Composite gel composition and preparation method thereof

Abstract

The invention provides a composite gel composition and a preparation method thereof, and belongs to the technical field of biomedical materials. The composite gel composition provided by the invention takes two proteins and a variety of polysaccharides as main components or preparation raw materials, and all the components can play a very good synergistic effect, so that the composite gel composition can better relieve skin damage symptoms, is beneficial to moisturizing of postoperative skin, promotes wound healing and repair of damaged skin, and has a good application prospect. The skin barrier function is improved; the health condition of damaged skin is improved; meanwhile, relatively good safety, stability and biocompatibility are realized.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a composite gel composition and a preparation method thereof. Background Art

[0002] To enhance skin beauty and / or health, exogenous interventions, such as cosmetic surgery, are a widespread and common practice today. These interventions often result in minor wounds or injuries, requiring postoperative repair. Composite gels are readily available and widely applicable, making them popular.

[0003] Human skin fibroblasts can synthesize and secrete extracellular matrices such as hyaluronic acid and collagen fibers. When the skin is damaged, such as after surgery, the cellular microenvironment in the skin will deteriorate, hyaluronic acid, collagen and other proteins will be damaged and reduced, and the skin barrier function will be reduced. To repair damaged skin after surgery, it is necessary to heal the wound, enhance the skin's barrier function, and improve the cellular microenvironment. However, the current repair gel is not ideal in promoting wound healing, enhancing the skin's barrier function, and improving the cellular microenvironment after surgery. Composite gels contain multiple "active ingredients" and are more powerful and better than single "active ingredients".

[0004] Therefore, it is very important and necessary for skin care and postoperative repair to provide a stable, safe and effective composite gel composition that helps to heal wounds after surgery, maintain or increase the level of extracellular matrix components, and repair damaged skin. Summary of the Invention

[0005] In light of the problems and needs in the prior art, the present invention provides a composite gel composition and a method for preparing the same. The composite gel composition provided by the present invention has the potential to promote skin health by maintaining a moist environment, protecting the skin from damage caused by external environmental pollution, promoting wound healing, repairing damaged skin, enhancing skin barrier function, stimulating and promoting fibroblast proliferation, and alleviating and improving adverse skin or mucosal conditions.

[0006] In a first aspect, the present invention provides a composite gel composition.

[0007] According to the present invention, a composite gel composition comprises, or its preparation raw materials comprise, the following ingredients: recombinant mussel mucin, recombinant fibronectin, chondroitin sulfate, sodium hyaluronate and carboxymethyl chitosan.

[0008] According to the present invention, the composite gel composition may include, or its preparation raw materials may include, 0.5%-5% of recombinant mussel mucin, by weight percentage; preferably, it includes, or its preparation raw materials include, 1%-5% of recombinant mussel mucin; preferably, it includes, or its preparation raw materials include, 2%-4% of recombinant mussel mucin; preferably, it includes, or its preparation raw materials include, 2.5%-3.5% of recombinant mussel mucin.

[0009] According to the present invention, the composite gel composition may include, or its preparation raw materials may include, 0.1%-2% recombinant fibronectin, by weight percentage; preferably, it includes, or its preparation raw materials include, 0.2%-1.5% recombinant fibronectin; preferably, it includes, or its preparation raw materials include, 0.5%-1% recombinant fibronectin.

[0010] According to the present invention, the composite gel composition may include, or its preparation raw materials may include, 0.1%-5% chondroitin sulfate, by weight percentage; preferably, it includes, or its preparation raw materials include, 0.5%-5% chondroitin sulfate; preferably, it includes, or its preparation raw materials include, 0.1%-3% chondroitin sulfate; preferably, it includes, or its preparation raw materials include, 0.5%-3% chondroitin sulfate; preferably, it includes, or its preparation raw materials include, 0.5%-2% chondroitin sulfate; preferably, it includes, or its preparation raw materials include, 0.5%-1.5% chondroitin sulfate.

[0011] According to the present invention, the composite gel composition may include, or its preparation raw materials may include, 0.05%-0.5% sodium hyaluronate, by weight percentage; preferably, it includes, or its preparation raw materials include, 0.05%-0.3% sodium hyaluronate; preferably, it includes, or its preparation raw materials include, 0.05%-0.15% sodium hyaluronate; preferably, it includes, or its preparation raw materials include, 0.08%-0.12% sodium hyaluronate.

[0012] According to the present invention, the composite gel composition may include, or its preparation raw materials may include, carboxymethyl chitosan 0.08%-0.5% by weight; preferably, it includes, or its preparation raw materials include, carboxymethyl chitosan 0.1%-0.5%; preferably, it includes, or its preparation raw materials include, carboxymethyl chitosan 0.1%-0.4%; preferably, it includes, or its preparation raw materials include, carboxymethyl chitosan 0.2%-0.4%.

[0013] According to the present invention, the composite gel composition may include, or its preparation raw materials may include, the following ingredients, calculated by weight percentage: 0.5%-5% recombinant mussel mucin, 0.1%-2% recombinant fibronectin, 0.1%-5% chondroitin sulfate, 0.05%-0.5% sodium hyaluronate, and 0.08%-0.5% carboxymethyl chitosan.

[0014] Preferably, the composite gel composition comprises, or its raw materials for preparation comprise, the following ingredients by weight: 1%-5% recombinant mussel mucin, 0.1%-1.5% recombinant fibronectin, 0.1%-3% chondroitin sulfate, 0.05%-0.3% sodium hyaluronate, and 0.1%-0.5% carboxymethyl chitosan. Further preferably, the composite gel composition comprises, or its raw materials for preparation comprise, the following ingredients by weight: 1%-4% recombinant mussel mucin, 0.2%-1% recombinant fibronectin, 0.5%-3% chondroitin sulfate, 0.08%-0.3% sodium hyaluronate, and 0.1%-0.4% carboxymethyl chitosan. Further preferably, the composite gel composition comprises, or its preparation raw materials comprise, the following ingredients in percentage by weight: 1.5%-3.5% recombinant mussel mucin, 0.4%-1% recombinant fibronectin, 0.5%-1.5% chondroitin sulfate, 0.08%-0.15% sodium hyaluronate, and 0.2%-0.4% carboxymethyl chitosan.

[0015] Further preferably, the composite gel composition comprises, or its preparation raw materials comprise, the following ingredients in percentage by weight: 3% recombinant mussel mucin, 0.5% recombinant fibronectin, 1% chondroitin sulfate, 0.12% sodium hyaluronate and 0.3% carboxymethyl chitosan.

[0016] Further preferably, the composite gel composition comprises, or its preparation raw materials comprise, the following ingredients in percentage by weight: 3% recombinant mussel mucin, 0.5% recombinant fibronectin, 3% chondroitin sulfate, 0.1% sodium hyaluronate and 0.3% carboxymethyl chitosan.

[0017] Further preferably, the composite gel composition comprises, or its preparation raw materials comprise, the following ingredients in percentage by weight: 3% recombinant mussel mucin, 1% recombinant fibronectin, 1% chondroitin sulfate, 0.12% sodium hyaluronate and 0.3% carboxymethyl chitosan.

[0018] Further preferably, the composite gel composition comprises, or its preparation raw materials comprise, the following ingredients in percentage by weight: 3% recombinant mussel mucin, 1% recombinant fibronectin, 3% chondroitin sulfate, 0.1% sodium hyaluronate and 0.4% carboxymethyl chitosan.

[0019] The peptide chain fragments of mussel mucin contain dopa groups (DOPA groups, 3,4-dihydroxyphenylalanine), which have the effects of promoting cell adhesion and crawling, promoting wound healing, anti-inflammatory and itching, repairing scars, and forming a water-resistant protective film. However, the source of mussel mucin is limited, the extraction is difficult, the preparation process is complicated, and the safety is difficult to control and ensure, making it difficult to meet application requirements. Recombinant mussel mucin can achieve standardized, high-quality fermentation production, taking into account both efficacy and safety. The mass content of the dopa group (DOPA group, 3,4-dihydroxyphenylalanine) in the recombinant mussel mucin can be 0.3wt%-5wt%.

[0020] Preferably, the mass content of dopa groups in the recombinant mussel mucin is 0.5 wt%-5 wt%. Further preferably, the mass content of dopa groups in the recombinant mussel mucin is 0.5 wt%-3 wt%. Further preferably, the mass content of dopa groups in the recombinant mussel mucin is 0.8 wt%-3 wt%. Further preferably, the mass content of dopa groups in the recombinant mussel mucin is 0.8 wt%-1.5 wt%. Further preferably, in some embodiments, the mass content of dopa groups in the recombinant mussel mucin is 1 wt%.

[0021] Fibronectin is widely distributed in the human body and is widely involved in processes such as cell migration, adhesion, proliferation, hemostasis and tissue repair. The extracted fibronectin has a high safety risk and high cost, which brings inconvenience to its application. Recombinant fibronectin is highly safe and has sufficient sources. It also has the functions of promoting cell proliferation, growth, adhesion, migration, etc., which is beneficial to the repair of skin barrier function and the improvement of skin health. The weight-average molecular weight of the recombinant fibronectin can be 50,000 Daltons to 300,000 Daltons; preferably, the weight-average molecular weight of the recombinant fibronectin is 100,000 Daltons to 200,000 Daltons.

[0022] Chondroitin sulfate has anti-inflammatory, lubricating, and wound-healing properties and has been used as an eye drop or a treatment for joint diseases. According to the present invention, a gel for skin repair includes an appropriate amount of chondroitin sulfate to enhance repair efficacy and user experience. The weight-average molecular weight of the chondroitin sulfate can range from 5,000 to 50,000 Daltons. Preferably, the weight-average molecular weight of the chondroitin sulfate is between 10,000 and 50,000 Daltons; more preferably, the weight-average molecular weight of the chondroitin sulfate is between 10,000 and 30,000 Daltons.

[0023] Sodium hyaluronate of an appropriate molecular weight helps form a moisturizing film, providing water retention, hydration, isolation, and sealing. Preferably, the weight-average molecular weight of the sodium hyaluronate is between 1 million and 1.5 million daltons; more preferably, the weight-average molecular weight of the sodium hyaluronate is between 1 million and 1.3 million daltons, which helps maintain skin moisture, promote wound healing, and improve skin health.

[0024] Carboxymethyl chitosan has good water solubility and has antibacterial and wound healing effects. Preferably, the weight-average molecular weight of the carboxymethyl chitosan is 1,000 to 100,000 Daltons, which helps promote wound healing; more preferably, the weight-average molecular weight of the carboxymethyl chitosan is 1,000 to 80,000 Daltons, which helps inhibit bacteria and promote wound healing.

[0025] More preferably, the carboxymethyl chitosan is a combination of carboxymethyl chitosan with a weight-average molecular weight of 50,000-100,000 Daltons and carboxymethyl chitosan with a weight-average molecular weight of 1,000-5,000 Daltons, with a mass ratio of 1:1-9:1, which is more conducive to antibacterial and wound healing. More preferably, the mass ratio of high and low molecular weight carboxymethyl chitosan is 1.5:1-6:1.

[0026] According to the present invention, the pH value of the composite gel composition can be 5.2-6.8. A pH that is too high is detrimental to the stability and application of the gel composition; a pH that is too low makes it difficult for the composition to form a gel. This pH range helps the composition be suitable for application to the skin and has good stability. Preferably, the pH value of the composite gel composition is 5.2-6.5. More preferably, the pH value of the composite gel composition is 5.4-6.0. Even more preferably, the pH value of the composite gel composition is 5.4-5.8. More preferably, the pH value of the composite gel composition is 5.5, 5.6, or 5.7.

[0027] According to the present invention, the composite gel composition or its preparation raw materials further include a pH regulator, an auxiliary agent and water.

[0028] The pH adjuster may be at least one of sodium hydroxide, potassium hydroxide, sodium carbonate and potassium carbonate.

[0029] The auxiliary agent is preferably trehalose, carbomer, p-hydroxyacetophenone, glycerol, 1,3-butylene glycol and 1,2-hexanediol.

[0030] Trehalose helps maintain the stability of the composition, improves the cell microenvironment, and promotes the recovery of damaged skin. According to the present invention, the trehalose content in the composite gel composition can be 0.1%-5%, preferably 0.5%-3%, by weight.

[0031] Carbomer can serve as a matrix and / or thickener, facilitating the uniform and stable presence of recombinant mussel mucin and recombinant fibronectin in the composition, resulting in a gel-like composition free of precipitates or insoluble matter. According to the present invention, the carbomer content of the composite gel composition can be 0.2% to 1%, preferably 0.4% to 0.8%, by weight.

[0032] Parahydroxyacetophenone is beneficial to the stability of the gel composition, making it less likely to deteriorate. According to the present invention, the parahydroxyacetophenone in the composite gel composition can be 0.1%-1%, preferably 0.3%-0.6%, by weight.

[0033] Glycerin helps retain moisture and maintain moisture. Combined with protein and polysaccharide components, it forms a membrane-like barrier on the skin / mucosal surface, keeping the skin moist and shielding it from the outside world. This reduces wound discomfort and accelerates the repair of damaged skin. According to the present invention, the composite gel composition may contain 0.5%-10% glycerin by weight, preferably 1%-10%, more preferably 2%-8%, and even more preferably 2%-6%.

[0034] 1,3-Butanediol and 1,2-hexanediol are beneficial to enhancing the moisturizing property of the composite gel composition and are also beneficial to stabilizing the composite gel composition so that the composite gel composition is not easily deteriorated.

[0035] In terms of weight percentage, the 1,3-butanediol in the composite gel composition may be 0.5%-5%, preferably 2%-5%.

[0036] In terms of weight percentage, the 1,2-hexanediol content in the composite gel composition may be 0.1%-1%, preferably 0.3%-0.6%.

[0037] In terms of weight percentage, the adjuvant preferably includes: 0.1%-5% trehalose, 0.2%-1% carbomer, 0.1%-1% p-hydroxyacetophenone, 0.5%-10% glycerol, 0.5%-5% 1,3-butanediol and 0.1%-1% 1,2-hexanediol.

[0038] Preferably, the adjuvant comprises, by weight percentage, 0.2%-4% trehalose, 0.2%-0.8% carbomer, 0.2%-0.8% p-hydroxyacetophenone, 1%-10% glycerol, 1%-5% 1,3-butylene glycol, and 0.1%-0.8% 1,2-hexanediol. Further preferably, the adjuvant comprises, by weight percentage, 0.5%-4% trehalose, 0.2%-0.8% carbomer, 0.3%-0.8% p-hydroxyacetophenone, 2%-8% glycerol, 1%-5% 1,3-butylene glycol, and 0.1%-0.6% 1,2-hexanediol. Further preferably, the adjuvant comprises, by weight percentage, 0.5%-3% trehalose, 0.4%-0.8% carbomer, 0.3%-0.6% p-hydroxyacetophenone, 2%-6% glycerol, 2%-5% 1,3-butanediol and 0.2%-0.6% 1,2-hexanediol.

[0039] More preferably, the adjuvant comprises, by weight percentage, 2% trehalose, 0.5% carbomer, 0.5% p-hydroxyacetophenone, 5% glycerol, 3% 1,3-butylene glycol, and 0.5% 1,2-hexanediol. More preferably, the adjuvant comprises, by weight percentage, 1% trehalose, 0.5% carbomer, 0.5% p-hydroxyacetophenone, 4% glycerol, 3% 1,3-butylene glycol, and 0.3% 1,2-hexanediol. More preferably, the adjuvant comprises, by weight percentage, 3% trehalose, 0.6% carbomer, 0.5% p-hydroxyacetophenone, 4% glycerol, 3% 1,3-butylene glycol, and 0.3% 1,2-hexanediol.

[0040] Further preferably, the composite gel composition comprises or its preparation raw materials comprise the following ingredients, calculated by weight percentage: 3% recombinant mussel mucin, 0.5% recombinant fibronectin, 1% chondroitin sulfate, 0.12% sodium hyaluronate and 0.3% carboxymethyl chitosan; and: 0.1%-5% trehalose, 0.2%-1% carbomer, 0.1%-1% parahydroxyacetophenone, 0.5%-10% glycerol, 0.5%-5% 1,3-butanediol and 0.1%-1% 1,2-hexanediol. Further preferably, the composite gel composition comprises or its preparation raw materials comprise the following ingredients, calculated by weight percentage: 3% recombinant mussel mucin, 0.5% recombinant fibronectin, 1% chondroitin sulfate, 0.12% sodium hyaluronate and 0.3% carboxymethyl chitosan; and: 0.5%-4% trehalose, 0.2%-0.8% carbomer, 0.3%-0.8% parahydroxyacetophenone, 2%-8% glycerol, 1%-5% 1,3-butanediol and 0.1%-0.6% 1,2-hexanediol. Further preferably, the composite gel composition comprises or its preparation raw materials comprise the following ingredients, calculated by weight percentage: 3% recombinant mussel mucin, 0.5% recombinant fibronectin, 1% chondroitin sulfate, 0.12% sodium hyaluronate and 0.3% carboxymethyl chitosan; and: 0.5%-3% trehalose, 0.4%-0.8% carbomer, 0.3%-0.6% parahydroxyacetophenone, 2%-6% glycerol, 2%-5% 1,3-butanediol and 0.2%-0.6% 1,2-hexanediol.

[0041] Further preferably, the composite gel composition comprises or its preparation raw materials comprise the following ingredients, calculated by weight percentage: 3% recombinant mussel mucin, 0.5% recombinant fibronectin, 3% chondroitin sulfate, 0.1% sodium hyaluronate and 0.3% carboxymethyl chitosan, as well as: 0.1%-5% trehalose, 0.2%-1% carbomer, 0.1%-1% parahydroxyacetophenone, 0.5%-10% glycerol, 0.5%-5% 1,3-butanediol and 0.1%-1% 1,2-hexanediol.

[0042] Further preferably, the composite gel composition comprises or its preparation raw materials comprise the following ingredients, calculated by weight percentage: 3% recombinant mussel mucin, 1% recombinant fibronectin, 1% chondroitin sulfate, 0.12% sodium hyaluronate and 0.3% carboxymethyl chitosan, as well as: 0.1%-5% trehalose, 0.2%-1% carbomer, 0.1%-1% parahydroxyacetophenone, 0.5%-10% glycerol, 0.5%-5% 1,3-butanediol and 0.1%-1% 1,2-hexanediol.

[0043] Further preferably, the composite gel composition comprises or its preparation raw materials comprise the following ingredients, calculated by weight percentage: 3% recombinant mussel mucin, 1% recombinant fibronectin, 3% chondroitin sulfate, 0.1% sodium hyaluronate and 0.4% carboxymethyl chitosan, as well as: 0.1%-5% trehalose, 0.2%-1% carbomer, 0.1%-1% parahydroxyacetophenone, 0.5%-10% glycerol, 0.5%-5% 1,3-butanediol and 0.1%-1% 1,2-hexanediol.

[0044] According to the present invention, the composite gel composition further has the following characteristics, and its preparation process includes: preparing a mixture of sodium hyaluronate and water, adding chondroitin sulfate, mixing, and placing at 15°C-40°C for 1 hour-12 hours to obtain a sodium hyaluronate-chondroitin sulfate mixture. Preferably, the composite gel composition further has the following characteristics, and its preparation process includes: preparing a mixture of sodium hyaluronate and water, adding chondroitin sulfate, mixing, and placing at 15°C-40°C for 1 hour-12 hours to obtain a sodium hyaluronate-chondroitin sulfate mixture; preparing a carboxymethyl chitosan aqueous solution, adding it to the sodium hyaluronate-chondroitin sulfate mixture, mixing, and placing at 15°C-40°C for 1 hour-8 hours to obtain a sodium hyaluronate-chondroitin sulfate-carboxymethyl chitosan mixture.

[0045] According to the present invention, in a suitably proportioned composite gel composition, chondroitin sulfate and sodium hyaluronate are micro-crosslinked, and then micro-crosslinked with carboxymethyl chitosan (chemical reaction crosslinking and / or physical crosslinking such as molecular chain entanglement, etc.), thereby obtaining a polysaccharide system with a complex structure, and the two proteins are dispersed and / or cross-linked and entangled in this complex polysaccharide system. The finally formed composite gel composition is more stable, safe and biocompatible, and has a relatively lower chance of sensitive discomfort. It can retain water and moisturize and can be applied or injected. It can form an "isolation film" on the skin surface, so that the proteins with different functions and the polysaccharides with different functions can play a synergistic role, better alleviate the symptoms of skin damage, promote moisturizing and wound healing of damaged skin (such as after cosmetic surgery), promote the repair of damaged skin, and improve the health of the skin.

[0046] In a second aspect, the present invention provides a method for preparing the aforementioned composite gel composition.

[0047] A method for preparing the aforementioned composite gel composition comprises the following steps: 1) preparing a mixture of sodium hyaluronate and water, adding chondroitin sulfate, mixing, and incubating at 15° C. to 40° C. for 1 to 12 hours to obtain a sodium hyaluronate-chondroitin sulfate mixture; 2) preparing a carboxymethyl chitosan aqueous solution, adding it to the sodium hyaluronate-chondroitin sulfate mixed solution, mixing, and standing at 15° C.-40° C. for 0.5 hour-8 hours to obtain a sodium hyaluronate-chondroitin sulfate-carboxymethyl chitosan mixed solution; 3) Mixing water, glycerol, and 1,3-butylene glycol, and then adding carbomer to completely swell; then, adding 1,2-hexanediol, p-hydroxyacetophenone, and trehalose, and mixing to obtain a carbomer mixture; 4) mixing the aforementioned carbomer mixture with the sodium hyaluronate-chondroitin sulfate-carboxymethyl chitosan mixture, and mixing them evenly to obtain a polysaccharide mixture; 5) Separately preparing a recombinant mussel mucin aqueous solution and a recombinant fibronectin aqueous solution, adding them to the aforementioned polysaccharide mixture, mixing, then defoaming and homogenizing; optionally adjusting the pH to 5.2-6.8 with a pH regulator to obtain a composite gel composition.

[0048] Preferably, the pH value is adjusted to 5.2-6.5. More preferably, the pH value is adjusted to 5.4-6.0. More preferably, the pH value is adjusted to 5.4-5.8.

[0049] Preferably, a method for preparing the aforementioned gel composition comprises: 1) Prepare a sodium hyaluronate aqueous solution, add chondroitin sulfate, mix well, and incubate at 15°C-40°C for 1-12 hours to obtain a sodium hyaluronate-chondroitin sulfate mixed solution; 2) preparing a carboxymethyl chitosan aqueous solution, adding it to the sodium hyaluronate-chondroitin sulfate mixed solution, mixing, and standing at 15° C.-40° C. for 0.5 hour-8 hours to obtain a sodium hyaluronate-chondroitin sulfate-carboxymethyl chitosan mixed solution; 3) Mix water, glycerol, and 1,3-butylene glycol, then add carbomer, stir at 50 rpm-300 rpm (revolutions / minute) for 1 min-30 min, then let it stand until it is completely swollen, then homogenize at 1000 rpm-5000 rpm for 1 min-10 min, and vacuum defoam; add 1,2-hexanediol, p-hydroxyacetophenone, and trehalose while stirring at 250 rpm-300 rpm, and stir for 10 min-30 min to obtain a carbomer mixture; 4) Mix the aforementioned carbomer mixture and the sodium hyaluronate-chondroitin sulfate-carboxymethyl chitosan mixture, and stir at 250-300 rpm for 15-60 minutes to obtain a polysaccharide mixture; 5) Separately preparing a recombinant mussel mucin aqueous solution and a recombinant fibronectin aqueous solution, adding the solution to the aforementioned polysaccharide mixture, stirring at 250-300 rpm for 5-30 minutes, vacuuming to defoam, and homogenizing at 1000-5000 rpm for 1-10 minutes; optionally adjusting the pH to 5.4-6.0 with a pH adjuster, adding water to a predetermined amount, and filtering with a 200-mesh filter cloth to obtain a composite gel composition.

[0050] In some embodiments, sodium hydroxide aqueous solution is used to adjust the pH. Preferably, sodium hydroxide aqueous solution is used to adjust the pH to 5.4-5.8.

[0051] According to the present invention, in the aforementioned preparation method, any appropriate amount of water can be used to obtain an aqueous solution or a water mixture of each component.

[0052] In some embodiments, 5 wt% to 30 wt% (mass fraction) of the total amount of water is used to obtain a mixture of sodium hyaluronate and water. In some embodiments, 10 wt% to 25 wt% of the total amount of water is used to obtain a mixture of sodium hyaluronate and water.

[0053] In some embodiments, the carboxymethyl chitosan aqueous solution is obtained by using 2 wt % to 20 wt % of the total amount of water. In some embodiments, the carboxymethyl chitosan aqueous solution is obtained by using 2 wt % to 10 wt % of the total amount of water.

[0054] In some embodiments, the recombinant mussel mucin aqueous solution is obtained by using 2 wt% to 20 wt% of the total amount of water. In some embodiments, the recombinant mussel mucin aqueous solution is obtained by using 2 wt% to 10 wt% of the total amount of water. In some embodiments, the recombinant mussel mucin aqueous solution is obtained by using 5 wt% to 10 wt% of the total amount of water.

[0055] In some embodiments, 2 wt% to 20 wt% of the total amount of water is used to obtain a recombinant fibronectin aqueous solution. In some embodiments, 2 wt% to 10 wt% of the total amount of water is used to obtain a recombinant fibronectin aqueous solution. In some embodiments, 5 wt% to 10 wt% of the total amount of water is used to obtain a recombinant fibronectin aqueous solution.

[0056] In some embodiments, in step 2), the mixture is placed at 20°C-40°C for 0.5-8 hours to obtain a sodium hyaluronate-chondroitin sulfate-carboxymethyl chitosan mixture. In some embodiments, in step 2), the mixture is placed at 25°C-40°C for 2-8 hours to obtain a sodium hyaluronate-chondroitin sulfate-carboxymethyl chitosan mixture.

[0057] According to the present invention, the preparation method may further include a sterilization process as needed to obtain a final product that meets the requirements of a sterile preparation or medical device.

[0058] In the aforementioned preparation method, sodium hyaluronate and chondroitin sulfate are first mixed and allowed to stand, so that the two can be micro-cross-linked. Then, carboxymethyl chitosan is added to further micro-cross-link the polysaccharide substance, thereby forming a stable matrix liquid. The two protein substances added subsequently are dispersed in the already formed micro-cross-linked matrix liquid to form a complex micro-cross-linked system. The final composite gel composition is more stable and more conducive to forming an "isolation film" on the skin surface, exerting the effects of water retention, moisturizing, isolation and repair for a relatively long time, thereby being more conducive to the repair of damaged skin.

[0059] The preparation method of the present invention is simple, controllable, and easy to operate, and can prevent the components in the composition from being degraded, denatured, or otherwise failing to meet quality requirements.

[0060] In another aspect, the present invention provides a use of the composite gel composition.

[0061] The invention discloses an application of a composite gel composition in preparing a skin preparation or a medical device.

[0062] In another aspect, the present invention provides a skin preparation or medical device.

[0063] A skin preparation or medical device comprising the composite gel composition. Preferably, the skin preparation or medical device is a skin repair preparation or medical device.

[0064] The skin preparation or medical device provided by the present invention can be used for applying to the surface of human skin, such as the surface of skin after cosmetic or medical treatment, the surface of skin with minor wounds, or the surface of skin after sunburn or burn; or can be injected into the skin, such as the epidermis or other suitable or required areas.

[0065] The composite gel composition provided by the present invention has excellent synergistic effects between its ingredients / raw material components, so that the composition not only has excellent safety, stability (can be stably placed at room temperature for at least 30 days) and biocompatibility, but can also form an "isolation membrane" on the skin surface to protect the skin from external pollution and damage, while facilitating the maintenance of a moist microenvironment for damaged skin (such as after cosmetic surgery), utilizing wound healing, reducing pigmentation or scar formation, promoting the repair of damaged skin, effectively alleviating skin damage symptoms, enhancing skin barrier function and improving the health of damaged skin. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1The results of the cell scratch test of control group 1, sample 1 and sample 2 in Example 4 are shown (magnified 40 times, the left side is 0 h, the right side is 48 h); Figure 2 The cell scratch test results of Samples 3 and 4 in Example 4 are shown (magnified 40 times, the left side is 0 h, and the right side is 48 h); Figure 3 Graph showing the results of the zebrafish irritation test in Example 5 (fluorescence image, the dotted area is the quantitative area); Figure 4 Graph showing the results of the zebrafish experiment for the repair efficacy test in Example 6 (fluorescence graph, the dotted area is the quantitative area). DETAILED DESCRIPTION

[0067] In order to make the technical solution of the present invention more clearly understood by those skilled in the art, the following examples are given for illustration. It should be noted that the following examples do not limit the scope of protection claimed by the present invention.

[0068] In the present invention, unless otherwise specified, the content of each component / raw material is expressed in percentage by weight based on the total mass of the composition.

[0069] In the present invention, the molecular weight of a polymer or a high molecular weight substance is measured as a weight average molecular weight.

[0070] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial channels or by existing known methods; among them, recombinant mussel mucin: dopa group mass content is 1wt%; recombinant fibronectin: weight average molecular weight is 136,000 Daltons; sodium hyaluronate: weight average molecular weight is 1.21 million Daltons; chondroitin sulfate: weight average molecular weight is 17,000 Daltons; carboxymethyl chitosan in formula 1 to formula 13: weight average molecular weight is 2,000 Daltons, carboxymethyl substitution degree is 95.4%; weight average molecular weight is 57,000 Daltons, carboxymethyl substitution degree is 93.1%; the mass ratio of high and low molecular weight carboxymethyl chitosan is 3:1; carboxymethyl chitosan in the control group: weight average molecular weight is 52,000 Daltons, carboxymethyl substitution degree is 93.1%; 10% sodium hydroxide: sodium hydroxide aqueous solution with a mass fraction of 10%.

[0071] Example 1 The composite gel composition was prepared according to the following formula table and preparation method.

[0072] Recipe List: Preparation method: 1) At room temperature, approximately 15 wt% of the total water was taken to prepare a sodium hyaluronate aqueous solution, and chondroitin sulfate was added to the sodium hyaluronate aqueous solution. The mixture was stirred at 250-300 rpm for approximately 30 minutes, and then allowed to stand at 20°C for 8 hours to obtain a sodium hyaluronate-chondroitin sulfate mixed solution. 2) At room temperature, approximately 5 wt% of the total water was taken to prepare a carboxymethyl chitosan aqueous solution, which was added to the aforementioned sodium hyaluronate-chondroitin sulfate mixture, stirred at 250-300 rpm for approximately 20 minutes, and allowed to stand at 30°C for 3 hours to obtain a sodium hyaluronate-chondroitin sulfate-carboxymethyl chitosan mixture; 3) Mixing approximately 60 wt% of water, glycerol, and 1,3-butylene glycol with stirring at 250-300 rpm; adding carbomer 980, stirring for 2 minutes, and allowing to stand until swelling is complete (approximately 12 hours); homogenizing at 4500 rpm for 5 minutes, and vacuum defoaming; adding 1,2-hexanediol, p-hydroxyacetophenone, and trehalose with stirring at 250-300 rpm, and stirring to obtain a carbomer mixed solution; 4) Mixing the aforementioned carbomer mixed solution with the sodium hyaluronate-chondroitin sulfate-carboxymethyl chitosan mixed solution at 250-300 rpm and stirring for 30 minutes to obtain a polysaccharide mixed solution; 5) taking about 5 wt % of the total amount of water to prepare a recombinant mussel mucin aqueous solution and a recombinant fibronectin aqueous solution, respectively, and adding them to the aforementioned polysaccharide mixture, stirring at 250 rpm-300 rpm for 15 minutes, vacuuming to defoam, and homogenizing at 3000 rpm for 10 minutes; then adjusting the pH to 5.6 with a pH regulator, sodium hydroxide aqueous solution, adding water to the total amount (i.e., to 100%), and filtering with a 200-mesh filter cloth to obtain a clear and transparent composite gel composition.

[0073] Recombinant mussel mucin with a dopa group content of 3 wt% can be used to replace the recombinant mussel mucin with a dopa group content of 1 wt%, with the other ingredients and preparation methods being the same to obtain a composite gel composition.

[0074] The resulting composite gel composition was subjected to a simple skin feel evaluation: 6 healthy adults washed and dried their hands, and marked two approximately 3 cm long squares on the inside of their left arm and two approximately 3 cm long squares on the inside of their right arm. Each person applied 0.3 mL of the composition obtained from Formula 1 and the control group to the square on their right hand, and 0.3 mL of the gel composition obtained from the two formulas (different for each person) to the square on their left hand. After evenly applying, skin feel evaluation was collected and again collected 2 hours after application (without washing). The evaluation results are as follows: After application: the compositions obtained from the control group, formulation 1, and formulation 9 were easy to apply and had slightly higher fluidity; the compositions obtained from formulations 5 and 6 were relatively thick and spreadable, but had a sticky feel on the skin; the compositions obtained from formulations 7 and 13 were relatively thick, easy to apply, and had a smooth feel on the skin; the compositions obtained from the other formulations were all easy to apply, and had a smooth and comfortable feel on the skin; After 2 hours of application: the application sites of the compositions obtained from Formula 1 and Formula 9 felt relatively dry, with less residue; the application site of the composition obtained from Formula 5 felt moisturized, with obvious residue; the application site of the composition obtained from Formula 6 felt slightly dry, with slightly less residue (considered to be more than that of Formula 1); the application site of the composition obtained from Formula 7 felt moisturized, with obvious residue, and the skin feel was acceptable; the application site of the composition obtained from the control group and Formula 13 felt slightly dry, with obvious residue; the application site of the compositions obtained from other formulas felt moisturized, with obvious residue, and the skin feel was acceptable; all testers reported no irritation or discomfort such as stinging or itching.

[0075] According to the skin sense evaluation results, when only the skin sense is considered, it is believed that Formula 2-Formula 4, Formula 8 and Formula 10-Formula 12 are more likely to be accepted than other formulas.

[0076] Example 2: Stability test Take appropriate amounts of the compositions obtained from each formulation in Example 1 and place them in small bottles, seal them and place them under the following conditions: 1) At 40°C and 75±5% relative humidity, take samples after 15 days and 30 days to observe whether there is any discoloration or delamination; 2) Freeze at -10℃ for 30 days, then return to room temperature and observe for discoloration, stratification, or the presence of precipitates.

[0077] Results: Under 40°C conditions, the compositions obtained from formula 7 and formula 13 turned yellow after 15 days, among which the composition obtained from formula 13 was relatively more obviously yellow, and the compositions obtained from other formulas showed no obvious color change; the compositions obtained from all formulas showed no stratification and no precipitates; after 30 days, the colors of the compositions obtained from formula 7 and formula 13 deepened relative to those after 15 days, the compositions obtained from the control group and formula 1 turned slightly yellow, and the compositions obtained from all formulas showed no stratification and no precipitates; under -10°C conditions, after 30 days, no stratification, no precipitates, and no color change were observed.

[0078] Example 3: Effect of the gel composition on the expression of inflammatory factors in human immortalized keratinocytes Preparation of test samples: 0.039 g of each composite gel composition prepared according to the corresponding formula and method of Example 1 was added with water to 100 g, and mixed evenly to obtain the prepared sample for immediate use.

[0079] Normal control group: cells were not UV-irradiated and did not contain samples; Model control group: cells were exposed to ultraviolet light (80mJ / cm 2 ), excluding samples.

[0080] Then, referring to the "Cosmetic Soothing Efficacy Test - In Vitro Skin Keratinocyte Inflammatory Factor Test Method T / GDCQMA 005-2024", the test was designed and conducted: Cell line: human immortalized keratinocytes; cell generation: at least 2 generations; cell number per experimental group: 6×10 5 pcs / hole; Cells were seeded in 6-well plates (6×10 5 / well), incubated at 37 ° C, 5% CO2 for 24 h; after the incubation, the culture medium was removed, and the cells were washed 1-2 times with D-Hanks. Then, the sample group and the model control group were exposed to ultraviolet light (80 mJ / cm 2 ); After the modeling was completed, the normal control group (not subjected to UV irradiation) and the model control group were replaced with fresh culture medium, and the sample group was added with fresh culture medium containing the sample, and continued to incubate at 37 ° C, 5% CO2 for 24 h; total RNA was extracted from each experimental group, cDNA was synthesized, and q-PCR (quantitative PCR) was used to detect the gene expression of β-actin (β-actin) and the target gene; β-actin was used as the internal reference for gene expression, and the relative RNA expression of the target gene was calculated according to the following formula (1); SPSS26.0 software was used for statistical analysis; RNA relative expression = 2 C(t) Formula (1) In the formula, C(t)  C(t) model control group  C(t) sample group, C(t)  target gene C(t)  β-actin C(t); where C(t) represents the cycle threshold.

[0081] The test results are shown in the table below.

[0082] The p values in the table are compared with the model control group, and p < 0.05 indicates a significant difference.

[0083] According to the results in the above table, compared with the normal control group, the relative expression levels of TNF-α gene, IL-1α gene and IL-6 gene in the model control group were significantly upregulated, indicating that the test model is effective; the composite gel composition provided by the present invention has a good effect on reducing the expression levels of inflammatory factors in damaged cells, respectively, for TNF-α gene, IL-1α gene and IL-6 gene, which is beneficial to the repair of damaged cells and skin repair; different formulas have different strengths of effect, and the compositions obtained from Formula 3 and Formula 8 are better than the compositions obtained from Formula 1 and the control group, and the composition obtained from Formula 1 is relatively better than the composition obtained from the control group.

[0084] Example 4: Effect on migration of skin fibroblasts (HFF-1 cells) Control group 1: complete culture medium; Sample 1-Sample 4: gel compositions obtained from Formula 1, Formula 3, Formula 8 and Formula 11 in Example 1, respectively; Preparation of test samples: Take 0.5 mL of the gel composition, add 4.5 mL of complete culture medium, mix well, and obtain each test sample.

[0085] Test method: Referring to GB / T 16886.5-2017 standard, the cell scratch test was used to evaluate the effect of the sample on the migration of HFF-1 cells. HFF-1 cells in the logarithmic growth phase were taken and 2×10 5 Cells were seeded at 100 μg / well in a 6-well plate and cultured at 37°C and 5% CO2 for 48 h. The wells of the culture plate were streaked with a 200 μL pipette tip and washed three times with PBS solution. According to the group design, each test sample (2 mL / well) was added, with 3 parallel wells in each group. The cells were cultured at 37°C and 5% CO2 for 48 h, and then the cell migration was observed under a microscope.

[0086] Experimental results: Samples 1 to 4 can promote the migration of HFF-1 cells to the scratch area, and their effects on HFF-1 cell migration are better than those of control group 1; and among them, sample 2 (formula 3) is relatively optimal, and sample 3 (formula 8) is slightly better than sample 4 (formula 11); see Appendix Figure 1 and Figure 2 ( Figure 1 For control group 1, sample 1 and sample 2, Figure 2 For samples 3 and 4), Figure 1 and attached Figure 2 The photo is magnified 40 times, the left side is 0h, and the right side is 48h.

[0087] Example 5: Irritation Test 1) Zebrafish experiments Preparation method of test sample: Take 1 g of the composite gel composition obtained in formula 3 of Example 1, add water to 100 g, and mix well to obtain the test sample, which is prepared immediately before use.

[0088] Zebrafish assays were conducted using transgenic neutrophil green fluorescent zebrafish (MPX), 2 days post fertilization (dpf). Adult fish were raised and bred according to standard laboratory husbandry and breeding methods that meet the requirements of AAALAC international accreditation.

[0089] Randomly select zebrafish in a 6-well plate, with 15 fish in each well; according to the set concentration, the test sample is dissolved (or dispersed) in water and added to the sample, and a normal control group (no test sample) is set up at the same time, with a volume of 3 mL per well, and three biological replicates are performed. The cells are incubated in the dark at 28°C for 18 hours; 10 zebrafish are randomly selected from each experimental group and photographed under a fluorescence microscope. Advanced image processing software is used to analyze and collect data, and the number of neutrophils in the zebrafish skin (N) is analyzed. The irritation of the sample is calculated according to the following formula (2) to determine whether the test product is mild and non-irritating; Irritation (%) = [(sample group N) - (normal control group N)] / (normal control group N) Formula (2) The results are shown in the table below and in the appendix Figure 3 (Normal control group sample, composite gel 1% concentration sample), the dotted area in the figure is the quantitative area.

[0090] In the table, p<0.05 indicates significant difference.

[0091] According to the results in the above table, it can be seen that the composite gel composition provided by the present invention is non-irritating.

[0092] 2) Subject testing Participant information: 45 individuals who self-identified as having sensitive skin, prone to redness, dry facial skin, and dehydrated skin were selected. They ranged in age from 28 to 54 years old, with an average age of 39.4 years. During the test, the temperature was 20°C to 22°C and the relative humidity was 46% to 53%. Product, Usage, and Testing Method: After cleansing, test the TEWL rate on the cheeks. Then, evenly apply an appropriate amount of the compositions obtained from Formulas 1 and 3 in Example 1 to the face, with each participant applying Formula 1 to the left side of the face and Formula 3 to the right side. Feedback was collected 8 hours after application (without washing the face), followed by rinsing with water. The TEWL rate on the cheeks was then tested again. If any severe discomfort or abnormality occurs during this process, wash the face immediately and / or seek medical attention.

[0093] Results: No one had severe discomfort (persistent stinging, severe itching, redness and swelling, etc.); 10 people reported dryness on the left side of the face, including 3 with mild itching on the left side, and 1 person with dryness and mild itching on both the left and right sides of the face; no other people reported dryness and / or itching on the right side of the face; no one reported discomfort such as redness or burning sensation.

[0094] The results of the transepidermal water loss rate of the cheeks tested using a vapometer are shown in the table below.

[0095] According to the results, it is believed that the composition obtained by formula 3 has better water retention and moisturizing effects than the composition obtained by formula 1, is less irritating, and is safer.

[0096] Example 6: Repair efficacy test Test sample preparation method: Take 1 g of the composite gel composition obtained by formula 3 in Example 1, add water to 100 g, and mix well to obtain a sample, which is prepared for immediate use; take 1 g of La Mer concentrated repair essence purchased from the market, add water to 100 g, and mix well to obtain a control sample.

[0097] The zebrafish test method was used, using transgenic neutrophil green fluorescent zebrafish (MPX), aged 3 days post fertilization (dpf); the adult fish were raised and bred according to laboratory standard breeding and reproduction methods that meet the requirements of international AAALAC accreditation.

[0098] Randomly select zebrafish, and use a scalpel to remove the tail fin of the zebrafish to establish a zebrafish tail fin injury and inflammation model; transfer the model zebrafish to a 6-well plate, with 15 fish per well; according to the set concentration, the test sample is dissolved (or dispersed) in water and added to the sample, and a normal control group (no test sample) and a model control group (i.e., injury and inflammation model) are set up at the same time, with a capacity of 3 mL per well, and three biological replicates are performed, and the cells are incubated in the dark at 28°C for 5 hours; 10 zebrafish are randomly selected from each experimental group and photographed under a fluorescence microscope, and the data are analyzed and collected using advanced image processing software, the number of neutrophils (N) in the zebrafish tail fin area is analyzed, and the irritation of the sample is calculated according to the following formula (3) to determine whether the test product has a repair effect; Repair efficacy (%) = [(model control group N) - (sample group N)] / [(model control group N) - (normal control group N)] * 100% Formula (3) The results are shown in the table below and in the appendix Figure 4 (Normal control group samples, model control group samples and 1% concentration samples of the composite gel obtained by formula 3 in Example 1). The dotted area in the figure is the quantitative area.

[0099] Repair efficacy test results In the table, p<0.05 indicates a significant difference.

[0100] According to the above results, the composition obtained by formula 3 in Example 1 of the present invention is stable and non-irritating, and can reduce the expression of inflammatory factors, promote cell migration, have a good repair effect on damaged skin, can enhance the skin barrier function, and is beneficial to improving the health of damaged skin.

[0101] The solutions of the present invention have been described through preferred embodiments. It is apparent that relevant persons can modify or appropriately change and combine the solutions or applications described herein within the content and scope of the present invention to implement and apply the technology of the present invention. Those skilled in the art can refer to the content of this document and appropriately improve the conditions / parameters to implement and / or apply the technology of the present invention. It is particularly important to point out that all similar substitutions and modifications that are obvious to those skilled in the art are considered to be included in the present invention.

Claims

1. A composite gel composition, characterized in that The invention comprises or its preparation raw materials comprise: recombinant mussel adhesive protein, recombinant fibronectin, chondroitin sulfate, sodium hyaluronate and carboxymethyl chitosan.

2. The composite gel composition according to claim 1, characterized in that Calculated by weight percentage, it includes or its preparation raw materials include: 0.5%-5% recombinant mussel mucin, 0.1%-2% recombinant fibronectin, 0.1%-5% chondroitin sulfate, 0.05%-0.5% sodium hyaluronate and 0.08%-0.5% carboxymethyl chitosan.

3. The composite gel composition according to claim 1, wherein The mass content of the dopa group in the recombinant mussel mucin is 0.3 wt%-5 wt%.

4. The composite gel composition according to claim 1, characterized in that The weight-average molecular weight of the sodium hyaluronate is 1 million to 1.5 million Daltons; and / or the carboxymethyl chitosan is a combination of carboxymethyl chitosan having a weight-average molecular weight of 50,000 to 100,000 Daltons and carboxymethyl chitosan having a weight-average molecular weight of 1,000 to 5,000 Daltons, and the mass ratio of the high-molecular-weight and low-molecular-weight carboxymethyl chitosans is 1:1 to 9:

1.

5. The composite gel composition according to claim 1, characterized in that The preparation process of the composite gel composition comprises: preparing a mixture of sodium hyaluronate and water, adding chondroitin sulfate, mixing, and placing at 15° C.-40° C. for 1 hour-12 hours to obtain a sodium hyaluronate-chondroitin sulfate mixture.

6. The composite gel composition according to claim 1, characterized in that It also includes, or its preparation raw materials also include: a pH regulator, an auxiliary agent and water; the auxiliary agent is trehalose, carbomer, p-hydroxyacetophenone, glycerol, 1,3-butanediol and 1,2-hexanediol.

7. The composite gel composition according to claim 6, characterized in that Calculated by weight percentage, the auxiliary agent includes: 0.1%-5% trehalose, 0.2%-1% carbomer, 0.1%-1% p-hydroxyacetophenone, 0.5%-10% glycerol, 0.5%-5% 1,3-butanediol and 0.1%-1% 1,2-hexanediol.

8. A method for preparing the composite gel composition according to any one of claims 1 to 7, characterized in that: The following steps are involved: 1) preparing a mixture of sodium hyaluronate and water, adding chondroitin sulfate, mixing, and incubating at 15° C. to 40° C. for 1 to 12 hours to obtain a sodium hyaluronate-chondroitin sulfate mixture; 2) preparing a carboxymethyl chitosan aqueous solution, adding it to the sodium hyaluronate-chondroitin sulfate mixed solution, mixing, and standing at 15° C.-40° C. for 0.5 hour-8 hours to obtain a sodium hyaluronate-chondroitin sulfate-carboxymethyl chitosan mixed solution; 3) Mixing water, glycerol, and 1,3-butylene glycol, and then adding carbomer to completely swell; then, adding 1,2-hexanediol, p-hydroxyacetophenone, and trehalose, and mixing to obtain a carbomer mixture; 4) mixing the aforementioned carbomer mixture with the sodium hyaluronate-chondroitin sulfate-carboxymethyl chitosan mixture, and mixing them evenly to obtain a polysaccharide mixture; 5) Separately preparing a recombinant mussel mucin aqueous solution and a recombinant fibronectin aqueous solution, adding them to the aforementioned polysaccharide mixture, mixing, then defoaming and homogenizing; optionally adjusting the pH to 5.2-6.8 with a pH regulator to obtain a composite gel composition.

9. The preparation method according to claim 8, wherein Adjust the pH to 5.4-5.8 using sodium hydroxide.

10. Use of the composite gel composition according to any one of claims 1 to 7 in the preparation of skin preparations or devices.

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