimetic collagen prepared in Example 1 is treated with HSF cells after moist heat sterilization. Among them, A is the experimental result diagram of the relative expression level of the CollagenⅠ gene, and B is the experimental result diagram of the relative expression level of the CollagenⅢ gene. Detailed implementation mode

[0093] The reagents and raw materials used in the following examples and experimental examples are all commercially available products unless otherwise specified.

[0094] Example 1 A transparent and highly thermostable biomimetic collagen

[0095] This example provides a biomimetic collagen with high biological activity that is transparent, has good thermal stability, and no salting out. It is a protein with a triple helix structure, and its structural formula is as follows:

[0096] H2N-(P-O-G) n -COOH

[0097] P, O, and G in the formula are composed of the following amino acid residues:

[0098] P: proline,

[0099] O: hydroxyproline,

[0100] G: glycine;

[0101] n ≥ 4. Since the triple-helix bionic collagen of this embodiment is a mixture of molecules with different degrees of polymerization, there are multiple possible values for n, and its range can be estimated based on the characterization of the molecular weight.

[0102] The preparation method of the above triple-helix collagen is as follows:

[0103] 1. Preparation of phosphate buffer solution

[0104] Dilute the commercially available PBS solution (100 mM Na2HPO4·12H2O, 18 mM KH2PO4, 27 mM KCl, 1370 mM NaCl, pH = 7.4) tenfold to obtain the following, hereinafter referred to as PB solution.

[0105] 2. Condensation aid solution

[0106] Weigh 2 g of the condensation aid 1-hydroxybenzotriazole (HOBt), add it to 500 mL of PB solution, and stir at 4 °C for 2 h to form a uniform suspension.

[0107] 3. Preparation of tripeptide solution

[0108] Add 15 g of the tripeptide P-O-G (manufactured by Uniqs Inc.) to the suspension, heat up to 25 °C, and stir for 2 h; then cool down to 4 °C and keep warm for 1 h to obtain.

[0109] 3. Preparation of dehydrating condensing agent solution

[0110] Weigh 30 g of the dehydrating condensing agent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), add it to 500 mL of PB solution to obtain.

[0111] 4. Polycondensation reaction

[0112] Under the condition of 4 °C, slowly add the prepared dehydrating condensing agent solution dropwise to the prepared tripeptide solution, and finish dropping all the solution in 1 h. Heat up to 40 °C and keep for 3 h; cool down to 25 °C and keep for 36 h. Add 1000 mL of deionized water to the reaction solution, cool down to 4 °C and keep for 1 h. Load the reaction solution into a homogenizer, homogenize at 5000 revolutions per minute for 1 h, and stop for 1 h. Repeat this process 3 cycles. During this period, ensure that the system does not exceed 40 °C through a temperature control device.

[0113] 5. Separation and purification

[0114] Add 8 L of deionized water to the reaction solution, and perform membrane filtration using a hollow fiber membrane with a molecular weight of 50,000, and repeat the membrane filtration 3 times. During the membrane filtration process, the volume of the solution gradually decreases. Each time it decreases to 2 L, add deionized water to make it up to 10 L. Finally, an aqueous solution containing the target product bionic collagen is obtained.

[0115] Example 2 A transparent, highly thermostable, and highly bioactive biomimetic collagen

[0116] According to the method of Example 1, the differences are as follows: the dosage of the condensation auxiliary 1-hydroxybenzotriazole (HOBt) is 2 g, the dosage of the tripeptide P-O-G is 10 g, and the dosage of the dehydrating condensing agent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) is 34 g. The biomimetic collagen prepared in this example has similar transparency and stability (salting-out stability and thermal stability) to that of Example 1.

[0117] Example 3 A transparent, highly thermostable, and highly bioactive biomimetic collagen

[0118] According to the method of Example 1, the differences are as follows: the dosage of the condensation auxiliary 1-hydroxybenzotriazole (HOBt) is 1 g, the dosage of the tripeptide P-O-G is 20 g, and the dosage of the dehydrating condensing agent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) is 30 g. The biomimetic collagen prepared in this example has similar transparency and stability (salting-out stability and thermal stability) to that of Example 1.

[0119] Example 4 A transparent, highly thermostable, and highly bioactive biomimetic collagen

[0120] According to the method of Example 1, the difference is that in Step 4, "heating to 40°C" is changed to "heating to 50°C". The biomimetic collagen prepared in this example has similar transparency and stability (salting-out stability and thermal stability) to that of Example 1.

[0121] Example 5 A transparent, highly thermostable, and highly bioactive biomimetic collagen

[0122] According to the method of Example 1, the difference is that in Step 4, "heating to 40°C" is changed to "heating to 30°C". The biomimetic collagen prepared in this example has similar transparency and stability (salting-out stability and thermal stability) to that of Example 1.

[0123] Example 6 A transparent, highly thermostable, and highly bioactive biomimetic collagen

[0124] According to the method of Example 1, the difference is that in Step 4, "cooling to 25°C and maintaining for 36 h" is changed to "cooling to 25°C and maintaining for 70 h". The biomimetic collagen prepared in this example has similar transparency and stability (salting-out stability and thermal stability) to that of Example 1.

[0125] Example 7 A transparent, highly thermostable, and highly bioactive biomimetic collagen

[0126] According to the method of Example 1, the difference is that in step 4, "cool down to 25 °C and maintain for 36 h" is changed to "cool down to 10 °C and maintain for 36 h". The bionic collagen prepared in this example has similar transparent properties and stability (salting-out stability and thermal stability) to those of Example 1.

[0127] Example 8 A bionic collagen with high bioactivity, being transparent and having high thermal stability

[0128] According to the method of Example 1, the difference is that in step 4, the temperature when the prepared dehydrating condensing agent solution is dropped into the prepared tripeptide solution is changed to 10 °C. The bionic collagen prepared in this example has similar transparent properties and stability (salting-out stability and thermal stability) to those of Example 1.

[0129] Example 9 A bionic collagen with high bioactivity, being transparent and having high thermal stability

[0130] According to the method of Example 1, the difference is that in step 4, the rotation speed of the homogenizer is changed to 9000. The bionic collagen prepared in this example has similar transparent properties and stability (salting-out stability and thermal stability) to those of Example 1.

[0131] Example 10 A bionic collagen with high bioactivity, being transparent and having high thermal stability

[0132] According to the method of Example 1, the difference is that the dehydrating condensing agent is N,N'-diisopropylcarbodiimide. The bionic collagen prepared in this example has similar transparent properties and stability (salting-out stability and thermal stability) to those of Example 1.

[0133] Example 11 Moisturizing lotion and emulsion for anti-wrinkle and firming

[0134] This example provides a moisturizing lotion and emulsion, and its composition includes:

[0135] The triple-helix bionic collagen prepared in Example 1

[0136] Glycerol 10 wt%,

[0137] Polyethylene glycol 15 wt%,

[0138] Hyaluronic acid 5 wt%,

[0139] The balance is water.

[0140] Example 12 Moisturizing lotion and emulsion for anti-wrinkle and firming

[0141] This example provides a moisturizing lotion and emulsion, and its composition includes:

[0142] The triple-helix bionic collagen prepared in Example 1

[0143] Glycerol 10 wt%,

[0144] Polyethylene glycol 15 wt%,

[0145] Hyaluronic acid 5 wt%,

[0146] The balance is water.

[0147] Moisturizing lotion and emulsion for anti-wrinkle and firming in Example 13

[0148] This example provides a moisturizing lotion and emulsion, and its composition includes:

[0149] Triple-helix bionic collagen prepared in Example 1

[0150] Glycerol 10 wt%,

[0151] Polyethylene glycol 15 wt%,

[0152] Hyaluronic acid 5 wt%,

[0153] The balance is water.

[0154] Comparative Example 1

[0155] This comparative example provides a control sample used in the experiment.

[0156] The control sample is collagen prepared according to the scheme described in the patent with the publication number CN 117903293 A. This control sample has the same sequence and similar structure as the bionic collagen prepared in Example 1, but the difference lies in that multi-stage temperature control is not carried out during the preparation process, the material feeding ratio is different, and the film-passing conditions after the reaction are different, etc.

[0157] The technical solution of the present invention will be further described through experiments below.

[0158] Experimental Example 1 Characterization of the product

[0159] I. Experimental method

[0160] This experimental example characterizes the bionic collagen prepared in Example 1, including:

[0161] 1. Circular dichroism spectroscopy characterization

[0162] The comparative collagen and the bionic collagen of Example 1 before and after high-temperature and high-humidity sterilization (121 °C, 15 min) were prepared into aqueous solutions with a concentration of 0.2 mg / ml, and heat treatment was carried out at 20 °C, 40 °C, 60 °C, 80 °C, and 95 °C for 1 hour. 5 ml was taken from each solution and analyzed on a circular dichroism spectrometer. It was confirmed whether there was a positive peak unique to triple-helix structure collagen in each sample.

[0163] 2. Molecular weight characterization

[0164] Determination was carried out using SEC-MALS technology. A Wyatt multi-angle light scattering detector DAWN HELEOS II was used, and a Shodex SB-806M column was used.

[0165] 3. Infrared characterization

[0166] The bionic collagen of Example 1 was detected by infrared spectroscopy using a Thermo Fisher Scientific Nicolet iS20 infrared spectrometer.

[0167] II. Experimental results

[0168] 1. Circular dichroism spectroscopy characterization

[0169] The results of circular dichroism spectroscopy characterization of bionic collagen before and after high-temperature and high-humidity sterilization are as Figure 1 shown. It can be seen from the figure that before high-temperature and high-humidity sterilization, a positive peak in the range of 220 - 230 nm could be detected in the bionic collagen sample, indicating that the bionic collagen prepared in Example 1 has a triple helix structure. After treatment at 20 °C, 40 °C, 60 °C, 80 °C, and 95 °C, the peak height of the positive peak in the range of 220 - 230 nm hardly changed, indicating that the bionic collagen sample has good thermal stability and its triple helix structure was not damaged by high temperature. After the bionic collagen sample was sterilized by high-temperature and high-humidity, the positive peak in the range of 220 - 230 nm still existed. After treatment at 20 °C, 40 °C, 60 °C, 80 °C, and 95 °C, the peak height of the positive peak in the range of 220 - 230 nm hardly changed. It shows that after high-temperature sterilization, the bionic collagen sample still has good thermal stability and its triple helix structure was not damaged by high temperature.

[0170] The characterization results of the collagen in the comparative example after moisture and heat sterilization are as Figure 2As shown, it can be seen from the figure that for the comparative collagen sample, a positive peak can also be detected in the range of 220 - 230 nm, but the peak height is greatly reduced. As the heat treatment temperature increases, the peak height of the positive peak in the range of 220 - 230 nm is further reduced. Especially for heat treatment above 40 °C, the influence on the peak height at this place is significant. This indicates that the triple helix structure of the comparative collagen sample is significantly damaged after moist heat sterilization, and when further heat treated above 40 °C, its triple helix structure is further damaged, and the thermal stability is poor.

[0171] The results of this experiment show that the bionic collagen prepared in Example 1 of the present invention has a triple helix structure, and the triple helix structure has good thermal stability. Even after high-temperature sterilization and heat treatment at 95 °C, its triple helix structure can still be stably maintained. Moreover, for bionic collagen, whether a stable triple helix structure can be formed is related to its preparation method. Through multi-stage temperature control, adjusting the feeding ratio, sequence and interval of materials, homogenizing conditions, and membrane-passing conditions after reaction, etc., the triple helix bionic collagen prepared in the present invention can meet the requirements for forming a thermally stable triple helix structure.

[0172] 2. Molecular weight characterization

[0173] As Figure 3 shown, the results show that the weight-average molecular weight Mw of the bionic collagen prepared in Example 1 is 1.409×10 5 (g / mol).

[0174] 3. Results of infrared characterization

[0175] The results are as Figure 4 and Table 1 shown. The results show that 3326.00 cm -1 is the stretching vibration absorption peak of -OH and -NH-; 2949.71 cm -1 is the stretching vibration absorption peak of saturated -C-H; 1628.42 cm -1 is the stretching vibration absorption peak of C=O on the amide; 1554.12 cm -1 is the bending vibration absorption peak of N-H on the amide; 1449.38 cm -1 is the bending vibration absorption peak of saturated -CH2; 1403.61 cm -1 is the bending vibration absorption peak of -C-H; 1335.73 cm -1 is the stretching vibration absorption peak of C-N; 1234.89 cm -1 is the stretching vibration absorption peak of C-O. The results indicate that the bionic collagen product has been successfully synthesized in the present invention.

[0176] Table 1 Data analysis results of infrared spectra

[0177] <![CDATA[Absorption peak (cm -1 )]]> Absorption peak intensity Functional group Vibration type 3326.00 m O-H, N-H Stretching vibration 2949.71 m -C-H Stretching vibration 1628.42 s C=O Stretching vibration 1554.12 m N-H Bending vibration 1449.38 m -CH2 Bending vibration 1403.61 m -C-H Bending vibration 1335.73 m C-N Stretching vibration 1234.89 m C-O Stretching vibration

[0178] Experimental Example 2 Influence of Preparation Method on Thermal Stability

[0179] I. Experimental Method

[0180] For the bionic collagen prepared in Examples 1 - 10 and Comparative Example 1 in this experimental example, after autoclaving (121 °C, 15 min), an aqueous solution with a concentration of 0.2 mg / ml was prepared, and then heat treatment was carried out at 20 °C, 40 °C, 60 °C, 80 °C, and 95 °C for 1 hour respectively. 5 ml was taken from each solution and analyzed on a circular dichroism spectrometer respectively to evaluate the thermal stability of the bionic collagen of the present invention.

[0181] II. Experimental Results

[0182] The results of circular dichroism spectroscopy characterization of the bionic collagen prepared in Example 1 and Comparative Example 1 after autoclaving are as shown in Figure 1 Figure B.2, and the results of circular dichroism spectroscopy characterization of the bionic collagen prepared in Examples 2 - 10 after autoclaving are as shown in Figures 5 - 13 shown: After autoclaving, the positive peak in the range of 220 - 230 nm still exists, and the peak height of the bionic collagen prepared in Examples 2, 6, and 9 is higher than that of the bionic collagen prepared in Example 1 ( Figure 5 , 9 , 12), and the peak height of the bionic collagen prepared in other examples is significantly higher than that of the collagen in the comparative example.

[0183] After further heat treatment, after the bionic collagen prepared in Examples 1 - 10 was heat treated at a temperature above 40 °C, the peak height of the positive peak in the range of 220 - 230 nm did not change significantly. In particular, after the bionic collagen prepared in Examples 8 and 9 was further heat treated at 20 °C, 40 °C, 60 °C, 80 °C, and 95 °C, the peak height of the positive peak in the range of 220 - 230 nm did not change significantly ( Figure 11 , 12 ). It shows that the material feeding ratio, multi - stage temperature control method, rotation speed of the homogenizer, etc. have an impact on the thermal stability of the sample.

[0184] Compared with the collagen in the comparative example, the triple - helix structure of the collagen prepared in Examples 2 - 10 was not significantly damaged after autoclaving, and still had good thermal stability after further heat treatment. The specific comparison data are shown in Table 2.

[0185] Table 2 Circular Dichroism Spectroscopy Results of Collagen after Autoclaving

[0186]

[0187] Therefore, after the bionic collagen prepared in the embodiments of the present invention is sterilized by moist heat, the triple helix structure is not significantly damaged and still has good thermal stability. In particular, the types of materials, their feeding ratios, reaction temperatures, reaction times, and homogenizer rotation speeds in the preparation method have an impact on the thermal stability of the product.

[0188] Experimental Example 3 Effects of Bionic Collagen on Cell Proliferation, Migration, and Collagen Expression

[0189] In this experimental example, human-derived cells were used to study the effects of the bionic collagen prepared in Example 1 on cell proliferation, migration, and collagen expression. To evaluate whether the test sample has biosafety, wound healing promotion, and anti-wrinkle and firming effects.

[0190] I. Experimental Methods

[0191] 1. Cytotoxicity Experiment

[0192] The Cell Counting Kit-8 (CCK8) detection method was used to determine the effects of bionic collagen or control collagen at concentrations ranging from 0.1% to 10% on the proliferation of human skin fibroblasts (HSF) cells.

[0193] 2. Cell Scratch Assay

[0194] The scratch assay was used to study cell migration ability. Human immortalized keratinocytes (HaCaT) were used. The experimental group cells were treated with 0.5 mg / mL bionic collagen or 0.5 mg / mL control collagen for 0, 12 h, and 24 h; the control group cells were treated with the same volume of PBS. Then, the scratch assay was performed respectively. The specific steps are as follows:

[0195] (1) Cell culture: First, culture HACAT cells in a culture dish or 96-well plate until they form a monolayer.

[0196] (2) Create a scratch: Use a special scratch tool to draw a straight line on the cell monolayer to form a "wound".

[0197] (3) Wash: Gently wash the cells to remove any potentially detached cells and ensure that there are no cells in the scratch area.

[0198] (4) Culture and observation: Return the cells to the incubator for continued culture, and observe and record the situation of cell migration covering the scratch area at different time points (0 h, 12 h, 24 h).

[0199] 3. Determination of Collagen Expression

[0200] (1) Main Reagents

[0201] Low-sugar DMEM culture medium (Solarbio), fetal bovine serum (Gibco), PBS (VivaCell), trypsin (Gibco), cell lysate (Novoprotein).

[0202] (2) Main equipment

[0203] CO2 incubator (Thermo, 160i), biological safety cabinet (Suzhou Jing'an Antai, BSC-1604ⅡA2), inverted microscope (Leica, DMi8), QPCR instrument (Roche), floor-standing half-body ultraviolet therapy instrument (SigmaGo, SS-03AB).

[0204] (3) Test methods

[0205] The specific settings of the test groups are as follows:

[0206] Set up a blank control group (BC), a negative control group (NC), a positive control group (PC), and a collagen group Among them, the blank control group is not given drugs and radiation treatment; the negative control group is only irradiated with 9J / cm 2 UVA; the positive control group is irradiated with 9J / cm 2 UVA after drug administration. The drugs are 25 μg / mL vitamin C and 7 μg / mL vitamin E; the collagen group is divided into a comparative collagen group and a biomimetic collagen group prepared in Example 1. The collagen group is irradiated with 9J / cm 2 UVA after drug administration. The drug in the comparative collagen group is The comparative collagen at a certain concentration; the drug in the biomimetic collagen group is The biomimetic collagen at a certain concentration (prepared according to the method of Example 1).

[0207] The specific operation steps are as follows:

[0208] 1) Cell seeding: Seed HaCaT cells or HSF cells into a 24-well plate at 8×10 4 / well and incubate overnight in an incubator (37°C, 5% CO2). The culture medium is DMEM culture medium.

[0209] 2) Drug administration: When the cell confluence rate in the 24-well plate reaches 40% - 60%, drug administration is carried out. The blank control group and the negative control group add 1 mL of cell culture medium (DMEM medium) to each well; the positive control group adds 1 mL of culture medium (DMEM medium) containing 25 μg / mL vitamin C and 7 μg / mL vitamin E to each well; the collagen group adds 1 mL of culture medium (DMEM medium) containing the corresponding concentration of the sample to each well.

[0210] 3) Radiation: 24 h after the administration was completed, the negative control group, the positive control group, and the collagen group received UVA radiation with a total dose of 9 J / cm 2 At the same time, the blank control group was placed in the same environment (UVA radiation dose: 0 J / cm 2 ).

[0211] 4) Cell collection: 24 h after incubation and culture, after the culture ended, 0.5 mL of lysis buffer was added to each well and left at room temperature for 5 min to allow sufficient lysis. Then it was transferred to a 1.5 mL RNase-free Eppendorf tube, and RNA was extracted according to the operating procedures.

[0212] 5) Reverse transcription: According to the product instruction manual of the RNA reverse transcription kit, cDNA was synthesized.

[0213] 6) Quantitative real-time PCR (qRT-PCR) detection was performed on CollagenⅠ and CollagenⅢ.

[0214] III. Experimental Results

[0215] 1. Toxicity of the bionic collagen to cells

[0216] The results are as Figures 14 - 16 shown. The results indicate that neither the bionic collagen nor the collagen of the comparative example has an impact on the proliferation behavior of HSF cells, suggesting that the bionic collagen prepared in the present invention or the collagen of the comparative example is non-toxic to HSF cells, and both of these products have good biosafety.

[0217] 2. Effect of the bionic collagen on cell migration

[0218] The results of the scratch assay are as Figure 17 , 18 shown: Compared with the control group, the cells treated with the bionic collagen showed significantly increased proliferation at the scratch site at 12 h, and the scratch was almost healed at 24 h, indicating that the bionic collagen promoted cell migration and was beneficial to wound healing. For the cells treated with the collagen of the comparative example, the proliferation behavior at the scratch site was less than that of the control group, and the scratch width at 24 h was greater than that of the control group, proving that the collagen of the comparative example inhibited cell migration and was not conducive to wound healing.

[0219] 3. Effect of the bionic collagen on collagen expression

[0220] The qPCR experimental results of HaCaT cells are as Figure 19 shown: For the CollagenⅠ gene, the content of the CollagenⅠ gene in the cells treated with the bionic collagen increased significantly. In particular, when the administration concentrations were and When the concentration is [specific concentration], the promotion effect of the CollagenⅠ gene expression in cells is the best, and the promotion effect is significantly better than that of the blank control group. For the CollagenⅢ gene, in the cells treated with the bionic collagen, the content of the CollagenⅢ gene is equivalent to or slightly lower than that of the negative control group. The results show that the bionic collagen prepared in Example 1 of the present invention promotes the expression of the CollagenⅠ gene in HaCaT cells.

[0221] The qPCR experimental results of HSF cells are as Figure 20 shown: For the CollagenⅢ gene, in the cells treated with the bionic collagen, compared with the negative control group, the content of the CollagenⅢ gene increases significantly. Particularly, when the administration concentrations are and the promotion effect of the CollagenⅢ gene expression in cells is the best. The results show that the bionic collagen prepared in Example 1 of the present invention has a good promoting effect on the expression of the CollagenⅢ gene in HSF cells.

[0222] The detection results of the contents of the CollagenⅠ gene and the CollagenⅢ gene in the cells treated with the comparative example collagen are as Figure 21 and 22 shown: Compared with the negative control group, the expression of CollagenⅠ in the HaCaT cells treated with the comparative example collagen increases slightly, but is significantly lower than that of the bionic collagen group; the expression of CollagenⅢ in the HSF cells treated with the comparative example collagen is significantly lower than that of the bionic collagen group. The results show that the promoting effect of the comparative example collagen on the CollagenⅠ gene and the CollagenⅢ gene in cells is significantly weaker than that of the collagen prepared in Example 1 of the present invention.

[0223] The above experimental results show that the bionic collagen of the present invention has good biological safety and good ability to promote wound healing. In terms of collagen expression, the bionic collagen of the present invention promotes the expression of the CollagenⅠ gene in HaCaT cells, especially when the administration concentrations are and ; it has a good promoting effect on the expression of the CollagenⅢ gene in HSF cells, especially when the administration concentrations are and This shows that the bionic collagen has the effect of promoting collagen expression in cells. Therefore, the collagen of the present invention has the efficacy of anti-wrinkle and firming. Moreover, from the different effects of the comparative example collagen and the bionic collagen prepared in Example 1 of the present invention on cell migration and promoting collagen expression, the preparation method has an obvious influence on the effect of the bionic collagen.

[0224] Experimental Example 4: Effects of Sterilization and Salt Concentration on the Properties of Bionic Collagen

[0225] The collagen of the comparative example and the bionic collagen prepared in Example 1 of the present invention were respectively subjected to high-temperature and humid heat sterilization (121 °C, 15 min) or PBS treatment, and the properties of the products were observed.

[0226] The specific steps of the PBS treatment were as follows: The collagen of the comparative example and the bionic collagen prepared in Example 1 of the present invention were respectively dissolved in water to prepare an aqueous solution with a concentration of 0.5%; then, one-tenth volume of 10×PBS solution was added thereto.

[0227] The properties of the products after high-temperature and humid heat sterilization were as Figure 23 shown: The bionic collagen prepared in Example 1 was clear and transparent after sterilization, and there was no visual change; while the collagen of the comparative example became a suspension after high-temperature sterilization, with a large amount of white flocculates appearing, and it could not recover at room temperature.

[0228] The properties of the products before and after PBS treatment were as Figure 24 shown: The bionic collagen prepared in Example 1 was clear and transparent after PBS treatment; while the collagen of the comparative example showed white flocculent precipitation after adding PBS, and it could not recover.

[0229] In this experimental example, bionic collagen was also prepared according to the method of Comparative Example 1, except that a series of buffer solutions with different ratios were used, and the products were not in a transparent state and there was a salting-out problem; in this experimental example, bionic collagen was also prepared according to the method of Example 1, except that the reaction temperature was 20 °C throughout the process, and precipitation would occur after the product was sterilized by humid heat.

[0230] The results showed that the bionic collagen prepared in the experimental example of the present invention was transparent in color, had no salting out, good thermal stability, and high-temperature sterilization operation would not denature it.

[0231] The above results showed that the bionic collagen prepared in Example 1 had more stable properties and remained stable and uniform in properties after treatment with salt solution and high temperature.

[0232] Experimental Example 5: Biological Activity of Collagen after Sterilization Treatment

[0233] In this experimental example, the cell scratch experiment and the experiment on collagen expression were carried out according to the method of Experimental Example 3, except that the collagen used was collagen after humid heat sterilization (121 °C) or filter sterilization.

[0234] The results of the cell scratch experiment were as Figure 25 shown. After the collagen was sterilized by humid heat or filter sterilization, it still had good cell migration activity and could be used to promote wound healing.

[0235] The qPCR experimental results of HaCaT cells are as Figure 26 shown. After being sterilized by moist heat, the bionic collagen prepared in Example 1 still has a significant effect on promoting the expression of CollagenⅠ gene.

[0236] The qPCR experimental results of HSF cells are as Figure 27 shown. After being sterilized by moist heat, the bionic collagen prepared in Example 1 still has a significant effect on promoting the expression of CollagenⅢ gene.

[0237] The above results show that the bionic collagen prepared in Example 1 of the present invention still has good effects on promoting cell proliferation, migration and collagen expression after being sterilized by moist heat, and has good biological activity.

[0238] From the above examples and experimental examples, it can be seen that the present invention provides a transparent and highly thermostable bionic collagen, and the structural formula of the bionic collagen is H2N-(P-O-G) n -COOH; wherein, P-O-G is a peptide chain composed of proline, hydroxyproline and glycine residues. The present invention has prepared a bionic collagen with a transparent texture through multi-stage temperature control and improvement of the material ratio. After being sterilized at high temperature (121°C), the bionic collagen still has a stable triple helix structure; after being treated with a salt solution, there is no salting-out phenomenon. The bionic collagen has good biological safety, promotes wound healing, and promotes cell collagen expression. The bionic collagen maintains a triple helix structure in the temperature range of 20°C - 95°C, has good thermal stability, and can be better applied to fields such as cosmetics, pharmaceuticals, medical devices, and biomaterials, with good application prospects. Especially in tissue engineering and regenerative medicine fields such as artificial skin, vascular scaffolds, cartilage repair matrices and wound dressings, targeted drug delivery carriers, surface coatings of surgical sutures or implant devices, cosmetics and high-end skin care products for anti-aging and skin barrier repair, bioink materials, organ model printing materials, engineering functional materials for antibacterial and anti-fouling, functional food fields such as anti-aging beverages, plant-based foods, and functional food additives, high-end textiles and consumer products such as medical textiles, skin care clothing, degradable plastics and packaging materials, and experimental tools for scientific research such as collagen substrates, etc., it has broad prospects.

Claims

1. A transparent and highly thermostable bionic collagen, characterized in that, It is a mixture of triple-helix bionic collagen molecules with different degrees of polymerization formed by the structure shown in Formula I: Formula I: H2N-(P-O-G) n -COOH; Among them, P is a proline residue, O is a hydroxyproline residue, G is a glycine residue, and n ≥ 4; The bionic collagen is prepared according to the following steps: A condensation reaction is carried out using a condensation aid, P-O-G tripeptide and a dehydrating condensing agent to obtain a crude product solution; the reaction procedure of the condensation reaction is to raise the temperature to 30-50 °C, keep it for 1.5-6 h, then cool it down to 10-25 °C, and keep it warm for 34-72 h; The crude product solution is homogenized; separated to obtain; The feeding weight ratio of the condensation aid, P-O-G tripeptide and dehydrating condensing agent is 0.7-2.3: 7.2-24.6: 27.4-36.2; the homogenization treatment is carried out in a homogenizer, and the rotation speed of the homogenizer is 5000-10000.

2. The transparent and high thermal stability bionic collagen according to claim 1, characterized in that: The feeding weight ratio of the condensation aid, P-O-G tripeptide and dehydrating condensing agent is 1-2:10-20:30-34.

3. The transparent and high heat-stable biomimetic collagen according to claim 1, characterized in that, The steps include the following content: Step 1, prepare a condensation aid solution; Step 2, add a tripeptide with the structural formula H2N-P-O-G-COOH to the condensation aid solution to obtain a tripeptide solution; Step 3, prepare a dehydrating condensing agent solution; Step 4, mix the tripeptide solution and the dehydrating condensing agent solution, carry out a condensation reaction to obtain a crude product solution; Step 5, homogenize the crude product solution; Step 6, separate and purify to obtain.

4. The transparent and high thermal stability bionic collagen according to claim 1, characterized in that: The temperature of the temperature rise is to raise the temperature to 40-50 °C; And / or, the temperature of the subsequent cooling is to cool it down to 25 °C again.

5. The transparent and high thermal stability bionic collagen according to claim 1, characterized in that: The rotation speed of the homogenizer is 9000.

6. The transparent and highly thermostable biomimetic collagen according to claim 1, wherein: The dehydrating condensing agent is selected from at least one of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide or 1,3-dicyclohexylcarbodiimide or N,N'-diisopropylcarbodiimide.

7. The transparent and high thermal stability bionic collagen according to claim 3, characterized in that: In Step 1, after the condensation aid solution is prepared, it is stirred at 0-10 °C for 1-4 h; And / or, in Step 2, the tripeptide solution is stirred at 15-30 °C for 1-4 h, then cooled down to 0-10 °C, and kept for 1-4 h; And / or, in Step 4, the mixing is that the dehydrating condensing agent solution is dropped into the tripeptide solution and dropped within 0.2-1.6 h; And / or, the temperature of the mixing in Step 4 is 0-10 °C; And / or, in Step 5, the homogenization treatment is carried out at ≤40 °C, homogenized for 0.25-1.2 h, stopped for 0.25-1.2 h; repeated 2-3 cycles like this; And / or, the specific steps of the separation and purification in Step 6 are: add water with a volume 2.5-6 times that of the reaction solution, and carry out membrane filtration using a hollow fiber membrane with a molecular weight of 3-5W; the membrane filtration is carried out 2-3 times.

8. The transparent and high heat-stable bionic collagen according to claim 3, characterized in that: In Step 1, the solvent of the condensation aid solution is a phosphate buffer solution; in Step 3, the solvent of the dehydrating condensing agent solution is a phosphate buffer solution; the phosphate buffer solution is prepared using raw materials with the following molar concentrations: Potassium chloride 3.2-4.3 mM, Disodium hydrogen phosphate 8.8-13.1 mM, Potassium dihydrogen phosphate 1.2-2.5 mM, Sodium chloride 124-151 mM; The pH of the phosphate buffer solution is 6.9 - 7.6; and / or, the condensation aid is selected from at least one of 1-hydroxybenzotriazole or 1-hydroxy-7-azabenzotriazole or N-hydroxysuccinimide.

9. The preparation method of the transparent and high heat stability bionic collagen according to any one of claims 1-8, characterized in that, comprising the following steps: Performing a condensation reaction using a condensation aid, P-O-G tripeptide, and a dehydrating condensing agent to obtain a crude product solution; the reaction procedure of the condensation reaction is to raise the temperature to 30 - 50 °C, hold for 1.5 - 6 h, then lower the temperature to 10 - 25 °C, and keep warm for 34 - 72 h; Performing homogenization treatment on the crude product solution; separating to obtain the product; The feeding weight ratio of the condensation aid, P-O-G tripeptide, and the dehydrating condensing agent is 0.7 - 2.3: 7.2 - 24.6: 27.4 - 36.2; The homogenization treatment is carried out in a homogenizer, and the rotation speed of the homogenizer is 5000 - 10000.

10. Use of the transparent, high thermal stability biomimetic collagen according to any one of claims 1 - 8 in the preparation of cosmetics or tissue engineering materials.

11. The use according to claim 10, characterized in that: The cosmetics are functional skin care products; the tissue engineering materials include tissue engineering materials for drug carriers, cosmetic plastic surgery or medical devices, and bio-based packaging materials; the medical devices include dressings, filling materials or engineering scaffolds for skin, bones, ligaments, peripheral nerves, and blood vessels.

12. The use according to claim 11, characterized in that: The functional skin care products are facial masks, essence liquids or facial cleansers.

13. A cosmetic, characterized in that, It is prepared by using the transparent, high thermal stability biomimetic collagen according to any one of claims 1 - 8 as an active ingredient and adding excipients acceptable in cosmetics.

14. A tissue engineering material, characterized in that, It is prepared by using the transparent, high thermal stability biomimetic collagen according to any one of claims 1 - 8 as an active ingredient and adding excipients acceptable in medicine.

15. A medical device, characterized in that: It is prepared by using the transparent, high thermal stability biomimetic collagen according to any one of claims 1 - 8 as an active ingredient and adding excipients acceptable in medicine.

16. The cosmetic according to claim 13, the tissue engineering material according to claim 14, or the medical device according to claim 15, characterized in that: The concentration of the transparent and high thermal stability bionic collagen is 0.047 wt -0.52 wt .

Images