Multi-crosslinking composite gel as well as preparation method and application thereof

Abstract

The invention relates to the technical field of polymer gels, and provides a multi-crosslinking composite gel and a preparation method and application thereof, the preparation method comprises the following steps: fully dissolving an activator in a buffer solution, and adjusting the pH value of the solution to 5.0-6.0 to obtain an activated solution; sequentially adding recombinant III-type humanized collagen, polydeoxyribonucleotide and chitosan into the activated solution, and uniformly dispersing to obtain a cross-linked precursor solution; adding a cross-linking agent into the cross-linking precursor solution, reacting for a period of time to obtain a cross-linking gel intermediate, and purifying to obtain the multi-cross-linking composite gel; the invention also provides the multi-crosslinking composite gel prepared by the preparation method, and application of the multi-crosslinking composite gel in the fields of beauty and plastic surgery. The material has biodegradability and good antibacterial performance, and can ensure slow release of the material and good filling effect and anti-enzymolysis effect at the same time.

Description

Technical Field

[0001] The present invention relates to the technical field of polymer gels, and in particular to a multi-crosslinked composite gel and a preparation method and application thereof. Background Art

[0002] Loss of extracellular matrix is ​​an important cause of skin aging. With age, photoaging and other environmental damage, the loss of collagen and chitosan in the skin causes the skin to lose elasticity and luster, the skin color becomes grayish yellow, wrinkles appear, and even cell tissues are damaged, resulting in aging. By supplementing the extracellular matrix and skin nutrients such as collagen, sodium hyaluronate, polydeoxyribonucleotides, etc. to the dermis or subcutaneous tissue, stimulating collagen production can effectively delay skin anti-aging.

[0003] However, on the one hand, collagen and polydeoxyribonucleotides degrade too quickly in the body and have a short maintenance time, which greatly limits their application; on the other hand, dermal fillers have delayed complications such as nodules, chronic inflammation, abscesses and other complications. Biofilm formation is the main cause of delayed implant complications. Similar to implants, dermal fillers have the risk of biofilm formation, which may lead to the development of nodules, chronic inflammatory reactions, abscesses and other complications. Biofilm formation is an important risk factor for late and delayed complications after filler injection and is caused by bacterial contamination and latent infection at the injection site.

[0004] Therefore, it is particularly important to improve the biodegradability and antibacterial properties of subdermal fillers while ensuring that the materials have good filling effects and anti-enzymatic effects. Summary of the invention

[0005] The purpose of the present invention is to provide a multi-cross-linked composite gel and a preparation method and application thereof, specifically a chitosan-polydeoxyribonucleotide-recombinant type III humanized collagen composite gel, which has biodegradability and good antibacterial properties, and can ensure the slow release of the material and good filling effect and anti-enzymatic effect.

[0006] The embodiments of the present invention are achieved through the following technical solutions:

[0007] A method for preparing a multi-crosslinked composite gel comprises the following steps:

[0008] S1. Preparation of activation solution: fully dissolving the activator in the buffer solution, and adjusting the pH value of the solution to 5.0-6.0 to obtain an activation solution;

[0009] S2. Preparation of cross-linking precursor solution: adding recombinant humanized type III collagen, polydeoxyribonucleotide and chitosan to the activation solution obtained in S1 in sequence, and fully dispersing them to obtain a cross-linking precursor solution;

[0010] S3, cross-linking: adding a cross-linking agent to the cross-linking precursor solution obtained in S2, continuing to stir and react for a period of time to obtain a cross-linked gel intermediate, and after the cross-linked gel intermediate is purified, a multi-cross-linked composite gel is obtained.

[0011] The present invention also provides a multi-crosslinked composite gel, which is prepared by the above preparation method.

[0012] The present invention also provides an application of the multi-crosslinked composite gel in the field of beauty and plastic surgery.

[0013] The technical solution of the embodiment of the present invention has at least the following advantages and beneficial effects:

[0014] 1. The multi-cross-linked composite gel prepared by the present invention has good resistance to mechanical stress and enzyme degradation, can prolong the retention time of the gel in the body, and improve the effect; and can achieve the slow release of recombinant type III humanized collagen and polydeoxyribonucleotides, continuously stimulate the proliferation of fibroblasts in the body, promote collagen hyperplasia, and synergize active ingredients to provide nutrients for skin regeneration, thereby improving wrinkles caused by the loss of extracellular matrix at the injection site;

[0015] 2. The multi-crosslinked composite gel prepared by the present invention can be used in the fields of beauty and plastic surgery, has good anti-inflammatory and antibacterial properties, can resist long-term inflammation of the material, destroy the formation of bacterial biofilm, and prevent the occurrence of adverse reactions such as nodules and abscesses. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0017] Figure 1 This is a microscope image of the multi-cross-linked composite gel obtained in Example 1 of the present invention after aniline blue staining;

[0018] Figure 2 The graphs show the in vitro antibacterial effects of the gel samples prepared in the embodiments and comparative examples of the present invention. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the 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 of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0020] The following is a detailed description of a multi-crosslinked composite gel provided by an embodiment of the present invention and its preparation method and application.

[0021] A method for preparing a multi-crosslinked composite gel comprises the following steps:

[0022] S1. Preparation of activation solution: fully dissolving the activator 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) in a buffer solution, and adjusting the pH value of the solution to 5.0-6.0 to obtain an activation solution; wherein the buffer solution is any one or more of sodium chloride, sodium dihydrogen phosphate, and disodium hydrogen phosphate, and the osmotic pressure of the buffer solution is 200-400 mOsmol / kg;

[0023] S2. Preparation of cross-linking precursor solution: adding recombinant type III humanized collagen, polydeoxyribonucleotide (PDRN) and chitosan to the activation solution obtained in S1 in sequence, and fully dispersing them to obtain a cross-linking precursor solution; wherein the addition concentrations of recombinant type III humanized collagen, polydeoxyribonucleotide and chitosan are all 0.1-50 mg / mL;

[0024] S3, cross-linking: adding a cross-linking agent N-hydroxysuccinimide (NHS) to the cross-linking precursor solution obtained in S2, and continuing to stir the reaction for a period of time to obtain a cross-linked gel intermediate, wherein the molar concentration ratio of N-hydroxysuccinimide to 1-ethyl-(3-dimethylaminopropyl) carbodiimide is 1:1; after the cross-linked gel intermediate is purified, a multi-cross-linked composite gel is obtained; specifically, the purification treatment includes the following steps:

[0025] S4, dialysis: placing the cross-linked gel intermediate obtained in S3 into a dialysis bag for dialysis; wherein the molecular weight cut-off (MWCO) of the dialysis bag is 3500Da, the dialysis time is 48h, and the water is changed 3 to 5 times;

[0026] S5, swelling: adding the dialyzed cross-linked gel intermediate obtained in S4 to a buffer solution containing an active ingredient for swelling to obtain an intermediate; wherein the active ingredient is sodium hyaluronate or an amino acid or an amino acid derivative, including but not limited to the following components: arginine, ornithine, citrulline, acetylmethionine, acetylcysteine, lysine, methionine, tyrosine, cystine, cysteine, α-aminobutyric acid, alanine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, hydroxyproline, isoleucine, leucine, phenylalanine, proline, serine, taurine, threonine, tryptophan, valine, etc.; the buffer solution is any one or more of sodium chloride, sodium dihydrogen phosphate, and sodium hydrogen phosphate, and the osmotic pressure of the buffer solution is 200-400 mOsmol / kg, and the pH value is 6.5-7.6;

[0027] S6. Homogenization: Use a homogenizer to homogenize the intermediate product obtained by swelling to obtain multi-cross-linked composite gel particles with uniform particle size, namely, chitosan-polydeoxyribonucleotide-recombinant type III humanized collagen composite gel particles.

[0028] The present invention also provides a multi-crosslinked composite gel, which is prepared by the above preparation method.

[0029] The present invention also provides an application of the multi-crosslinked composite gel in the field of beauty and plastic surgery.

[0030] The present invention makes full use of the carboxyl and amino group characteristics on the molecular chains of chitosan, polydeoxyribonucleotides and recombinant type III humanized collagen, and uses a 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide system to cross-link the three polymer compounds to form a chitosan-polydeoxyribonucleotide-recombinant type III humanized collagen multi-cross-linked gel;

[0031] Specifically, the present invention first prepares an activation solution with 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) to activate the carboxyl groups in collagen and chitosan molecules to form a highly active but easily hydrolyzed O-acylisourea intermediate, and then adds N-hydroxysuccinimide (NHS) to stabilize the formed O-acylisourea intermediate to form an active ester that is not easily hydrolyzed. The ester is easy to react with the primary amine group at the N-terminus of polydeoxyribonucleotide or collagen to form a stable amide bond, so that chitosan is cross-linked with the polydeoxyribonucleotide and recombinant type III humanized collagen, thereby enhancing their structure and mechanical properties, forming a multi-cross-linked material, having good antibacterial properties, preventing long-term inflammation, slowing down the degradation rate of collagen and PDRN, and prolonging the action time.

[0032] Then, during the swelling process, the active ingredients are loaded into a cross-linked gel with a three-dimensional space. The dense cross-linked network structure of the gel is used to improve the mechanical strength and stability of the material, and enhance the resistance to mechanical stress and enzyme degradation. On the one hand, it plays a role in supporting and shaping after injection. On the other hand, it can achieve the slow release of recombinant type III humanized collagen and polydeoxyribonucleotides during the degradation process in the body, continuously stimulating the body to promote the proliferation of fibroblasts and promote collagen proliferation. At the same time, the loaded active ingredients are also essential nutrients for the skin, providing nutrients for skin regeneration. The components synergistically improve wrinkles caused by the loss of extracellular matrix at the injection site.

[0033] At the same time, the material has good antibacterial properties, which can resist long-term inflammation of the material and destroy the formation of bacterial biofilm, thereby preventing the occurrence of long-term inflammation of the material at the injection site and preventing sequelae such as chronic inflammation, nodules, and abscesses.

[0034] Example 1

[0035] A method for preparing a multi-crosslinked composite gel comprises the following steps:

[0036] S1. Preparation of activation solution: dissolve 115 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide in 100 mL of sodium chloride having an osmotic pressure of 300 mOsmol / kg at 37° C. until fully dissolved, and adjust the pH value of the solution to 6.0 to obtain an activation solution;

[0037] S2. Preparation of cross-linking precursor solution: 5 g of recombinant humanized type III collagen, 5 g of polydeoxyribonucleotides and 0.5 g of carboxymethyl chitosan were sequentially added to the activation solution obtained in S1, and fully dispersed at 37° C. to obtain a cross-linking precursor solution;

[0038] S3, cross-linking: at 37°C, add 80 mg of N-hydroxysuccinimide to the cross-linking precursor solution obtained in S2, and continue stirring for 30 minutes to obtain a cross-linked gel intermediate; wherein the molar concentration ratio of N-hydroxysuccinimide to 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:1;

[0039] S4, dialysis: the cross-linked gel intermediate obtained in S3 is placed in a dialysis bag with a molecular weight cut-off of 3500 Da for dialysis for 48 h;

[0040] S5, swelling: adding the dialyzed cross-linked gel intermediate obtained in S4 to a buffer solution containing sodium hyaluronate, proline, glycine and alanine to obtain an intermediate, adjusting the pH of the solution to 7.0, and performing swelling. The final product contains 7 mg / mL sodium hyaluronate, 5 mg / mL polydeoxyribonucleotide, 5 mg / mL recombinant type III humanized collagen, 0.5 mg / mL chitosan, 0.2 mg / mL proline, 0.1 mg / mL alanine, and 0.1 mg / mL glycine;

[0041] S6. Homogenization: Use a homogenizer to homogenize the intermediate product obtained by swelling to obtain a multi-cross-linked composite gel with uniform particle size, namely, chitosan-polydeoxyribonucleotide-recombinant type III humanized collagen composite gel particles.

[0042] Example 2

[0043] A multi-crosslinked composite gel is prepared by the following method:

[0044] S1. Preparation of activation solution: at 37° C., dissolve 184 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide in 100 mL of sodium dihydrogen phosphate having an osmotic pressure of 200 to 400 mOsmol / kg, fully dissolve, and adjust the pH value of the solution to 6.0 to obtain an activation solution;

[0045] S2. Preparation of cross-linking precursor solution: 5 g of recombinant humanized type III collagen, 5 g of polydeoxyribonucleotides and 0.5 g of carboxymethyl chitosan were sequentially added to the activation solution obtained in S1, and fully dispersed at 37° C. to obtain a cross-linking precursor solution;

[0046] S3, cross-linking: at 37°C, add 128 mg of N-hydroxysuccinimide to the cross-linking precursor solution obtained in S2, and continue stirring for 30 minutes to obtain a cross-linked gel intermediate; wherein the molar concentration ratio of N-hydroxysuccinimide to 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:1;

[0047] S4, dialysis: the cross-linked gel intermediate obtained in S3 is placed in a dialysis bag with a molecular weight cut-off of 3500 Da for dialysis for 48 h;

[0048] S5, swelling: add the dialyzed cross-linked gel intermediate obtained in S4 to a buffer solution containing sodium hyaluronate, proline, glycine and alanine to obtain an intermediate, adjust the solution pH to 7.0, and perform swelling. The final product contains 7 mg / mL sodium hyaluronate, 5 mg / mL polydeoxyribonucleotide, 5 mg / mL recombinant type III humanized collagen, 0.5 mg / mL chitosan, 0.2 mg / mL proline, 0.1 mg / mL alanine, and 0.1 mg / mL glycine;

[0049] S6. Homogenization: Use a homogenizer to homogenize the intermediate product obtained by swelling to obtain a multi-cross-linked composite gel with uniform particle size, namely, chitosan-polydeoxyribonucleotide-recombinant type III humanized collagen composite gel particles.

[0050] Example 3

[0051] A multi-crosslinked composite gel is prepared by the following method:

[0052] S1, preparation of activation solution: dissolve 253 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide in 100 mL of disodium hydrogen phosphate having an osmotic pressure of 200 to 400 mOsmol / kg at 37° C. until fully dissolved, and adjust the pH value of the solution to 6.0 to obtain an activation solution;

[0053] S2, preparation of cross-linking precursor solution: 5 g of recombinant humanized type III collagen, 5 g of polydeoxyribonucleotides and 0.5 g of carboxymethyl chitosan are sequentially added to the activation solution obtained in S1, and fully dispersed at 37° C. to obtain a cross-linking precursor solution;

[0054] S3, cross-linking: at 37°C, 176 mg of N-hydroxysuccinimide was added to the cross-linking precursor solution obtained in S2, and the mixture was stirred for 30 min to obtain a cross-linked gel intermediate; wherein the molar concentration ratio of N-hydroxysuccinimide to 1-ethyl-(3-dimethylaminopropyl)carbodiimide was 1:1;

[0055] S4, dialysis: the cross-linked gel intermediate obtained in S3 is placed in a dialysis bag with a molecular weight cut-off of 3500 Da for dialysis for 48 h;

[0056] S5, swelling: add the dialyzed cross-linked gel intermediate obtained in S4 to a buffer solution containing sodium hyaluronate, proline, glycine and alanine to obtain an intermediate, adjust the solution pH to 7.0, and perform swelling. The final product contains 7 mg / mL sodium hyaluronate, 5 mg / mL polydeoxyribonucleotide, 5 mg / mL recombinant type III humanized collagen, 0.5 mg / mL chitosan, 0.2 mg / mL proline, 0.1 mg / mL alanine, and 0.1 mg / mL glycine;

[0057] S6, homogenization: homogenize the intermediate product obtained by swelling using a homogenizer to obtain multi-cross-linked composite gel particles with uniform particle size, namely, chitosan-polydeoxyribonucleotide-recombinant type III humanized collagen composite gel particles.

[0058] Comparative Example 1

[0059] A multi-crosslinked composite gel is prepared by the following method:

[0060] S1, preparation of activation solution: at 37° C., dissolve 115 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide in 100 mL of sodium chloride having an osmotic pressure of 200 to 400 mOsmol / kg, fully dissolve, and adjust the pH value of the solution to 6.0 to obtain an activation solution;

[0061] S2, preparation of cross-linking precursor solution: 5 g of recombinant humanized type III collagen and 5 g of polydeoxyribonucleotide were sequentially added to the activation solution obtained in S1, and fully dispersed at 37°C to obtain a cross-linking precursor solution;

[0062] S3, cross-linking: at 37°C, add 80 mg of N-hydroxysuccinimide to the cross-linking precursor solution obtained in S2, and continue stirring for 30 min to obtain a cross-linked gel intermediate; wherein the molar concentration ratio of N-hydroxysuccinimide to 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:1;

[0063] S4, dialysis: the cross-linked gel intermediate obtained in S3 is placed in a dialysis bag with a molecular weight cut-off of 3500 Da and dialyzed for 48 h;

[0064] S5, swelling: add the dialyzed cross-linked gel intermediate obtained in S4 to a buffer solution containing sodium hyaluronate, proline, glycine and alanine to obtain an intermediate, adjust the solution pH to 7.0, and perform swelling. The final product contains 7 mg / mL sodium hyaluronate, 5 mg / mL polydeoxyribonucleotide, 5 mg / mL recombinant type III humanized collagen, 0.2 mg / mL proline, 0.1 mg / mL alanine, and 0.1 mg / mL glycine;

[0065] S6, homogenization: homogenize the swollen intermediate product using a homogenizer to obtain a multi-cross-linked composite gel with uniform particle size, namely, polydeoxyribonucleotide-recombinant type III humanized collagen composite gel particles.

[0066] Experimental Example 1

[0067] 1. The multi-crosslinked composite gel sample prepared in Example 1 of the present invention was stained with aniline blue and its microscopic image was observed under a microscope. The results are shown in FIG. Figure 1 .

[0068] Experimental Example 2

[0069] The physical and chemical properties of the gel samples prepared in each embodiment and comparative example were tested, specifically including:

[0070] 1. In vitro antibacterial effect: The antibacterial activity was measured by the coating method. Staphylococcus aureus (S. aureus) was used to test the antibacterial activity of the gel samples prepared in each embodiment and comparative example, and the corresponding colony forming units (CFU) were determined to evaluate their antibacterial ability. The results are shown in Figure 2 .Depend on Figure 2 It can be seen that the chitosan-polydeoxyribonucleotide-recombinant type III humanized collagen composite gels prepared in Examples 1 to 3 have good antibacterial effects on Staphylococcus aureus, and the in vitro antibacterial rates are all above 85%, while the polydeoxyribonucleotide-recombinant type III humanized collagen composite gel without chitosan prepared in Comparative Example 1 shows an unsatisfactory in vitro antibacterial effect, which confirms that the presence of chitosan can significantly improve the antibacterial properties of the gel.

[0071] 2. The syringe handle pushing force, swelling degree, particle size distribution and in vitro degradation performance of the gel samples prepared in each embodiment and comparative example were tested, as follows:

[0072] 2.1 Pushing force of syringe handle: Install a 27G injection needle on the syringe and install the push rod to simulate actual use. Push the push rod at a constant speed of 30mm / min, and the sample in the syringe is pushed out through the needle to obtain the pushing force curve;

[0073] 2.2 Swelling degree: Weigh 0.5 g of gel sample into a stainless steel sieve, soak it in PBS solution for 30 min, take it out and put the sieve into a drying oven at 80°C until constant weight is obtained. The ratio of the weight of the swollen gel to the weight of the dried gel powder is the swelling degree.

[0074] 2.3 Particle size distribution: The particle size distribution of gel samples D(50) and D(90) was measured using a laser particle size analyzer;

[0075] 2.4 In vitro degradation: Weigh 1 g of gel sample, add 10 mL of PBS solution to fully disperse it, then add a mixed solution of deoxyribonucleotidase and collagenase, and place it in a water bath at 37°C for different times (1, 2, 5, 10, 16, 20, 24, 30, 36 h), and then use 0.22 μm filter to collect the supernatant. Determination of A 260nm The in vitro degradation rate of polydeoxyribonucleotides was detected by measuring the absorbance at 40 °C. The degradation rate of polydeoxyribonucleotides in vitro was detected by using the biuret reagent method based on the absorption of more than two peptide bonds in collagen molecules with Cu in alkaline solution. 2+ A purple-red complex is formed, and the color depth is proportional to the concentration within a certain range. The recombinant type III humanized collagen reference solution is used as a standard curve, and the content of recombinant type III humanized collagen in each gel sample is determined by colorimetry; the results are shown in Table 1:

[0076] Table 1-Syringe handle pushing force, swelling degree, particle size distribution and in vitro degradation test results of gel samples prepared in various embodiments and comparative examples

[0077]

[0078] As shown in Table 1, the chitosan-polydeoxyribonucleotide-recombinant type III humanized collagen composite gel prepared by the present invention has suitable syringe handle pushing force, swelling degree, particle size distribution, and good resistance to enzymatic hydrolysis. And with the increase of cross-linking agent, the degree of cross-linking increases, although the syringe handle pushing force increases moderately, the swelling degree decreases, the particle size increases slightly, and the in vitro complete degradation time of polydeoxyribonucleotide and recombinant type III humanized collagen is also prolonged. The appropriate particle size and pushing force can make the material easy to inject and degrade more slowly after injection. Rather than the smaller the better, for example, the polydeoxyribonucleotide-recombinant type III humanized collagen composite gel sample prepared in Comparative Example 1 has a low mechanical strength, so the syringe handle pushing force is small and the particle size is small, but at the same time its swelling degree is higher and the in vitro degradation rate is faster. It can be seen that the multi-cross-linked gel product of the present invention can not only increase the mechanical strength and anti-enzymatic ability of the material, but also prolong the retention time of the gel in the body and improve the effect; and can achieve the slow release of recombinant type III humanized collagen and polydeoxyribonucleotides, continuously stimulate the proliferation of fibroblasts in the body, promote collagen proliferation, and synergize the active ingredients to provide nutrients for skin regeneration, thereby improving wrinkles caused by the loss of extracellular matrix at the injection site.

[0079] In summary, the chitosan-polydeoxyribonucleotide-recombinant type III humanized collagen composite gel of the present invention can achieve effective cross-linking and form a triple cross-linked product, and the gel has good biocompatibility, in vitro antibacterial property, uniform particle size distribution, suitable swelling degree, and good injectability. Through the cross-linking method, the degradation of polydeoxyribonucleotide and recombinant type III humanized collagen is effectively delayed, the action time of the material in vivo is prolonged, and the gel has a good antibacterial effect, can resist the long-term inflammation of the material, destroy the formation of bacterial biofilm, and prevent the occurrence of adverse reactions such as nodules and abscesses. The preparation method is simple and controllable, and is suitable for industrial production.

[0080] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a multi-crosslinked composite gel, characterized in that: The following steps are involved: S1. Preparation of activation solution: fully dissolving the activator in the buffer solution, and adjusting the pH value of the solution to 5.0-6.0 to obtain an activation solution; S2. Preparation of cross-linking precursor solution: adding recombinant humanized type III collagen, polydeoxyribonucleotide and chitosan to the activation solution obtained in S1 in sequence, and fully dispersing them to obtain a cross-linking precursor solution; S3, cross-linking: adding a cross-linking agent to the cross-linking precursor solution obtained in S2, continuing to stir and react for a period of time to obtain a cross-linked gel intermediate, and after the cross-linked gel intermediate is purified, a multi-cross-linked composite gel is obtained.

2. The method for preparing the multi-crosslinked composite gel according to claim 1, characterized in that: The purification process comprises the following steps: S4, dialysis: placing the cross-linked gel intermediate obtained in S3 into a dialysis bag for dialysis; S5, swelling: adding the dialyzed cross-linked gel intermediate obtained in S4 to a buffer solution containing an active ingredient for swelling to obtain an intermediate; S6. Homogenization: homogenize the intermediate product obtained after swelling to obtain a multi-crosslinked composite gel.

3. The method for preparing the multi-crosslinked composite gel according to claim 1, characterized in that: In S1, the activator is 1-ethyl-(3-dimethylaminopropyl)carbodiimide; the buffer solution is any one or more of sodium chloride, sodium dihydrogen phosphate, and disodium hydrogen phosphate, and the osmotic pressure of the buffer solution is 200-400 mOsmol / kg.

4. The method for preparing the multi-crosslinked composite gel according to claim 1, characterized in that: In S2, the addition concentrations of the recombinant humanized type III collagen, polydeoxyribonucleotides and chitosan are all 0.1-50 mg / mL.

5. The method for preparing the multi-crosslinked composite gel according to claim 1, characterized in that: The cross-linking agent in S3 is N-hydroxysuccinimide; the molar concentration ratio of the cross-linking agent to the activator is 1:

1.

6. The method for preparing the multi-crosslinked composite gel according to claim 2, characterized in that: In S4, the molecular weight cutoff of the dialysis bag is 3500Da, and the dialysis time is 36-50h.

7. The method for preparing the multi-crosslinked composite gel according to claim 2, characterized in that: In S5, the active ingredient is sodium hyaluronate or amino acid.

8. The method for preparing a multi-crosslinked composite gel according to claim 2, characterized in that: In S5, the buffer solution is any one or more of sodium chloride, sodium dihydrogen phosphate, and disodium hydrogen phosphate. The osmotic pressure of the buffer solution is 200-400 mOsmol / kg, and the pH value is 6.5-7.

6.

9. A multi-crosslinked composite gel, characterized in that: Prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the multi-crosslinked composite gel prepared by the preparation method according to any one of claims 1 to 8 in the fields of beauty and plastic surgery.

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