A biomimetic collagen with high thermal stability, a cosmetic, a drug, a tissue engineering material or a medical device, and a preparation method and use thereof

Abstract

The present invention belongs to the technical fields of medical and cosmetic raw materials, and particularly relates to a biomimetic collagen with high thermal stability, a cosmetic, a drug, a tissue engineering material or a medical device, and a preparation method and use thereof. The structural formula of the biomimetic 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. Through multi-stage temperature control and improvement of the material input ratio, the prepared biomimetic collagen still has a stable triple helix structure after moist heat sterilization; there is no salting-out phenomenon. The biomimetic collagen has good biological safety, promotes wound healing, and promotes the expression of cellular collagen. The biomimetic collagen maintains a triple helix structure and a fiber network structure within a relatively large temperature range, has good thermal stability, and can be better applied to the fields of cosmetics, drugs, medical devices, biological materials, 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 biomimetic collagen with high thermal stability, a cosmetic, a drug, a tissue engineering material or a medical device, and a preparation method and use thereof. Background Art

[0002] Biomimetic Collagen Protein is a synthetic material that mimics the structure and function of natural collagen. It replicates or simulates the characteristics of collagen in nature through scientific methods, usually having good biocompatibility and biodegradability, being able to interact well with human tissues, and being effectively utilized by the human body. The efficacy of biomimetic collagen products has been supported by clinical research. By mimicking the structure and function of natural collagen, biomimetic collagen provides new solutions for fields such as medicine, beauty, and biomaterials.

[0003] When applied, biomimetic collagen has sufficient plasticity; it can stimulate the production of collagen by fibroblast cells in the body; it can effectively promote the activity of mitochondria, accelerate the elimination of aging molecules and organelles by cells, achieving the effect of anti-aging; and its production process is not restricted by the natural environment, and it can avoid the raw material safety risks of traditional collagen with deep-sea fish, cattle, and pigs as the main sources. Therefore, the research and preparation of biomimetic collagen have received enthusiastic pursuit in the market and have also become a research hotspot for scientific researchers.

[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 research has 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 biomimetic 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, the "Gly–Pro–Hyp" that constitutes the key triple helix region in biomimetic collagen can be completely consistent with that in natural collagen, that is, achieving 100% homology in this part.

[0005] However, there are some problems in the preparation of biomimetic collagen, such as salting out phenomenon, and it is difficult to compound and prepare a medical aesthetic product preparation; the thermal stability is poor, and precipitation occurs after high-temperature or sterilization treatment. These problems seriously reduce the efficacy of biomimetic collagen and limit the wide application of biomimetic collagen.

[0006] Therefore, developing a bioactive bionic collagen with good thermal stability and no salting out 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 bionic collagen with high thermal stability, a cosmetic, a drug, a tissue engineering material or a medical device, and a preparation method and use thereof.

[0008] The present invention provides a bionic collagen with high thermal stability, and its 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 heat to 30-50°C and then cool 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, the tripeptide and the dehydrating condensing agent is 0.7-2.3:7.2-24.6:27.4-36.2.

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

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

[0017] Preferably, the temperature of the heating is heated to 30°C.

[0018] Preferably, the temperature of the subsequent cooling is cooled to 10°C.

[0019] Preferably, the homogenization treatment is carried out in a homogenizer, and the rotation speed of the homogenizer is greater than or equal to 2000 and less than 5000.

[0020] Preferably, the rotation speed of the homogenizer is 2000-4500.

[0021] Preferably, after cooling to 10-25°C, heat preservation is carried out for 34-72h.

[0022] Preferably, the dehydrating condensing agent is selected from at least one of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, 1,3-dicyclohexylcarbodiimide, and N,N'-diisopropylcarbodiimide.

[0023] Preferably, the dehydrating condensing agent is N,N'-diisopropylcarbodiimide.

[0024] Preferably, the steps include the following:

[0025] Step 1: Prepare a condensation aid solution.

[0026] Step 2: Add a tripeptide with the structural formula H2N-P-O-G-COOH to the condensation aid solution to obtain a tripeptide solution.

[0027] Step 3: Prepare a dehydrating condensing agent solution.

[0028] Step 4: Mix the tripeptide solution and the dehydrating condensing agent solution to carry out a condensation reaction to obtain a crude product solution.

[0029] Step 5: Carry out homogenization treatment on the crude product solution.

[0030] Step 6: Separate and purify to obtain the product.

[0031] Preferably, the temperature of the mixing in Step 4 is 0 - 10°C.

[0032] Preferably, in Step 1, after the condensation aid solution is prepared, it is stirred at 0 - 10°C for 1 - 4 h.

[0033] And / or, in Step 2, the tripeptide solution is stirred at 15 - 30°C for 1 - 4 h, then cooled to 0 - 10°C and kept for 1 - 4 h.

[0034] And / or, in Step 4, the mixing is that the dehydrating condensing agent solution is added dropwise to the tripeptide solution and dropped within 0.2 - 1.6 h.

[0035] And / or, in Step 4, it is kept at 30 - 50°C for 1.5 - 6 h.

[0036] And / or, the specific process of the homogenization treatment in Step 5 is to homogenize at ≤10°C for 0.25 - 1.2 h and stop for 0.25 - 1.2 h; repeat this 2 - 3 cycles.

[0037] 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.

[0038] 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 with raw materials in the following molar parts:

[0039] Potassium chloride 3.2 - 4.3 mM,

[0040] Disodium hydrogen phosphate 8.8 - 13.1 mM,

[0041] Potassium dihydrogen phosphate 1.2 - 2.5 mM,

[0042] Sodium chloride 124 - 151 mM;

[0043] The pH of the phosphate buffer is 6.9 - 7.6;

[0044] And / or, the condensation aid is selected from at least one of 1-hydroxybenzotriazole or 1-hydroxy-7-azabenzotriazole or N-hydroxysuccinimide.

[0045] The present invention provides a method for preparing the above-mentioned high thermal stability bionic collagen, comprising the following steps:

[0046] Performing a condensation reaction with 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;

[0047] Performing homogenization treatment on the crude product solution; separating to obtain the product.

[0048] The feeding weight ratio of the condensation aid, the tripeptide and the dehydrating condensing agent is 0.7 - 2.3:7.2 - 24.6:27.4 - 36.2.

[0049] The present invention provides the use of the above-mentioned high thermal stability bionic collagen in the preparation of cosmetics, drugs or tissue engineering materials.

[0050] Preferably, 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.

[0051] Preferably, the functional skin care products are facial masks, essence or facial cleansers.

[0052] The present invention provides a cosmetic or a drug, which is prepared by using the above-mentioned high thermal stability bionic collagen as an active ingredient and adding cosmetically or pharmaceutically acceptable excipients.

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

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

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

[0056] The present invention provides a bionic collagen with high thermal stability. Through multi-stage temperature control and material feeding ratio, the prepared bionic collagen still has a stable triple helix structure after being sterilized by moist heat (121 °C); there is no salting-out phenomenon after being treated with a salt solution. This bionic collagen has good biological safety, promotes wound healing, and promotes the expression of cell collagen. This bionic collagen maintains the triple helix structure and fiber network structure in the temperature range of 20 °C - 95 °C, has good thermal stability, and can be better applied in the fields of cosmetics, drugs, medical devices, biomaterials, etc. Its effect is similar to that of human collagen, and it has good application prospects.

[0057] Bionic collagen is an artificial synthetic collagen material designed and constructed through a bionic technology that crosses multiple disciplines. Its core feature is to precisely simulate 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 multi-level structure of Poly(POG)n is consistent with that of natural collagen. The multi-level 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 the formation of the triple helix structure of collagen; 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 is based on the tripeptide repeating unit and forms a stable triple helix structure through hydrogen bonds and hydrophobic interactions, etc. 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 multi-functionality; 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 characteristics of natural collagen.

[0059] In addition, the function of Poly(POG)n is consistent with that 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. 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 cross-linking 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 National Medical Products Administration (NMPA) of China 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 Cosmetic Safety" (2021 Edition) defines "collagen" as "proteins or oligopeptides containing a collagen triple helix structure". Poly(POG)n meets this definition, has functional consistency with natural collagen, and has no source risks. China's "Medical Device Classification Rules and Classification Catalog" (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 raw material safety and skin repair promotion effects 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 trauma 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 declarations 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 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 the molecular design or preparation method, it solves the performance bottlenecks of animal collagen such as easy degradation, poor thermal stability, and difficult chemical modification, as well as the problem of insufficient stability of the triple helix structure 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, does not rely on animal extraction at all, and avoids the potential risks of animal-derived collagen; (2) It has a high degree of intelligent controllability. According to specific application requirements, the molecular weight, molecular structure and amino acid sequence, degradation rate, crosslinking density and pore structure, functional modification, etc. can be precisely regulated; An artificial blood vessel patch with high tensile toughness and stability can be designed to meet the physiological conditions of blood flow impact and stress load, and sufficient mechanical strength can be obtained without secondary crosslinking treatment. The flexibility of the material is close to that of natural tissues, which is convenient for 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, and at the same time promotes the repair of endothelial cells and surrounding blood vessel tissues; (4) It can be produced efficiently in batches. Compared with the complexity of the natural extraction process, the bionic collagen of the present invention can achieve a highly controllable molecular weight and chemical consistency, ensuring uniformity in batch production; (5) It has a 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 a variety of 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 technical 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 above content of the present invention will be further described in detail below 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 belong to the scope of the present invention. Brief Description of the Drawings

[0066] Figure 1 The circular dichroism spectrum of the bionic collagen prepared for Example 1 after heat treatment at 20 - 95°C, where A is the circular dichroism spectrum of the bionic collagen before moist heat sterilization after heat treatment at 20 - 95°C, and B is the circular dichroism spectrum of the bionic collagen after moist heat 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 Molecular weight result graph of the biomimetic collagen prepared in Example 1;

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

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

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

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

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

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

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

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

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

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

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

[0080] Figure 15It is a graph of cell viability results when the bionic collagen is incubated with cells for 4 days. Among them, A is the graph of cell viability results when the bionic collagen prepared in Example 1 is incubated with cells for 4 days, and B is the graph of cell viability results when the bionic collagen prepared in Comparative Example 1 is 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 the cells treated with the 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 the 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 the HaCat cells treated with the bionic collagen in Experimental Example 3. Among them, A is the experimental result graph of the relative expression level of the CollagenⅠ gene, and B is the experimental result 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 the HSF cells treated with the bionic collagen in Experimental Example 3. Among them, A is the experimental result graph of the relative expression level of the CollagenⅠ gene, and B is the experimental result 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 the HaCat cells treated with the comparative collagen in Experimental Example 3. Among them, A is the experimental result graph of the relative expression level of the CollagenⅠ gene, and B is the experimental result 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 the HSF cells treated with the comparative collagen in Experimental Example 3. Among them, A is the experimental result graph of the relative expression level of the CollagenⅠ gene, and B is the experimental result graph of the relative expression level of the CollagenⅢ gene;

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

[0089] Figure 24 It is a state diagram of collagen in aqueous solution after being treated with PBS. Among them, the solution diagram in the left test tube shows the state diagram of the collagen in the comparative example in aqueous solution, and the solution diagram in the right 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 being sterilized by moist heat 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] For the reagents and raw materials used in the following examples and experimental examples, those not specifically stated are all commercially available products.

[0094] Example 1 A biomimetic collagen with good thermal stability, no salting out and high biological activity

[0095] This example provides a biomimetic collagen with good thermal stability, no salting out and high biological activity. 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 in this example 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. Condensation aid solution

[0104] (1) Dilute the commercially available PBS solution (100 mM Na2HPO4·12H2O, 18 mM KH2PO4, 27 mM KCl, 1370 mM NaCl, pH = 7.4) ten times to obtain the phosphate buffer solution, hereinafter referred to as PB solution.

[0105] (2) 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.

[0106] 2. Preparation of tripeptide solution

[0107] Add 15 g of the tripeptide P-O-G (manufactured by Uniqs Inc.) to the suspension, raise the temperature to 25 °C, and stir for 2 h; then lower the temperature to 4 °C and keep it warm for 1 h to obtain it.

[0108] 3. Preparation of dehydrating condensing agent solution

[0109] Weigh 30 g of the dehydrating condensing agent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), and add it to 500 mL of PB solution to obtain it.

[0110] 4. Polycondensation reaction

[0111] At 4 °C, add the prepared dehydrating condensing agent solution dropwise to the prepared tripeptide solution, and finish dropping all the solution in 1 h. Raise the temperature to 40 °C and keep it for 3 h; lower the temperature to 25 °C and keep it for 36 h. Add 1000 mL of deionized water to the reaction solution, lower the temperature to 4 °C, and keep it for 1 h. Load the reaction solution into a homogenizer, homogenize at 2000 revolutions per minute for 1 h, and stop for 1 h. Repeat this 3 cycles. During this period, ensure that the system does not exceed 10 °C through a temperature control device.

[0112] 5. Separation and purification

[0113] 8 L of deionized water was added to the reaction solution, and membrane filtration was carried out using a hollow fiber membrane with a molecular weight of 5 W. The membrane filtration was repeated 3 times. During the membrane filtration process, the volume of the solution gradually decreased. Each time it decreased to 2 L, deionized water was added to make it up to 10 L. Finally, an aqueous solution containing the target product, bionic collagen, was obtained.

[0114] Example 2 A bionic collagen with good thermal stability, no salting out, and high biological activity

[0115] According to the method of Example 1, the difference is that: the amount of the condensation auxiliary 1-hydroxybenzotriazole (HOBt) is 2 g, the amount of the tripeptide P-O-G is 10 g, and the amount of the dehydrating condensing agent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) is 34 g. The bionic collagen prepared in this example has a stability similar to that of Example 1.

[0116] Example 3 A bionic collagen with good thermal stability, no salting out, and high biological activity

[0117] According to the method of Example 1, the difference is that: the amount of the condensation auxiliary 1-hydroxybenzotriazole (HOBt) is 1 g, the amount of the tripeptide P-O-G is 20 g, and the amount of the dehydrating condensing agent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) is 30 g. The bionic collagen prepared in this example has a stability similar to that of Example 1.

[0118] Example 4 A bionic collagen with good thermal stability, no salting out, and high biological activity

[0119] According to the method of Example 1, the difference is that: in Step 4, “heating to 40 °C” was changed to “heating to 50 °C”. The bionic collagen prepared in this example has a stability similar to that of Example 1.

[0120] Example 5 A bionic collagen with good thermal stability, no salting out, and high biological activity

[0121] According to the method of Example 1, the difference is that: in Step 4, “heating to 40 °C” was changed to “heating to 30 °C”. The bionic collagen prepared in this example has a stability similar to that of Example 1.

[0122] Example 6 A bionic collagen with good thermal stability, no salting out, and high biological activity

[0123] According to the method of Example 1, the difference is that: in Step 4, “cooling to 25 degrees and maintaining for 36 h” was changed to “cooling to 25 degrees and maintaining for 70 h”. The bionic collagen prepared in this example has a stability similar to that of Example 1.

[0124] Example 7. A bionic collagen with good thermal stability, no salting out, and high biological activity

[0125] According to the method of Example 1, the difference is that in Step 4, "cool down to 25°C and hold for 36 h" is changed to "cool down to 10°C and hold for 36 h". The bionic collagen prepared in this example has a stability similar to that of Example 1.

[0126] Example 8. A bionic collagen with good thermal stability, no salting out, and high biological activity

[0127] 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 a stability similar to that of Example 1.

[0128] Example 9. A bionic collagen with good thermal stability, no salting out, and high biological activity

[0129] According to the method of Example 1, the difference is that in Step 4, the rotation speed of the homogenizer is changed to 4500. The bionic collagen prepared in this example has a stability similar to that of Example 1.

[0130] Example 10. A bionic collagen with good thermal stability, no salting out, and high biological activity

[0131] 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 a stability similar to that of Example 1.

[0132] Example 11. A moisturizing lotion and emulsion for anti-wrinkle and firming

[0133] This example provides a moisturizing lotion and emulsion, and its composition includes:

[0134] The triple-helix bionic collagen prepared in Example 1

[0135] Glycerol 10 wt%,

[0136] Polyethylene glycol 15 wt%,

[0137] Hyaluronic acid 5 wt%,

[0138] The balance is water.

[0139] Example 12. A moisturizing lotion and emulsion for anti-wrinkle and firming

[0140] This example provides a moisturizing lotion and emulsion, and its composition includes:

[0141] The triple-helix bionic collagen prepared in Example 1

[0142] Glycerin 10 wt%,

[0143] Polyethylene glycol 15 wt%,

[0144] Hyaluronic acid 5 wt%,

[0145] The balance is water.

[0146] Moisturizing Lotion for Anti-Wrinkle and Firming in Example 13

[0147] This example provides a moisturizing lotion, and its composition includes:

[0148] Triple-Helix Bionic Collagen Prepared in Example 1

[0149] Glycerin 10 wt%,

[0150] Polyethylene glycol 15 wt%,

[0151] Hyaluronic acid 5 wt%,

[0152] The balance is water.

[0153] Comparative Example 1

[0154] This comparative example provides a control sample used in the experiment.

[0155] The control sample is collagen prepared according to the scheme of Example 1 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 is that there is no multi-stage temperature control during the preparation process, the material feeding ratio is different, and the membrane-passing conditions after the reaction are different.

[0156] The technical solution of the present invention will be further described through experiments below.

[0157] Experimental Example 1 Characterization of the Product

[0158] I. Experimental Method

[0159] This experimental example characterizes the bionic collagen prepared in Example 1, including:

[0160] 1. Circular Dichroism Spectroscopy Characterization

[0161] The collagen in the comparative example and the bionic collagen in Example 1 before and after moist heat sterilization (121 °C, 15 min) were prepared into an aqueous solution 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. For each solution, 5 ml was taken 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.

[0162] 2. Molecular weight characterization

[0163] It is measured using SEC-MALS technology. The Wyatt multi-angle light scattering detector DAWN HELEOS II is used, and the column is Shodex SB-806M.

[0164] 3. Infrared characterization

[0165] The bionic collagen of Example 1 is detected by infrared spectrum using the Thermo Fisher Scientific NicoletiS20 infrared spectrometer.

[0166] II. Experimental results

[0167] 1. Circular dichroism spectrum characterization

[0168] The results of circular dichroism spectrum characterization of the bionic collagen prepared in Example 1 before and after moist heat sterilization are as Figure 1 shown. It can be seen from the figure that before moist heat sterilization, a positive peak in the range of 220 - 230 nm can be detected for 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, and 80 °C, the peak height of the positive peak in the range of 220 - 230 nm hardly changes. After heat treatment at 95 °C, the peak height decreases slightly, indicating that the bionic collagen sample has good thermal stability and its triple helix structure is not damaged by high temperature. After the bionic collagen sample is sterilized by moist heat, the positive peak in the range of 220 - 230 nm still exists. After treatment at 20 °C, 40 °C, and 60 °C, the peak height of the positive peak in the range of 220 - 230 nm hardly changes. After heat treatment at 80 °C, the peak height decreases slightly, indicating that after being sterilized by moist heat, the bionic collagen sample still has good thermal stability and its triple helix structure is not damaged by high temperature.

[0169] The characterization results of the collagen in the comparative example after moist heat sterilization are as Figure 2 shown. It can be seen from the figure that for the collagen sample in the comparative example, a positive peak can also be detected in the range of 220 - 230 nm, but the peak height decreases greatly. As the heat treatment temperature increases, the peak height of the positive peak in the range of 220 - 230 nm further decreases, especially for heat treatment above 40 °C, which has a great impact on the peak height at this place. This indicates that the triple helix structure of the collagen sample in the comparative example is damaged after moist heat sterilization, and when heat treated above 40 °C, its triple helix structure is further damaged and the thermal stability is poor.

[0170] The results of this experiment show that the bionic collagen prepared in the embodiments of the present invention has a triple helix structure, and the triple helix structure has good thermal stability. Even after autoclaving and heat treatment at 95 °C, its triple helix structure can still be stably maintained. Moreover, for bionic collagen, whether it can form a stable triple helix structure is related to its preparation method. By controlling the temperature in multiple stages, adjusting the feeding ratio, sequence and interval of materials, and the conditions for membrane filtration after reaction, etc., the triple helix bionic collagen prepared by the present invention can meet the requirements for forming a triple helix structure with high thermal stability.

[0171] 2. Molecular weight characterization

[0172] As Figure 3 shown, the results show that the weight-average molecular weight Mw of the bionic collagen prepared in Example 1 is 6.646×10 6 (g / mol).

[0173] 3. Results of infrared characterization

[0174] The results are as Figure 4 and Table 1 shown. The results show that 3327.16 cm -1 is the stretching vibration absorption peak of -OH and -NH-; 2949.38 cm -1 is the stretching vibration absorption peak of saturated -C-H; 1626.35 cm -1 is the stretching vibration absorption peak of C=O on the amide; 1551.99 cm -1 is the bending vibration absorption peak of N-H on the amide; 1446.35 cm -1 is the bending vibration absorption peak of saturated -CH2; 1402.73 cm -1 is the bending vibration absorption peak of -C-H; 1334.43 cm -1 is the stretching vibration absorption peak of C-N; 1233.78 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.

[0175] Table 1 Data analysis results of infrared spectra

[0176]

[0177]

[0178] Influence of the preparation method in Experimental Example 2 on thermal stability

[0179] I. Experimental method

[0180] In this experimental example, the biomimetic collagen prepared in Examples 1-10 and Comparative Example 1 was prepared into an aqueous solution with a concentration of 0.2 mg / ml after moist heat sterilization (121 °C, 15 min), and then heat-treated 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 to evaluate the thermal stability of the biomimetic collagen of the present invention.

[0181] II. Experimental Results

[0182] The circular dichroism spectral characterization results of the biomimetic collagen prepared in Example 1 and Comparative Example 1 after moist heat sterilization are as Figure 1 shown in B, 2. The circular dichroism spectral characterization results of the biomimetic collagen prepared in Examples 2-10 after moist heat sterilization are as Figures 5 - 13 shown: After moist heat sterilization, the positive peak in the range of 220-230 nm still exists, and the peak height hardly changes. The peak height of the biomimetic collagen prepared in Examples 4-8, 10 in the range of 220-230 nm is higher than that of the biomimetic collagen prepared in Example 1, indicating that the biomimetic collagen prepared by the present invention still has a stable triple helix structure after high-temperature moist heat sterilization.

[0183] After further heat treatment at 20 °C, 40 °C, 60 °C, 80 °C, and 95 °C for the biomimetic collagen prepared in Examples 2 and 9, the peak height of the positive peak in the range of 220-230 nm hardly changes ( Figure 5 , 12 ). After further heat treatment at 20 °C, 40 °C, 60 °C, 80 °C for the biomimetic collagen prepared in Examples 1, 5, 7, the peak height of the positive peak in the range of 220-230 nm hardly changes ( Figure 1 B, 8, 10).

[0184] After further heat treatment at 20 °C, 40 °C, 60 °C for the biomimetic collagen prepared in Examples 3, 4, 6, 8, 10, the peak height of the positive peak in the range of 220-230 nm hardly changes ( Figure 7 , 9 , 11, 13), indicating that the thermal stability of the biomimetic collagen prepared by the present invention is better than that of the biomimetic collagen in the comparative example; the raw material composition and ratio, and temperature control have a certain influence on the thermal stability of the biomimetic collagen. Compared with the collagen in the comparative example, the triple helix structure of the collagen prepared in Examples 2-10 is not damaged after moist heat sterilization, and after further heat treatment, it still has good thermal stability. The specific comparison data are shown in Table 2.

[0185] Table 2 Circular dichroism spectral results of collagen after moist heat sterilization

[0186]

[0187]

[0188] Therefore, after the bionic collagen prepared in the embodiments of the present invention is sterilized by moist heat, the triple helix structure is not damaged and still has good thermal stability. In particular, the raw material composition and ratio, the multi-stage temperature control method, the homogenizer rotation speed, etc. in the preparation method have an impact on the thermal stability of the product.

[0189] Experimental Example 3 Effects of Bionic Collagen on Cell Proliferation, Migration and Collagen Expression

[0190] 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, so as to evaluate whether the sample to be tested has biological safety, wound healing promotion and anti-wrinkle and firming effects.

[0191] I. Experimental Methods

[0192] 1. Cytotoxicity Experiment

[0193] The Cell Counting Kit-8 (CCK8) detection method was used to determine the effects of bionic collagen or collagen in the control example at concentrations in the range of 0.1%-10% on the proliferation of human skin fibroblasts (HSF) cells.

[0194] 2. Cell Scratch Experiment

[0195] The scratch experiment was used to study the cell migration ability. Human immortalized keratinocytes (HaCaT) were used. The cells in the experimental group were treated with bionic collagen at a concentration of 0.5 mg / mL or collagen in the control example at a concentration of 0.5 mg / mL for 0, 12 h, and 24 h; the cells in the control group were treated with the same volume of PBS. Then the scratch experiment was carried out respectively. The specific steps are as follows:

[0196] (1) Cell culture: First, culture HACAT cells in a culture dish or 96-well plate until they form a monolayer.

[0197] (2) Create a scratch: Use a special scratch tool to draw a straight line on the cell monolayer to form a "wound".

[0198] (3) Cleaning: Gently clean the cells to remove any cells that may have fallen off to ensure that there are no cells in the scratch area.

[0199] (4) Culture and observation: Put the cells back into the incubator for continued culture, and observe and record the situation of cell migration covering the scratch area at different time points (0 hour, 12 hours, 24 hours).

[0200] 3. Determination of Collagen Expression

[0201] (1) Main Reagents

[0202] Low-glucose DMEM culture medium (Solarbio), fetal bovine serum (Gibco), PBS (VivaCell), trypsin (Gibco), cell lysate (Novoprotein).

[0203] (2) Main Equipment

[0204] 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 (Sigma High, SS-03AB).

[0205] (3) Test Methods

[0206] The specific settings of the test groups are as follows:

[0207] 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 25μg / mL vitamin C and 7μg / 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).

[0208] The specific operation steps are as follows:

[0209] 1) Cell seeding: Seed HaCaT cells or HSF cells at 8×10 4 / well into a 24-well plate and incubate overnight in an incubator (37°C, 5% CO2). The culture medium is DMEM culture medium.

[0210] 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 1mL of cell culture medium (DMEM medium) to each well; the positive control group adds 1mL of culture medium (DMEM medium) containing 25μg / mL vitamin C and 7μg / mL vitamin E to each well; the comparative collagen group and the collagen group add 1mL of culture medium (DMEM medium) containing the corresponding concentration of samples to each well respectively.

[0211] 3) Radiation: 24 h after the administration was completed, the negative control group, positive control group, comparative collagen group and bionic collagen group received UVA radiation with a total dose of 9 J / cm 2 , while at the same time, the blank control group was placed in the same environment (UVA radiation dose was 0 J / cm 2 ).

[0212] 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 it was placed at room temperature for 5 min to allow sufficient lysis, then transferred to a 1.5 mL RNase-free Eppendorf tube, and RNA was extracted according to the operating procedures.

[0213] 5) Reverse transcription: According to the product instructions of the RNA reverse transcription kit, cDNA was synthesized.

[0214] 6) qRT-PCR detection was performed on CollagenⅠ and CollagenⅢ.

[0215] III. Experimental results

[0216] 1. Toxicity of bionic collagen to cells

[0217] The results are as Figures 14 - 16 shown. The results show that neither bionic collagen nor comparative collagen has an impact on the proliferation behavior of HSF cells, indicating that raw collagen or comparative collagen is non-toxic to HSF cells, and both of these products have good biosafety.

[0218] 2. Effect of bionic collagen on cell migration

[0219] The results of the scratch assay are as Figure 17 , 18 shown: Compared with the control group, the cells treated with bionic collagen showed significantly increased proliferation at the scratch site at 12 h, and the scratch was almost healed at 24 h, indicating that bionic collagen promoted cell migration and was beneficial to wound healing. For the cells treated with comparative collagen, 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 comparative collagen would inhibit cell migration and was not conducive to wound healing.

[0220] 3. Effect of bionic collagen on collagen expression

[0221] 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 bionic collagen increased significantly. In particular, when the administration concentrations were and At this time, 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.

[0222] 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 and increases with the increase of the concentration of the bionic collagen. 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.

[0223] The detection results of the CollagenⅠ gene and the CollagenⅢ gene contents in the cells treated with the comparative collagen are as Figure 21 、 22 shown: Compared with the negative control group, the expression of CollagenⅠ in HaCaT cells treated with the comparative collagen increases slightly, but is significantly lower than that of the bionic collagen group; the expression of CollagenⅢ in HSF cells treated with the comparative collagen is also significantly lower than that of the bionic collagen group. The results show that the promoting effect of the comparative 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.

[0224] 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 concentration is and ; it has a good promoting effect on the expression of the CollagenⅢ gene in HSF cells. It 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 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.

[0225] Experimental Example 4 Effects of sterilization and salt concentration on the properties of bionic collagen

[0226] The comparative collagen and the biomimetic collagen prepared in Example 1 of the present invention were respectively subjected to moist heat sterilization (121 °C, 15 min) or PBS treatment, and the properties of the products were observed.

[0227] The specific steps of PBS treatment were as follows: The comparative collagen and the biomimetic collagen prepared in Example 1 of the present invention were respectively dissolved in water to form an aqueous solution with a concentration of 0.5%; then one-tenth volume of 10×PBS solution was added thereto respectively.

[0228] The properties of the products after moist heat sterilization were as Figure 23 shown: The biomimetic collagen prepared in Example 1 was in a clear state after sterilization, and there was no visual change; while the comparative collagen became a suspension with a large amount of white flocculants after moist heat sterilization and could not recover at room temperature.

[0229] The properties of the products before and after PBS treatment were as Figure 24 shown: The biomimetic collagen prepared in Example 1 was in a clear state after PBS treatment, and there was no visual change; while the comparative collagen showed white flocculent precipitation after adding PBS and could not recover.

[0230] In this experimental example, biomimetic 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 salting out problems existed in all products; in this experimental example, biomimetic 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 moist heat sterilization of the product.

[0231] The results showed that the biomimetic collagen prepared in the examples of the present invention had no salting out, good thermal stability, and moist heat sterilization operation would not denature it.

[0232] The above results showed that the biomimetic collagen prepared in Example 1 had more stable properties and remained stable and uniform in properties after treatment with salt solution and high temperature.

[0233] Biological activity of collagen after sterilization treatment in Experimental Example 5

[0234] 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 moist heat sterilization (121 °C) or filter sterilization.

[0235] The results of the cell scratch experiment were as Figure 25 shown. After moist heat sterilization or filter sterilization, the collagen still had good cell migration activity and could be used to promote wound healing.

[0236] The qPCR experiment results of the cells were as Figure 26 、 27As shown in the figure. After the bionic collagen prepared in Example 1 was sterilized by moist heat, it still had a significant effect on promoting the expression of CollagenⅠ gene.

[0237] The above results indicate 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 bionic collagen with high thermal stability, 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. By multi-stage temperature control and improvement of the material ratio in the present invention, the prepared bionic collagen still has a stable triple helix structure after being sterilized by moist heat (121 °C); there is no salting-out phenomenon after being treated with a salt solution. This bionic collagen has good biological safety, promotes wound healing, and promotes cell collagen expression. This 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, drugs, medical devices, and biomaterials, with good application prospects. Especially in the fields of tissue engineering and regenerative medicine such as artificial skin, vascular scaffolds, cartilage repair matrices and wound dressings, targeted drug delivery carriers, surface coatings of surgical suture materials 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 biomimetic collagen with high thermal stability, 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; Wherein, 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: Using a condensation aid, P-O-G tripeptide and a dehydrating condensing agent to carry out a condensation reaction to obtain a crude product solution; the reaction procedure of the condensation reaction is to raise the temperature to 30-50°C, maintain for 1.5-6h, and then lower the temperature to 10-25°C and keep warm for 34-72h; The crude product solution is subjected to homogenization treatment; separation is carried out to obtain the product; 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.

2. The biomimetic collagen with high thermal stability according to claim 1, wherein: 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 biomimetic collagen with high thermal stability according to claim 1, characterized in that: The temperature of the temperature increase is to increase the temperature to 30°C; And / or, the temperature of the subsequent temperature decrease is to decrease the temperature to 10°C.

4. The biomimetic collagen with high thermal stability according to claim 1, characterized in that: The homogenization treatment is carried out in a homogenizer, and the rotation speed of the homogenizer is greater than or equal to 2000 and less than 5000.

5. The biomimetic collagen with high thermal stability according to claim 4, characterized in that: The rotation speed of the homogenizer is 2000-4500.

6. The biomimetic collagen with high thermal stability according to claim 1, characterized in that: 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 biomimetic collagen with high thermal stability 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 to carry out a condensation reaction to obtain a crude product solution; Step 5, carry out homogenization treatment on the crude product solution; Step 6, carry out separation and purification to obtain the product.

8. The bionic collagen with high thermal stability according to claim 7, characterized in that: In step 1, after the condensation aid solution is prepared, it is stirred at 0-10°C for 1-4h; And / or, in step 2, the tripeptide solution is stirred at 15-30°C for 1-4h, and then the temperature is lowered to 0-10°C and kept for 1-4h; And / or, the mixing in step 4 is that the dehydrating condensing agent solution is added dropwise to the tripeptide solution and dropped within 0.2-1.6h; And / or, the temperature of the mixing in step 4 is 0-10°C; And / or, the specific process of the homogenization treatment in step 5 is to homogenize at ≤10°C for 0.25-1.2h and stop for 0.25-1.2h; repeat this 2-3 times; 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.

9. The bionic collagen with high thermal stability according to claim 7, characterized in that: In Step 1, the solvent of the condensation auxiliary solution is phosphate buffer; in Step 3, the solvent of the dehydrating condensing agent solution is phosphate buffer; the phosphate buffer is prepared with raw materials having 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 is 6.9 - 7.6; And / or, the condensation auxiliary is selected from at least one of 1-hydroxybenzotriazole or 1-hydroxy-7-azabenzotriazole or N-hydroxysuccinimide.

10. The preparation method of the biomimetic collagen with high thermal stability according to any one of claims 1-9, characterized in that, It includes the following steps: Performing a condensation reaction using a condensation auxiliary, 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 auxiliary, P-O-G tripeptide, and the dehydrating condensing agent is 0.7 - 2.3: 7.2 - 24.6: 27.4 - 36.

2.

11. Use of the high thermal stability bionic collagen according to any one of claims 1 - 9 in the preparation of cosmetics or tissue engineering materials.

12. Use of the biomimetic collagen with high thermal stability according to claim 11 in the preparation of cosmetics or tissue engineering materials, 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.

13. Use of the biomimetic collagen with high thermal stability according to claim 12 in the preparation of cosmetics or tissue engineering materials, characterized in that: The functional skin care products are facial masks, essence, or facial cleansers.

14. A cosmetic, characterized in that, It is prepared by using the high thermal stability bionic collagen according to any one of claims 1 - 9 as an active ingredient and adding excipients acceptable in cosmetics.

15. A tissue engineering material, characterized in that: It is prepared by using the high thermal stability bionic collagen according to any one of claims 1 - 9 as an active ingredient and adding excipients acceptable in medicine.

16. A medical device, characterized in that: It is prepared by using the high thermal stability bionic collagen according to any one of claims 1 - 9 as an active ingredient and adding excipients acceptable in medicine.

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

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