Recombinant collagen repair fluid and preparation method and use thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a recombinant collagen repair solution and a preparation method and application thereof. The application comprises the following steps: preparation of a protein activity enhancer; preparation of a recombinant humanized collagen fiber sol; and photo-polymerization construction of a recombinant humanized collagen hydrogel. The application is modified by sulfate esterification of pollen of apiaceae polysaccharide, and is compounded with the recombinant humanized collagen. The application can not only form a stable complex with the positively charged collagen amino acid residues through electrostatic interaction, maintain the natural triple helix conformation, reduce the aggregation and denaturation of the collagen, and enhance the activity of the collagen, but also can assist the recombinant humanized collagen in activating the cell surface receptors, enhancing the adhesion and proliferation of the cells, thereby enhancing the epidermal regeneration capacity of the recombinant humanized collagen, effectively improving the bioavailability of the recombinant humanized collagen at the wound site, and improving the repair and treatment effect on the wounded skin.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a recombinant collagen repair solution, its preparation method and uses. Background Technology

[0002] Collagen, as a major component of the extracellular matrix, plays a crucial role in structural support and biological signal transduction during skin tissue repair. In recent years, recombinant humanized collagen technology has achieved efficient expression of type III collagen through genetic engineering. Its biocompatibility and epidermal regeneration-promoting ability are significantly superior to animal-derived collagen, making it a research hotspot in the field of wound repair. However, recombinant collagen is prone to conformational changes in molecular aggregates during storage and use, leading to the dissociation of the triple helix structure and a decrease in biological activity, which seriously affects its effective concentration and repair efficacy in wounds.

[0003] Existing technologies for maintaining the activity of recombinant collagen often employ low-temperature preservation or the addition of stabilizers, but these methods still have significant limitations in practical applications: conventional stabilizers are unable to prevent the hydrophobic aggregation between collagen molecules and cannot work synergistically with recombinant collagen to enhance its activity.

[0004] Furthermore, existing technologies mostly construct hydrogel systems to utilize recombinant collagen. The construction methods of hydrogel systems are generally divided into physical cross-linking and chemical cross-linking. Physically cross-linked hydrogels are formed through physical interactions such as electrostatic interactions and hydrogen bonding, resulting in poor stability and low strength. Although chemically cross-linked hydrogels can bind hydrogel monomers through stronger chemical bonds, the amount of chemical cross-linking agents used is small to avoid the toxicity problems of various chemical cross-linking agents. This leads to insufficient cross-linking degree of chemically cross-linked hydrogels, thus their stability and strength still do not reach a high level. Summary of the Invention

[0005] To address the aforementioned technical deficiencies, this invention presents a recombinant collagen repair solution and its preparation method. The recombinant collagen repair solution contains recombinant collagen with high bioavailability, and the gel in the repair solution has the characteristics of high water retention, resistance to high and low temperatures, and good mechanical properties, which can effectively and stably treat skin wounds or inflammation.

[0006] The technical solution of the present invention is as follows:

[0007] A method for preparing a recombinant collagen repair solution includes the following steps:

[0008] Preparation of S1 protein activity enhancer

[0009] After pulverizing the turnip pollen, it was extracted by reflux with citric acid solution. Then, chitosan was added, stirred evenly, and allowed to stand. The supernatant was collected by centrifugation, concentrated, and freeze-dried to obtain the turnip pollen extract. The extract was then redissolved in distilled water and precipitated with alcohol to obtain crude turnip pollen polysaccharide. The extract was then purified by gel chromatography to obtain turnip pollen polysaccharide. The polysaccharide was dissolved in anhydrous formamide, and a sulfonating agent prepared from pyridine and chlorosulfonic acid was added to react with it. The reaction solution was dialyzed with deionized water and then freeze-dried to obtain a protein activity enhancer.

[0010] Preparation of S2 recombinant humanized collagen fibrinolytic sol

[0011] After adjusting the pH by adding NaOH solution to neutral PBS buffer, NaCl was added to obtain a sol base solution. Recombinant type III humanized collagen, protein activity enhancer and lupin dietary fiber were added to the sol base solution and stirred evenly. Then, it was placed in a hot water bath for treatment and rapidly cooled to room temperature to obtain recombinant humanized collagen fiber sol.

[0012] S3 photopolymerization constructs recombinant humanized collagen hydrogel

[0013] Acryloyloxyethyltrimethylammonium chloride, acrylamide, and choline chloride were mixed in a container to obtain a mixed monomer. Deionized water and recombinant type III humanized collagen sol were added, followed by polyethylene glycol diacrylate and phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide, which were mixed evenly. The mixture was then cured under ultraviolet light to obtain a recombinant humanized collagen hydrogel. Sodium parabens and disodium ethylenediaminetetraacetate were added and stirred evenly. The mixture was then filled, capped, and sterilized to obtain a recombinant collagen repair solution.

[0014] Further, the preparation of the protein activity enhancer in step S1 includes the following steps:

[0015] S1.1: After pulverizing turnip pollen through a 60-70 mesh sieve, dry it at 60-65℃ for 2-3 hours. Then, add it to citric acid solution at a solid-liquid ratio of 1:(30-40) g / mL and mix well. Transfer it to a container equipped with a reflux device and reflux extract it at 100-105℃ for 3-5 hours. Filter to remove the residue. Take the filtrate and add 0.2-0.3wt% chitosan and stir well. Then stir at 65-70℃ for 1-1.5 hours. After standing and cooling to room temperature, centrifuge at 6000-6500 rpm for 12-15 minutes. Take the supernatant and concentrate and freeze dry it to obtain turnip pollen extract.

[0016] S1.2: The turnip pollen extract was redissolved in distilled water at a solid-liquid ratio of 1:(8-10) g / mL, and then anhydrous ethanol was added and stirred to make the volume concentration of anhydrous ethanol 60%. After ultrasonic dispersion for 6-8 minutes, it was allowed to stand for 12-15 hours. The precipitate was collected by centrifugation at 8000-8500 rpm for 10-12 minutes to obtain crude turnip pollen polysaccharide. The crude turnip pollen polysaccharide was purified by gel chromatography to obtain turnip pollen polysaccharide.

[0017] S1.3: Place the grinding dish on ice and put it in a fume hood. Add pyridine to the grinding dish, then slowly add chlorosulfonic acid along the wall while continuously stirring rapidly. When a white solid forms in the grinding dish, remove the ice bath to obtain the sulfonating reagent. Dissolve turnip pollen polysaccharide in anhydrous formamide at a solid-liquid ratio of 1:(40-50) g / mL. Add 50-60% of the volume of the sulfonating reagent to the anhydrous formamide and stir until homogeneous. Then, react at 42-45℃ for 4-5 hours to obtain the reaction solution. Add 2-2.5 mol / L sodium hydroxide solution to the reaction solution to adjust it to neutral. Then, put it into a dialysis bag with a molecular weight cutoff of 10 kD and dialyze it with deionized water for 4-5 days, changing the deionized water every 8 hours. Then, take out the liquid from the dialysis bag and freeze-dry it to obtain the protein activity enhancer.

[0018] Further, the preparation of recombinant humanized collagen fibrillary sol in step S2 includes the following steps:

[0019] S2.1: Take PBS buffer with pH 7 and a concentration of 20mM, add 0.1M NaOH solution to adjust the pH to 7.5-8, then add NaCl to dissolve it so that the concentration of NaCl in the PBS buffer is 0.2-0.3M, to obtain the sol-based solution;

[0020] S2.2: Add recombinant type III humanized collagen, protein activity enhancer and lupin dietary fiber to the sol base solution and stir evenly to make the mass concentration of recombinant type III humanized collagen 8-10%, the mass concentration of protein activity enhancer 0.8-1%, and the mass concentration of lupin dietary fiber 2-3% to obtain a composite suspension.

[0021] S2.3: The composite suspension is treated in a water bath at 55-60℃ for 30-35 minutes, and then transferred to an ice-water bath to cool rapidly to room temperature to obtain recombinant humanized collagen fiber sol.

[0022] Further, step S3, photopolymerization, constructs a recombinant humanized collagen hydrogel, including the following steps:

[0023] S3.1: Acryloyloxyethyltrimethylammonium chloride, acrylamide and choline chloride are mixed in a molar ratio of 1:(0.8-1):(2-3) and placed in a container to obtain mixed monomers. Deionized water and recombinant type III humanized collagen sol are added, followed by polyethylene glycol diacrylate and phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide. The mixture is then poured into a mold and cured under ultraviolet light for 6-8 minutes to obtain recombinant humanized collagen hydrogel.

[0024] S3.2: Place the recombinant humanized collagen hydrogel in a container, add 0.05-0.1wt% sodium paraben and 0.08-0.1wt% disodium EDTA, stir thoroughly, and then fill into vials, 10mL per vial. After capping, sterilize at Co60 to obtain the recombinant collagen repair solution.

[0025] Furthermore, the citric acid solution in step S1.1 has a mass concentration of 0.02-0.03%.

[0026] Further, in step S1.2, the gel packing material in the gel chromatography column is dextran gel G-100. When purifying the crude polysaccharide of turnip pollen, the crude polysaccharide of turnip pollen and the gel packing material are loaded at a mass ratio of 1:50, and pure water is used for elution at a flow rate of 1.5 mL / min. The eluent with the largest elution peak is collected and dried with nitrogen to obtain turnip pollen polysaccharide.

[0027] Furthermore, the sulfonating agent in step S1.3 is prepared by mixing pyridine and chlorosulfonic acid at a solid-liquid ratio of 1:1 g / mL.

[0028] Further, in step S3.1, based on the mass of the mixed monomers, deionized water accounts for 80-100%, recombinant type III humanized collagen sol accounts for 40-50%, polyethylene glycol diacrylate accounts for 1-1.5%, and phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide accounts for 0.5-0.7%.

[0029] A recombinant collagen repair solution is prepared by the above-mentioned method for preparing a recombinant collagen repair solution.

[0030] Another aspect of this application provides a method for applying the above-mentioned recombinant collagen repair solution, which involves applying the recombinant collagen repair solution to a skin wound or site of skin inflammation for the purpose of repairing and treating skin wounds and inflammation.

[0031] The beneficial effects are as follows: 1. This invention obtains turnip pollen polysaccharide by extracting and purifying turnip pollen, and then modifies it by sulfation with a sulfonating agent to obtain a protein activity enhancer. When combined with recombinant humanized collagen, it can not only form a stable complex with positively charged collagen amino acid residues through electrostatic interaction, maintaining its natural triple helix conformation, reducing collagen aggregation and denaturation, and enhancing its activity, but also assist recombinant humanized collagen in activating cell surface receptors, enhancing cell adhesion and proliferation, thereby strengthening the epidermal regeneration ability of recombinant humanized collagen. It can effectively improve the bioavailability of recombinant humanized collagen at the wound site and the repair and treatment effect on wounded skin.

[0032] 2. This invention involves heating recombinant type III humanized collagen, a protein activity enhancer, and lupin dietary fiber in a sol-based solution to form a recombinant humanized collagen fiber sol with a primary gel structure. Lupin dietary fiber possesses high water-holding capacity and a network skeletal structure, forming physical cross-links with collagen molecules through hydrogen bonds and van der Waals forces. Simultaneously, its abundant hydroxyl groups on its surface electrostatically attract the sulfonic acid groups of the protein activity enhancer. Under the conditions of the sol-based solution and water bath heating, the three components jointly construct a network structure, increasing the contact area between lupin dietary fiber and water, further enhancing its high water-holding capacity. This significantly improves the sol's water retention and mechanical properties, preventing performance degradation due to water loss at body surface temperature, and enabling effective and stable wound treatment.

[0033] 3. This invention uses acryloyloxyethyltrimethylammonium chloride, acrylamide, and choline chloride as monomers. These are mixed with recombinant humanized collagen fiber sol, and then crosslinked with polyethylene glycol diacrylate and photoinitiator phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide. The mixture is then photocured. During this process, polyethylene glycol diacrylate binds to the double bonds of acrylamide and acryloyloxyethyltrimethylammonium chloride. The hydroxyl groups and quaternary ammonium salt structure of choline chloride act as hydrogen bond acceptors, forming hydrogen bonds with the recombinant humanized collagen fiber sol, acrylamide, and acryloyloxyethyltrimethylammonium chloride. This results in an interpenetrating hydrogel formed by the interpenetrating network structure of the recombinant humanized collagen fiber sol within the monomer-formed gel structure. This significantly increases the mechanical properties of the hydrogel, preventing gel breakage caused by external physical factors (such as joint extension and friction). It also provides antibacterial effects, allowing the recombinant collagen repair solution to stably release effective components over time when applied to wounds, promoting wound healing and preventing infection. Detailed Implementation

[0034] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0035] Example 1: A method for preparing a recombinant collagen repair solution, comprising the following steps:

[0036] Preparation of S1 protein activity enhancer

[0037] S1.1: Turnip pollen was pulverized through a 60-mesh sieve and dried at 60℃ for 2 hours. Then, it was added to a 0.02% citric acid solution at a solid-liquid ratio of 1:30 g / mL and mixed evenly. The mixture was then transferred to a container equipped with a reflux device and extracted by reflux at 100℃ for 3 hours. The residue was removed by filtration. 0.2 wt% chitosan was added to the filtrate and stirred evenly. The mixture was then stirred at 65℃ for 1 hour. After standing and cooling to room temperature, the mixture was centrifuged at 6000 rpm for 12 minutes. The supernatant was collected, concentrated, and freeze-dried to obtain the turnip pollen extract.

[0038] S1.2: The turnip pollen extract was redissolved in distilled water at a solid-liquid ratio of 1:8 g / mL, and then anhydrous ethanol was added and stirred to make the volume concentration of anhydrous ethanol 60%. After ultrasonic dispersion for 6 minutes, it was allowed to stand for 12 hours. The precipitate was collected by centrifugation at 8000 rpm for 10 minutes to obtain crude turnip pollen polysaccharide. A gel chromatography column was prepared, and the crude turnip pollen polysaccharide and dextran gel G-100 were loaded at a mass ratio of 1:50. Pure water was used for elution at a flow rate of 1.5 mL / min. The eluent with the largest elution peak was collected and dried with nitrogen to obtain turnip pollen polysaccharide.

[0039] S1.3: Place the grinding dish on ice and put it in a fume hood. Add pyridine to the grinding dish, then slowly add chlorosulfonic acid along the wall while continuously stirring rapidly. When a white solid forms in the grinding dish, remove the ice bath to obtain the sulfonating reagent. The solid-liquid ratio of pyridine to chlorosulfonic acid is 1:1 g / mL. Dissolve turnip pollen polysaccharide in anhydrous formamide at a solid-liquid ratio of 1:40 g / mL. Then add sulfonating reagent accounting for 50% of the volume of anhydrous formamide and stir evenly. Then react at 42℃ for 4 hours to obtain the reaction solution. Add 2 mol / L sodium hydroxide solution to the reaction solution to adjust to neutrality. Then put it into a dialysis bag with a molecular weight cutoff of 10 kD and dialyze it with deionized water for 4 days, changing the deionized water every 8 hours. Then take the liquid out of the dialysis bag and freeze-dry it to obtain the protein activity enhancer.

[0040] Preparation of S2 recombinant humanized collagen fibrinolytic sol

[0041] S2.1: Take PBS buffer with pH 7 and a concentration of 20mM, add 0.1M NaOH solution to adjust the pH to 7.5, then add NaCl to dissolve it so that the concentration of NaCl in the PBS buffer is 0.2M, to obtain the sol-based solution;

[0042] S2.2: Add recombinant type III humanized collagen, protein activity enhancer and lupin dietary fiber to the sol base solution and stir evenly to make the mass concentration of recombinant type III humanized collagen 8%, the mass concentration of protein activity enhancer 0.8% and the mass concentration of lupin dietary fiber 2% to obtain a composite suspension.

[0043] S2.3: The composite suspension was treated in a water bath at 55°C for 30 minutes, and then transferred to an ice-water bath to cool rapidly to room temperature to obtain recombinant humanized collagen fiber sol.

[0044] S3 photopolymerization constructs recombinant humanized collagen hydrogel

[0045] S3.1: Acryloyloxyethyltrimethylammonium chloride, acrylamide, and choline chloride were mixed in a molar ratio of 1:0.8:2 in a container to obtain a mixed monomer. Deionized water and recombinant type III humanized collagen sol were added, followed by polyethylene glycol diacrylate and phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide, which were mixed evenly. By mass of the mixed monomer, deionized water accounted for 80%, recombinant type III humanized collagen sol accounted for 40%, polyethylene glycol diacrylate accounted for 1%, and phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide accounted for 0.5%. The mixture was then poured into a mold and cured under ultraviolet light for 6 minutes to obtain a recombinant humanized collagen hydrogel.

[0046] S3.2: Place the recombinant humanized collagen hydrogel in a container, add 0.05wt% sodium paraben and 0.08wt% disodium EDTA, stir thoroughly, and then fill into vials, 10mL per vial. After capping, sterilize at Co60 to obtain the recombinant collagen repair solution.

[0047] Example 2: Preparation of S1 protein activity enhancer

[0048] S1.1: Turnip pollen was pulverized through a 60-mesh sieve and dried at 63℃ for 2.5h. Then, it was added to a 0.025% citric acid solution at a solid-liquid ratio of 1:35g / mL and mixed evenly. The mixture was then transferred to a container equipped with a reflux device and extracted by reflux at 102℃ for 4h. The residue was removed by filtration. 0.25wt% chitosan was added to the filtrate and stirred evenly. The mixture was then stirred at 67℃ for 1.2h. After standing and cooling to room temperature, it was centrifuged at 6250rpm for 14min. The supernatant was collected, concentrated, and freeze-dried to obtain turnip pollen extract.

[0049] S1.2: The turnip pollen extract was redissolved in distilled water at a solid-liquid ratio of 1:9 g / mL, and then anhydrous ethanol was added and stirred to make the volume concentration of anhydrous ethanol 60%. After ultrasonic dispersion for 7 minutes, it was allowed to stand for 13 hours. The precipitate was collected by centrifugation at 8250 rpm for 11 minutes to obtain crude turnip pollen polysaccharide. A gel chromatography column was prepared, and the crude turnip pollen polysaccharide and dextran gel G-100 were loaded at a mass ratio of 1:50. Pure water was used for elution at a flow rate of 1.5 mL / min. The eluent with the largest elution peak was collected and dried with nitrogen to obtain turnip pollen polysaccharide.

[0050] S1.3: Place the grinding dish on ice and put it in a fume hood. Add pyridine to the grinding dish, then slowly add chlorosulfonic acid along the wall while continuously stirring rapidly. When a white solid forms in the grinding dish, remove the ice bath to obtain the sulfonating reagent. The solid-liquid ratio of pyridine to chlorosulfonic acid is 1:1 g / mL. Dissolve turnip pollen polysaccharide in anhydrous formamide at a solid-liquid ratio of 1:45 g / mL. Then add sulfonating reagent accounting for 55% of the volume of anhydrous formamide and stir evenly. Then react at 43℃ for 4.5 h to obtain the reaction solution. Add 2.5 mol / L sodium hydroxide solution to the reaction solution to adjust to neutrality. Then put it into a dialysis bag with a molecular weight cutoff of 10 kD and dialyze it with deionized water for 4 days, changing the deionized water every 8 h. Then take out the liquid from the dialysis bag and freeze-dry it to obtain the protein activity enhancer.

[0051] Preparation of S2 recombinant humanized collagen fibrinolytic sol

[0052] S2.1: Take PBS buffer with pH 7 and a concentration of 20mM, add 0.1M NaOH solution to adjust the pH to 7.7, then add NaCl to dissolve it so that the concentration of NaCl in the PBS buffer is 0.2M, to obtain the sol-based solution;

[0053] S2.2: Add recombinant type III humanized collagen, protein activity enhancer and lupin dietary fiber to the sol base solution and stir evenly to make the mass concentration of recombinant type III humanized collagen 9%, the mass concentration of protein activity enhancer 0.9% and the mass concentration of lupin dietary fiber 2.5% to obtain a composite suspension.

[0054] S2.3: The composite suspension was treated in a water bath at 57°C for 33 minutes, and then transferred to an ice-water bath to be rapidly cooled to room temperature to obtain recombinant humanized collagen fiber sol.

[0055] S3 photopolymerization constructs recombinant humanized collagen hydrogel

[0056] S3.1: Acryloyloxyethyltrimethylammonium chloride, acrylamide, and choline chloride were mixed in a molar ratio of 1:0.9:2.5 in a container to obtain a mixed monomer. Deionized water and recombinant type III humanized collagen sol were added, followed by polyethylene glycol diacrylate and phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide, which were mixed evenly. By mass of the mixed monomer, deionized water accounted for 90%, recombinant type III humanized collagen sol accounted for 45%, polyethylene glycol diacrylate accounted for 1.2%, and phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide accounted for 0.6%. The mixture was then poured into a mold and cured under ultraviolet light for 7 minutes to obtain a recombinant humanized collagen hydrogel.

[0057] S3.2: Place the recombinant humanized collagen hydrogel in a container, add 0.08wt% sodium paraben and 0.09wt% disodium EDTA, stir thoroughly, and then fill into vials, 10mL per vial. After capping, sterilize at Co60 to obtain the recombinant collagen repair solution.

[0058] Example 3: A method for preparing a recombinant collagen repair solution, comprising the following steps:

[0059] Preparation of S1 protein activity enhancer

[0060] S1.1: Turnip pollen was pulverized through a 70-mesh sieve and dried at 65℃ for 3 hours. Then, it was added to a 0.03% citric acid solution at a solid-liquid ratio of 1:40 g / mL and mixed evenly. The mixture was then transferred to a container equipped with a reflux device and extracted by reflux at 105℃ for 5 hours. The residue was removed by filtration. 0.3 wt% chitosan was added to the filtrate and stirred evenly. The mixture was then stirred at 70℃ for 1.5 hours. After standing and cooling to room temperature, it was centrifuged at 6500 rpm for 15 minutes. The supernatant was collected, concentrated, and freeze-dried to obtain turnip pollen extract.

[0061] S1.2: The turnip pollen extract was redissolved in distilled water at a solid-liquid ratio of 1:10 g / mL, and then anhydrous ethanol was added and stirred to make the volume concentration of anhydrous ethanol 60%. After ultrasonic dispersion for 8 minutes, it was allowed to stand for 15 hours. The precipitate was collected by centrifugation at 8500 rpm for 12 minutes to obtain crude turnip pollen polysaccharide. A gel chromatography column was prepared, and the crude turnip pollen polysaccharide and dextran gel G-100 were loaded at a mass ratio of 1:50. Pure water was used for elution at a flow rate of 1.5 mL / min. The eluent with the largest elution peak was collected and dried with nitrogen to obtain turnip pollen polysaccharide.

[0062] S1.3: Place the grinding dish on ice and put it in a fume hood. Add pyridine to the grinding dish, then slowly add chlorosulfonic acid along the wall while continuously stirring rapidly. When a white solid forms in the grinding dish, remove the ice bath to obtain the sulfonating reagent. The solid-liquid ratio of pyridine to chlorosulfonic acid is 1:1 g / mL. Dissolve turnip pollen polysaccharide in anhydrous formamide at a solid-liquid ratio of 1:50 g / mL. Then add sulfonating reagent accounting for 60% of the volume of anhydrous formamide and stir evenly. Then react at 45℃ for 5 hours to obtain the reaction solution. Add 2.5 mol / L sodium hydroxide solution to the reaction solution to adjust to neutrality. Then put it into a dialysis bag with a molecular weight cutoff of 10 kD and dialyze it with deionized water for 5 days, changing the deionized water every 8 hours. Then take the liquid out of the dialysis bag and freeze-dry it to obtain the protein activity enhancer.

[0063] Preparation of S2 recombinant humanized collagen fibrinolytic sol

[0064] S2.1: Take PBS buffer with pH 7 and a concentration of 20mM, add 0.1M NaOH solution to adjust the pH to 8, then add NaCl to dissolve it so that the concentration of NaCl in the PBS buffer is 0.3M, to obtain the sol-based solution;

[0065] S2.2: Add recombinant type III humanized collagen, protein activity enhancer and lupin dietary fiber to the sol base solution and stir evenly to make the mass concentration of recombinant type III humanized collagen 10%, the mass concentration of protein activity enhancer 1%, and the mass concentration of lupin dietary fiber 3% to obtain a composite suspension.

[0066] S2.3: The composite suspension was treated in a water bath at 60°C for 35 minutes, and then transferred to an ice-water bath to cool rapidly to room temperature to obtain recombinant humanized collagen fiber sol.

[0067] S3 photopolymerization constructs recombinant humanized collagen hydrogel

[0068] S3.1: Acryloyloxyethyltrimethylammonium chloride, acrylamide, and choline chloride were mixed in a molar ratio of 1:1:3 in a container to obtain a mixed monomer. Deionized water and recombinant type III humanized collagen sol were added, followed by polyethylene glycol diacrylate and phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide. The mixture was then thoroughly mixed. By mass of the mixed monomer, deionized water accounted for 100%, recombinant type III humanized collagen sol accounted for 50%, polyethylene glycol diacrylate accounted for 1.5%, and phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide accounted for 0.7%. The mixture was then poured into a mold and cured under ultraviolet light for 8 minutes to obtain a recombinant humanized collagen hydrogel.

[0069] S3.2: Place the recombinant humanized collagen hydrogel in a container, add 0.1 wt% sodium paraben and 0.1 wt% disodium EDTA, stir thoroughly, and then fill into vials, 10 mL per vial. After capping, sterilize at Co60 to obtain the recombinant collagen repair solution.

[0070] Comparative Example 1: A recombinant collagen repair solution and its preparation method. Compared with the implementation method of Example 1, the difference is that step S1 is removed and a protein activity enhancer is not added in step S2.2. All other steps are the same as those in Example 1.

[0071] Comparative Example 2: A recombinant collagen repair solution and its preparation method. Compared with the implementation method of Example 1, the difference is that the lupin dietary fiber in step S2.2 is replaced with an equal mass of carboxymethyl cellulose, and the remaining steps are the same as those in Example 1.

[0072] Comparative Example 3: A recombinant collagen repair solution and its preparation method. Compared with the implementation method of Example 1, the difference is that steps S2.1 and S2.3 are removed, the sol base solution in step S2.2 is replaced with deionized water to obtain a composite suspension, and the recombinant type III humanized collagen sol in step S3.1 is replaced with an equal mass of composite suspension. The remaining steps are the same as in Example 1.

[0073] Comparative Example 4: A recombinant collagen repair solution and its preparation method. Compared with the implementation method of Example 1, the difference is that step S3.1 is removed, and the recombinant humanized collagen hydrogel in step S3.2 is replaced with recombinant humanized collagen fibrinosol. The remaining steps are the same as those in Example 1.

[0074] Wound healing test

[0075] Fifteen SD rats (150-180g) were selected and divided into 5 groups of 3 rats each. All SD rats were anesthetized with isoflurane, and two 5mm diameter wounds were made on their backs. Then, 10μL of Staphylococcus aureus solution (10...) was applied. 8 (CFU / mL) was injected into rat wounds, and left for 10 minutes to simulate bacterial infection of the wound. Recombinant collagen repair solutions prepared in Examples 1-3 and Comparative Example 1 were then taken from different groups of rats and uniformly applied to the rat wounds, with a coverage area of ​​1 cm². 2 One group of rats was designated as the application group, and another group of rats had sterile gauze applied to the wound and fixed with medical tape, which was designated as the blank group. All rats were cultured separately, and the dressing was changed every 24 hours. Wound images were taken on days 3, 7 and 14. The wound area was observed and the percentage relative to the initial wound area was calculated. The average value of each group was taken, and the data were recorded and made into a table, as shown in Table 1.

[0076] Table 1: Wound area of ​​recombinant collagen repair solution

[0077] Wound area on day 3 / % Wound area on day 7 / % Wound area on day 14 / % Blank group 78.5 50.3 18.5 Example 1 52.4 16.3 0 Example 2 51.8 16.1 0 Example 3 51.6 15.7 0 Comparative Example 1 59.2 24.7 4.6

[0078] As can be seen from the data in Examples 1-3 in Table 1, the recombinant collagen repair solution prepared in this application has excellent therapeutic and repair effects on wounds. However, as can be seen from the data in Comparative Example 1, without the addition of a protein activity enhancer, the ability of the recombinant collagen repair solution to treat wounds decreases. This proves that the protein activity enhancer can enhance cell adhesion and proliferation, thereby strengthening the epidermal regeneration ability of recombinant humanized collagen and effectively improving the bioavailability of recombinant humanized collagen at the wound site and its repair and treatment effects on wounded skin.

[0079] Water retention performance test

[0080] Take 10g of the recombinant collagen repair solution prepared in Examples 1-3 and Comparative Examples 2-3 as samples, and prepare three samples for each. Then place them in an open environment at 36℃ and 35% relative humidity for 3 days. After weighing, record the mass as w. Calculate the weight loss rate = (10-w) / 10×100%. The lower the weight loss rate, the better the water retention performance of the sample. The test results are shown in Table 2.

[0081] Table 2: Weight loss rate of recombinant collagen repair solution

[0082] First weight loss rate / % Second weight loss rate / % Third weight loss rate / % Example 1 13.4 13.7 13.3 Example 2 13.1 12.9 13.2 Example 3 12.8 12.5 12.6 Comparative Example 2 24.5 24.3 24.6 Comparative Example 3 20.4 19.7 20.2

[0083] As can be seen from the data of Examples 1-3 in Table 2, the repair solutions prepared in Examples 1-3 had a low weight loss rate after being placed in an open environment at 36°C and 35% relative humidity for three days, proving their good water retention performance. However, as can be seen from the data of Comparative Examples 2-3, the water retention performance of the repair solution decreased after replacing lupin dietary fiber with carboxymethyl cellulose and after not preparing recombinant humanized collagen fiber sol. This proves that recombinant type III humanized collagen, protein activity enhancer, and lupin dietary fiber can jointly construct a network structure to form a sol, and then form an interpenetrating structure with mixed monomers, which can significantly improve the water retention performance of the repair solution.

[0084] Mechanical property testing

[0085] The recombinant collagen repair solutions prepared in Examples 1-3 and Comparative Examples 2-4 were used to make dumbbell-shaped samples (10mm×40mm×1mm). The dumbbell-shaped samples were stretched using a tensile testing machine at a running speed of 50mm / min to test their tensile strength. Each dumbbell-shaped sample was tested three times and the average value was taken. The recombinant collagen repair solution was made into cylindrical samples (15mm in diameter and 10mm in height). The cylindrical samples were compressed at room temperature at a running speed of 5mm / s to test their compressive strength at 90% strain. Each cylindrical sample was tested three times and the average value was taken. The test results are shown in Table 3.

[0086] Table 3: Mechanical properties of recombinant collagen repair solution

[0087] Tensile strength / kPa Compressive strength at 90% strain / kPa Example 1 406.16 462.53 Example 2 408.27 467.62 Example 3 410.34 470.14 Comparative Example 2 382.57 433.79 Comparative Example 3 313.85 362.13 Comparative Example 4 262.89 297.81

[0088] As can be seen from the data in Examples 1-3 in Table 3, the tensile strength of the repair solution prepared in this application reached over 400 kPa, and the compressive strength at 90% strain reached over 460 kPa, demonstrating excellent mechanical properties. Furthermore, the data from Comparative Examples 2-4 show that using lupin dietary fiber to prepare recombinant humanized collagen fiber sol, and using acryloyloxyethyltrimethylammonium chloride, acrylamide, and choline chloride as monomers, and interpenetrating the recombinant humanized collagen fiber sol network structure within the monomer-formed gel structure to create an interpenetrating hydrogel, significantly increases the mechanical properties of the hydrogel, preventing gel breakage caused by external physical factors (such as joint extension and friction), thereby stably releasing the effective components to promote wound healing and prevent infection.

[0089] Biocompatibility testing

[0090] The recombinant collagen repair solutions prepared in Examples 1-3 were sterilized under ultraviolet light, and then each solution was extracted by immersing it in distilled water to obtain a 1 g / mL gel extract. 2×10 4200 μL of L929 cell suspension (approximately 100 μL / mL) was injected into each well of a 96-well plate, followed by 100 μL of gel extraction buffer. The plates were incubated at 37°C in a 5% CO2 incubator for 24 h. Then, 10 μL of LCK-8 solution was added to each well, and the plates were incubated for 1 h, with absorbance measured at 450 nm. L929 cells were labeled with a live / dead cell staining kit for 30 min, and cell viability was monitored using an inverted confocal fluorescence microscope after 48 h. Each recombinant collagen repair solution was tested in triplicate, and cell viability was calculated and averaged. The results are shown in Table 4.

[0091] Table 4: Survival of L929 cells after 48 hours of co-culturing with recombinant collagen repair medium

[0092] Cell viability / % Live / dead staining fluorescence images Example 1 85 The vast majority are green. Example 2 88 The vast majority are green. Example 3 87 The vast majority are green.

[0093] As can be seen from the data of Examples 1-3 in Table 4, the cell survival rate is above 85%, which is within the standard range of cell survival rate in nutrient-free culture. Furthermore, the vast majority of the cell fluorescence staining images are green, which confirms that the recombinant collagen repair solution prepared in this application has good biocompatibility and meets the basic requirements for human application.

[0094] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for preparing a recombinant collagen repair solution, characterized in that, Includes the following steps: Preparation of S1 protein activity enhancer After pulverizing the turnip pollen, it was extracted by reflux with citric acid solution. Then, chitosan was added, stirred evenly, and allowed to stand. The supernatant was collected by centrifugation, concentrated, and freeze-dried to obtain the turnip pollen extract. The extract was then redissolved in distilled water and precipitated with alcohol to obtain crude turnip pollen polysaccharide. The extract was then purified by gel chromatography to obtain turnip pollen polysaccharide. The polysaccharide was dissolved in anhydrous formamide, and a sulfonating agent prepared from pyridine and chlorosulfonic acid was added to react with it. The reaction solution was dialyzed with deionized water and then freeze-dried to obtain a protein activity enhancer. Preparation of S2 recombinant humanized collagen fibrinolytic sol After adjusting the pH by adding NaOH solution to neutral PBS buffer, NaCl was added to obtain a sol base solution. Recombinant type III humanized collagen, protein activity enhancer and lupin dietary fiber were added to the sol base solution and stirred evenly. Then, it was placed in a hot water bath for treatment and rapidly cooled to room temperature to obtain recombinant humanized collagen fiber sol. S3 photopolymerization constructs recombinant humanized collagen hydrogel Acryloyloxyethyltrimethylammonium chloride, acrylamide, and choline chloride were mixed in a container to obtain a mixed monomer. Deionized water and recombinant type III humanized collagen sol were added, followed by polyethylene glycol diacrylate and phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide, which were mixed evenly. The mixture was then cured under ultraviolet light to obtain a recombinant humanized collagen hydrogel. Sodium parabens and disodium ethylenediaminetetraacetate were added and stirred evenly. The mixture was then filled, capped, and sterilized to obtain a recombinant collagen repair solution.

2. The method for preparing a recombinant collagen repair solution according to claim 1, characterized in that, The preparation of the protein activity enhancer in step S1 specifically includes the following steps: S1.1: After pulverizing turnip pollen through a 60-70 mesh sieve, dry it at 60-65℃ for 2-3 hours. Then, add it to citric acid solution at a solid-liquid ratio of 1:(30-40) g / mL and mix well. Transfer it to a container equipped with a reflux device and reflux extract it at 100-105℃ for 3-5 hours. Filter to remove the residue. Take the filtrate and add 0.2-0.3wt% chitosan and stir well. Then stir at 65-70℃ for 1-1.5 hours. After standing and cooling to room temperature, centrifuge at 6000-6500 rpm for 12-15 minutes. Take the supernatant and concentrate and freeze dry it to obtain turnip pollen extract. S1.2: The turnip pollen extract was redissolved in distilled water at a solid-liquid ratio of 1:(8-10) g / mL, and then anhydrous ethanol was added and stirred to make the volume concentration of anhydrous ethanol 60%. After ultrasonic dispersion for 6-8 minutes, it was allowed to stand for 12-15 hours. The precipitate was collected by centrifugation at 8000-8500 rpm for 10-12 minutes to obtain crude turnip pollen polysaccharide. The crude turnip pollen polysaccharide was purified by gel chromatography to obtain turnip pollen polysaccharide. S1.3: Place the grinding dish on ice and put it in a fume hood. Add pyridine to the grinding dish, then slowly add chlorosulfonic acid along the wall while continuously stirring rapidly. When a white solid forms in the grinding dish, remove the ice bath to obtain the sulfonating reagent. Dissolve turnip pollen polysaccharide in anhydrous formamide at a solid-liquid ratio of 1:(40-50) g / mL. Add 50-60% of the volume of the sulfonating reagent to the anhydrous formamide and stir until homogeneous. Then, react at 42-45℃ for 4-5 hours to obtain the reaction solution. Add 2-2.5 mol / L sodium hydroxide solution to the reaction solution to adjust it to neutral. Then, put it into a dialysis bag with a molecular weight cutoff of 10 kD and dialyze it with deionized water for 4-5 days, changing the deionized water every 8 hours. Then, take out the liquid from the dialysis bag and freeze-dry it to obtain the protein activity enhancer.

3. The method for preparing a recombinant collagen repair solution according to claim 2, characterized in that, Step S2, the preparation of recombinant humanized collagen fiber sol, specifically includes the following steps: S2.1: Take PBS buffer with pH 7 and a concentration of 20mM, add 0.1M NaOH solution to adjust the pH to 7.5-8, then add NaCl to dissolve it so that the concentration of NaCl in the PBS buffer is 0.2-0.3M, to obtain the sol-based solution; S2.2: Add recombinant type III humanized collagen, protein activity enhancer and lupin dietary fiber to the sol base solution and stir evenly to make the mass concentration of recombinant type III humanized collagen 8-10%, the mass concentration of protein activity enhancer 0.8-1%, and the mass concentration of lupin dietary fiber 2-3% to obtain a composite suspension. S2.3: The composite suspension is treated in a water bath at 55-60℃ for 30-35 minutes, and then transferred to an ice-water bath to cool rapidly to room temperature to obtain recombinant humanized collagen fiber sol.

4. The method for preparing a recombinant collagen repair solution according to claim 3, characterized in that, Step S3, photopolymerization, constructs a recombinant humanized collagen hydrogel, specifically including the following steps: S3.1: Acryloyloxyethyltrimethylammonium chloride, acrylamide and choline chloride are mixed in a molar ratio of 1:(0.8-1):(2-3) and placed in a container to obtain mixed monomers. Deionized water and recombinant type III humanized collagen sol are added, followed by polyethylene glycol diacrylate and phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide. The mixture is then poured into a mold and cured under ultraviolet light for 6-8 minutes to obtain recombinant humanized collagen hydrogel. S3.2: Place the recombinant humanized collagen hydrogel in a container, add 0.05-0.1wt% sodium paraben and 0.08-0.1wt% disodium EDTA, stir thoroughly, and then fill into vials, 10mL per vial. After capping, sterilize at Co60 to obtain the recombinant collagen repair solution.

5. The method for preparing a recombinant collagen repair solution according to claim 2, characterized in that, The citric acid solution in step S1.1 has a mass concentration of 0.02-0.03%.

6. The method for preparing a recombinant collagen repair solution according to claim 2, characterized in that, In step S1.2, the gel packing material in the gel chromatography column is dextran gel G-100. When purifying crude polysaccharide from turnip pollen, crude polysaccharide from turnip pollen and gel packing material are loaded at a mass ratio of 1:

50. Pure water is used for elution at a flow rate of 1.5 mL / min. The eluent with the largest elution peak is collected and dried with nitrogen to obtain turnip pollen polysaccharide.

7. The method for preparing a recombinant collagen repair solution according to claim 2, characterized in that, The sulfonating agent in step S1.3 is prepared by mixing pyridine and chlorosulfonic acid at a solid-liquid ratio of 1:1 g / mL.

8. The method for preparing a recombinant collagen repair solution according to claim 4, characterized in that, In step S3.1, based on the mass of the mixed monomers, deionized water accounts for 80-100%, recombinant type III humanized collagen sol accounts for 40-50%, polyethylene glycol diacrylate accounts for 1-1.5%, and phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide accounts for 0.5-0.7%.

9. A recombinant collagen repair solution, characterized in that, It is prepared by any one of the recombinant collagen repair solution methods of claims 1-8.