Collagen-hyaluronic acid cross-linking product, and preparation method and application thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to a collagen-hyaluronic acid cross-linking product and a preparation method and application thereof.The collagen-hyaluronic acid cross-linking product provided by the present application comprises the following components by weight percentage: 0.05%-2.5% collagen, 1%-3.5% hyaluronic acid, 0.005%-0.05% cross-linking agent, 0.2%-0.5% cross-linking agent solvent, and water for injection as a solvent.The collagen-hyaluronic acid cross-linking product provided by the present application has strong stability and good heat resistance, and can be widely applied in the fields of tissue engineering, cosmetics, medicines and medical devices.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a collagen-hyaluronic acid cross-linked product, its preparation method, and its application. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Collagen is the most abundant protein in the human body, widely found in tissues such as skin, bones, tendons, and blood vessels, playing a vital structural support role. Its typical triple helix structure endows it with low immunogenicity, good biocompatibility, biodegradability, and the ability to promote blood clotting, making it a biomaterial with broad application value. In functional skincare products, collagen can strengthen the skin barrier and improve skin texture; in medical dressings, it can promote wound healing and is used to repair burns, ulcers, and other wounds; in cosmetic medicine, collagen injections can fill soft tissue defects and induce the body's own collagen regeneration. Furthermore, collagen can be used in biomedical fields such as dental implant materials, and its small molecule peptide products can be used as dietary supplements for joint health and immune regulation. Cross-linked collagen is a structure formed by collagen molecules through a cross-linking reaction, which can increase the structural stability and mechanical strength of collagen, improve its tensile strength and abrasion resistance, reduce its degradation rate in vivo, and prolong its retention time in tissues.

[0004] Hyaluronic acid is a naturally occurring acidic mucopolysaccharide found in all tissues and body fluids of the human body. Primarily acting as an intercellular filler, it possesses properties that help retain moisture, maintain extracellular space, regulate osmotic pressure, lubricate, and promote cell repair. Small-molecule hyaluronic acid can penetrate the dermis, promoting local blood circulation and enhancing the skin's absorption of nutrients, thus exerting its physiological regulatory functions for beauty and anti-wrinkle effects. Through chemical cross-linking reactions, hyaluronic acid can combine with cross-linking agents to form cross-linked hyaluronic acid. This structure effectively slows down the degradation rate of hyaluronic acid in the body, prolongs its existence, and provides more durable structural support.

[0005] In existing technologies, most medical aesthetic products use single cross-linked collagen preparations or cross-linked hyaluronic acid preparations, such as the collagen implants from Shuangmei Biotechnology Co., Ltd., and Juvederm from Juvederm. ®Series of products, etc. The cross-linking agents, acids, and alkalis used in the preparation of formulations using cross-linked collagen and hyaluronic acid can affect the cross-linking effect, and the products have certain biological toxicity; freeze-drying during the cross-linking process of collagen and hyaluronic acid can cause structural collapse, severely affecting its performance. Summary of the Invention

[0006] In view of this, the present invention provides a collagen-hyaluronic acid crosslinked product, its preparation method and application.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution: In a first aspect, the present invention provides a collagen-hyaluronic acid crosslinked product, wherein the raw materials comprise the following components by weight percentage: 0.05%~2.5% collagen, 1%~3.5% hyaluronic acid, 0.005%~0.05% crosslinking agent, 0.2%~0.5% crosslinking agent solvent, and water for injection as solvent.

[0008] The collagen-hyaluronic acid crosslinking product provided by this invention stably and firmly links collagen and hyaluronic acid together, resulting in an injectable collagen-sodium hyaluronate crosslinking product. By chemically grafting and modifying collagen and then crosslinking it with hyaluronic acid, the advantages of both can be simultaneously utilized. This results in high biocompatibility, good mechanical properties and injectability, as well as significantly enhanced resistance to enzymatic degradation. When injected into depressed skin defects, it not only provides support and filling but also significantly reduces the in vivo degradation of both components, increasing their retention time in the body and providing long-lasting support.

[0009] Preferably, the collagen is animal-derived collagen or recombinant collagen; the recombinant collagen is recombinant type III collagen.

[0010] Preferably, the hyaluronic acid is sodium hyaluronate produced by bacterial fermentation, with a relative molecular mass of 600,000 to 2,600,000 Daltons. When the relative molecular mass of hyaluronic acid is less than 600,000, the hyaluronic acid is softer, has less extrusion force, and weaker filling capacity.

[0011] Preferably, the cross-linking agent is genipin. Genipin, as a natural biological cross-linking agent, is relatively safe and has low cytotoxicity.

[0012] Preferably, the crosslinking agent solvent is dimethyl sulfoxide (DMSO). Low concentrations of DMSO not only have anti-inflammatory and analgesic effects, but also act as a cryoprotectant during freeze-drying, protecting the crosslinking product structure from damage and ensuring no DMSO residue remains in the product. As a polar solvent, DMSO facilitates the dispersion and dissolution of genipin in solution, thereby enhancing its crosslinking effect. The permeability of DMSO helps genipin penetrate more effectively into the crosslinking product and biological tissues, improving the efficiency of the crosslinking reaction and enhancing the filling effect. Furthermore, after freeze-drying, the DMSO evaporates with the water, resulting in a final product with zero DMSO content.

[0013] Secondly, the present invention provides a method for preparing the above-mentioned collagen-hyaluronic acid crosslinked product, comprising the following steps: (1) Collagen and sodium hyaluronate were dissolved in water for injection to obtain collagen aqueous solution and sodium hyaluronate aqueous solution, respectively; (2) Dissolve the crosslinking agent in the crosslinking agent solvent, and then add water for injection to prepare a crosslinking agent solution; (3) Mix the collagen aqueous solution and the sodium hyaluronate aqueous solution evenly, and add the cross-linking agent solution to the collagen and sodium hyaluronate mixed aqueous solution under homogeneous conditions; (4) After the mixture has been left to stand for a set time, it is dried, reconstituted with water for injection, and then freeze-dried to obtain the final product.

[0014] Preferably, in step (1), the collagen content in the collagen aqueous solution is 0.23~4.6%, and the sodium hyaluronate content in the sodium hyaluronate aqueous solution is 2.3~6.9%.

[0015] Preferably, in step (2), the crosslinking agent is genipin, and the genipin content in the crosslinking agent solution is 0.038%~0.077%; the crosslinking agent solvent is dimethyl sulfoxide, and the dimethyl sulfoxide content in the crosslinking agent solution is 2.3%~3.07%.

[0016] Preferably, in step (3), the crosslinking agent solution is slowly added to the collagen-hyaluronic acid mixed aqueous solution under homogeneous conditions with a stirring speed of 5000~15000 rpm / min, and maintained for 8~12 min.

[0017] Preferably, in step (4), the mixture is left to stand at 5~8 ℃ for 18~24 h.

[0018] Preferably, in step (4), the drying operation is: gradient drying at 50±2 ℃, 45±2 ℃, 40±2 ℃ and 35±2 ℃ for 5~7 h each, and gradient drying protects the microstructure of the crosslinking product to the maximum extent.

[0019] Thirdly, the present invention provides the application of the collagen-sodium hyaluronate cross-linked product described in the first aspect in the field of skin filling.

[0020] Preferably, the collagen-sodium hyaluronate cross-linked product is used as an injectable preparation.

[0021] Compared with the prior art, the present invention has achieved the following beneficial effects: (1) This invention uses genipin as a crosslinking agent, and the product has no biological toxicity, thereby reducing the damage to the injection site and the human body. No acid or alkali is used to adjust the pH during the preparation process of this invention, which has little impact on collagen activity and the crosslinking process; dimethyl sulfoxide is used as a crosslinking agent solvent. Dimethyl sulfoxide can not only play an anti-inflammatory and analgesic role, but also act as a cryoprotectant during the freeze-drying process, protecting the structure of the crosslinked product from being destroyed during the freeze-drying process, and dimethyl sulfoxide will not remain in the crosslinked product.

[0022] (2) The collagen-sodium hyaluronate cross-linked product prepared by this invention undergoes intramolecular and intermolecular cross-linking, thereby reducing the degradation rate of collagen and sodium hyaluronate and increasing their retention time in vivo. Therefore, the injectable collagen-sodium hyaluronate cross-linked composite filler of this invention can be used in the medical device industry. Attached Figure Description

[0023] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0024] Figure 1 This is an electron micrograph of the collagen-sodium hyaluronate cross-linked product in Example 1; Figure 2 This is an electron micrograph of the collagen-sodium hyaluronate cross-linked product in Comparative Example 7. Detailed Implementation

[0025] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0026] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0027] Example 1 (1) Weigh 2.3g of recombinant type III collagen and dissolve it in 997.7g of water for injection to obtain a collagen aqueous solution. Weigh 23g of sodium hyaluronate with a molecular weight of 2.6 million Daltons and dissolve it in 977g of water for injection to obtain a sodium hyaluronate aqueous solution.

[0028] (2) Dissolve 0.23g genipin in 6.9g dimethyl sulfoxide, and then add 292.87g water for injection to prepare a crosslinking agent solution.

[0029] (3) Mix the collagen aqueous solution and sodium hyaluronate aqueous solution evenly, and slowly add the crosslinking agent solution into the collagen and hyaluronic acid mixed aqueous solution under homogeneous conditions with a stirring speed of 5000 rpm / min, and keep for 10 min.

[0030] (4) The mixture was allowed to stand at 6 °C for 24 h to allow the collagen and sodium hyaluronate to fully crosslink. It was then dried in a gradient of 50 °C-45 °C-40 °C-35 °C for 6 h each time. After reconstitution with an appropriate amount of water for injection, it was freeze-dried to obtain the collagen-sodium hyaluronate crosslinked product. The electron micrograph of the collagen-sodium hyaluronate crosslinked product prepared in this example is shown below. Figure 1 As shown.

[0031] Example 2 (1) Weigh 46g of recombinant type III collagen and dissolve it in 954g of water for injection to obtain a collagen aqueous solution. Weigh 69g of sodium hyaluronate with a molecular weight of 2.6 million Daltons and dissolve it in 931g of water for injection to obtain a sodium hyaluronate aqueous solution.

[0032] (2) Dissolve 0.115g genipin in 9.2g dimethyl sulfoxide, and then add 290.685g water for injection to prepare a crosslinking agent solution.

[0033] (3) Mix the collagen aqueous solution and sodium hyaluronate aqueous solution evenly, and slowly add the crosslinking agent solution into the collagen and hyaluronic acid mixed aqueous solution under homogeneous conditions with a stirring speed of 5000 rpm / min, and keep for 10 min.

[0034] (4) The mixture was left to stand at 6 °C for 24 h to allow collagen and sodium hyaluronate to fully cross-link. It was then dried at a gradient of 50 °C-45 °C-40 °C-35 °C for 6 h each time. After being reconstituted with an appropriate amount of water for injection, it was freeze-dried to obtain the collagen-sodium hyaluronate cross-linked product.

[0035] Example 3 (1) Weigh 34.5g of recombinant type III collagen and dissolve it in 965.5g of water for injection to obtain a collagen aqueous solution. Weigh 46g of sodium hyaluronate with a molecular weight of 2.6 million Daltons and dissolve it in 954g of water for injection to obtain a sodium hyaluronate aqueous solution.

[0036] (2) Dissolve 0.23g genipin in 9.2g dimethyl sulfoxide, and then add 290.57g water for injection to prepare a crosslinking agent solution.

[0037] (3) Mix the collagen aqueous solution and sodium hyaluronate aqueous solution evenly, and slowly add the crosslinking agent solution into the collagen and hyaluronic acid mixed aqueous solution under homogeneous conditions with a stirring speed of 5000 rpm / min, and keep for 10 min.

[0038] (4) The mixture was left to stand at 6 °C for 24 h to allow collagen and sodium hyaluronate to fully cross-link. It was then dried at a gradient of 50 °C-45 °C-40 °C-35 °C for 6 h each time. After being reconstituted with an appropriate amount of water for injection, it was freeze-dried to obtain the collagen-sodium hyaluronate cross-linked product.

[0039] Example 4 (1) Weigh 46g of recombinant type III collagen and dissolve it in 954g of water for injection to obtain a collagen aqueous solution. Weigh 69g of sodium hyaluronate with a molecular weight of 1.8 million Daltons and dissolve it in 931g of water for injection to obtain a sodium hyaluronate aqueous solution.

[0040] (2) Dissolve 0.23g genipin in 9.2g dimethyl sulfoxide, and then add 290.57g water for injection to prepare a crosslinking agent solution.

[0041] (3) Mix the collagen aqueous solution and sodium hyaluronate aqueous solution evenly, and slowly add the crosslinking agent solution into the collagen and hyaluronic acid mixed aqueous solution under homogeneous conditions with a stirring speed of 5000 rpm / min, and keep for 10 min.

[0042] (4) The mixture was left to stand at 6 °C for 24 h to allow collagen and sodium hyaluronate to fully cross-link. It was then dried at a gradient of 50 °C-45 °C-40 °C-35 °C for 6 h each time. After being reconstituted with an appropriate amount of water for injection, it was freeze-dried to obtain the collagen-sodium hyaluronate cross-linked product.

[0043] Example 5 (1) Weigh 46g of recombinant type III collagen and dissolve it in 954g of water for injection to obtain a collagen aqueous solution. Weigh 69g of sodium hyaluronate with a molecular weight of 600,000 Daltons and dissolve it in 931g of water for injection to obtain a sodium hyaluronate aqueous solution.

[0044] (2) Dissolve 0.23g genipin in 9.2g dimethyl sulfoxide, and then add 290.57g water for injection to prepare a crosslinking agent solution.

[0045] (3) Mix the collagen aqueous solution and sodium hyaluronate aqueous solution evenly, and slowly add the crosslinking agent solution into the collagen and hyaluronic acid mixed aqueous solution under homogeneous conditions with a stirring speed of 5000 rpm / min, and keep for 10 min.

[0046] (4) The mixture was left to stand at 6 °C for 24 h to allow collagen and sodium hyaluronate to fully cross-link. It was then dried at a gradient of 50 °C-45 °C-40 °C-35 °C for 6 h each time. After being reconstituted with an appropriate amount of water for injection, it was freeze-dried to obtain the collagen-sodium hyaluronate cross-linked product.

[0047] Example 6 (1) Weigh 34.5g of recombinant type III collagen and dissolve it in 965.5g of water for injection to obtain a collagen aqueous solution. Weigh 46g of sodium hyaluronate with a molecular weight of 1.8 million Daltons and dissolve it in 954g of water for injection to obtain a sodium hyaluronate aqueous solution.

[0048] (2) Dissolve 0.23g genipin in 9.2g dimethyl sulfoxide, and then add 290.57g water for injection to prepare a crosslinking agent solution.

[0049] (3) Mix the collagen aqueous solution and sodium hyaluronate aqueous solution evenly, and slowly add the crosslinking agent solution into the collagen and hyaluronic acid mixed aqueous solution under homogeneous conditions with a stirring speed of 5000 rpm / min, and keep for 10 min.

[0050] (4) The mixture was left to stand at 6 °C for 24 h to allow collagen and sodium hyaluronate to fully cross-link. It was then dried at a gradient of 50 °C-45 °C-40 °C-35 °C for 6 h each time. After being reconstituted with an appropriate amount of water for injection, it was freeze-dried to obtain the collagen-sodium hyaluronate cross-linked product.

[0051] Example 7 (1) Weigh 34.5g of recombinant type III collagen and dissolve it in 965.5g of water for injection to obtain a collagen aqueous solution. Weigh 46g of sodium hyaluronate with a molecular weight of 600,000 Daltons and dissolve it in 954g of water for injection to obtain a sodium hyaluronate aqueous solution.

[0052] (2) Dissolve 0.23g genipin in 9.2g dimethyl sulfoxide, and then add 290.57g water for injection to prepare a crosslinking agent solution.

[0053] (3) Mix the collagen aqueous solution and sodium hyaluronate aqueous solution evenly, and slowly add the crosslinking agent solution into the collagen and hyaluronic acid mixed aqueous solution under homogeneous conditions with a stirring speed of 5000 rpm / min, and keep for 10 min.

[0054] (4) The mixture was left to stand at 6 °C for 24 h to allow collagen and sodium hyaluronate to fully cross-link. It was then dried at a gradient of 50 °C-45 °C-40 °C-35 °C for 6 h each time. After being reconstituted with an appropriate amount of water for injection, it was freeze-dried to obtain the collagen-sodium hyaluronate cross-linked product.

[0055] Comparative Example 1 (1) Weigh 0.92g of recombinant type III collagen and dissolve it in 999.08g of water for injection to obtain a collagen aqueous solution. Weigh 20.7g of sodium hyaluronate with a molecular weight of 2.6 million Daltons and dissolve it in 979.3g of water for injection to obtain a sodium hyaluronate aqueous solution.

[0056] (2) Dissolve 0.23g genipin in 9.2g dimethyl sulfoxide, and then add 290.57g water for injection to prepare a crosslinking agent solution.

[0057] (3) Mix the collagen aqueous solution and sodium hyaluronate aqueous solution evenly, and slowly add the crosslinking agent solution into the collagen and hyaluronic acid mixed aqueous solution under homogeneous conditions with a stirring speed of 5000 rpm / min, and keep for 10 min.

[0058] (4) The mixture was left to stand at 6 °C for 24 h to allow collagen and sodium hyaluronate to fully cross-link. It was then dried at a gradient of 50 °C-45 °C-40 °C-35 °C for 6 h each time. After being reconstituted with an appropriate amount of water for injection, it was freeze-dried to obtain the collagen-sodium hyaluronate cross-linked product.

[0059] Comparative Example 2 (1) Weigh 46g of recombinant type III collagen and dissolve it in 954g of water for injection to obtain a collagen aqueous solution. Weigh 92g of sodium hyaluronate with a molecular weight of 2.6 million Daltons and dissolve it in 908g of water for injection to obtain a sodium hyaluronate aqueous solution.

[0060] (2) Dissolve 0.23g genipin in 9.2g dimethyl sulfoxide, and then add 290.57g water for injection to prepare a crosslinking agent solution.

[0061] (3) Mix the collagen aqueous solution and sodium hyaluronate aqueous solution evenly, and slowly add the crosslinking agent solution into the collagen and hyaluronic acid mixed aqueous solution under homogeneous conditions with a stirring speed of 5000 rpm / min, and keep for 10 min.

[0062] (4) The mixture was allowed to stand at 2~10 ℃ for 24 h to allow collagen and sodium hyaluronate to fully cross-link. It was then dried at a gradient of 50 ℃-45 ℃-40 ℃-35 ℃ for 6 h each time. After reconstitution with an appropriate amount of water for injection, it was freeze-dried to obtain the collagen-sodium hyaluronate cross-linked product.

[0063] Comparative Example 3 (1) Weigh 34.5g of recombinant type III collagen and dissolve it in 965.5g of water for injection to obtain a collagen aqueous solution. Weigh 20.7g of sodium hyaluronate with a molecular weight of 1.8 million Daltons and dissolve it in 979.3g of water for injection to obtain a sodium hyaluronate aqueous solution.

[0064] (2) Dissolve 0.23g genipin in 9.2g dimethyl sulfoxide, and then add 290.57g water for injection to prepare a crosslinking agent solution.

[0065] (3) Mix the collagen aqueous solution and sodium hyaluronate aqueous solution evenly, and slowly add the crosslinking agent solution into the collagen and hyaluronic acid mixed aqueous solution under homogeneous conditions with a stirring speed of 5000 rpm / min, and keep for 10 min.

[0066] (4) The mixture was allowed to stand at 2~10 ℃ for 24 h to allow collagen and sodium hyaluronate to fully cross-link. It was then dried at a gradient of 50 ℃-45 ℃-40 ℃-35 ℃ for 6 h each time. After reconstitution with an appropriate amount of water for injection, it was freeze-dried to obtain the collagen-sodium hyaluronate cross-linked product.

[0067] Comparative Example 4 (1) Weigh 69g of recombinant type III collagen and dissolve it in 931g of water for injection to obtain a collagen aqueous solution. Weigh 69g of sodium hyaluronate with a molecular weight of 600,000 Daltons and dissolve it in 931g of water for injection to obtain a sodium hyaluronate aqueous solution.

[0068] (2) Dissolve 0.23g genipin in 9.2g dimethyl sulfoxide, and then add 290.57g water for injection to prepare a crosslinking agent solution.

[0069] (3) Mix the collagen aqueous solution and sodium hyaluronate aqueous solution evenly, and slowly add the crosslinking agent solution into the collagen and hyaluronic acid mixed aqueous solution under homogeneous conditions with a stirring speed of 5000 rpm / min, and keep for 10 min.

[0070] (4) The mixture was left to stand at 6 °C for 24 h to allow collagen and sodium hyaluronate to fully cross-link. It was then dried at a gradient of 50 °C-45 °C-40 °C-35 °C for 6 h each time. After being reconstituted with an appropriate amount of water for injection, it was freeze-dried to obtain the collagen-sodium hyaluronate cross-linked product.

[0071] Comparative Example 5 (1) Weigh 34.5g of recombinant type III collagen and dissolve it in 965.5g of water for injection to obtain a collagen aqueous solution. Weigh 46g of sodium hyaluronate with a molecular weight of 2.6 million Daltons and dissolve it in 954g of water for injection to obtain a sodium hyaluronate aqueous solution.

[0072] (2) Dissolve 2.3g of genipin in 13.8g of dimethyl sulfoxide, and then add 283.9g of water for injection to prepare a crosslinking agent solution.

[0073] (3) Mix the collagen aqueous solution and sodium hyaluronate aqueous solution evenly, and slowly add the crosslinking agent solution into the collagen and hyaluronic acid mixed aqueous solution under homogeneous conditions with a stirring speed of 5000 rpm / min, and keep for 10 min.

[0074] (4) The mixture was left to stand at 6 °C for 24 h to allow collagen and sodium hyaluronate to fully cross-link. It was then dried at a gradient of 50 °C-45 °C-40 °C-35 °C for 6 h each time. After being reconstituted with an appropriate amount of water for injection, it was freeze-dried to obtain the collagen-sodium hyaluronate cross-linked product.

[0075] Comparative Example 6 (1) Weigh 34.5g of recombinant type III collagen and dissolve it in 965.5g of water for injection to obtain a collagen aqueous solution. Weigh 46g of sodium hyaluronate with a molecular weight of 2.6 million Daltons and dissolve it in 954g of water for injection to obtain a sodium hyaluronate aqueous solution.

[0076] (2) Dissolve 0.115g genipin in 2.3g dimethyl sulfoxide, and then add 297.585g water for injection to prepare a crosslinking agent solution.

[0077] (3) Mix the collagen aqueous solution and sodium hyaluronate aqueous solution evenly, and slowly add the crosslinking agent solution into the collagen and hyaluronic acid mixed aqueous solution under homogeneous conditions with a stirring speed of 5000 rpm / min, and keep for 10 min.

[0078] (4) The mixture was left to stand at 6 °C for 24 h to allow collagen and sodium hyaluronate to fully cross-link. It was then dried at a gradient of 50 °C-45 °C-40 °C-35 °C for 6 h each time. After being reconstituted with an appropriate amount of water for injection, it was freeze-dried to obtain the collagen-sodium hyaluronate cross-linked product.

[0079] Comparative Example 7 (1) Weigh 34.5g of recombinant type III collagen and dissolve it in 965.5g of water for injection to obtain a collagen aqueous solution. Weigh 46g of sodium hyaluronate with a molecular weight of 1.8 million Daltons and dissolve it in 954g of water for injection to obtain a sodium hyaluronate aqueous solution.

[0080] (2) Dissolve 2.3g of genipin in 297.7g of water for injection to prepare a crosslinking agent solution.

[0081] (3) Mix the collagen aqueous solution and sodium hyaluronate aqueous solution evenly, and slowly add the crosslinking agent solution into the collagen and hyaluronic acid mixed aqueous solution under homogeneous conditions with a stirring speed of 5000 rpm / min, and keep for 10 min.

[0082] (4) The mixture was allowed to stand at 6 °C for 24 h to allow the collagen and sodium hyaluronate to fully crosslink. It was then dried in a gradient of 50 °C-45 °C-40 °C-35 °C for 6 h each time. After reconstitution with an appropriate amount of water for injection, it was freeze-dried to obtain the collagen-sodium hyaluronate crosslinked product. The electron micrograph of the collagen-sodium hyaluronate crosslinked product prepared in this comparative example is shown below. Figure 2 As shown.

[0083] The component types and formulations of Examples 1-7 and Comparative Examples 1-7 are shown in Table 1; and the performance of the products of Examples 1-7 and Comparative Examples 1-7 were tested, including elastic modulus, viscous modulus, extrusion force, etc. The test results are shown in Table 2.

[0084] Table 1. Component types and ratios of Examples 1-7 and Comparative Examples 1-7

[0085] Table 2 Main test indicators for Examples 1-7 and Comparative Examples 1-7

[0086] Note: The optimal range for pushing force is generally 10-30N, which is convenient for clinical use.

[0087] As shown in Tables 1 and 2, the cross-linking of collagen and sodium hyaluronate, within a certain range, resulted in excellent water absorption properties, higher elastic and viscous moduli, better swelling properties, and relatively better filling effect when the molecular weight and amount of sodium hyaluronate increased. In Comparative Example 1, with lower collagen and sodium hyaluronate content, the product had no obvious particles and less cross-linking, leading to lower elastic and viscous moduli, poor swelling effect, and poor filling effect. In Comparative Example 2, with both the hyaluronic acid content and molecular weight increasing (exceeding 4%), the product was pale yellow with larger particles. Simultaneously, the elastic and viscous moduli increased, resulting in excessive extrusion force, making it unsuitable for clinical use. In Comparative Example 3, the sodium hyaluronate content was only 0.9%, below 1%, resulting in a slightly lower elastic modulus compared to the examples, leading to a worse filling effect. In Comparative Example 4, with a higher collagen content, the product particles were larger than in Examples 5 and 7, resulting in excessive extrusion force and affecting the product's performance. In Comparative Example 5, the amounts of genipin and dimethyl sulfoxide were increased. Compared to Example 6, the product of Comparative Example 5 was yellow, making it less suitable for clinical use. In Comparative Example 6, the amounts of genipin and dimethyl sulfoxide were reduced, resulting in a lower degree of crosslinking, a slightly lower viscous modulus of the product compared to the examples, and poorer filling effect.

[0088] Comparative Example 7 used genipin crosslinking alone. During the freeze-drying process, the absence of the protective agent dimethyl sulfoxide caused structural collapse, resulting in uneven particle aggregation and uneven pushing force. Figure 1 This is an electron micrograph of the collagen-sodium hyaluronate cross-linked product in Example 1; Figure 2 This is an electron micrograph of the collagen-sodium hyaluronate cross-linked product from Comparative Example 7. Dimethyl sulfoxide was not used in Comparative Example 7, such as... Figure 1-2 As shown, with dimethyl sulfoxide (DMSO) as the crosslinking agent solvent, the product network structure is complete, uniform, and does not collapse; the product without DMSO as the crosslinking agent solvent has an uneven network structure and collapses. This demonstrates that DMSO not only acts as a crosslinking agent solvent but also serves as a protective agent during freeze-drying, ensuring complete dissolution of the final product during reconstitution and improving its performance.

[0089] Experimental Example 1: Efficacy Test Skin wrinkle change rate During the trial, volunteers were divided into an experimental group (Examples 1-7) and a control group (Comparative Examples 1-7). Ten female volunteers aged 40 to 50 were randomly selected from each group. The volunteers underwent the following tests, and the average test results of each group were recorded.

[0090] Volunteer Recruitment Criteria: 1) Problems include dry skin, fine lines, dull skin tone, large pores, and pigmentation; 2) No other facial treatments were received within the past month; 3) Agree to complete the follow-up visit.

[0091] Those with the following conditions should be excluded: a. Those who have used antihistamines within the past week or immunosuppressants within the past month; b. Those who have applied any anti-inflammatory drugs to the test site within the past two months; c. Subjects with clinically unhealed inflammatory skin diseases; d. Patients with insulin-dependent diabetes mellitus; e. Patients currently receiving treatment for asthma or other chronic respiratory diseases; f. Those who have received anticancer chemotherapy within the past 6 months; g. Patients with immunodeficiency or autoimmune diseases; h. Breastfeeding or pregnant women; i. Those who have undergone bilateral mastectomy and bilateral axillary lymph node dissection; j. Those whose test results are affected by scars, pigmentation, atrophy, port-wine stains, or other blemishes at the skin test site; k. Those participating in other clinical trials; l. Those with highly sensitive constitutions; m. Those who are not participating voluntarily or are unable to complete the prescribed content as required by the experiment.

[0092] Injection method: A single-use sterile injection needle (nine needles, purchased from Suzhou Hannuoxin Biotechnology Co., Ltd.) was used to inject the volunteer into the neck using a syringe-assisted propulsion device (BZ-1, purchased from Shaoxing Yannuo Medical Technology Co., Ltd.). The needle length was 0.8-1.2 mm, and the injection volume was 1 mL.

[0093] Experimental method: Three consecutive neck injections were administered, with a four-week interval between the two treatments. Initial values ​​of each indicator were measured before the first injection (T0), and all indicators were measured two weeks after the first injection (T1), two weeks after the third injection (T2), and four weeks after the third injection (T3).

[0094] Effect Test 1: Improvement in Skin Wrinkles Skin wrinkles in volunteers were detected at various time points using the VISIA CR skin analyzer (lower values ​​indicated fewer wrinkles). The percentage change in skin wrinkles was calculated and recorded, as shown in Table 3. The percentage change in skin wrinkles was calculated as follows: Change rate (%) = [(skin wrinkles at each time point after treatment - initial skin wrinkles before treatment) / initial skin wrinkles before treatment] * 100%.

[0095] Table 3. Change rate of skin wrinkles (%)

[0096] The product in Comparative Example 2 has larger particles, is pale yellow, and has excessive extrusion force, making it difficult to use clinically. The product in Comparative Example 5 has a higher content of cross-linking agent, resulting in a yellow appearance, which is inconvenient for clinical use. Therefore, no efficacy test was conducted.

[0097] The results in Table 3 show that the products in Examples 1-7 have good effects on improving skin wrinkles, with Example 3 showing the best improvement effect. This indicates that the combination of recombinant collagen and sodium hyaluronate in a certain ratio results in the best filling and wrinkle-reducing effect. Referring to Tables 2 and 3, the products in Comparative Examples 1 and 6 have no obvious particles, and their elastic modulus and viscosity modulus are low. Compared to the examples, their swelling effect is poor, resulting in a poorer filling and wrinkle-reducing effect. Comparative Example 3 has a lower elastic modulus than the examples due to the low amount of sodium hyaluronate added, thus affecting its filling and wrinkle-reducing effect. Comparative Example 4 has excessive amounts of both collagen and sodium hyaluronate added, making the product preparation process difficult and viscous, affecting the final effect. The product in Comparative Example 7 exhibits uneven granular structure. Due to the lack of dimethyl sulfoxide, the product structure collapsed during freeze-drying, severely affecting its performance.

[0098] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A collagen-hyaluronic acid cross-linked product, characterized in that, By weight percentage, the raw materials include the following components: 0.05%~2.5% collagen, 1%~3.5% hyaluronic acid, 0.005%~0.05% cross-linking agent, 0.2%~0.5% cross-linking agent solvent, and water for injection as solvent; The crosslinking agent is genipin; The crosslinking agent solvent is dimethyl sulfoxide; The collagen mentioned is animal-derived collagen or recombinant collagen; The collagen-hyaluronic acid crosslinking product is prepared by the following method, including the following steps: (1) Collagen and sodium hyaluronate were dissolved in water for injection to obtain collagen aqueous solution and sodium hyaluronate aqueous solution, respectively; (2) Dissolve the crosslinking agent in the crosslinking agent solvent, and then add water for injection to prepare a crosslinking agent solution; (3) Mix the collagen aqueous solution and the sodium hyaluronate aqueous solution evenly, and add the cross-linking agent solution to the collagen and hyaluronic acid mixed aqueous solution under homogeneous conditions; (4) After the mixture has been allowed to stand for a set time, it is dried, reconstituted with water for injection, and then freeze-dried to obtain the final product. In step (4), the mixture is left to stand at 5-8 ℃ for 18-24 h.

2. The collagen-hyaluronic acid crosslinked product as described in claim 1, characterized in that, The recombinant collagen is recombinant type III collagen.

3. The collagen-hyaluronic acid crosslinked product as described in claim 1, characterized in that, The hyaluronic acid is sodium hyaluronate produced by bacterial fermentation, with a relative molecular mass of 600,000 to 2,600,000 Daltons.

4. The collagen-hyaluronic acid crosslinked product as described in claim 1, characterized in that, In step (1), the collagen content in the collagen aqueous solution is 0.23~4.6%, and the sodium hyaluronate content in the sodium hyaluronate aqueous solution is 2.3~6.9%.

5. The collagen-hyaluronic acid crosslinked product as described in claim 1, characterized in that, In step (2), the content of genipin in the crosslinking agent solution is 0.038%~0.077%; the content of dimethyl sulfoxide in the crosslinking agent solution is 2.3%~3.07%.

6. The collagen-hyaluronic acid crosslinked product as described in claim 1, characterized in that, In step (3), the crosslinking agent solution is slowly added to the collagen-hyaluronic acid mixed aqueous solution under homogeneous conditions with a stirring speed of 5000~15000 rpm / min, and maintained for 8~12 min.

7. The collagen-hyaluronic acid crosslinked product as described in claim 1, characterized in that, In step (4), the drying operation is as follows: gradient drying at 50±2 ℃, 45±2 ℃, 40±2 ℃ and 35±2 ℃ for 5~7 h each.

8. The use of the collagen-hyaluronic acid crosslinking product as described in claim 1 in the preparation of dermal filler injections.

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