Protein-sugar cross-linked matrix as well as preparation method and application thereof

Abstract

The invention relates to a protein-sugar cross-linked matrix as well as a preparation method and application thereof, and particularly provides a method for preparing the protein-sugar cross-linked matrix, which comprises the following steps: firstly, performing carboxyl activation treatment on glycosaminoglycan, and then adding protein to perform a first cross-linking reaction to form a prepolymer; secondly, carrying out carboxyl activation treatment on the prepolymer and a polyamino compound, and carrying out a second cross-linking reaction, so as to prepare the protein-sugar cross-linking matrix. According to the preparation method, carboxyl of glycosaminoglycan is activated preferentially, self-crosslinking of protein is reduced, and then a secondary crosslinking technology is introduced, so that the density of a crosslinking network is improved, and a stable and uniform protein-sugar crosslinking matrix is obtained. Also provided are uses of the protein-sugar cross-linked matrix in the preparation of medical, plastic, cosmetic, or daily products.

Description

[0001] This application claims the priority of Chinese patent application No. 202411166426.4 filed on August 23, 2024. This application cites the entire text of the above Chinese patent application. Technical Field

[0002] The present invention relates to the technical field of biomedicine or polymer materials, and more specifically, to a protein-sugar cross-linked matrix and a preparation method and application thereof. Background Art

[0003] Collagen and hyaluronic acid, as the main components of the extracellular matrix of the dermis of human skin, are widely used in various tissue repair treatments. Collagen, as the main structural component of the dermis and the most abundant protein in the human body, gives the skin a certain strength and has a supporting role. It is the main connective tissue protein of animals. Collagen is mainly used clinically to improve facial tissue depressions, static wrinkles on the skin and tissue contours. It is especially suitable for facial soft, well-stretched, smooth-edged skin lesions and static wrinkles such as aging wrinkles on the forehead, between the eyebrows, corners of the eyes, and nasolabial folds. Hyaluronic acid is a natural glycosaminoglycan, also known as mucopolysaccharide, present in the skin and other tissues. Hyaluronic acid can hold up to 500 to 1000 times its own weight in water. Therefore, injecting hyaluronic acid directly into the dermis can increase the hyaluronic acid content in the skin, enhance hydration, and restore skin volume.

[0004] In recent years, many researchers have tried to simulate the state of collagen and hyaluronic acid in the skin, forming an amide bond between the amino group on collagen and the carboxyl group on hyaluronic acid, thereby developing a new dermal bionic implant that has the advantages of both collagen and hyaluronic acid.

[0005] For example, patent US11844878B2 dissolves hyaluronic acid in a collagen solution, mixes and hydrates, and then adds an activator to cause a cross-linking reaction between hyaluronic acid and collagen to obtain a gel implant. However, since both collagen and hyaluronic acid are high molecular weight natural compounds, the steric hindrance when the two react is extremely large, and this method is difficult to obtain a stable and uniform implant.

[0006] Patent CN115279330A mixes collagen, lysine and hyaluronic acid evenly, and then adds a cross-linking agent for cross-linking reaction. The implant obtained by this method faces the problem of uneven composition, containing not only cross-linked collagen hyaluronic acid, but also a large proportion of cross-linked collagen.

[0007] In summary, obtaining cross-linked collagen hyaluronic acid implants with stable, uniform performance and excellent indicators will provide more options to meet clinical tissue repair, filling and shaping needs. Summary of the invention

[0008] In view of the above problems, the present application preferentially activates the carboxyl groups of glycosaminoglycans and then reacts with proteins. At this time, the amino groups on the proteins will preferentially react with the activated carboxyl groups on the glycosaminoglycans, thereby reducing the self-crosslinking of collagen; and then introduces a second crosslinking technology to deepen the crosslinking of hyaluronic acid and collagen, thereby increasing the density of the crosslinking network and obtaining a stable and uniform protein-sugar crosslinked matrix. The following aspects are provided:

[0009] In a first aspect, the present application provides a method for preparing a protein-sugar cross-linked matrix, which comprises firstly subjecting glycosaminoglycan to carboxyl activation treatment, and then adding protein to perform a first cross-linking reaction to form a prepolymer; secondly, subjecting the prepolymer and a polyamine compound to carboxyl activation treatment, and then performing a second cross-linking reaction to obtain a protein-sugar cross-linked matrix;

[0010] Wherein, the polyamine compound comprises a polyamine compound without carboxyl group and / or a polyamine compound containing carboxyl group;

[0011] The carboxyl activation treatment includes activating at least one of the carboxyl groups of glycosaminoglycans, the carboxyl groups of proteins or the carboxyl groups of carboxyl-containing polyamine compounds to form carboxyl derivatives, so that the carboxyl derivatives can form amide bonds with the amino groups of proteins and the amino groups of polyamine compounds; and / or, so that the activated carboxyl groups can form amide bonds with the amino groups of proteins; and / or, so that the activated carboxyl groups can form amide bonds with the amino groups of polyamine compounds;

[0012] The cross-linking reaction includes cross-links containing amide bonds formed between the carboxyl groups of glycosaminoglycans and the amino groups of proteins, and / or, cross-links containing amide bonds formed between the amino groups of polyamine compounds and the carboxyl groups of glycosaminoglycans and the carboxyl groups of proteins, and / or, cross-links containing amide bonds formed between the amino groups of polyamine compounds and the carboxyl groups of glycosaminoglycans, and / or, cross-links containing amide bonds formed between the amino groups of polyamine compounds and the carboxyl groups of proteins, and / or, cross-links containing amide bonds formed between the carboxyl groups of carboxyl-containing polyamine compounds and the amino groups of proteins.

[0013] In certain embodiments, the carboxyl-containing polyamine compound comprises a basic amino acid; in certain preferred embodiments, the basic amino acid is selected from at least one of arginine, citrulline, glutamine, and L-asparagine; in certain preferred embodiments, the basic amino acid is arginine.

[0014] In certain embodiments, the carboxyl-free polyamine compound comprises at least one of a diamine and a triamine compound; in certain preferred embodiments, the diamine is hexamethylenediamine.

[0015] In certain embodiments, the "polyamine compound" used in the present application refers to a compound containing at least two amino groups; wherein, the "carboxyl-containing polyamine compound" refers to a compound containing at least two amino groups and a carboxyl group, and the "non-carboxyl-containing polyamine compound" refers to a compound containing at least two amino groups but no carboxyl group. In certain embodiments, the "carboxyl-containing polyamine compound" is preferably a basic amino acid, that is, an amino acid having two amino groups and one carboxyl group, such as arginine, citrulline, glutamine, and L-asparagine. In certain embodiments, the "non-carboxyl-containing polyamine compound" is preferably a diamine with a molecular formula of A-(NH2)2 or a triamine with a molecular formula of A-(NH2)3, wherein A is a C2-C10 straight or branched alkyl chain, preferably a C2-C6 chain, which may be optionally substituted with a hydroxyl, a halogen, or an alkoxy group; in certain preferred embodiments, A in the diamine molecular formula is a non-substituted C6 straight chain, such as hexamethylenediamine.

[0016] In certain embodiments, the first cross-linking reaction step includes dissolving the glycosaminoglycan, adding a reagent for carboxyl activation treatment to form a first reaction solution; dissolving the protein to form a second reaction solution; mixing the first reaction solution with the second reaction solution, and performing a cross-linking reaction to form a prepolymer. In the first cross-linking reaction, the carboxyl groups of the glycosaminoglycan are first activated, and then the protein is added. At this time, the amino groups on the protein will preferentially react with the activated carboxyl groups on the glycosaminoglycan, which can reduce the self-cross-linking of the protein and form a sheet network structure.

[0017] "Activation" in the present application can include intermediate forms of molecules that can be susceptible to and / or initiate nucleophilic attack and / or nucleophilic substitution by nucleophilic compounds. For example, in certain embodiments, carboxyl-containing molecules (e.g., glycosaminoglycans and / or proteins containing carboxyl groups) can be activated to form activated intermediates, such as activated esters, when treated with reagents for carboxyl activation treatment, wherein the activated intermediates can be susceptible to or initiate nucleophilic attack and / or nucleophilic substitution by nucleophilic compounds (e.g., amines), forming a bond, such as an amide bond, between the carboxyl-containing molecule and the nucleophilic compound.

[0018] The "carboxyl derivative" in this application refers to the activated carboxyl group formed after the carboxyl-containing molecule (for example, glycosaminoglycans and / or proteins containing carboxyl groups) is treated with a reagent for carboxyl activation treatment, which is easy to form an amide bond with the amino group of the amino-containing molecule (for example, polyamine compounds and / or proteins containing amino groups).

[0019] In certain embodiments, the reagent for the carboxyl activation process can include the use of an activator, such as carbodiimide, such as EDC; or include the use of an activator, such as an epoxide, such as allyl glycidyl ether. The reagent for the carboxyl activation process can further include the use of a modifier, such as N-succinimide (NHS) or a halide (e.g., bromine). For example, hyaluronic acid and / or chondroitin sulfate can be activated by an activator such as carbodiimide, and can be further modified by a modifier such as N-succinimide. For example, hyaluronic acid and / or chondroitin sulfate can be activated by an activator (e.g., EDC) as an o-acyl isourea group, and can be further modified by a modifier (e.g., NHS), thereby forming a carboxylate intermediate activated by NHS, which can react with the primary amino group of protein to form an amide bond.

[0020] In certain embodiments, the reagent for performing the carboxyl activation treatment comprises an activator, wherein the activator is selected from at least one of DMT-MM, EDC, DCC, CDI, or epoxides (eg, allyl glycidyl ether).

[0021] In certain preferred embodiments, the reagent for performing carboxyl activation treatment further comprises a modifying agent, wherein the modifying agent is selected from at least one of NHS or a halide (eg, bromine).

[0022] In certain embodiments, the reagent for the carboxyl activation treatment in the first cross-linking reaction can activate at least 80% of the carboxyl groups on the glycosaminoglycan, so that the activated carboxyl groups can form amide bonds with the amino groups. In certain preferred embodiments, the reagent for the carboxyl activation treatment can activate at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the carboxyl groups on the glycosaminoglycan, so that the activated carboxyl groups can form amide bonds with the amino groups.

[0023] In certain embodiments, the reagents for the carboxyl activation treatment in the first cross-linking reaction step are EDC and NHS, and the molar ratio of the NHS to the carboxyl groups in the glycosaminoglycan is 0.8 to 1.5. In certain preferred embodiments, the molar ratio of the NHS to the carboxyl groups in the glycosaminoglycan is 1.0 to 1.5, for example, the molar ratio is 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, or any sub-range or numerical point therein. In certain embodiments, the molar ratio of the EDC to the NHS is 1 to 5, for example, the molar ratio is 1, 2, 3, 4, 5, or any sub-range or numerical point therein. In certain preferred embodiments, the molar ratio of the EDC to the NHS is 2 to 4, for example, the molar ratio is 2.

[0024] In certain embodiments, the mass ratio of protein to glycosaminoglycan in the first cross-linking reaction is 10:1 to 1:10, for example, a mass ratio of 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, or any sub-range or numerical point thereof. In certain preferred embodiments, the mass ratio of protein to glycosaminoglycan is 5:1 to 1:1, for example, a mass ratio of 1:1.

[0025] In some embodiments, the first cross-linking reaction step further comprises adjusting the pH of the first reaction solution to 6-8 after the first reaction solution is mixed with the second reaction solution; for example, adjusting the pH to 6, 7, 8, or any sub-range or numerical point thereof. In some preferred embodiments, the pH is adjusted to 6.5-7.5.

[0026] In certain embodiments, the first cross-linking reaction is cross-linked at a temperature of 10-37° C., for example, the cross-linking temperature is 10° C., 20° C., 25° C., 30° C., 35° C., 37° C., or any sub-range or numerical point therein. In certain preferred embodiments, the cross-linking reaction is stirred and cross-linked at 25-30° C. for 12 hours.

[0027] In certain embodiments, the second cross-linking reaction step includes adding the polyamine compound and the reagent for carboxyl activation treatment to the prepolymer for cross-linking reaction to form a protein-sugar cross-linked matrix. Understandably, in the second cross-linking reaction step, the polyamine compound and the reagent for carboxyl activation treatment are added to the prepolymer in a mixed manner or separately, for example, the reagent for carboxyl activation treatment is first added, and then the polyamine compound is added, or the polyamine compound is first added, and then the reagent for carboxyl activation treatment is added. In the second cross-linking reaction, the glycosaminoglycans and proteins on the prepolymer are further cross-linked by the polyamine compound, so that the cross-linking network density is increased, and a stable and uniform protein-sugar cross-linked matrix is ​​obtained.

[0028] In certain embodiments, the molar ratio of the amino groups of the polyamine compound to the carboxyl groups and carboxyl derivatives in the prepolymer in the second crosslinking reaction is 0.5 to 10, for example, the molar ratio is 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any sub-range or numerical point thereof. In certain preferred embodiments, the molar ratio of the amino groups of the polyamine compound to the carboxyl groups and carboxyl derivatives in the prepolymer is 1.0 to 5.0, for example, the molar ratio is 1.0.

[0029] The "molar amount of carboxyl groups and carboxyl derivatives in the prepolymer" in this application refers to the content of carboxyl groups and carboxyl derivatives in the prepolymer that do not form cross-links including amide bonds with amino groups of proteins. In certain embodiments, when the molar amount of carboxyl groups on the glycosaminoglycan is greater than the molar amount of amino groups on the protein, the "molar amount of carboxyl groups and carboxyl derivatives in the prepolymer" is calculated by subtracting the "molar amount of protein amino groups" from the "molar amount of glycosaminoglycan carboxyl groups" and adding the "molar amount of protein carboxyl groups". In certain embodiments, when the molar amount of carboxyl groups on the glycosaminoglycan is less than the molar amount of amino groups on the protein, the "molar amount of carboxyl groups and carboxyl derivatives in the prepolymer" is the molar amount of carboxyl groups on the protein.

[0030] In certain embodiments, the reagent for carboxyl activation treatment in the second cross-linking step can activate at least 80% of the carboxyl groups of the unactivated carboxyl groups in the carboxyl-containing polyamine compound and / or prepolymer, so that the activated carboxyl groups can form amide bonds with the amino groups. In certain embodiments, the reagent for carboxyl activation treatment can activate at least 80% of the carboxyl groups of the unactivated carboxyl groups in the carboxyl-containing polyamine compound and prepolymer, so that the activated carboxyl groups can form amide bonds with the amino groups. In certain embodiments, the reagent for carboxyl activation treatment can activate at least 80% of the carboxyl groups of the carboxyl-containing polyamine compound, so that the activated carboxyl groups can form amide bonds with the amino groups. In certain embodiments, the reagent for carboxyl activation treatment can activate at least 80% of the carboxyl groups of the unactivated carboxyl groups in the prepolymer, so that the activated carboxyl groups can form amide bonds with the amino groups. In certain preferred embodiments, the reagent for the carboxyl activation treatment can activate at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the carboxyl groups of the unactivated carboxyl groups in the carboxyl-containing polyamine compound and / or prepolymer, so that the activated carboxyl groups can form amide bonds with the amino groups. In certain preferred embodiments, the reagent for the carboxyl activation treatment can activate at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the carboxyl groups of the unactivated carboxyl groups in the carboxyl-containing polyamine compound and prepolymer, so that the activated carboxyl groups can form amide bonds with the amino groups. In certain preferred embodiments, the reagent for the carboxyl activation treatment can activate at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the carboxyl groups of the unactivated carboxyl groups in the prepolymer, so that the activated carboxyl groups can form amide bonds with the amino groups. In certain preferred embodiments, the reagent for the carboxyl activation treatment can activate at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the carboxyl groups of the carboxyl-containing polyamine compound, so that the activated carboxyl groups can form amide bonds with the amino groups.

[0031] In some embodiments, the pH of the second cross-linking reaction is 6-8; for example, the pH is 6, 7, 8, or any sub-range or numerical point therein. In some preferred embodiments, the pH of the second cross-linking reaction is 6.5-7.5.

[0032] In certain embodiments, the second cross-linking reaction is cross-linked at a temperature of 10-37° C., for example, the cross-linking temperature is 10° C., 20° C., 25° C., 30° C., 35° C., 37° C., or any sub-range or numerical point therein. In certain preferred embodiments, the cross-linking reaction is stirred and cross-linked at 25-30° C. for 12 hours.

[0033] In certain embodiments, the reagents for the carboxyl activation treatment in the step of the second cross-linking reaction are EDC and NHS, and the molar ratio of the NHS to the unactivated carboxyl groups in the carboxyl-containing polyamine compound and / or prepolymer is 0.8 to 1.5. In certain preferred embodiments, the molar ratio of the NHS to the unactivated carboxyl groups in the carboxyl-containing polyamine compound and / or prepolymer is 1.0 to 1.5, for example, the molar ratio is 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, or any sub-range or numerical point therein. In certain preferred embodiments, the molar ratio of the NHS to the unactivated carboxyl groups in the carboxyl-containing polyamine compound and prepolymer is 1.0. In certain embodiments, the molar ratio of the EDC to the NHS is 1 to 5, for example, the molar ratio is 1, 2, 3, 4, 5, or any sub-range or numerical point therein. In certain preferred embodiments, the molar ratio of the EDC to the NHS is 2 to 4, for example, the molar ratio is 2.

[0034] The "molar amount of unactivated carboxyl groups in the prepolymer" in the present application refers to the molar amount of carboxyl groups in the prepolymer that cannot form amide bond crosslinks with the amino groups of the protein. In certain embodiments, when the reagent for the carboxyl activation treatment contains only an activator (for example, one or more of DMT-MM, EDC, DCC, CDI or epoxide), when the molar amount of the activator in the first crosslinking step is greater than or equal to the molar amount of the glycosaminoglycan carboxyl groups, the "molar amount of unactivated carboxyl groups in the prepolymer" is the "molar amount of carboxyl groups in the protein"; when the molar amount of the activator in the first crosslinking step is less than the molar amount of the glycosaminoglycan carboxyl groups, the "molar amount of unactivated carboxyl groups in the prepolymer" is calculated by subtracting the "molar amount of the activator" from the "molar amount of the glycosaminoglycan carboxyl groups" and adding the "molar amount of the carboxyl groups in the protein". In certain embodiments, when the reagent for carboxyl activation treatment contains, in addition to the activator, a modifier (e.g., one or more NHS or halides), when the molar amount of the modifier in the first cross-linking step is greater than or equal to the molar amount of the glycosaminoglycan carboxyl groups, the "molar amount of unactivated carboxyl groups in the prepolymer" is the "molar amount of carboxyl groups in the protein"; when the molar amount of the modifier in the first cross-linking step is less than the molar amount of the glycosaminoglycan carboxyl groups, the "molar amount of unactivated carboxyl groups in the prepolymer" is calculated by subtracting the "molar amount of the modifier" from the "molar amount of the glycosaminoglycan carboxyl groups" and adding the "molar amount of the carboxyl groups in the protein".

[0035] In certain specific embodiments, the prepolymer is 20 ml, 25 mg / ml hyaluronic acid (its carboxyl molar weight is 1.24 mmol) treated with a reagent for carboxyl activation treatment (2.48 mmol EDC and 1.24 mmol NHS), and then 50 ml, 10 mg / ml collagen (its amino molar weight is 0.165 mmol, carboxyl molar weight is 0.575 mmol) is added for the first cross-linking. The carboxyl-containing polyamine compound in the second cross-linking step is 0.825 mmol arginine (its amino molar weight is 1.65 mmol, carboxyl molar weight is 0.825 mmol), and a reagent for carboxyl activation treatment (2.8 mmol EDC and 1.4 mmol NHS) is added for cross-linking. In the first cross-linking step, the molar amount of NHS is equal to the molar amount of hyaluronic acid, that is, the "molar amount of carboxyl groups and carboxyl derivatives in the prepolymer" is the molar amount of carboxyl groups of hyaluronic acid in the first cross-linking step, 1.24mmol, minus the molar amount of collagen amino groups, 0.165mmol, plus the molar amount of collagen carboxyl groups, 0.575mmol, a total of 1.65mmol. The molar amount of arginine amino groups is 1.65mmol; the "molar amount of unactivated carboxyl groups in the prepolymer" is the molar amount of carboxyl groups in the protein, a total of 0.575mmol, that is, the molar amount of NHS in the second cross-linking step is the molar amount of arginine carboxyl groups, 0.825mmol, plus the "molar amount of unactivated carboxyl groups in the prepolymer", 0.575mmol, a total of 1.4mmol. Therefore, in this embodiment, the "mass ratio of protein to glycosaminoglycan" in the first cross-linking step is 1:1; the "molar ratio of NHS to carboxyl groups in glycosaminoglycans" is 1; and the "molar ratio of EDC to NHS" is 2.0. In the second crosslinking step, the "molar ratio of amino groups of the polyamine compound to carboxyl groups and carboxyl derivatives in the prepolymer" is 1.0. The "molar ratio of NHS to carboxyl-containing polyamine compounds and unactivated carboxyl groups in the prepolymer" is 1; and the "molar ratio of EDC to NHS" is 2.

[0036] In some specific embodiments, the prepolymer is 50 ml, 10 mg / ml hyaluronic acid (its carboxyl molar weight is 1.24 mmol) treated with a reagent for carboxyl activation treatment (7.44 mmol EDC and 1.488 mmol NHS), and then 500 ml, 10 mg / ml collagen (its amino molar weight is 0.825 mmol, carboxyl molar weight is 2.875 mmol) is added for the first cross-linking. The carboxyl-containing polyamine compound in the second cross-linking step is 16.45 mmol citrulline (its amino molar weight is 32.9 mmol, carboxyl molar weight is 16.45 mmol), and a reagent for carboxyl activation treatment (115.8 mmol EDC and 23.19 mmol NHS) is added for cross-linking. In the first cross-linking step, the molar amount of NHS is greater than the molar amount of hyaluronic acid, that is, the "molar amount of carboxyl groups and carboxyl derivatives in the prepolymer" is the molar amount of carboxyl groups of hyaluronic acid in the first cross-linking step, 1.24mmol, minus the molar amount of collagen amino groups, 0.825mmol, plus the molar amount of collagen carboxyl groups, 2.875mmol, totaling 3.29mmol. The amino molar amount of citrulline is 32.9mmol; the "molar amount of inactivated carboxyl groups in the prepolymer" is the molar amount of carboxyl groups in the protein, totaling 2.875mmol, that is, the molar amount of carboxyl-containing polyamine compounds and inactivated carboxyl groups in the prepolymer is the molar amount of carboxyl groups of citrulline, 16.45mmol, plus the "molar amount of inactivated carboxyl groups in the prepolymer", 2.875mmol, totaling 19.325mmol. Therefore, in this embodiment, the "mass ratio of protein to glycosaminoglycan" in the first cross-linking step is 1:5; the "molar ratio of NHS to carboxyl groups in glycosaminoglycan" is 1.2; and the "molar ratio of EDC to NHS" is 5.0. In the second cross-linking step, the "molar ratio of amino groups in polyamine compounds to carboxyl groups and carboxyl derivatives in prepolymers" is 10.0. The "molar ratio of NHS to carboxyl-containing polyamine compounds and unactivated carboxyl groups in prepolymers" is 1.2; and the "molar ratio of EDC to NHS" is 5.

[0037] In some embodiments, the protein is selected from at least one of collagen, bovine serum albumin, and elastin. In some preferred embodiments, the collagen is selected from at least one of type I collagen, type II collagen, and type III collagen. In some preferred embodiments, the collagen is selected from animal-derived collagen or recombinant collagen, such as recombinant human collagen or recombinant human-like collagen.

[0038] In certain embodiments, the concentration of the protein is 2 to 10 mg / mL, such as 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, or any subrange or numerical point therein. In certain preferred embodiments, the concentration of the protein is 10 mg / mL.

[0039] In certain embodiments, the glycosaminoglycan is selected from at least one of hyaluronic acid, heparan sulfate, heparin, chondroitin sulfate, dermatan sulfate, keratan sulfate, or salts thereof.

[0040] In certain embodiments, the molecular weight of the glycosaminoglycan is 10KD to 3000KD, for example, a molecular weight of 10KD, 100KD, 500KD, 1000KD, 2000KD, 2500KD, 3000KD, or any sub-range or numerical point therein. In certain preferred embodiments, the molecular weight of the glycosaminoglycan is 100KD.

[0041] In certain embodiments, the concentration of the glycosaminoglycan is 1 to 25 mg / mL, for example, a concentration of 1 mg / mL, 2 mg / mL, 2.5 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, or any subrange or numerical point therein. In certain preferred embodiments, the concentration of the glycosaminoglycan is 25 mg / mL.

[0042] In a second aspect, the present application provides a protein-sugar cross-linked matrix, which is prepared by the preparation method described in the first aspect.

[0043] In a third aspect, the present application provides a freeze-dried product, which is obtained by freeze-drying the protein-sugar cross-linked matrix described in the second aspect.

[0044] In a fourth aspect, the present application provides a composition comprising the protein-sugar cross-linked matrix described in the second aspect or the lyophilized product described in the third aspect.

[0045] In certain embodiments, the composition further comprises a buffer and hyaluronic acid. In certain preferred embodiments, the hyaluronic acid comprises free hyaluronic acid.

[0046] In certain embodiments, the hyaluronic acid molecular weight is 100KD~1500KD, for example 100KD, 150KD, 200KD, 300KD, 400KD, 500KD, 1000KD, 1500KD, or any subrange or numerical point therein. In some preferred embodiments, the hyaluronic acid molecular weight is 500KD~1500KD, for example 500KD, 1000KD, 1500KD.

[0047] In certain embodiments, the concentration of the hyaluronic acid in the composition is 5% to 25%, for example, a concentration of 5%, 10%, 15%, 20%, 25%, or any sub-range or numerical point therein. In certain preferred embodiments, the concentration of the hyaluronic acid in the composition is 5% to 15%, for example, 5%.

[0048] In certain embodiments, the concentration of the protein-sugar cross-linked matrix or lyophilized product in the composition is 1 to 100 mg / mL, for example, 1 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, or any sub-range or numerical point thereof. In certain preferred embodiments, the concentration of the protein-sugar cross-linked matrix or lyophilized product in the composition is 25 to 60 mg / mL, for example, 25, 35, 40 or 60 mg / mL.

[0049] In certain embodiments, the storage modulus of the composition is 300 Pa to 4000 Pa. In certain preferred embodiments, the storage modulus of the composition is 1500 to 3000 Pa.

[0050] In certain embodiments, the loss modulus of the composition is 50 Pa to 800 Pa. In certain preferred embodiments, the loss modulus of the composition is 300 to 600 Pa.

[0051] In certain embodiments, the dielectric loss tangent parameter (G" / G') of the composition is 0.05 to 0.35. In certain preferred embodiments, the dielectric loss tangent parameter (G" / G') of the composition is 0.15 to 0.30.

[0052] In some embodiments, the shear viscosity of the composition is 20 to 300 Pa.s. In some preferred embodiments, the shear viscosity of the composition is 100 to 250 Pa.s.

[0053] In certain embodiments, the composition has degradable properties.

[0054] In certain embodiments, the composition has the effect of promoting cell adhesion or cell proliferation.

[0055] In certain embodiments, the composition has the effect of promoting collagen regeneration.

[0056] In the fifth aspect, the present application provides the use of the protein-sugar cross-linked matrix described in the second aspect, the lyophilized product described in the third aspect, or the composition described in the fourth aspect in the preparation of medical, plastic surgery, beauty, or daily use products.

[0057] In certain embodiments, the medical, plastic surgery, cosmetic, or daily use products include products for swelling and / or enlarging tissues, surgical operations, restorative operations, cosmetic operations, dermatological operations, plastic surgery, promoting wound healing, promoting tissue regeneration, promoting bone formation, cosmetic operations, filling skin or tissues; or preventing and / or treating skin aging, skin diseases, and scars. In certain preferred embodiments, the filling of skin or tissues includes filling facial wrinkles or filling skin depressions.

[0058] In a sixth aspect, the present application provides a kit comprising the protein-sugar cross-linked matrix described in the second aspect, the lyophilized product described in the third aspect, or the composition described in the fourth aspect.

[0059] It should be understood that within the scope of this application, the above-mentioned technical features of this application and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0060] Beneficial Effects

[0061] The protein-sugar cross-linked matrix prepared by the method provided in this application has the following good effects:

[0062] (1) The present invention preferentially activates the carboxyl groups of glycosaminoglycans and then reacts with proteins. At this time, the amino groups on the proteins will preferentially react with the activated carboxyl groups on the glycosaminoglycans, thereby reducing the self-crosslinking reaction of collagen. Then, through the secondary crosslinking technology, the density of the collagen hyaluronic acid network of the crosslinked matrix is ​​increased to form a sheet network, and the obtained product performance is more uniform and stable, and the mechanical properties and maintenance time are better.

[0063] (2) The cross-linked matrix of the present application can be used to prepare a novel dermal biomimetic implant, the structure of which is conducive to the adhesion of fibroblasts, thereby achieving the effect of collagen regeneration;

[0064] (3) The cross-linked matrix of the present application can be compounded into implants of different concentrations according to clinical use requirements to meet multiple clinical requirements for tissue repair, filling and shaping;

[0065] (4) The final product of the present application does not require any additional chemical cross-linking agent and has higher biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 The SEM network morphology of the gel implant in Experimental Example 1 at different magnifications is shown.

[0067] Figure 2 The infrared structure of the gel implant in Experimental Example 2 is shown.

[0068] Figure 3A , 3B The mechanical property data of the gel implant in Experimental Example 3 are shown.

[0069] Figure 4 The in vitro water absorption and swelling of the gel implant in Experimental Example 4 is shown.

[0070] Figure 5 The in vitro swelling data of the gel implant in Experimental Example 4 are shown.

[0071] Figure 6 The properties of the gel implant in Experimental Example 4 after implantation in vivo are shown.

[0072] Figure 7 The in vitro degradation data of the gel implant in Experimental Example 5 are shown.

[0073] Figure 8 The in vitro cytotoxicity data of the gel implant in Experimental Example 6 are shown.

[0074] Fig. 9 The data of Experimental Example 7 on the promotion of in vitro cell adhesion by the gel implant are shown.

[0075] Fig.10 The fluorescence images of the gel implant promoting cell adhesion in vitro in Experimental Example 7 are shown.

[0076] Fig.11 The data of the gel implant promoting in vitro cell proliferation in Experimental Example 8 are shown.

[0077] Fig.12 The anatomical diagram of the animal in Experimental Example 9 is shown.

[0078] Fig.13 The height data of the filler in Experimental Example 9 is shown.

[0079] Fig.14 The results of inflammation after in vivo implantation of the gel implant in Experimental Example 9 are shown.

[0080] Fig.15 The pathological filling height data of the gel implant in Experimental Example 9 are shown.

[0081] Figures 16A to 16H The microscopic anatomy of the gel implant in Experimental Example 9 is shown.

[0082] Fig.17 The Masson diagram of the subcutaneous implantation of the gel implant in vivo in Experimental Example 10 is shown.

[0083] Fig.18 The graph shows the results of the gel implant shaping evaluation of Experimental Example 11.

[0084] Abbreviations

[0085] NHS: N-hydroxysuccinimide

[0086] EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide

[0087] DMT-MM: 4-(4,6-dimethoxytriazine)-4-methylmorpholinium chloride

[0088] DCC: dicyclohexylcarbodiimide

[0089] CDI: N,N-Carbonyldiimidazole

[0090] HA: Hyaluronic acid

[0091] COL: collagen

[0092] MES: Morpholineethanesulfonic acid DETAILED DESCRIPTION

[0093] The embodiments of the present disclosure will be described in detail below in conjunction with the drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only used to illustrate the present application, rather than to limit the scope of the present application. According to the following detailed description of the drawings and preferred embodiments, various objects and advantages of the present application will become apparent to those skilled in the art.

[0094] The present application is now described with reference to the following embodiments which are intended to illustrate the present application (but not to limit the present application).

[0095] Those skilled in the art will appreciate that the embodiments describe the present application by way of example and are not intended to limit the scope of protection claimed in the present application. The experimental methods in the embodiments are conventional methods unless otherwise specified. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0096] Table 1: Main materials and sources

[0097]

[0098] Comparative Example 1

[0099] 100 mM MES was used as the dissolution medium. 100 KD hyaluronic acid was weighed and stirred to dissolve to form a 20 ml homogeneous solution. The concentration of hyaluronic acid was 25 mg / mL. Then 2.48 mmol 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 1.24 mmol N-hydroxysuccinimide were added. The hyaluronic acid was activated by stirring at 30 °C for 3 h as the first reaction solution. 0.01 M HCl was used as the medium to dissolve collagen, the concentration of collagen was 10 mg / mL, and the collagen solution was used as the second reaction solution; after the dissolution was completed, 2.5 times the volume of the second reaction solution was added to the first reaction solution, and the mass ratio of collagen to HA was 1:1, wherein the amino molar amount of collagen was 0.165 mmol, the carboxyl molar amount was 0.575 mmol, and the carboxyl molar amount of hyaluronic acid was 1.24 mmol; the pH was adjusted to 6.5-7.5, and the cross-linking was stirred at 30°C for 12 hours; after the cross-linking was completed, the cross-linked matrix was dialyzed in ultrapure water at 2°C-8°C for 3 days, during which the ultrapure water was replaced several times to remove the processing aids. After the dialysis was completed, the cross-linked collagen hyaluronic acid solid sponge was obtained by freeze drying, and sterilized by irradiation, sterile phosphate buffer and 500KD sterile hyaluronic acid were added, and the concentration of hyaluronic acid was 5%. Finally, a 35 mg / mL sterile implant was compounded and recorded as: COL-HA-Ⅰ.

[0100] Comparative Example 2

[0101] Collagen was dissolved in 0.01M HCl as a medium, the concentration of collagen was 10 mg / mL, the volume was 50 ml (the amino molar weight of collagen was 0.165 mmol, and the carboxyl molar weight was 0.575 mmol), and then 500 mg of 100KD hyaluronic acid (the carboxyl molar weight of hyaluronic acid was 1.24 mmol), 5.28 mmol 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, 2.64 mmol N-hydroxysuccinimide, and 0.825 mmol arginine were weighed, and the solution was dissolved by stirring to form a homogeneous solution. The pH was adjusted to 6.5-7.5, and the solution was cross-linked at 30°C for 12 h. After the cross-linking was completed, the cross-linked matrix was dialyzed in ultrapure water at 2°C-8°C for 3 days, during which the ultrapure water was replaced several times to remove the processing aids. After dialysis, freeze-drying was performed to obtain a cross-linked collagen hyaluronic acid solid sponge, which was sterilized by irradiation and then added with sterile phosphate buffer and 500KD sterile hyaluronic acid, with a hyaluronic acid concentration of 5%. Finally, a 35 mg / mL sterile implant was prepared, which was recorded as: COL-HA-Ⅱ.

[0102] Example 1

[0103] 100 mM MES was used as the dissolution medium. 100 KD hyaluronic acid was weighed and stirred to dissolve to form a 20 ml homogeneous solution. The concentration of hyaluronic acid was 25 mg / mL. Then 2.48 mmol 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 1.24 mmol N-hydroxysuccinimide were added. The hyaluronic acid was activated by stirring at 30 °C for 3 h as the first reaction solution. 0.01 M HCl was used as a medium to dissolve collagen, the concentration of collagen was 10 mg / mL, and the collagen solution was used as the second reaction solution; after the dissolution was completed, 2.5 times the volume of the second reaction solution was added to the first reaction solution, the mass ratio of collagen to HA was 1:1, wherein the amino molar amount of collagen was 0.165 mmol, the carboxyl molar amount was 0.575 mmol, and the carboxyl molar amount of hyaluronic acid was 1.24 mmol; the pH was adjusted to 6.5-7.5, and the mixture was stirred and cross-linked at 30°C for 12 hours to form a prepolymer; 0.825 mmol arginine, 2.80 mmol 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, and 1.40 mmol N-hydroxysuccinimide powder were weighed and added to the above prepolymer, the pH was adjusted to 6.5-7.5, and the mixture was stirred and cross-linked at 30°C for 12 hours; after the cross-linking was completed, the cross-linked matrix was dialyzed in ultrapure water at 2°C-8°C for 3 days, during which the ultrapure water was replaced several times to remove the processing aid. After dialysis, freeze-drying was performed to obtain a cross-linked collagen hyaluronic acid solid sponge, which was sterilized by irradiation and then added with sterile phosphate buffer and 500KD sterile hyaluronic acid, with a hyaluronic acid concentration of 5%. Finally, a 35 mg / mL sterile implant was prepared, which was recorded as: COL-HA-Ⅲ.

[0104] Take 10 ml of the secondary cross-linking reaction solution, place it in a boiling activated dialysis bag, the molecular weight cutoff of the dialysis bag is 1400D, seal the dialysis bag and place it in a light-proof nitrogen container, add 30 ml of ultrapure water for dialysis, take the external solution after dialysis for 12 hours, and refer to the literature "Determination of 18 Amino Acids in Rana Lepis Collagen Peptide by Pre-column Derivatization RP-HPLC" to detect the residual amount of arginine after the secondary cross-linking reaction, and calculate the mass proportion of arginine in the cross-linked matrix. The mass proportion of arginine in the cross-linked matrix is ​​3.20%.

[0105] Example 2

[0106] With 100mM MES as the dissolution medium, weigh 1000KD hyaluronic acid and stir to dissolve to form 50ml homogeneous solution, the concentration of hyaluronic acid is 10mg / mL, then add 6.20mmol 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 1.24mmol N-hydroxysuccinimide, stir at 30℃ for 3h to activate hyaluronic acid as the first reaction solution; with 0.01M HCl was used as a medium to dissolve collagen, the concentration of collagen was 10 mg / mL, and the collagen solution was used as the second reaction solution; after the dissolution was completed, 10 times the volume of the second reaction solution was added to the first reaction solution, the mass ratio of collagen to HA was 10:1, wherein the amino molar amount of collagen was 1.65 mmol, the carboxyl molar amount was 5.75 mmol, and the carboxyl molar amount of hyaluronic acid was 1.24 mmol; the pH was adjusted to 6.5-7.5, and the mixture was stirred and cross-linked at 30°C for 12 hours to form a prepolymer; 13.35 mmol hexamethylenediamine, 28.75 mmol 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 5.75 mmol N-hydroxysuccinimide powder were weighed and added to the above prepolymer, the pH was adjusted to 6.5-7.5, and the mixture was stirred and cross-linked at 30°C for 12 hours; after the cross-linking was completed, the cross-linked matrix was dialyzed in ultrapure water at 2°C-8°C for 3 days, during which the ultrapure water was replaced several times to remove the processing aid. After dialysis, freeze-drying was performed to obtain a cross-linked collagen hyaluronic acid solid sponge, which was sterilized by irradiation and then added with sterile phosphate buffer and 500KD sterile hyaluronic acid, with a hyaluronic acid concentration of 5%. Finally, a 35 mg / mL sterile implant was prepared, which was recorded as: COL-HA-IV.

[0107] Take 10 ml of the secondary cross-linking reaction solution and place it in a boiling activated dialysis bag with a molecular weight cutoff of 1400D. After the dialysis bag is sealed, place it in a light-proof nitrogen container, add 30 ml of ultrapure water for dialysis, and take the external liquid after dialysis for 12 hours. Refer to "2,4-Dinitrochlorobenzene Spectrophotometric Determination of Hexamethylenediamine in Water" to detect the residual amount of hexamethylenediamine and calculate the mass proportion of hexamethylenediamine in the cross-linked matrix. The mass proportion of hexamethylenediamine in the cross-linked matrix is ​​3.44%.

[0108] Example 3

[0109] With 100mM MES as the dissolution medium, weigh 1500KD hyaluronic acid and stir and dissolve to form 100ml homogeneous solution, the concentration of hyaluronic acid is 5mg / mL, then add 7.44mmol 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 1.488mmol N-hydroxysuccinimide, stir at 30℃ for 3h to activate hyaluronic acid as the first reaction solution; with 0.01M HCl was used as a medium to dissolve collagen, the concentration of collagen was 5 mg / mL, and the collagen solution was used as the second reaction solution; after the dissolution was completed, 5 times the volume of the second reaction solution was added to the first reaction solution, the mass ratio of collagen to HA was 5:1, wherein the amino molar amount of collagen was 0.825 mmol, the carboxyl molar amount was 2.875 mmol, and the carboxyl molar amount of hyaluronic acid was 1.24 mmol; the pH was adjusted to 6.5-7.5, and the mixture was stirred and cross-linked at 30°C for 12 hours to form a prepolymer; 16.45 mmol citrulline, 115.8 mmol 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, and 23.19 mmol N-hydroxysuccinimide powder were weighed and added to the above prepolymer, the pH was adjusted to 6.5-7.5, and the mixture was stirred and cross-linked at 30°C for 12 hours; after the cross-linking was completed, the cross-linked matrix was dialyzed in ultrapure water at 2°C-8°C for 3 days, during which the ultrapure water was replaced several times to remove the processing aid. After dialysis, freeze-drying was performed to obtain a cross-linked collagen hyaluronic acid solid sponge, which was sterilized by irradiation and then added with sterile phosphate buffer and 500KD sterile hyaluronic acid, with a hyaluronic acid concentration of 5%. Finally, a 35 mg / mL sterile implant was prepared, which was recorded as: COL-HA-V.

[0110] Take 10 ml of the secondary cross-linking reaction solution, place it in a boiling activated dialysis bag, the molecular weight cutoff of the dialysis bag is 1400D, seal the dialysis bag and place it in a light-proof nitrogen container, add 30 ml of ultrapure water for dialysis, take the external solution after dialysis for 12 hours, and refer to the literature "Determination of 18 Amino Acids in Rana Lepis Collagen Peptide by Pre-column Derivatization RP-HPLC" to detect the residual amount of citrulline after the secondary cross-linking reaction, and calculate the mass proportion of citrulline in the cross-linked matrix. The mass proportion of citrulline in the cross-linked matrix is ​​5.65%.

[0111] Example 4

[0112] Using 100mM MES as the dissolution medium, weigh 2000KD hyaluronic acid and stir to dissolve to form 200ml homogeneous solution. The concentration of hyaluronic acid is 2.5mg / mL. Then add 1.860mmol 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.860mmol N-hydroxysuccinimide, stirred at 30°C for 3h to activate hyaluronic acid as the first reaction solution; 0.01M HCl was used as the medium to dissolve collagen, the concentration of collagen was 5mg / mL, and the collagen solution was used as the second reaction solution; after the dissolution was completed, 0.1 times the volume of the second reaction solution was added to the first reaction solution, and the mass ratio of collagen to HA was 1:5, wherein the amino molar weight of collagen was 0.033mmol, the carboxyl molar weight was 0.115mmol, and the carboxyl molar weight of hyaluronic acid was 1.24mmol; the pH was adjusted to 7-8, and the prepolymer was formed by stirring and cross-linking at 30°C for 12h; then 0.331mmol glutamine, 0.669mmol 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 0.669mmol N-hydroxysuccinimide powder was added to the above prepolymer, pH was adjusted to 7-8, and secondary cross-linking was performed at 30℃ with stirring for 12h. After cross-linking, the cross-linked matrix was dialyzed in ultrapure water at 2℃-8℃ for 3d, during which the ultrapure water was replaced several times to remove the processing aids. After dialysis, freeze-dried solid sponge of cross-linked collagen hyaluronic acid was obtained, and sterilized by irradiation, sterile phosphate buffer and 500KD sterile hyaluronic acid were added, and the concentration of hyaluronic acid was 5%. Finally, a 35mg / mL sterile implant was compounded and recorded as: COL-HA-VI.

[0113] Take 10 ml of the secondary cross-linking reaction solution, place it in a boiling activated dialysis bag, the molecular weight cutoff of the dialysis bag is 1400D, seal the dialysis bag and place it in a light-proof nitrogen container, add 30 ml of ultrapure water for dialysis, take the external liquid after dialysis for 12 hours, and refer to the literature "Determination of 18 Amino Acids in Rana Lepis Collagen Peptide by Pre-column Derivatization RP-HPLC" to detect the residual amount of glutamine after the secondary cross-linking reaction, and calculate the mass proportion of glutamine in the cross-linked matrix. The mass proportion of glutamine in the cross-linked matrix is ​​1.31%.

[0114] Example 5

[0115] Using 100mM MES as the dissolution medium, weigh 3000KD hyaluronic acid, stir and dissolve to form 500ml homogeneous solution, the concentration of hyaluronic acid is 1mg / mL, then add 2.976mmol 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, 1.488mmol N-hydroxysuccinimide, stir at 30℃ for 3h to activate hyaluronic acid as the first reaction solution; use 0.01M HCl as the medium to dissolve collagen, the concentration of collagen is 2mg / mL, and the collagen solution is used as the second reaction solution; after the dissolution is completed, add 0.05 times the volume of the second reaction solution to the first reaction solution, the mass ratio of collagen to HA is 1:10, wherein the amino molar amount of collagen is 0.017mmol, the carboxyl molar amount is 0.058mmol, and the carboxyl molar amount of hyaluronic acid is 1.24mmol; adjust the pH to 7-8, stir at 30℃ for 12h to form a prepolymer; then weigh 1.601mmol L-asparagine, 3.982mmol 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, 1.991mmol N-hydroxysuccinimide powder were added to the above prepolymer, the pH was adjusted to 7-8, and the secondary cross-linking was stirred at 30℃ for 12h; after the cross-linking, the cross-linked matrix was dialyzed in ultrapure water at 2℃-8℃ for 3d, and the ultrapure water was changed several times during this period to remove the processing aids. After the dialysis was completed, freeze-dried to obtain the cross-linked collagen hyaluronic acid solid sponge, and sterilized by irradiation, sterile phosphate buffer and 500KD sterile hyaluronic acid were added, and the concentration of hyaluronic acid was 5%. Finally, a 35mg / mL sterile implant was compounded and recorded as: COL-HA-VII.

[0116] Take 10ml of the secondary cross-linking reaction solution and place it in a boiling activated dialysis bag with a molecular weight cutoff of 1400D. After the dialysis bag is sealed, place it in a light-proof nitrogen container, add 30ml of ultrapure water for dialysis, and take the external solution after dialysis for 12 hours. Refer to the literature "Determination of 18 Amino Acids in Rana Lepis Collagen Peptides by Pre-column Derivatization RP-HPLC" to detect the residual amount of L-asparagine after the secondary cross-linking reaction, and calculate the mass proportion of L-asparagine in the cross-linked matrix. The mass proportion of L-asparagine in the cross-linked matrix is ​​5.14%.

[0117] Example 6

[0118] 100mM MES was used as the dissolution medium, 50KD chondroitin sulfate was weighed and stirred to dissolve to form 50ml of homogeneous solution, the concentration of chondroitin sulfate was 10mg / mL, and then 2.158mmol 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 1.079mmol N-hydroxysuccinimide were added, and the chondroitin sulfate was activated by stirring at 30℃ for 3h as the first reaction solution; 0.01M HCl was used as a medium to dissolve collagen, the concentration of collagen was 10 mg / mL, and the collagen solution was used as the second reaction solution; after the dissolution was completed, an equal volume of the second reaction solution was added to the first reaction solution, the mass ratio of collagen to chondroitin sulfate was 1:1, wherein the amino molar amount of collagen was 0.165 mmol, the carboxyl molar amount was 0.575 mmol, and the carboxyl molar amount of chondroitin sulfate was 1.079 mmol; the pH was adjusted to 6.5-7.5, and the mixture was stirred and cross-linked at 30°C for 12 hours to form a prepolymer; 0.745 mmol arginine, 2.64 mmol 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1.32 mmol N-hydroxysuccinimide powder were weighed and added to the above prepolymer, the pH was adjusted to 6.5-7.5, and the mixture was stirred and cross-linked at 30°C for 12 hours; after the cross-linking was completed, the cross-linked matrix was dialyzed in ultrapure water at 2°C-8°C for 3 days, during which the ultrapure water was replaced several times to remove the processing aid. After dialysis, freeze-drying was performed to obtain cross-linked collagen chondroitin sulfate solid sponge, which was sterilized by irradiation and added with sterile phosphate buffer and 1500KD sterile hyaluronic acid, with the concentration of hyaluronic acid being 25%. Finally, a 60 mg / mL sterile implant was prepared, which was recorded as: COL-CS-Ⅰ.

[0119] Example 7

[0120] Use 100mM MES as the dissolution medium, weigh 15KD heparin and stir to dissolve to form 50ml homogeneous solution, the concentration of heparin is 10mg / mL, and then add 0.882mmol 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, 0.441mmol N-hydroxysuccinimide, and activated heparin by stirring at 30°C for 3h as the first reaction solution; collagen was dissolved in 0.01M HCl as the medium, and the concentration of collagen was 10mg / mL, and the collagen solution was used as the second reaction solution; after the dissolution was completed, an equal volume of the second reaction solution was added to the first reaction solution, and the mass ratio of collagen to heparin was 1:1, wherein the amino molar amount of collagen was 0.165mmol, the carboxyl molar amount was 0.575mmol, and the carboxyl molar amount of heparin was 0.441mmol; the pH was adjusted to 6.5-7.5, and the prepolymer was formed by stirring and crosslinking at 30°C for 12h; then 0.426mmol arginine, 2.002mmol 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, and 1.001mmol N-hydroxysuccinimide powder was added to the above prepolymer, pH was adjusted to 6.5-7.5, and secondary cross-linking was performed at 30°C for 12 hours. After cross-linking, the cross-linked matrix was dialyzed in ultrapure water at 2°C-8°C for 3 days, during which the ultrapure water was replaced several times to remove the processing aids. After dialysis, the cross-linked collagen heparin solid sponge was obtained by freeze drying. After irradiation sterilization, sterile phosphate buffer and 1000KD sterile hyaluronic acid were added, and the concentration of hyaluronic acid was 15%. Finally, a 40mg / mL sterile implant was compounded and recorded as: COL-HP-Ⅰ.

[0121] Experimental Example 1

[0122] In this experimental example, a Phenom Pro scanning electron microscope was used to test the network morphology of the implants COL-HA-Ⅰ, COL-HA-Ⅱ, and COL-HA-Ⅲ.

[0123] The cross-linked collagen hyaluronic acid solid sponge after freeze-drying in Comparative Example 1, Comparative Example 2, and Example 1 was accurately weighed, and 3 mL of 0.3 mg / mL hydrogel was prepared in a 6 mL vial with ultrapure water as the medium. After freeze-drying, the columnar sponge was taken out and placed in liquid nitrogen for freezing treatment for 30 min. After taking out, the cross section was cut longitudinally with a blade and fixed on a copper mesh. 10 nm of gold was vertically sprayed as a conductive coating under vacuum and then scanned with an electron microscope. The results are as follows Figure 1 shown.

[0124] Comparative Example 1: The carboxyl groups of hyaluronic acid are first activated and then mixed with collagen for cross-linking. Since both collagen and hyaluronic acid are high molecular weight natural compounds, the steric hindrance during the reaction is extremely large and the reaction efficiency is low. The electron microscopic morphology shows that the sheet network of cross-linked collagen hyaluronic acid is loose and the network density is low.

[0125] In comparative example 2, collagen, arginine and hyaluronic acid were mixed and then EDC / NHS was added for cross-linking reaction. Electron microscopic morphology showed that it contained not only a lamellar network of cross-linked collagen and hyaluronic acid, but also a large proportion of cross-linked collagen fibrous network. The implant obtained by this method had uneven composition.

[0126] Example 1: By preferentially activating the carboxyl groups of glycosaminoglycans and then reacting with proteins, the amino groups on the proteins will preferentially react with the activated carboxyl groups on the glycosaminoglycans, thereby reducing the self-crosslinking of collagen; and then introducing a second crosslinking technology to deepen the crosslinking of hyaluronic acid and collagen. Electron microscopic morphology shows that the obtained crosslinked collagen hyaluronic acid is a more uniform sheet network with a higher network density.

[0127] Experimental Example 2

[0128] In this experimental example, the infrared structure images of the implants COL-HA-Ⅰ, COL-HA-Ⅱ, and COL-HA-Ⅲ were tested using Shimadzu IRAffinity-1S Fourier transform infrared spectrometer.

[0129] Accurately weigh 4 mg of the freeze-dried cross-linked collagen hyaluronic acid solid sponge in Comparative Example 1, Comparative Example 2, and Example 1, place it in a mortar, add 0.5 ml of water, grind it evenly, add 400 mg of potassium bromide, grind it evenly, dry it under reduced pressure at 37°C to constant weight, and take the dried powder for infrared detection using the potassium bromide tablet method.

[0130] Referring to the literature "Determination of 18 Amino Acids in Rana sylvestris Skin Collagen Peptides by Pre-column Derivatization RP-HPLC", the glycine content in the cross-linked collagen hyaluronic acid matrix in the gel implant was tested, and then the collagen content in the matrix was further calculated by the proportion of glycine in the collagen raw material; referring to Appendix C of YY / T 0962-2021 "Cross-linked Sodium Hyaluronate Gel for Plastic Surgery" to test the hyaluronic acid content; finally, the proportion of collagen components in the cross-linked collagen hyaluronic acid matrix was calculated by the formula: collagen content / (collagen content + hyaluronic acid content)*100%. The results are shown in Table 2. The collagen proportions of the three implants COL-HA-Ⅰ, COL-HA-Ⅱ, and COL-HA-Ⅲ are all close to 65%; the infrared detection results are shown in Figure 2 As shown, the amide bands (1655cm-1, 1541cm-1, 1450cm-1) of the COL-HA-Ⅲ cross-linked collagen hyaluronic acid matrix are more intense, indicating that more amide bonds are generated in the structure.

[0131] Table 2: Proportion of collagen components in the cross-linked collagen hyaluronic acid matrix of gel implants

[0132] batch number Gel implant collagen % COL-HA-Ⅰ 66.5 COL-HA-Ⅱ 67.2 COL-HA-Ⅲ 66.3

[0133] Experimental Example 3

[0134] In this experimental example, the mechanical properties of the implants COL-HA-Ⅰ, COL-HA-Ⅱ, and COL-HA-Ⅲ were tested.

[0135] The storage modulus and loss modulus of the implant were tested using an RVN2200 rheometer. Test parameters: 1) Test temperature 20.0°C; 2) Shear strain: 0.01-100%; 3) Angular frequency: 10 rad / s; 4) Gap: 1.0 mm; 5) Plate diameter: 40 mm.

[0136] The proportion of collagen components in the cross-linked collagen hyaluronic acid matrix in the gel implant is shown in Table 2. The compositions of the three implants, COL-HA-Ⅰ, COL-HA-Ⅱ, and COL-HA-Ⅲ, are basically the same. The mechanical properties are shown in Table 3 and Appendix. Figure 3A-3B As shown, the storage modulus and loss modulus of COL-HA-Ⅲ are significantly higher than those of COL-HA-Ⅰ and COL-HA-Ⅱ, indicating that the mechanical properties of the gel implant of the present application are significantly higher.

[0137] Table 3: Storage modulus and loss modulus of gel implants

[0138] batch number Storage modulus (pa) Loss modulus (pa) COL-HA-Ⅰ 396.1 126.6 COL-HA-Ⅱ 1490 130.7 COL-HA-Ⅲ 2280 576.5

[0139] Experimental Example 4

[0140] In this experimental example, the in vitro swelling resistance of the implants COL-HA-Ⅱ, COL-HA-Ⅲ, COL-HA-VI, COL-HA-Ⅶ, COL-CS-Ⅰ, Juvederm Enrichment (commercially available product, pure cross-linked hyaluronic acid gel implant), and Double Beauty (commercially available product, pure cross-linked collagen gel implant) was investigated, and the swelling of the implants after absorbing liquid in vitro and absorbing body fluids after being implanted subcutaneously was evaluated.

[0141] In vitro fluid absorption evaluation: Accurately weigh a certain amount of the above implants, add physiological saline at a ratio of 0.05g / ml, shake at 60rpm at 37℃ for 72h, and observe and record the water absorption and swelling of the implants. Figure 4 , Figure 5 As shown, the swelling resistance of the implant prepared by the present invention is close to that of the pure collagen cross-linked implant (Shuangmeifu Limei).

[0142] In vivo fluid absorption evaluation: Female rats aged 6 to 8 weeks were used as experimental animals, and Juvederm Enlarge (commercially available, pure cross-linked hyaluronic acid gel implant) and COL-HA-Ⅲ gel implant were quickly injected into the rat skull using a 27G needle, with a single-point injection volume of 0.2 ml. During the experiment, the feeding environment was set to a temperature of 20°C, a relative humidity of 50%, and a lighting time of 12 hours, with free access to food. The animals were killed after 3 days of feeding, and the fillers were separated to test the mechanical properties of the gel implant. The results are shown in Table 4. Compared with Juvederm Enlarge, COL-HA-Ⅲ gel implant is tighter and has a smaller tendency to diffuse. It has sufficient viscosity for injection and initial molding, can maintain its contour in the tissue, and its elastic behavior under low shear stress is better than its viscous behavior; at the same time, combined with the attached Figure 6 It can be seen that the performance of COL-HA-Ⅲ gel implant is stable after being implanted into the skull, and it is translucent. It can provide filling support while avoiding the phenomenon of light transmission displacement, making the effect more natural.

[0143] Table 4: In vivo fluid absorption performance of gel implants

[0144]

[0145] Experimental Example 5

[0146] In this experimental example, the in vitro degradation performance of the implants COL-HA-Ⅰ, COL-HA-Ⅱ, COL-HA-Ⅲ, COL-HA-Ⅳ, COL-HA-Ⅴ and COL-HA-Ⅵ was investigated.

[0147] Accurately weigh a certain amount of the above implant into a centrifuge tube, add 1 mol / L HCl solution at a mass ratio of 3:1, and completely immerse the implant. After sealing, place it in a 37°C oven, observe and record the in vitro degradation endpoint. The results are shown in the attached Figure 7 As shown, the gel implant of the present application has a higher cross-linking network density, significantly improved in vitro degradation resistance, and significantly improved maintenance time.

[0148] Experimental Example 6

[0149] In this experimental example, the in vitro cytotoxicity of the implants COL-HA-Ⅰ, COL-HA-Ⅱ, COL-HA-Ⅲ, COL-HA-Ⅳ, COL-HA-Ⅴ, COL-HA-Ⅵ, COL-HA-Ⅶ, COL-CS-Ⅰ, and COL-HP-Ⅰ was evaluated.

[0150] Accurately weigh a certain mass of the above implants, and soak them in a culture medium at 37°C, 120rpm for 24 hours at a ratio of 0.2g / ml to allow the gel to fully swell. Add the culture medium absorbed by the swollen gel, and continue to soak in the culture medium at 37°C, 120rpm for 72±2h. Transfer the extracts of each group to a 15ml centrifuge tube and centrifuge at 3000rpm for 10min. The product extract was gradiently diluted and incubated with L929 cells for 24h. After incubating the cells with 10% CCK-8 for 0.5h, the absorbance value was detected with an enzyme marker and the cell viability was calculated. The results are shown in the attached figure. Figure 8 As shown, the gel implant directly reacts with the amino group on collagen or amino acid and the carboxyl group on polysaccharide, and the final product does not require additional chemical cross-linking agents and has no cytotoxicity.

[0151] Experimental Example 7

[0152] In this experimental example, the in vitro cell adhesion promoting function of the implants COL-HA-Ⅰ, COL-HA-Ⅱ, COL-HA-Ⅲ, COL-HA-Ⅳ, COL-HA-Ⅴ, COL-HA-Ⅵ, COL-HA-Ⅶ, COL-CS-Ⅰ, and COL-HP-Ⅰ was investigated.

[0153] Take the implant and the collagen raw material of the same concentration as the positive control, and inject about 50μL of sample into the bottom of the well plate in each well to ensure that the bottom area is covered with a thin layer of sample. Solidify in a 37℃, 5% CO2 incubator for 1h. Add 500μL of 1% BSA-PBS solution to each well and incubate in a 37℃, 5% CO2 incubator for 1h. Remove the liquid in the well. Wash 3 times with PBS, discard the washing solution, and store at 4℃ for later use. When the confluence of 3t3 cells reaches 80% to 90%, digest the cells. Adjust the cell density to about 4W / well, mix the cell suspension thoroughly, and inoculate the cells in a 24-well plate at an inoculation volume of 500μL per well. After inoculation, let the biosafety cabinet stand for a few minutes to allow the cells to settle. Observe whether the cells are evenly distributed under a microscope. After incubation for 2h, discard the supernatant. Fix the cells with 4% paraformaldehyde for 15min. After Hoechst 1:100 dilution (PBS dilution), add 300μL per well and incubate in the dark for 20min. Wash twice with PBS. Add 300 μL PBS to each well and observe under a fluorescence microscope. Take 5 fluorescence images of each well in the upper, lower, left, right and middle fields under a 10x microscope. Use image J to automatically count the number of cells in each image, with a total of 10 data in each group. The average value of each group of data is taken as the number of adherent cells. The data of each group are compared and analyzed with the positive collagen group and the negative group. The results are shown in the attached figure. Fig. 9 , Attachment Fig.10 shown.

[0154] Experimental Example 8

[0155] In this experimental example, the in vitro cell proliferation promoting function of the implants COL-HA-Ⅰ, COL-HA-Ⅱ, COL-HA-Ⅲ, COL-HA-Ⅳ, COL-HA-Ⅴ, COL-HA-Ⅵ, COL-HA-Ⅶ, COL-CS-Ⅰ, and COL-HP-Ⅰ was investigated.

[0156] An ultra-low adhesion 24-well plate was selected, and the above-mentioned implants and collagen raw materials of the same concentration were taken as positive controls. According to the sample plating volume of 0.2mL / well, a 1mL syringe equipped with a 5mL syringe needle was used to plate each test sample into the ultra-low adhesion 24-well plate. After spreading the bottom evenly, it was placed in a 37°C incubator to solidify overnight. The negative group did not perform any synchronous operations. Approximately 4W cells were inoculated in each well, with 1mL of suspension in each well. Cell proliferation on the 5th day after inoculation was detected as follows: 100μL CCK-8 was added to each well, shaken to mix, and returned to the 37°C incubator for continued incubation for 3h. After the incubation, 100μL of culture supernatant was aspirated from each well to detect the absorbance value at 450nm. The results are shown in the attached Fig.11 As shown in the figure, combined with Experimental Example 7, compared with COL-HA-I and COL-HA-II, the gel implant of the present application, as a new type of dermal bionic implant, has a complete network space structure, and polysaccharides such as collagen and hyaluronic acid serve as extracellular matrix to provide a microenvironment for cell adhesion and proliferation. Therefore, the gel implant has obvious effects of promoting cell adhesion and proliferation.

[0157] Experimental Example 9

[0158] In this experimental example, the in vivo filling efficacy of the implants COL-HA-Ⅰ, COL-HA-Ⅱ, COL-HA-Ⅲ, COL-HA-Ⅳ, COL-HA-Ⅴ, COL-HA-Ⅵ, COL-CS-Ⅰ, and COL-HP-Ⅰ was investigated.

[0159] The above implants were implanted into the subcutaneous tissue of female rats and injected rapidly using a 27G needle. Injection method: Each rat was injected with different samples, and each rat was injected at 4 injection sites on the back, with a single subcutaneous injection of 0.2 mL at each site. The injection sites were marked with a non-fading marker. During the experiment, the feeding environment was set at a temperature of 20°C, a relative humidity of 50%, and a lighting time of 12 hours, and free feeding was allowed.

[0160] After a certain period of feeding, the experimental animals were killed at the corresponding time point. The animal anatomical structures of the samples 6 months after gel implantation are shown in the attached figure. Fig.12 Separate the filling area, observe and record the shape, color and touch of the filling; cut open the filling from the highest point and measure the height of the filling with a vernier caliper, as shown in the attached figure. Fig.13 As shown, combined with the attached Fig.12It can be seen that the gel has good supporting strength. After 6 months of implantation, the filling height of the secondary cross-linked gel implant is significantly maintained. At the same time, the filler is separated from the subcutaneous tissue, and the filler and the skin tissue of the remaining samples are fixed with 10% neutral buffered formalin solution, embedded in paraffin and solidified, and slices are prepared. The slices are stained with eosin (HE), and the tissue inflammatory response and filler height are observed under an optical microscope. The observation time points are 3 months (3M) and 6 months (6M). The inflammation results of COL-HA-Ⅲ, COL-HA-Ⅴ, COL-HP-Ⅰ, and COL-CS-Ⅰ are shown in the attached Fig.14 As shown: the epidermis of the skin tissue has a complete and clear structure, with an obvious keratinized layer, a thin stratified squamous epithelium, and cells arranged neatly and tightly; the dermis hair follicle structure is clearly visible, and the hair papilla formed by the connective tissue rich in capillaries and nerves at the lower end of the hair follicle has normal morphology and structure, and collagen fibers are arranged in an interlaced manner; different amounts of implants are seen in clusters in the subcutaneous tissue, and collagen fibers are seen proliferating in the implants. The implants are surrounded by dense fibrous connective tissue, which is mainly collagen fibers and contains abundant capillaries. The central area of ​​all sample fillers has a clear boundary with the tissue periphery, and there are different numbers of lymphocytes infiltrating in the central area of ​​the fillers, and the inflammatory cells infiltrating in the central area are loose. The gel implant has good biocompatibility with tissues.

[0161] Pathological filling height as attached Fig.15 , 16A As shown in Figure 16H, the filler was found in the loose connective tissue below the thin layer of subcutaneous muscle tissue under the microscope. The filling height was maintained after 3 months (3M) and 6 months (6M) of implantation.

[0162] Experimental Example 10

[0163] In this experimental example, the efficacy of the implants COL-HA-Ⅲ, COL-HA-Ⅴ, COL-HP-Ⅰ, and COL-CS-Ⅰ in promoting collagen regeneration was investigated.

[0164] The filler in Experimental Example 9 was separated from the subcutaneous tissue, and the filler and the skin tissue of the remaining samples were fixed with 10% neutral buffered formalin solution, embedded and solidified with paraffin, and sliced ​​and then stained with Masson staining. The collagen regeneration of the tissue was observed using an optical microscope. The results are shown in the attached figure. Fig.17 As shown, microscopically, connective tissue grows from the edge of the filler to the inside to varying degrees, and there is obvious new collagen.

[0165] Experimental Example 11

[0166] In this experimental example, the body shaping effect of the implant COL-HA-Ⅲ was investigated.

[0167] Female rats aged 6 to 8 weeks were used as experimental animals. COL-HA-Ⅲ gel implants were injected into the rat skull. The single-point injection volume was 0.2 ml. Micro-CT scans were used to scan the head images. Micro-CT was then used to reconstruct the filler and detect the height and volume of the filler. The observation time point was 6 months. The results are shown in the attached Fig.18 As shown, the implant prepared by the present invention maintains a good shaping effect.

[0168] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. A method for preparing a protein-sugar cross-linked matrix, characterized in that: The method comprises firstly subjecting glycosaminoglycan to carboxyl activation treatment, and then adding protein to carry out a first cross-linking reaction to form a prepolymer; Secondly, the prepolymer and the polyamine compound are treated with carboxyl activation to carry out a second cross-linking reaction to obtain a protein-sugar cross-linked matrix; Wherein, the polyamine compound includes a polyamine compound without carboxyl group and / or a polyamine compound containing carboxyl group; The carboxyl activation treatment includes activating at least one of the carboxyl groups of glycosaminoglycans, the carboxyl groups of proteins or the carboxyl groups of carboxyl-containing polyamine compounds to form carboxyl derivatives, so that the carboxyl derivatives can form amide bonds with the amino groups of proteins and / or the amino groups of polyamine compounds; The cross-linking reaction includes cross-links containing amide bonds that can be formed between the carboxyl groups of glycosaminoglycans and the amino groups of proteins, and / or, includes cross-links containing amide bonds that can be formed between the amino groups of polyamine compounds and the carboxyl groups of glycosaminoglycans and / or the carboxyl groups of proteins, and / or, includes cross-links containing amide bonds that can be formed between the carboxyl groups of carboxyl-containing polyamine compounds and the amino groups of proteins.

2. The preparation method according to claim 1, characterized in that: The first cross-linking reaction comprises dissolving the glycosaminoglycan, adding a reagent for carboxyl activation treatment to form a first reaction solution; dissolving the protein to form a second reaction solution; mixing the first reaction solution with the second reaction solution, and performing a cross-linking reaction to form a prepolymer; wherein the reagent for carboxyl activation treatment can activate at least 80% of the carboxyl groups on the glycosaminoglycan, so that the activated carboxyl groups can form amide bonds with amino groups; and the mass ratio of the protein to the glycosaminoglycan is 10:1 to 1:10; Preferably, the reagent for the carboxyl activation treatment can activate at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the carboxyl groups on the glycosaminoglycan, so that the activated carboxyl groups can form amide bonds with the amino groups; Preferably, the mass ratio of the protein to the glycosaminoglycan is 5:1 to 1:1; further preferably, the mass ratio of the protein to the glycosaminoglycan is 1:1; Preferably, the first cross-linking reaction step further comprises adjusting the pH to 6 to 8 after the first reaction solution and the second reaction solution are mixed; further preferably, adjusting the pH to 6.5 to 7.5; Preferably, the first cross-linking reaction is carried out at a temperature of 10 to 37° C.; more preferably, the cross-linking reaction is stirred at 25 to 30° C. for 12 hours.

3. The preparation method according to claim 1 or 2, characterized in that: The second cross-linking reaction step comprises adding the polyamine compound and a reagent for carboxyl activation treatment to the prepolymer to carry out a cross-linking reaction to form a protein-sugar cross-linked matrix; wherein the molar ratio of the amino group of the polyamine compound to the carboxyl group and the carboxyl derivative in the prepolymer is 0.5 to 10; the reagent for carboxyl activation treatment can activate at least 80% of the carboxyl groups of the unactivated carboxyl groups in the carboxyl-containing polyamine compound and / or the prepolymer, so that the activated carboxyl groups can form amide bonds with the amino groups; Preferably, the molar ratio of the amino group of the polyamine compound to the carboxyl group and the carboxyl derivative in the prepolymer is 1.0 to 5.0 (e.g., 1.0, 2.5 or 5.0); Preferably, the reagent for the carboxyl activation treatment can activate at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the unactivated carboxyl groups in the carboxyl-containing polyamine compound and / or prepolymer, so that the activated carboxyl groups can form amide bonds with amino groups; Preferably, the pH of the second cross-linking reaction is 6 to 8; further preferably, the cross-linking pH is 6.5 to 7.5; Preferably, the second cross-linking reaction is carried out at a temperature of 10 to 37° C.; more preferably, the cross-linking reaction is stirred at 25 to 30° C. for 12 hours.

4. The preparation method according to any one of claims 1 to 3, characterized in that: The reagent for the carboxyl activation treatment comprises an activator, wherein the activator is selected from at least one of DMT-MM, EDC, DCC, CDI or epoxide (e.g., allyl glycidyl ether); Preferably, the reagent for performing the carboxyl activation treatment further comprises a modifying agent, wherein the modifying agent is selected from at least one of NHS and a halide (eg, bromine).

5. The preparation method according to any one of claims 1 to 4, characterized in that: The reagents for the carboxyl activation treatment are EDC and NHS, wherein, in the step of the first cross-linking reaction, the molar ratio of NHS to the carboxyl group in the glycosaminoglycan is 0.8-1.5; the molar ratio of EDC to NHS is 1-5; Preferably, the molar ratio of the NHS to the carboxyl groups in the glycosaminoglycan is 1.0 to 1.5 (e.g., 1.0, 1.2 or 1.5); Preferably, the molar ratio of EDC to NHS is 2 to 4 (e.g., 2); and / or, in the step of the second cross-linking reaction, the molar ratio of the NHS to the carboxyl-containing polyamine compound and / or the unactivated carboxyl groups in the prepolymer is 0.8 to 1.5; the molar ratio of the EDC to the NHS is 1 to 5; Preferably, the molar ratio of the NHS to the unactivated carboxyl groups in the carboxyl-containing polyamine compound and / or prepolymer is 1.0 to 1.5 (e.g., 1.0, 1.2 or 1.5); Preferably, the molar ratio of EDC to NHS is 2 to 4 (eg, 2).

6. The preparation method according to any one of claims 1 to 5, characterized in that: The protein is selected from at least one of collagen, bovine serum albumin and elastin; Preferably, the collagen is selected from at least one of type I collagen, type II collagen, and type III collagen; Preferably, the collagen is selected from animal-derived collagen or recombinant collagen (such as recombinant human collagen or recombinant human-like collagen); Preferably, the concentration of the protein is 2-10 mg / mL (eg, 2, 5 or 10 mg / mL).

7. The preparation method according to any one of claims 1 to 6, characterized in that: The glycosaminoglycan is selected from at least one of hyaluronic acid, heparan sulfate, heparin, chondroitin sulfate, dermatan sulfate, keratan sulfate or salts thereof; Preferably, the molecular weight of the glycosaminoglycan is 10KD to 3000KD (e.g., 15, 50, 100, 1000, 1500, 2000 or 3000KD); Preferably, the concentration of the glycosaminoglycan is 1 to 25 mg / mL (eg, 1, 2.5, 5, 10 or 25 mg / mL).

8. The preparation method according to any one of claims 1 to 7, characterized in that: The carboxyl-containing polyamine compound comprises a basic amino acid; the non-carboxyl-containing polyamine compound comprises at least one of a diamine and a triamine compound; Preferably, the basic amino acid is selected from at least one of arginine, citrulline, glutamine and L-asparagine; preferably arginine; Preferably, the diamine is hexamethylenediamine.

9. A protein-sugar cross-linked matrix, characterized in that The method is prepared according to any one of claims 1 to 8.

10. A freeze-dried product, characterized in that: The method comprises freeze-drying the protein-sugar cross-linked matrix according to claim 9.

11. A composition, characterized in that It comprises the protein-sugar cross-linked matrix according to claim 9 or the lyophilized product according to claim 10.

12. The composition according to claim 11, characterized in that: It also contains buffer and hyaluronic acid; Preferably, the hyaluronic acid comprises free hyaluronic acid; Preferably, the molecular weight of the hyaluronic acid is 100KD to 1500KD; further preferably, the molecular weight of the hyaluronic acid is 500KD to 1500KD (e.g., 500KD, 1000KD or 1500KD); Preferably, the concentration of the hyaluronic acid in the composition is 5% to 25%; further preferably, the concentration of the hyaluronic acid in the composition is 5% to 15% (eg, 5% or 15%).

13. The composition according to claim 11 or 12, characterized in that The concentration of the protein-sugar cross-linked matrix or lyophilized product in the composition is 1 to 100 mg / mL; Preferably, the concentration of the protein-sugar cross-linked matrix or lyophilized product in the composition is 25-60 mg / mL (eg, 25, 35, 40 or 60 mg / mL).

14. The composition according to any one of claims 11 to 13, comprising any one of the following features: (a) its storage modulus is 300Pa to 4000Pa; (b) its loss modulus is 50Pa to 800Pa; (c) its dielectric loss tangent parameter (G" / G') is 0.05 to 0.35; (d) its shear viscosity is 20 to 300Pa.s; (e) it has degradable properties; (f) it has the effect of promoting cell adhesion or cell proliferation; or, (g) it has the effect of promoting collagen regeneration; Preferably, it includes any of the following features: (I) its storage modulus is 1500-3000 Pa; (II) its loss modulus is 300-600 Pa; (III) its dielectric loss tangent parameter (G" / G') is 0.15-0.30; or, (IV) its shear viscosity is 100-250 Pa.s.

15. Use of the protein-sugar cross-linked matrix according to claim 9, the freeze-dried product according to claim 10, or the composition according to any one of claims 11 to 14 in the preparation of medical, plastic surgery, cosmetic, or daily use products.

16. The use according to claim 15, characterized in that The medical, plastic surgery, cosmetic, or daily use products include those used for expanding and / or enlarging tissues, surgical operations, restorative operations, cosmetic operations, dermatological operations, plastic surgery, promoting wound healing, promoting tissue regeneration, promoting bone formation, cartilage repair, cosmetic operations, filling skin or tissues; or preventing and / or treating skin aging, skin diseases, and scars; Preferably, the filling of skin or tissue includes filling facial wrinkles or filling skin depressions.

17. A kit comprising the protein-sugar cross-linking matrix according to claim 9, The freeze-dried product according to claim 10 or the composition according to any one of claims 11 to 14.

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