Preparation method of high-stability I-type collagen solution for injection and obtained product

Abstract

The invention belongs to the field of collagen extraction, and particularly relates to a preparation method of a high-stability I-type collagen solution for injection and an obtained product. The method is realized by the following steps: firstly, pretreating a type I collagen freeze-dried product; and sequentially adding a sodium hyaluronate mixed solution and a chitosan mixed solution 3, fully and uniformly mixing, and adding a disodium hydrogen phosphate buffer solution to obtain the I-type collagen solution for injection. The preparation method disclosed by the invention is simple and easy to operate, the prepared animal-derived I-type collagen solution for injection is relatively good in uniformity and stability, and the selected components are safe and relatively excellent in effectiveness. The type I collagen and the chitosan have good effects of promoting cell proliferation, migration and repair, and related repair products are sold on the market; the type I collagen and the sodium hyaluronate have a good moisturizing effect; the trehalose component has good moisturizing and anti-oxidation effects, and particularly, the trehalose has excellent characteristics of keeping cell viability and biomacromolecule activity; the mannitol component has a good effect of improving the osmotic pressure molar concentration of the solution, the osmotic pressure of the solution can be basically kept consistent with the osmotic pressure of body fluid in the solution, and side effects such as edema are not prone to occurring.

Description

Technical Field

[0001] This invention belongs to the field of collagen extraction, specifically relating to a method for preparing a highly stable type I collagen solution for injection and the resulting product. Background Technology

[0002] Collagen is a vital biological macromolecular fibrous protein with a unique triple helix structure, giving it a crucial role as a key structural component of the extracellular matrix. Collagen constitutes approximately 6% of a person's body weight and is essential for maintaining the structural integrity of the dermis. Its dense network provides a scaffold for organs and determines the skin's structural characteristics, strength, and elasticity.

[0003] Collagen loss due to various reasons is one of the main causes of skin aging. The loss of collagen eventually manifests as stiff skin, reduced elasticity, and a yellowish, dull complexion. The collagen in the dermis is mainly composed of type I and type III collagen. Type I collagen has a robust, tightly packed bundle structure, providing strong support and structural strength to the skin. It also possesses powerful moisturizing, nourishing, hemostatic, and repairing abilities.

[0004] Therefore, timely replenishment of lost type I collagen in the early stages helps maintain youthful skin. Direct injection of type I collagen is considered the most direct and effective way to replenish collagen. Currently, most collagen implants on the market are designed to fill dynamic and static wrinkles. Injectable type I collagen solutions can be used for facial hyaluronic acid injections. Multiple collagen hyaluronic acid injections can effectively whiten, nourish, moisturize, repair, and stimulate the regeneration of the body's own collagen, repairing problem skin and improving skin condition, such as improving skin texture, fine lines, and skin tone.

[0005] Because injectable type I collagen homogeneous and stable dilute solutions must not irritate the skin after injection, the osmolar concentration of the product must be close to that of body fluids, and the pH must be neutral and non-irritating. Type I collagen raw materials are slightly acidic, and white protein flocculents easily precipitate in neutral dilute solutions. Preparing injectable type I collagen homogeneous and stable dilute solutions is quite difficult, and currently there are no commercially available type I collagen mesotherapy products for facial dermal tissue injection. Therefore, how to ensure the bioactivity of collagen, the safety and effectiveness of the solution, and maintain the homogeneity and stability of the product solution is a pressing problem that needs to be solved. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a method for preparing a highly stable type I collagen solution for injection. The type I collagen solution prepared by this invention ensures the bioactivity of the collagen and the safety and effectiveness of the product, while maintaining the homogeneity and stability of the solution throughout the product's shelf life.

[0007] The present invention also provides a highly stable type I collagen for injection prepared using the above-described preparation method.

[0008] The technical solution adopted by the present invention to achieve the above objectives is as follows: This invention provides a method for preparing a highly stable type I collagen solution for injection, comprising the following steps: (1) Accurately weigh type I collagen freeze-dried product, first pre-treat it, wash it until neutral and freeze-dry it to obtain pre-treated collagen; wash the pre-treated collagen, freeze-dry it and then sterilize it by electron beam irradiation, and add sterile water for injection to obtain a homogeneous mixture 1. (2) Slowly add sodium hyaluronate solution to the solution containing trehalose and mannitol, mix thoroughly to obtain mixture 2; (3) Weigh chitosan powder, add sterile water for injection and lactic acid to dissolve it until the solution is homogeneous. Sterilize the solution by terminal electron beam irradiation to obtain mixture 3; (4) Slowly add mixture 3 to mixture 2 and mix thoroughly to obtain mixture 4; (5) Add mixture 1 to mixture 4, mix thoroughly, then add disodium hydrogen phosphate buffer, mix thoroughly, and obtain type I collagen solution for injection.

[0009] Furthermore, in step (1), the pretreatment adopts a segmented treatment method, specifically as follows: at a stirring speed of 80-100 r / min, firstly, treat with a 0.1-0.15 mol / L tromethorphan solution for 20-30 min; then adjust the concentration of tromethorphan to 0.18-0.2 mol / L and continue treatment for 15-20 min; after treatment, repeatedly rinse with sterile water for injection until neutral, centrifuge, and then ultrafilter; the concentration of the mixture 1 is 1-6 mg / mL. Furthermore, in step (1), the electron beam irradiation sterilization dose is 23 kGy.

[0010] Furthermore, in step (2), the concentrations of trehalose and mannitol in the solution are 0.5-5 mg / mL and 1-10 mg / mL, respectively; the concentration of the sodium hyaluronate solution is 10 mg / mL.

[0011] Furthermore, in step (2), the volume ratio of the sodium hyaluronate solution to the solution containing trehalose and mannitol is 20:19.

[0012] Furthermore, in step (3), the concentration of chitosan in mixture 3 is 0.2-2 mg / mL, and the concentration of lactic acid is 0.01-0.1%.

[0013] Furthermore, in step (4), the volume ratio of mixture 2 to mixture 3 is 3.9:1.

[0014] Furthermore, in step (5), the volume ratio of mixture 1 to mixture 4 is 50:49.

[0015] Furthermore, in step (5), the pH value of the disodium hydrogen phosphate buffer is 11.

[0016] The present invention also provides a highly stable type I collagen solution for injection prepared by the above preparation method.

[0017] The disodium hydrogen phosphate buffer used in this invention is prepared by the following method: (a) Accurately weigh NaOH granules, transfer them to a 100mL beaker, add 10mL of sterile water for injection to dissolve them completely, and set aside for later use; (b) Accurately weigh anhydrous disodium hydrogen phosphate, transfer it to a 100mL beaker, add 50mL of sterile water for injection to dissolve it completely, then add the NaOH solution prepared in (a) above to adjust the pH value to 11, filter it through a 0.22μm filter to sterilize it, and set it aside for use.

[0018] The injectable collagen solution provided by this invention comprises: animal-derived type I collagen, sodium hyaluronate, trehalose, mannitol, chitosan, lactic acid, sodium hydroxide, disodium hydrogen phosphate, sodium chloride injection, and sterile water for injection. This invention, through modification of the collagen and adjustment of the order of addition of each raw material, produces a collagen solution with good homogeneity and stability, without stratification. If the order of addition of the ingredients is adjusted, components may become difficult to dissolve, and flocculent precipitates may appear in the solution, resulting in a stratified and heterogeneous state.

[0019] The homogeneous and stable type I collagen solution for injection described in this invention, based on a volume of 1000 mL, has the following components and dosages: Type I collagen solution, 500 mL, final concentration 0.5-3 mg / mL; 200 mL of sodium hyaluronate solution, with a final concentration of 0.5-3 mg / mL; 190 mL of trehalose & mannitol solution, with final concentrations of 0.5-5 mg / mL and 1-10 mg / mL, respectively; 99.5 mL of chitosan solution, with a final concentration of 0.2-2 mg / mL; 0.5 mL of lactic acid solution: final concentration 0.01-0.1%; 2 mL of sodium hydroxide solution, with a final concentration of 0.1-1 mg / mL; 8 mL of disodium hydrogen phosphate solution yields a final concentration of 0.1-1 mg / mL.

[0020] The preparation method of the homogeneous and stable type I collagen injection solution of the present invention is as follows (based on 1000 mL): (1) Accurately weigh the pretreated type I collagen freeze-dried product, aseptically tear it in a clean bench, tear it with tweezers and transfer it to a 1000mL sterile blue cap bottle, add 600mL sterile water for injection to fully dissolve it until the solution is homogeneous, and set aside for later use.

[0021] (2) Accurately weigh sodium hyaluronate powder and aseptically transfer it to a 1000mL sterile blue-capped bottle in a clean bench. Add 200mL of sodium chloride injection solution and dissolve it thoroughly until the solution is homogeneous.

[0022] (3) Accurately weigh trehalose and mannitol powder, transfer them to a 200mL beaker, add 200mL sodium chloride injection to dissolve them completely, filter them through a 0.22μm filter to remove bacteria, and set aside for later use.

[0023] (4) Accurately weigh the chitosan powder, transfer it to a 200mL sterile blue cap bottle, add 199mL of sterile water for injection and 1mL of lactic acid to dissolve it until the solution is homogeneous. The solution is then sterilized by terminal electron beam irradiation and set aside.

[0024] (5) Accurately weigh the NaOH particles, transfer them to a 100mL beaker, add 10mL of sterile water for injection to dissolve them completely, and set aside for later use.

[0025] (6) Accurately weigh anhydrous disodium hydrogen phosphate, transfer it to a 100mL beaker, add 50mL of sterile water for injection to dissolve it completely, then add the NaOH solution prepared in (5) above to adjust the pH value to 11, filter it through a 0.22μm filter to remove bacteria, and set it aside for later use.

[0026] (7) Perform aseptic operation, transfer 190 mL of the solution prepared in (3) to (2) and mix thoroughly.

[0027] (8) Perform aseptic operation, transfer 100 mL of the solution prepared in (4) to (7) and mix thoroughly.

[0028] (9) Perform aseptic operation, transfer 500 mL of the solution prepared in (1) to (8) and mix thoroughly.

[0029] (10) Aseptic operation: Transfer 10 mL of the solution prepared in (6) to (9) and mix thoroughly to obtain a neutral, homogeneous and stable solution with a pH range of 6.8-7.2. It can then be aseptically dispensed into suitable containers such as sterile pre-filled syringes and stored at 2-8℃. After passing the test, it can be used for facial water light injection.

[0030] The beneficial effects of this invention are as follows: (1) The present invention first pre-treats collagen, which can improve the purity of injectable collagen on the one hand; on the other hand, through gradient treatment, hydrogen bonds in the terminal peptide region can be destroyed, thereby reducing the risk of immune response after injection, and the stability of collagen in the system is improved after treatment. (2) The preparation method disclosed in this invention is simple and easy to operate. The resulting animal-derived type I collagen solution for injection has good uniformity and stability, and the selected components have good safety and efficacy. Type I collagen and chitosan have good cell proliferation, migration and repair effects; (3) In the injection solution provided by the present invention, type I collagen and sodium hyaluronate have good moisturizing effects; trehalose has good moisturizing and antioxidant effects, especially trehalose has excellent properties of maintaining cell vitality and biomolecular activity; mannitol has a good effect of increasing the osmotic pressure molar concentration of the solution, so that the osmotic pressure of the solution can be basically consistent with the osmotic pressure of body fluid, and side effects such as edema are not easy to occur; the raw materials work together to prepare an injection solution with excellent stability and reduce the occurrence of side effects during use. Attached Figure Description

[0031] Figure 1 This is a bottled image of the type I collagen solution for injection prepared in Example 1 of this invention; Figure 2 This is a pre-filling and sealing diagram of the type I collagen solution for injection prepared in Example 1 of the present invention; Figure 3 This is a bottled image of the type I collagen solution for injection prepared in Example 2 of this invention; Figure 4 The images show SDS-PAGE images of aqueous solutions of type I collagen and injectable type I collagen solutions prepared in Examples 1 and 2 of this invention; where Marker is protein Marker PR1930; Control is aqueous solution of type I collagen with a concentration of 6 mg / mL; 1-1, 1-2, and 1-3 are respectively the 3-fold dilution, 2-fold dilution, and stock solution of injectable type I collagen solution prepared in Example 1; 2-1, 2-2, and 2-3 are respectively the 3-fold dilution, 2-fold dilution, and stock solution of injectable type I collagen solution prepared in Example 2. Figure 5This is a comparison chart showing the results of cell adhesion and proliferation promoted by the injection type I collagen solution prepared in Example 1 of this invention; Figure 6 This is a comparison chart showing the cell migration promotion results of the injectable type I collagen solution prepared in Example 1 of this invention; Figure 7 This is an immunofluorescence staining image. Detailed Implementation

[0032] The technical solution and effects of the present invention will be further described below with reference to specific embodiments and accompanying drawings. Unless otherwise specified, the methods described in this invention are conventional methods in the art, and the reagents described are also conventional reagents in the art and can be obtained commercially.

[0033] Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0034] In some embodiments, the animal collagen (type I collagen) used in this invention has a molecular weight of 50,000 to 300,000.

[0035] Example 1 A method for preparing a homogeneous and stable solution of animal-derived type I collagen for injection, with a volume of 1000 mL, comprises the following steps: (1) Accurately weigh 3.6000g of type I collagen freeze-dried product, aseptically shred it, and transfer it to a 1000mL sterile blue cap bottle with ophthalmic forceps. At a stirring speed of 100r / min, first treat it with 600mL of 0.1mol / L tromethorphan solution for 30min; then adjust the concentration of tromethorphan to 0.2mol / L and continue treatment for 15min; after treatment, wash the pretreated collagen three times with sterile water for injection, freeze-dry it, and then sterilize the pretreated type I collagen freeze-dried tablets by electron beam (23kGy) irradiation; accurately weigh 3.6000g of sterilized type I collagen freeze-dried product, aseptically shred it, and transfer it to a 1000mL sterile blue cap bottle with ophthalmic forceps. Add 600mL of sterile water for injection to fully dissolve it until the solution is homogeneous and set aside.

[0036] (2) Accurately weigh 2.0000g of sodium hyaluronate powder, aseptically transfer it to a 1000mL sterile blue cap bottle, add 200mL of sodium chloride injection solution and dissolve it thoroughly until the solution is homogeneous.

[0037] (3) Accurately weigh 2.1100g of trehalose and 10.23g of mannitol, transfer them to a 200mL beaker, add 200mL of sodium chloride injection to dissolve them completely, filter them through a 0.22μm filter to remove bacteria, and set aside for later use.

[0038] (4) Accurately weigh 2.0000g of chitosan, transfer it to a 200mL blue cap bottle, add 199mL of sterile water for injection and 1mL of lactic acid to dissolve it until the solution is homogeneous. Sterilize the solution by terminal electron beam irradiation and set aside.

[0039] (5) Accurately weigh 8.0000g of NaOH granules, transfer them to a 100mL beaker, add 20mL of sterile water for injection to dissolve them completely, and set aside for later use.

[0040] (6) Accurately weigh 1.4200g of anhydrous disodium hydrogen phosphate, transfer it to a 100mL beaker, add 50mL of sterile water for injection to dissolve it completely, then add 12.5mL of the NaOH solution prepared in (5) above to adjust the pH value to 11, filter it through a 0.22μm filter to sterilize it, and set it aside for later use.

[0041] (7) Perform aseptic operation, transfer 190 mL of the solution prepared in (3) to (2) and mix thoroughly.

[0042] (8) Perform aseptic operation, transfer 100 mL of the solution prepared in (4) to (7) and mix thoroughly.

[0043] (9) Perform aseptic operation, transfer 500 mL of the solution prepared in (1) to (8) and mix thoroughly.

[0044] (10) Under aseptic conditions, transfer 10 mL of the solution prepared in (6) to (9) and mix thoroughly to obtain a neutral, homogeneous, and stable solution of animal-derived type I collagen for injection with a pH of approximately 7.01 and a type I collagen concentration of 3 mg / mL (see [link to solution]). Figure 1 It can be aseptically dispensed into sterile pre-filled syringes (see...). Figure 2 Store in suitable containers at 2-8℃.

[0045] Example 2 A method for preparing a homogeneous and stable solution of animal-derived type I collagen for injection, with a volume of 1000 mL, comprises the following steps: (1) Accurately weigh 1.2000g of type I collagen freeze-dried product (pretreatment as in Example 1), aseptically tear it into pieces, and transfer it to a 1000mL sterile blue cap bottle after tearing it into pieces with ophthalmic forceps. Add 600mL of sterile water for injection to fully dissolve it until the solution is homogeneous, and set it aside for later use.

[0046] (2) Accurately weigh 2.0000g of sodium hyaluronate powder, aseptically transfer it to a 1000mL sterile blue cap bottle, add 200mL of sodium chloride injection solution and dissolve it thoroughly until the solution is homogeneous.

[0047] (3) Accurately weigh 2.1100g of trehalose and 10.23g of mannitol, transfer them to a 200mL beaker, add 200mL of sodium chloride injection to dissolve them completely, filter them through a 0.22μm filter to remove bacteria, and set aside for later use.

[0048] (4) Accurately weigh 2.0000g of chitosan, transfer it to a 200mL blue cap bottle, add 199mL of sterile water for injection and 1mL of lactic acid to dissolve it until the solution is homogeneous. Sterilize the solution by terminal electron beam irradiation and set aside.

[0049] (5) Accurately weigh 8.0000g of NaOH granules, transfer them to a 100mL beaker, add 20mL of sterile water for injection to dissolve them completely, and set aside for later use.

[0050] (6) Accurately weigh 1.4200g of anhydrous disodium hydrogen phosphate, transfer it to a 100mL beaker, add 50mL of sterile water for injection to dissolve it completely, then add 12.5mL of the NaOH solution prepared in (5) above to adjust the pH value to 11, filter it through a 0.22μm filter to sterilize it, and set it aside for later use.

[0051] (7) Perform aseptic operation, transfer 190 mL of the solution prepared in (3) to (2) and mix thoroughly.

[0052] (8) Perform aseptic operation, transfer 100 mL of the solution prepared in (4) to (7) and mix thoroughly.

[0053] (9) Perform aseptic operation, transfer 500 mL of the solution prepared in (1) to (8) and mix thoroughly.

[0054] (10) Under aseptic conditions, transfer 10 mL of the solution prepared in (6) to (9) and mix thoroughly to obtain a neutral, homogeneous, and stable solution of animal-derived type I collagen for injection with a pH of approximately 7.10 and a type I collagen concentration of 1 mg / mL (see Appendix). Figure 3 ).

[0055] Comparative Example 1 A method for preparing a homogeneous and stable solution of animal-derived type I collagen for injection, with a volume of 1000 mL, comprises the following steps: (1) Accurately weigh 3.6000g of type I collagen freeze-dried product (untreated), aseptically shred it, and transfer it to a 1000mL sterile blue cap bottle after shredding it with ophthalmic forceps. Add 600mL of sterile water for injection to fully dissolve it until the solution is homogeneous, and set it aside for later use.

[0056] The other steps are the same as in Example 1.

[0057] The injection solution prepared in Comparative Example 1 is prone to intermolecular aggregation during storage, forming visible flocculent matter or precipitate. Especially when the temperature fluctuates, the clarity of the injection solution decreases significantly, making it unable to pass through sterile filtration and clogging the injection needle during further use.

[0058] Example 1 An SDS-PAGE analysis was performed using a 6 mg / mL aqueous solution of type I collagen prepared in step (1) of Example 1 of this invention, and a homogeneous and stable solution of animal-derived type I collagen for injection prepared in Examples 1 and 2 of this invention (see [link]). Figure 4 Analysis showed that the molecular weight of collagen peptide chains in the homogeneous and stable solution prepared in Example 1 of the present invention was increased compared with that in the aqueous solution of type I collagen, indicating that its homogeneity and stability are due to the stable binding with amino acids in sodium hyaluronate, which keeps the solution in a homogeneous and stable state.

[0059] Detailed operation steps: (1) Take the 6 mg / mL type I collagen aqueous solution prepared in step (1) of Example 1 and label it as Control.

[0060] (2) Take the neutral, homogeneous and stable animal-derived type I collagen solution with a pH of about 7.01 and a type I collagen concentration of 3 mg / mL prepared in Example 1, and prepare 3-fold dilution, 2-fold dilution and stock solution, which are labeled as 1-1, 1-2 and 1-3 respectively.

[0061] (3) Take the neutral, homogeneous and stable animal-derived type I collagen solution with a pH of about 7.10 and a type I collagen concentration of 1 mg / mL prepared in Example 2, and prepare a 3-fold dilution, a 2-fold dilution and a stock solution, which are labeled as 2-1, 2-2 and 2-3, respectively.

[0062] (4) In accordance with the fifth method of electrophoresis in Section 4, General Chapter 0541 of the 2020 edition of the Pharmacopoeia of the People's Republic of China, the SDS-polyacrylamide gel electrophoresis method was used, with a separating gel concentration of 6%, a stacking gel concentration of 5%, and a sample loading volume of 30µL. After running the gel, the gel was scanned and analyzed by electrophoresis.

[0063] Depend on Figure 4 It can be seen that the electrophoretic bands of Control are located at 120 kDa, 110 kDa, and 230 kDa, respectively. The electrophoretic bands corresponding to the solutions prepared in Examples 1 and 2 of this invention are located at 125 kDa, 115 kDa, and 240 kDa, respectively.

[0064] Example 2 Treating cell culture dishes with the homogeneous and stable solution of animal-derived type I collagen for injection prepared in Example 1 of this invention can improve the adhesion rate and proliferation rate of human epidermal cells.

[0065] Experimental Materials and Methods Cells: Human epidermal cells, specifically P3 passage.

[0066] Experimental Groups: Blank control group: cell culture dishes without coating treatment; Collagen group: Cell culture dishes coated with 3 mg / mL collagen aqueous solution; Injectable collagen solution group: cell culture dishes coated with the injectable collagen solution prepared in Example 1.

[0067] (1) Take cell culture dishes without any coating treatment and label them as blank control group.

[0068] (2) Take the 6 mg / mL type I collagen aqueous solution prepared in step (1) of Example 1 and dilute it twice with sterile 0.006 mol / L acetic acid to obtain a 3 mg / mL collagen aqueous solution. Coat cell culture dishes with the 3 mg / mL collagen aqueous solution and label the cell culture dishes coated with the 3 mg / mL collagen aqueous solution as the collagen group.

[0069] (3) Take the collagen solution for injection prepared in Example 1 and coat the cell culture dish. The cell culture dish coated with the collagen solution for injection is labeled as the collagen solution for injection group.

[0070] (4) Collect the passaged P2 generation human epidermal cells, resuspend the cells using Keratinocyte-SFM, seed them at 1.2 million per dish in the above culture dishes, and continue to culture them in a 5% CO2, 37℃ incubator. Take photos daily to record cell adhesion and proliferation.

[0071] (5) Results Analysis: Cells are suitable for passage when they grow to 80%-90%. The cell adhesion and proliferation effects of cells passaged in cell culture dishes coated with the homogeneous and stable collagen solution prepared in this invention were significantly improved compared to the blank control group and the collagen group. By the third day, epidermal cells had densely covered the entire cell culture dish, while the blank control group and the collagen group had not yet completely covered the cells. The experiment shows that the homogeneous and stable collagen solution prepared in this invention has a good effect on promoting cell adhesion and proliferation. The results are shown in the figure below. Figure 5 The collagen aqueous solution group showed better cell adhesion and proliferation than the blank control group, and the collagen homogeneous and stable solution group for injection was superior to the collagen aqueous solution group.

[0072] Example 3 Treatment of 6-well cell culture plates with the homogeneous and stable solution of animal-derived type I collagen for injection prepared in Example 1 of this invention can improve the migration rate of human epidermal cells.

[0073] Experimental Materials and Methods Cells: Human epidermal cells, specifically P3; Experimental Groups: Blank control group: 6-well cell culture plates without coating treatment; Collagen group: 6-well cell culture plates were coated with 3 mg / mL collagen aqueous solution; Injectable collagen solution group: The injectable collagen solution prepared in Example 1 was used to coat 6-well cell culture plates.

[0074] (1) Take a 6-well cell culture plate without any coating treatment and label it as a blank control group.

[0075] (2) Take the 6 mg / mL type I collagen aqueous solution prepared in step (1) of Example 1 and dilute it twice with sterile 0.006 mol / L acetic acid to obtain a 3 mg / mL collagen aqueous solution. Coat a 6-well cell culture plate with the 3 mg / mL collagen aqueous solution and label the 6-well cell culture plate coated with the 3 mg / mL collagen aqueous solution as the collagen group.

[0076] (3) Take the collagen solution for injection prepared in Example 1 and coat it with a 6-well cell culture plate. The 6-well cell culture plate coated with the collagen solution for injection is labeled as the collagen solution for injection group.

[0077] (4) Collect the passaged P2 generation human epidermal cells, resuspend the cells using Keratinocyte-SFM, and seed them into the above 6-well cell culture plates at a rate of 1.6 million cells / well.

[0078] (5) After the cells adhere to the plate and completely cover it, use a 200 μL sterile pipette tip to quickly glide along the bottom of the 6-well plate as perpendicularly as possible to create scratches. Change the cell culture medium and remove any epidermal cells detached during the scratching process. Incubate in a 5% CO2, 37℃ incubator and continue culturing. Take photos at 0, 3, 6, 9, and 12 hours for observation.

[0079] (6) Results Analysis: In 6-well cell culture plates coated with the homogeneous and stable collagen solution prepared according to the present invention, the cell migration effect was significantly enhanced compared with the blank control group and the collagen group. At 9 hours after the scratch test, the cell migration rate reached 100%, while the cell migration rates of the blank control group and the collagen group were approximately 25% and 55%, respectively. The experiment shows that the homogeneous and stable collagen solution prepared in Example 1 of the present invention has a good effect on promoting cell migration. The results are shown in […]. Figure 6Among them, the collagen aqueous solution group showed better cell migration promotion effect than the blank control group, and the homogeneous and stable collagen injection solution group prepared in Example 1 of this invention was better than the collagen aqueous solution group.

[0080] Effect Experiment Example 4 When the homogeneous and stable solution of animal-derived type I collagen for injection prepared in Example 1 was added to the recombinant human 3D skin model, the content of moisturizing protein in the recombinant human 3D skin model increased.

[0081] Experimental Materials and Methods Cells: dermal fibroblasts and keratinocytes, specifically P3; Experimental Groups: Blank control group: a conventionally prepared recombinant human 3D skin model; Collagen group: A recombinant human 3D skin model was constructed using a 3 mg / mL collagen aqueous solution. Injectable collagen solution group: The recombinant human 3D skin model was constructed using the injectable collagen solution prepared in Example 1.

[0082] (1) Take a bovine type I collagen solution containing dermal fibroblasts and add it to a six-well plate transwell insert chamber. Place it in an incubator to gel, and compress the thickness of the collagen gel in the middle to 1.0 mm.

[0083] (2) Add dermal culture medium to the inside and outside of the chamber respectively, so that the interface between the inside and outside of the chamber is flat, and culture for 2-4 days to allow dermal fibroblasts and collagen to interact and form the dermal layer.

[0084] (3) The chambers with no special treatment on the dermis were marked as blank control group; the chambers with 3 mg / mL collagen aqueous solution added to the dermis were marked as collagen group; the chambers with injectable collagen solution added to the dermis were marked as injectable collagen solution group. They were placed in an incubator and left to stand for 1-2 hours to allow the solution to soak in.

[0085] (4) Inoculate the keratinocyte culture medium containing keratinocytes onto the dermis of the above-mentioned chambers and place them in an incubator for 2-4 hours to allow the keratinocytes to adhere to the chamber walls.

[0086] (5) Add keratinocyte culture medium to the inner and outer surfaces of the small chamber respectively, so that the inner and outer surfaces of the small chamber are level, and immerse and culture for 2 to 4 days.

[0087] (6) Add gas-liquid culture medium to the outside of the model and incubate for 10-12 days, changing the medium daily. Collect the model for immunofluorescence staining to label moisturizing proteins.

[0088] (7) Results Analysis: When the homogeneous and stable injectable animal-derived type I collagen solution prepared in Example 1 of this invention was added to a recombinant human 3D skin model, the thickness of the recombinant human 3D skin model increased, and the content of moisturizing proteins in the model was significantly increased compared with the blank control group and the collagen group. The experiment shows that the homogeneous and stable injectable collagen solution prepared in Example 1 of this invention can appropriately increase the thickness of the skin model and has a good moisturizing effect. The results are shown in […]. Figure 7 The moisturizing effect of the collagen aqueous solution group was better than that of the blank control group, and the homogeneous and stable collagen injection solution group prepared in Example 1 of the present invention was better than that of the collagen aqueous solution group.

[0089] The data above demonstrates that the method for preparing a homogeneous and stable type I collagen injection solution according to this invention ensures the homogeneity and stability of the solution. While maintaining the triple helix structure of the original collagen, the interaction of the components appropriately stabilizes the collagen peptide chain structure, increases the molecular weight of each peptide chain, and enhances the solution stability. Furthermore, the homogeneous and stable collagen injection solution prepared by this invention effectively promotes epidermal cell adhesion, proliferation, and migration, and also has good moisturizing properties, which is beneficial for the proliferation and migration of human skin cells and the improvement of skin condition.

[0090] The above description is merely an example and illustration of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described, or use similar methods to replace them, as long as they do not deviate from the structure of the invention or exceed the scope defined in the claims, all of which should fall within the protection scope of the present invention.

Claims

1. A method for preparing a highly stable type I collagen solution for injection, characterized in that, Includes the following steps: (1) Accurately weigh type I collagen freeze-dried product, pre-treat and wash until neutral, freeze-dry to obtain pre-treated collagen; The pretreated collagen was washed, freeze-dried, and then sterilized by electron beam irradiation. After that, sterile water for injection was added again to obtain a homogeneous mixture 1. (2) Slowly add sodium hyaluronate solution to the solution containing trehalose and mannitol, mix thoroughly to obtain mixture 2; (3) Weigh chitosan powder, add sterile water for injection and lactic acid to dissolve it until the solution is homogeneous. Sterilize the solution by terminal electron beam irradiation to obtain mixture 3; (4) Slowly add mixture 3 to mixture 2 and mix thoroughly to obtain mixture 4; (5) Add mixture 1 to mixture 4, mix thoroughly, then add disodium hydrogen phosphate buffer, mix thoroughly, and obtain type I collagen solution for injection.

2. The preparation method according to claim 1, characterized in that, In step (1), the pretreatment adopts a segmented treatment method. The specific process is as follows: at a stirring speed of 80-100 r / min, first treat with 0.1-0.15 mol / L tromethorphan solution for 20-30 min; then adjust the concentration of tromethorphan solution to 0.18-0.2 mol / L and continue treatment for 15-20 min; after treatment, rinse repeatedly with sterile water for injection until neutral, centrifuge and then ultrafilter; the concentration of the mixture 1 is 1-6 mg / mL.

3. The preparation method according to claim 1 or 2, characterized in that, In step (1), the electron beam irradiation sterilization dose is 23 kGy.

4. The preparation method according to claim 1, characterized in that, In step (2), the concentrations of trehalose and mannitol in the solution are 0.5-5 mg / mL and 1-10 mg / mL, respectively; the concentration of the sodium hyaluronate solution is 10 mg / mL.

5. The preparation method according to claim 1 or 4, characterized in that, In step (2), the volume ratio of the sodium hyaluronate solution to the solution containing trehalose and mannitol is 20:

19.

6. The preparation method according to claim 1, characterized in that, In step (3), the concentration of chitosan in mixture 3 is 0.2-2 mg / mL, and the concentration of lactic acid is 0.01-0.1%.

7. The preparation method according to claim 1, characterized in that, In step (4), the volume ratio of mixture 2 to mixture 3 is 3.9:

1.

8. The preparation method according to any one of claims 1-7, characterized in that, In step (5), the volume ratio of mixture 1 to mixture 4 is 50:

49.

9. The preparation method according to claim 1 or 8, characterized in that, In step (5), the pH value of the disodium hydrogen phosphate buffer is 11.

10. A highly stable type I collagen solution for injection prepared by the preparation method according to any one of claims 1-9.

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