Medical collagen liquid for wound repair and preparation process thereof

Abstract

The invention provides a medical collagen liquid for wound repair and a preparation process thereof, and belongs to the technical field of collagen liquid preparation. The preparation process comprises the following steps: preparing the tea polyphenol zinc complex; preparing a tea polyphenol zinc complex liposome; preparing a thermo-sensitive composite microsphere; and preparing a collagen liquid. The collagen liquid contains the thermo-sensitive composite microspheres which are prepared by firstly carrying out lipidosome coating on the tea polyphenol zinc complex and then compounding with the recombinant human collagen III, and the thermo-sensitive composite microspheres have thermo-sensitivity and can realize intelligent release; the microspheres can quickly release the recombinant human collagen III type and tea polyphenol zinc complex liposome, continuously provide nutrition and support for wound repair, and are beneficial to long-term repair and healing of wounds.

Description

Technical Field

[0001] The invention relates to the technical field of collagen liquid preparation, and in particular to a medical collagen liquid for wound repair and a preparation process thereof. Background Art

[0002] In the field of trauma treatment, wound repair is a crucial link. Effective wound repair is not only related to the patient's physical recovery, but also directly affects their quality of life. At present, there are many products for wound repair on the market, such as traditional vaseline gauze, various antibacterial ointments, and some biological agents containing growth factors.

[0003] However, these existing wound repair products still have many defects. Although traditional vaseline gauze can protect the wound to a certain extent, it has poor air permeability, is prone to infection, and is not conducive to wound healing. Although antibacterial ointments can inhibit the growth of some bacteria, they are often unable to effectively remove the excessive free radicals produced during the wound repair process. Excessive oxidative stress can damage the wound tissue, leading to an increase in inflammatory response and hindering the normal healing of the wound. Although biological preparations containing growth factors can promote cell proliferation, they have poor stability, high cost, and limited absorption and utilization efficiency in the body, making it difficult to provide long-term support for wound repair.

[0004] During the wound repair process, it is necessary to effectively inhibit pathogens and reduce the risk of infection, provide a good growth environment for cells, promote cell proliferation and tissue regeneration, and at the same time solve the problem of drug absorption and sustained release in the wound tissue to achieve long-term and effective repair of the wound.

[0005] Therefore, developing a safe, efficient, multifunctional and intelligently released medical collagen solution for wound repair and its preparation process has important practical significance and clinical application value. Summary of the invention

[0006] In view of the deficiencies in the prior art, the object of the present invention is to provide a medical collagen solution for wound repair and a preparation process thereof.

[0007] A medical collagen solution for wound repair and a preparation process thereof, comprising the following steps:

[0008] S1: Preparation of tea polyphenol zinc complex

[0009] The tea polyphenols extract is obtained by desorbing with tea powder and then extracting with a desorbent, and then the tea polyphenols solution and a zinc chloride solution are mixed, and NaHCO3 is added to adjust the mixed solution to a pH of 6, and then reacting to prepare a tea polyphenols zinc complex;

[0010] S2: Preparation of tea polyphenol zinc complex liposomes

[0011] Soybean lecithin and cholesterol were used as raw materials to embed tea polyphenol zinc complex to prepare tea polyphenol zinc complex liposome;

[0012] S3: Preparation of thermosensitive composite microspheres

[0013] Sodium alginate was grafted with N-isopropylacrylamide to prepare grafted modified sodium alginate, and then tea polyphenol zinc complex liposomes and recombinant human collagen type III were embedded in grafted modified sodium alginate and chitosan to prepare thermosensitive composite microspheres.

[0014] S4: Preparation of collagen solution

[0015] To 80-100 parts by weight of deionized water, add 1-3 parts by weight of sodium hyaluronate, 2-3 parts by weight of trehalose, 2-3 parts by weight of glycerol, 3-5 parts by weight of glyceryl caprylate, 1-2 parts by weight of caprylhydroxamic acid, 1-2 parts by weight of hexylene glycol and 10-12 parts by weight of thermosensitive composite microspheres, stir and mix, and then sterilize to obtain a collagen solution.

[0016] Furthermore, step S1 of preparing the tea polyphenol zinc complex specifically comprises the following steps:

[0017] S1.1: 3-5 parts by weight of tea powder are uniformly moistened with 15-20 parts by weight of desorbent, and desorbed for 8-10 minutes to allow the desorbent to fully penetrate the tea powder, and then 80-100 parts by weight of pure water at 90-100°C are added, and extraction is performed for 10-12 minutes. After the extraction is completed, centrifuge at 4000-4200 r / min for 10-12 minutes, and the supernatant is taken to obtain a tea polyphenol extract;

[0018] S1.2: Mix the tea polyphenol solution and zinc chloride solution, then adjust the mixed solution to pH 6 with 0.1 mol / L NaHCO3, stir at a constant speed for 20-30 min, then collect the precipitate by centrifugation, wash the precipitate with distilled water and anhydrous ethanol 2-3 times in turn, and vacuum freeze-dry to obtain a solid powder, i.e., tea polyphenol zinc complex.

[0019] Furthermore, step S2 of preparing tea polyphenol zinc complex liposomes specifically comprises the following steps:

[0020] S2.1: 0.7-1 parts by weight of soybean lecithin and 0.25-0.3 parts by weight of cholesterol are dissolved in 20-30 parts by weight of a chloroform-ethanol mixed solvent, and after mixing evenly, 0.05-0.08 parts by weight of a tea polyphenol zinc complex is added, and the mixture is stirred for 20-30 minutes, and then a rotary evaporator is used to rotary evaporate at 40-60° C. for 1-2 hours to obtain a lipid film;

[0021] S2.2: adding 1-2 parts by weight of the lipid film to 10-12 parts by weight of PBS buffer preheated to 55-60°C, pH 6.5, and then magnetically stirring at 300-500 r / min for 1-2 hours to obtain a hydrated suspension;

[0022] S2.3: The hydrated suspension is ultrasonically treated in an ice bath for 5-8 minutes, and the ultrasonic suspension is filtered through a 0.45 μm microporous filter membrane and then transferred to a high-pressure homogenizer for 4-5 cycles of homogenization at a pressure of 800 bar to obtain a homogenized suspension;

[0023] S2.4: The homogenized suspension is centrifuged at 3000-4000 centrifugal force for 10-15 min using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa, and then filtered through a 0.22 μm sterile filter membrane to obtain tea polyphenol zinc complex liposomes.

[0024] Furthermore, step S3 of preparing the temperature-sensitive composite microspheres specifically comprises the following steps:

[0025] S3.1: 1-2 parts by weight of 1wt% sodium alginate solution is magnetically stirred and heated at 70-80°C for 20-30min, and then 0.23-0.4 parts by weight of initiator is added, and the temperature is kept at 40-50°C for 20-30min, and then 1-2 parts by weight of N-isopropylacrylamide is added, and the reaction is continued for 3-4h. At the end of the reaction, the reactant is poured into 200-230 parts by weight of ethanol to terminate the chemical reaction, and then washed 2-3 times and dried to obtain grafted modified sodium alginate;

[0026] S3.2: Disperse 1.5-2 parts by weight of grafted modified sodium alginate in 100-120 parts by weight of distilled water, stir magnetically for 20-30 minutes, then add 5-10 parts by weight of calcium carbonate, ultrasonicate for 5-10 minutes, mix as the water phase, add 3-4 parts by weight of emulsifier Span-80 into 200-220 parts by weight of liquid paraffin oil, stir and mix evenly as the oil phase;

[0027] S3.3: Disperse 2-3 parts by weight of chitosan in 100-120 parts by weight of 0.5wt% acetic acid solution, magnetically stir for 20-30 minutes to obtain a cross-linking agent, add the aqueous phase to the oil phase, mechanically stir and emulsify for 5-8 minutes, then add 2-3 parts by weight of tea polyphenol zinc complex liposomes and 2-3 parts by weight of recombinant human collagen type III, continue emulsification for 10-12 minutes, then add 1-2 parts by weight of acetic acid and 2-3 parts by weight of calcium carbonate to react for 2-3 hours to obtain an emulsion, add the emulsion dropwise into the cross-linking agent, cross-link reaction for 20-30 minutes, then centrifuge, filter, wash and dry to obtain thermosensitive composite microspheres.

[0028] Furthermore, the desorbent in step S1.1 is a 40-60 wt % ethanol aqueous solution.

[0029] Furthermore, in step S1.2, the tea polyphenol solution and the zinc chloride solution are mixed in a molar ratio of 1:2-3.

[0030] Furthermore, the volume ratio of chloroform to ethanol in step S2.1 is 4:1.

[0031] Furthermore, in step S2.3, the power of the ultrasonic treatment is 200-220 W, working for 2 seconds / resting for 3 seconds.

[0032] Furthermore, in step S3.1, the initiator is potassium persulfate.

[0033] A medical collagen solution for wound repair, which is prepared by the preparation process of a medical collagen solution for wound repair as described in any one of claims 1 to 9.

[0034] Compared with the prior art, the present invention has at least the following beneficial effects:

[0035] 1. The collagen liquid of the present invention contains a tea polyphenol zinc complex. Tea polyphenols themselves have a strong antioxidant capacity. The catechins and other ingredients contained therein can remove excess free radicals generated in the process of wound repair. After forming a complex with zinc, it helps to reduce the damage of oxidative stress to wound tissue, prevent excessive inflammatory response, create a good microenvironment for wound repair, and promote cell proliferation and tissue regeneration. The complex formed by the two can produce a synergistic effect, enhance the ability to inhibit common pathogens on wounds such as Staphylococcus aureus and Escherichia coli, reduce the risk of wound infection, thereby alleviating inflammatory response and facilitating normal healing of wounds. At the same time, the zinc ions in the tea polyphenol zinc complex can participate in the intracellular signal transduction pathway, regulate cell growth and proliferation, promote fibroblast proliferation and collagen synthesis, and contribute to wound healing and tissue repair.

[0036] 2. The present invention compounds tea polyphenol zinc complex with recombinant human collagen type III. As an important component of the extracellular matrix, recombinant human collagen type III can provide a good growth scaffold for cells, promote cell adhesion, proliferation and differentiation, especially for fibroblasts and other cells closely related to wound repair, it can accelerate their proliferation and lay the foundation for wound repair. Zinc ions participate in the synthesis and activation of various enzymes, can regulate the signal transduction pathways in cells, further promote the mitosis and proliferation process of cells, and work together with collagen type III to more significantly increase the cell proliferation rate and accelerate the wound healing process.

[0037] 3. The present invention first encapsulates the tea polyphenol zinc complex into liposomes and then compound it with recombinant human collagen type III to prepare thermosensitive composite microspheres. The structure of the liposome is similar to that of the biological membrane and has good biocompatibility. The liposome encapsulating the tea polyphenol zinc complex can more easily fuse with cells or be taken up by cells, thereby promoting the tea polyphenol zinc complex to enter the cells, improving its absorption and utilization efficiency in the wound tissue, and better exerting its role in wound repair. In addition, the liposome can be used as a sustained-release carrier to slowly release the tea polyphenol zinc complex into the wound tissue. In the process of wound repair, it can continuously provide tea polyphenol zinc complex to the wound, maintain effective drug concentration, prolong its action time, and then compound with recombinant human collagen type III for embedding to prepare thermosensitive composite microspheres. The thermosensitive composite microspheres prepared by grafted modified sodium alginate and chitosan as raw materials have temperature sensitivity and can realize intelligent release. Under the human body temperature environment, the microspheres will quickly release recombinant human collagen type III and tea polyphenol zinc complex liposomes, continuously provide nutrition and support for wound repair, which is beneficial to the long-term repair and healing of the wound. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The accompanying drawings, which are incorporated herein and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, further serve to explain the principles of the invention and to enable those skilled in the relevant art to make and use the invention.

[0039] Figure 1 This is a process flow chart for preparing a medical collagen solution for wound repair adopted in an embodiment of the present invention. DETAILED DESCRIPTION

[0040] The following is a detailed description of the preparation process of a medical collagen solution for wound repair provided by the present invention in conjunction with the accompanying drawings and specific embodiments. At the same time, it is explained here that in order to make the embodiments more detailed, the following embodiments are the best and preferred embodiments, and those skilled in the art may also adopt other alternatives to implement some known technologies; and the accompanying drawings are only for a more specific description of the embodiments, and are not intended to specifically limit the present invention.

[0041] Example 1

[0042] A medical collagen solution for wound repair and a preparation process thereof, such as Figure 1 As shown, the following steps are included:

[0043] S1: Preparation of tea polyphenol zinc complex

[0044] S1.1: 3 parts by weight of tea powder are uniformly moistened with 15 parts by weight of a 40wt% ethanol aqueous solution desorbent, and desorbed for 8 minutes to allow the desorbent to fully penetrate the tea powder, and then 80 parts by weight of 90°C pure water are added for 10 minutes of extraction. After the extraction is completed, centrifuge at 4000r / min for 10 minutes, and take the supernatant to obtain a tea polyphenol extract;

[0045] S1.2: Mix the tea polyphenol solution and the zinc chloride solution in a molar ratio of 1:2, then adjust the mixed solution to pH 6 with 0.1 mol / L NaHCO3, stir at a constant speed for 20 min, then collect the precipitate by centrifugation, wash the precipitate with distilled water and anhydrous ethanol twice in sequence, and freeze-dry in vacuum to obtain a solid powder, i.e., the tea polyphenol zinc complex;

[0046] S2: Preparation of tea polyphenol zinc complex liposomes

[0047] S2.1: 0.7 parts by weight of soybean lecithin and 0.25 parts by weight of cholesterol were dissolved in 20 parts by weight of a chloroform-ethanol mixed solvent, with the volume ratio of chloroform to ethanol being 4:1. After mixing evenly, 0.05 parts by weight of tea polyphenol zinc complex was added, and the mixture was stirred for 20 minutes. Then, a rotary evaporator was used to rotary evaporate the mixture at 40°C for 1 hour to obtain a lipid film.

[0048] S2.2: adding 1 part by weight of the lipid film to 10 parts by weight of PBS buffer preheated to 55°C, pH 6.5, and then magnetically stirring at 300 r / min for 1 hour to obtain a hydrated suspension;

[0049] S2.3: The hydrated suspension was placed in an ice bath at 200 W, working for 2 seconds / resting for 3 seconds, and ultrasonically treated for 5 minutes. The ultrasonic suspension was filtered through a 0.45 μm microporous membrane and then transferred to a high-pressure homogenizer for 4 cycles of homogenization at a pressure of 800 bar to obtain a homogenized suspension;

[0050] S2.4: The homogenized suspension was centrifuged at 3000 centrifugal force for 10 min using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa, and then filtered through a 0.22 μm sterile filter membrane to obtain tea polyphenol zinc complex liposomes;

[0051] S3: Preparation of thermosensitive composite microspheres

[0052] S3.1: 1 part by weight of 1 wt% sodium alginate solution was magnetically stirred and heated at 70°C for 20 min, and then 0.23 parts by weight of initiator potassium persulfate was added and kept at 40°C for 20 min. After that, 1 part by weight of N-isopropylacrylamide was added and the reaction was continued for 3 h. At the end of the reaction, the reactant was poured into 200 parts by weight of ethanol to terminate the chemical reaction, and then washed twice and dried to obtain grafted modified sodium alginate;

[0053] S3.2: 1.5 parts by weight of grafted modified sodium alginate was dispersed in 100 parts by weight of distilled water, and magnetically stirred for 20 minutes. Then, 5 parts by weight of calcium carbonate was added, and ultrasonicated for 5 minutes. After mixing, the mixture was used as the water phase. 3 parts by weight of emulsifier Span-80 was added to 200 parts by weight of liquid paraffin oil, and stirred to mix well as the oil phase.

[0054] S3.3: Disperse 2 parts by weight of chitosan in 100 parts by weight of 0.5wt% acetic acid solution, stir magnetically for 20 minutes to obtain a crosslinker, add the aqueous phase to the oil phase, stir mechanically for emulsification for 5 minutes, then add 2 parts by weight of tea polyphenol zinc complex liposomes and 2 parts by weight of recombinant human collagen type III, continue emulsification for 10 minutes, then add 1 part by weight of acetic acid and 2 parts by weight of calcium carbonate to react for 2 hours to obtain an emulsion, add the emulsion dropwise to the crosslinker, crosslink for 20 minutes, then centrifuge, filter, wash and dry to obtain thermosensitive composite microspheres;

[0055] S4: Preparation of collagen solution

[0056] To 80 parts by weight of deionized water, 1 part by weight of sodium hyaluronate, 2 parts by weight of trehalose, 2 parts by weight of glycerol, 3 parts by weight of glyceryl caprylate, 1 part by weight of caprylhydroxamic acid, 1 part by weight of hexylene glycol and 10 parts by weight of thermosensitive composite microspheres were added, the mixture was stirred and mixed, and then sterilized to obtain a collagen solution.

[0057] Example 2

[0058] A preparation process of medical collagen solution for wound repair, such as Figure 1 As shown, the following steps are included:

[0059] S1: Preparation of tea polyphenol zinc complex

[0060] S1.1: 5 parts by weight of tea powder are uniformly moistened with 20 parts by weight of a 60wt% ethanol aqueous solution desorbent, and desorbed for 8 minutes to allow the desorbent to fully penetrate the tea powder, and then 100 parts by weight of 90°C pure water are added and extracted for 10 minutes. After the extraction is completed, centrifuge at 4000r / min for 10 minutes, and take the supernatant to obtain a tea polyphenol extract;

[0061] S1.2: Mix the tea polyphenol solution and the zinc chloride solution in a molar ratio of 1:3, then adjust the mixed solution to pH 6 with 0.1 mol / L NaHCO3, stir at a constant speed for 20 min, then collect the precipitate by centrifugation, wash the precipitate with distilled water and anhydrous ethanol 2-3 times in turn, and freeze-dry in vacuum to obtain a solid powder, i.e., the tea polyphenol zinc complex;

[0062] S2: Preparation of tea polyphenol zinc complex liposomes

[0063] S2.1: 1 part by weight of soybean lecithin and 0.3 parts by weight of cholesterol were dissolved in 30 parts by weight of a chloroform-ethanol mixed solvent, with the volume ratio of chloroform to ethanol being 4:1. After mixing evenly, 0.08 parts by weight of tea polyphenol zinc complex was added, and the mixture was stirred for 20 minutes. Then, a rotary evaporator was used to rotary evaporate the mixture at 40°C for 1 hour to obtain a lipid film.

[0064] S2.2: adding 2 parts by weight of the lipid film to 12 parts by weight of PBS buffer preheated to 55°C, pH 6.5, and then magnetically stirring at 300 r / min for 1 hour to obtain a hydrated suspension;

[0065] S2.3: The hydrated suspension was placed in an ice bath at 200 W, working for 2 seconds / resting for 3 seconds, and ultrasonically treated for 5 minutes. The ultrasonic suspension was filtered through a 0.45 μm microporous membrane and then transferred to a high-pressure homogenizer for 4 cycles of homogenization at a pressure of 800 bar to obtain a homogenized suspension;

[0066] S2.4: The homogenized suspension was centrifuged at 3000 centrifugal force for 10 min using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa, and then filtered through a 0.22 μm sterile filter membrane to obtain tea polyphenol zinc complex liposomes;

[0067] S3: Preparation of thermosensitive composite microspheres

[0068] S3.1: 2 parts by weight of 1 wt% sodium alginate solution are magnetically stirred and heated at 70°C for 20 min, and then 0.4 parts by weight of initiator potassium persulfate are added and kept at 40°C for 20 min. Thereafter, 2 parts by weight of N-isopropylacrylamide are added and the reaction is continued for 3 h. At the end of the reaction, the reactant is poured into 230 parts by weight of ethanol to terminate the chemical reaction, and then washed twice and dried to obtain grafted modified sodium alginate;

[0069] S3.2: 2 parts by weight of grafted modified sodium alginate was dispersed in 120 parts by weight of distilled water, and magnetically stirred for 20 minutes. Then, 10 parts by weight of calcium carbonate was added, and ultrasonicated for 5 minutes. After mixing, the mixture was used as the water phase. 4 parts by weight of emulsifier Span-80 was added to 220 parts by weight of liquid paraffin oil, and stirred to mix well as the oil phase.

[0070] S3.3: Disperse 3 parts by weight of chitosan in 120 parts by weight of 0.5wt% acetic acid solution, stir magnetically for 20 minutes to obtain a crosslinking agent, add the aqueous phase to the oil phase, stir mechanically for emulsification for 5 minutes, then add 3 parts by weight of tea polyphenol zinc complex liposomes and 3 parts by weight of recombinant human collagen type III, continue emulsification for 10 minutes, then add 2 parts by weight of acetic acid and 3 parts by weight of calcium carbonate to react for 2 hours to obtain an emulsion, add the emulsion dropwise to the crosslinking agent, crosslink for 20 minutes, then centrifuge, filter, wash and dry to obtain thermosensitive composite microspheres;

[0071] S4: Preparation of collagen solution

[0072] To 100 parts by weight of deionized water, 3 parts by weight of sodium hyaluronate, 3 parts by weight of trehalose, 3 parts by weight of glycerol, 5 parts by weight of glyceryl caprylate, 2 parts by weight of caprylhydroxamic acid, 2 parts by weight of hexylene glycol and 12 parts by weight of thermosensitive composite microspheres were added, the mixture was stirred and mixed, and then sterilized to obtain a collagen solution.

[0073] Example 3

[0074] A preparation process of medical collagen solution for wound repair, such as Figure 1 As shown, the following steps are included:

[0075] S1: Preparation of tea polyphenol zinc complex

[0076] S1.1: 3 parts by weight of tea powder are uniformly moistened with 15 parts by weight of a 40-60wt% ethanol aqueous solution desorbent, and desorbed for 10 minutes to allow the desorbent to fully penetrate the tea powder, and then 80 parts by weight of 100°C pure water are added and extracted for 12 minutes. After the extraction is completed, centrifuge at 4200r / min for 12 minutes, and take the supernatant to obtain a tea polyphenol extract;

[0077] S1.2: Mix the tea polyphenol solution and the zinc chloride solution in a molar ratio of 1:2, then adjust the mixed solution to pH 6 with 0.1 mol / L NaHCO3, stir at a constant speed for 30 min, then collect the precipitate by centrifugation, wash the precipitate with distilled water and anhydrous ethanol three times in sequence, and freeze-dry in vacuum to obtain a solid powder, i.e., the tea polyphenol zinc complex;

[0078] S2: Preparation of tea polyphenol zinc complex liposomes

[0079] S2.1: 0.7 parts by weight of soybean lecithin and 0.25 parts by weight of cholesterol were dissolved in 20 parts by weight of a chloroform-ethanol mixed solvent, with the volume ratio of chloroform to ethanol being 4:1. After mixing evenly, 0.05 parts by weight of tea polyphenol zinc complex was added, and the mixture was stirred for 30 minutes. Then, a rotary evaporator was used to evaporate the mixture at 60°C for 2 hours to obtain a lipid film.

[0080] S2.2: adding 1 part by weight of the lipid film to 10 parts by weight of PBS buffer preheated to 60°C, pH 6.5, and then magnetically stirring at 500 r / min for 2 h to obtain a hydrated suspension;

[0081] S2.3: The hydrated suspension was placed in an ice bath at 220W, working for 2s / resting for 3s, and ultrasonically treated for 8min. The ultrasonic suspension was filtered through a 0.45μm microporous filter membrane and then transferred to a high-pressure homogenizer for 5 cycles of homogenization at a pressure of 800bar to obtain a homogenized suspension;

[0082] S2.4: The homogenized suspension was centrifuged at 4000 centrifugal force for 15 min using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa, and then filtered through a 0.22 μm sterile filter membrane to obtain tea polyphenol zinc complex liposomes;

[0083] S3: Preparation of thermosensitive composite microspheres

[0084] S3.1: 1 part by weight of 1 wt% sodium alginate solution was magnetically stirred and heated at 80°C for 30 min, and then 0.23 parts by weight of initiator potassium persulfate was added and kept at 50°C for 30 min. After that, 1 part by weight of N-isopropylacrylamide was added and the reaction was continued for 4 h. At the end of the reaction, the reactant was poured into 200 parts by weight of ethanol to terminate the chemical reaction, and then washed 3 times and dried to obtain grafted modified sodium alginate;

[0085] S3.2: 1.5 parts by weight of grafted modified sodium alginate was dispersed in 100 parts by weight of distilled water, and magnetically stirred for 30 minutes. Then, 5 parts by weight of calcium carbonate was added, and ultrasonicated for 10 minutes. After mixing, the mixture was used as the water phase. 3 parts by weight of emulsifier Span-80 was added to 200 parts by weight of liquid paraffin oil, and stirred to mix well as the oil phase.

[0086] S3.3: Disperse 2 parts by weight of chitosan in 100 parts by weight of 0.5wt% acetic acid solution, stir magnetically for 30 minutes to obtain a crosslinker, add the aqueous phase to the oil phase, stir mechanically for emulsification for 8 minutes, then add 2 parts by weight of tea polyphenol zinc complex liposomes and 2 parts by weight of recombinant human collagen type III, continue emulsification for 12 minutes, then add 1 part by weight of acetic acid and 2 parts by weight of calcium carbonate to react for 3 hours to obtain an emulsion, add the emulsion dropwise to the crosslinker, crosslink for 30 minutes, then centrifuge, filter, wash and dry to obtain thermosensitive composite microspheres;

[0087] S4: Preparation of collagen solution

[0088] To 80 parts by weight of deionized water, 1 part by weight of sodium hyaluronate, 2 parts by weight of trehalose, 2 parts by weight of glycerol, 3 parts by weight of glyceryl caprylate, 1 part by weight of caprylhydroxamic acid, 1 part by weight of hexylene glycol and 10 parts by weight of thermosensitive composite microspheres were added, the mixture was stirred and mixed, and then sterilized to obtain a collagen solution.

[0089] Comparative Example 1

[0090] Compared with Example 1, the difference of Comparative Example 1 is that, in Comparative Example 1, the tea polyphenol zinc complex liposomes in Steps S1-S2 and Step S3.3 are removed, and the remaining steps are unchanged to prepare the collagen solution, which is recorded as Comparative Example 1.

[0091] Comparative Example 2

[0092] Compared with Example 1, the difference of Comparative Example 2 is that, in Comparative Example 2, the recombinant human collagen type III in step S3.3 is removed, and the remaining steps remain unchanged to prepare the collagen solution, which is recorded as Comparative Example 2.

[0093] Comparative Example 3

[0094] Compared with Example 1, the difference of Comparative Example 3 is that step S3.1 is removed in Comparative Example 3, the grafted modified sodium alginate in step S3.2 is replaced with sodium alginate, and the remaining steps remain unchanged to prepare the collagen solution, which is recorded as Comparative Example 3.

[0095] Antimicrobial test results:

[0096] Table 1. Antibacterial performance test results of Example 1 and Comparative Example 1

[0097]

[0098] From the data in Table 1, it can be seen that the tea polyphenol zinc complex added in the present invention can enhance the inhibitory ability against common pathogens on wound surfaces, such as Staphylococcus aureus and Escherichia coli, so that the collagen solution has an antibacterial effect.

[0099] Determination of healing rate of rat burn model:

[0100] Healthy adult rats were selected, and their body weight was usually between 200-250g. Adaptive feeding was performed for 1 week before the experiment to ensure that the rats were in good health. The rats were anesthetized with an anesthetic, and the hair was shaved on the back to ensure that the skin was exposed. 90°C hot water was used to contact the skin for a certain period of time to cause standardized deep II degree burns. The rats were randomly divided into seven groups, with 10 rats in each group. The first six groups were sprayed with the collagen solution prepared in Examples 1-3 and Comparative Examples 1-3 for treatment, and the seventh group was a blank control group treated only with physiological saline, and the healing rate was determined.

[0101] Healing rate (%) = [(initial wound area - current wound area) / initial wound area] × 100%. The measurement results are shown in Table 3.

[0102] Table 2. Healing rate test results of Examples and Comparative Examples 1-3

[0103]

[0104] It can be seen from the data of comparative examples 1-2 in Table 2 that the compounding of tea polyphenol zinc complex and recombinant human collagen type III can significantly increase the cell proliferation rate and accelerate the wound healing process.

[0105] From the data of Comparative Example 3, it can be seen that the healing rate of Comparative Example 3 is lower than that of the embodiment, because the composite microspheres prepared with sodium alginate and chitosan as raw materials are not temperature-sensitive and cannot be released at a faster speed after contacting the human body, but can only be released slowly, resulting in some recombinant human collagen type III and tea polyphenol zinc complex liposomes not being released, and thus unable to play a role, resulting in a low healing rate.

[0106] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may 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 a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A preparation process of medical collagen solution for wound repair, characterized in that: The steps include: S1: Preparation of tea polyphenol zinc complex The tea polyphenols extract is obtained by desorbing with tea powder and then extracting with a desorbent, and then the tea polyphenols solution and a zinc chloride solution are mixed, and NaHCO3 is added to adjust the mixed solution to a pH of 6, and then reacting to prepare a tea polyphenols zinc complex; S2: Preparation of tea polyphenol zinc complex liposomes Soybean lecithin and cholesterol were used as raw materials to embed tea polyphenol zinc complex to prepare tea polyphenol zinc complex liposome; S3: Preparation of thermosensitive composite microspheres Sodium alginate was grafted with N-isopropylacrylamide to prepare grafted modified sodium alginate, and then tea polyphenol zinc complex liposomes and recombinant human collagen type III were embedded in grafted modified sodium alginate and chitosan to prepare thermosensitive composite microspheres. S4: Preparation of collagen solution To 80-100 parts by weight of deionized water, add 1-3 parts by weight of sodium hyaluronate, 2-3 parts by weight of trehalose, 2-3 parts by weight of glycerol, 3-5 parts by weight of glyceryl caprylate, 1-2 parts by weight of caprylhydroxamic acid, 1-2 parts by weight of hexylene glycol and 10-12 parts by weight of thermosensitive composite microspheres, stir and mix, and then sterilize to obtain a collagen solution.

2. The preparation process of a medical collagen solution for wound repair according to claim 1, characterized in that: Step S1: Preparation of tea polyphenol zinc complex, specifically comprising the following steps: S1.1: 3-5 parts by weight of tea powder are uniformly moistened with 15-20 parts by weight of desorbent, and desorbed for 8-10 minutes to allow the desorbent to fully penetrate the tea powder, and then 80-100 parts by weight of pure water at 90-100°C are added, and extraction is performed for 10-12 minutes. After the extraction is completed, centrifuge at 4000-4200 r / min for 10-12 minutes, and the supernatant is taken to obtain a tea polyphenol extract; S1.2: Mix the tea polyphenol solution and zinc chloride solution, then adjust the mixed solution to pH 6 with 0.1 mol / L NaHCO3, stir at a constant speed for 20-30 min, then collect the precipitate by centrifugation, wash the precipitate with distilled water and anhydrous ethanol 2-3 times in turn, and vacuum freeze-dry to obtain a solid powder, i.e., tea polyphenol zinc complex.

3. The preparation process of a medical collagen solution for wound repair according to claim 2, characterized in that: Step S2: Preparation of tea polyphenol zinc complex liposomes, specifically comprising the following steps: S2.1: 0.7-1 parts by weight of soybean lecithin and 0.25-0.3 parts by weight of cholesterol are dissolved in 20-30 parts by weight of a chloroform-ethanol mixed solvent, and after mixing evenly, 0.05-0.08 parts by weight of a tea polyphenol zinc complex is added, and the mixture is stirred for 20-30 minutes, and then a rotary evaporator is used to rotary evaporate at 40-60° C. for 1-2 hours to obtain a lipid film; S2.2: adding 1-2 parts by weight of the lipid film to 10-12 parts by weight of PBS buffer preheated to 55-60°C, pH 6.5, and then magnetically stirring at 300-500 r / min for 1-2 hours to obtain a hydrated suspension; S2.3: The hydrated suspension is ultrasonically treated in an ice bath for 5-8 minutes, and the ultrasonic suspension is filtered through a 0.45 μm microporous filter membrane and then transferred to a high-pressure homogenizer for 4-5 cycles of homogenization at a pressure of 800 bar to obtain a homogenized suspension; S2.4: The homogenized suspension is centrifuged at 3000-4000 centrifugal force for 10-15 min using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa, and then filtered through a 0.22 μm sterile filter membrane to obtain tea polyphenol zinc complex liposomes.

4. The preparation process of a medical collagen solution for wound repair according to claim 3, characterized in that: Step S3: Preparation of thermosensitive composite microspheres, specifically comprising the following steps: S3.1: 1-2 parts by weight of 1wt% sodium alginate solution is magnetically stirred and heated at 70-80°C for 20-30min, and then 0.23-0.4 parts by weight of initiator is added, and the temperature is kept at 40-50°C for 20-30min, and then 1-2 parts by weight of N-isopropylacrylamide is added, and the reaction is continued for 3-4h. At the end of the reaction, the reactant is poured into 200-230 parts by weight of ethanol to terminate the chemical reaction, and then washed 2-3 times and dried to obtain grafted modified sodium alginate; S3.2: Disperse 1.5-2 parts by weight of grafted modified sodium alginate in 100-120 parts by weight of distilled water, stir magnetically for 20-30 minutes, then add 5-10 parts by weight of calcium carbonate, ultrasonicate for 5-10 minutes, mix as the water phase, add 3-4 parts by weight of emulsifier Span-80 into 200-220 parts by weight of liquid paraffin oil, stir and mix evenly as the oil phase; S3.3: Disperse 2-3 parts by weight of chitosan in 100-120 parts by weight of 0.5wt% acetic acid solution, magnetically stir for 20-30 minutes to obtain a cross-linking agent, add the aqueous phase to the oil phase, mechanically stir and emulsify for 5-8 minutes, then add 2-3 parts by weight of tea polyphenol zinc complex liposomes and 2-3 parts by weight of recombinant human collagen type III, continue emulsification for 10-12 minutes, then add 1-2 parts by weight of acetic acid and 2-3 parts by weight of calcium carbonate to react for 2-3 hours to obtain an emulsion, add the emulsion dropwise into the cross-linking agent, cross-link reaction for 20-30 minutes, then centrifuge, filter, wash and dry to obtain thermosensitive composite microspheres.

5. The preparation process of a medical collagen solution for wound repair according to claim 2, characterized in that: The desorbent in step S1.1 is a 40-60 wt % ethanol aqueous solution.

6. The preparation process of a medical collagen solution for wound repair according to claim 2, characterized in that: In step S1.2, the tea polyphenol solution and the zinc chloride solution are mixed in a molar ratio of 1:2-3.

7. The preparation process of a medical collagen solution for wound repair according to claim 3, characterized in that: The volume ratio of chloroform to ethanol in step S2.1 is 4:

1.

8. The preparation process of a medical collagen solution for wound repair according to claim 3, characterized in that: The power of the ultrasonic treatment in step S2.3 is 200-220 W, working for 2 s / resting for 3 s.

9. The preparation process of a medical collagen solution for wound repair according to claim 4, characterized in that: In step S3.1, the initiator is potassium persulfate.

10. A medical collagen solution for wound repair, characterized in that: The collagen liquid is prepared by the preparation process of a medical collagen liquid for wound repairing as described in any one of claims 1 to 9.

Images