Method for preparing composite injectable gel, composite injectable gel and application thereof

Abstract

The invention belongs to the technical field of biological materials. The preparation steps of the existing composite injectable gel are tedious, and the prepared composite injectable gel has the problems of poor injectability, low swelling rate, low mechanical strength, low drug loading rate, high cytotoxicity and the like. On the basis, the invention provides a method for preparing the composite injectable gel, and the method comprises the following steps: 1) dissolving hyaluronic acid and sodium chloride in an aqueous solution of NaOH or KOH; (2) adding a cross-linking agent 1, 4-butanediol diglycidyl ether or genipin, and mixing; 3) carrying out a cross-linking reaction; 4) purifying a reaction product; and 5) performing vacuum freeze-drying to obtain the final composite injectable gel. According to the method, the preparation process is simplified, and the prepared composite injectable gel has significantly improved injectability, swelling ratio, mechanical strength and drug loading rate, and lower cytotoxicity, and has good application prospects in the fields of drug delivery, tissue engineering, skin wound healing and repair, medical cosmetology and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomaterials, and in particular relates to a method for preparing a composite injectable gel, a composite injectable gel and uses thereof. BACKGROUND

[0002] Biomaterials are a kind of special functional materials, natural or artificially synthesized, used for contact and interaction with life systems, and capable of diagnosing, treating, replacing, repairing or inducing regeneration of cells, tissues and organs thereof. According to material properties, biomaterials can be classified into medical metal materials, medical inorganic materials, medical polymer materials, medical composite materials, etc.; according to material functions, biomaterials can be classified into hard tissue compatible materials, soft tissue compatible materials, blood compatible materials, biodegradable materials, polymer drugs, etc.; according to material sources, biomaterials can be classified into autologous tissues, allogeneic organs and tissues, xenogeneic organs and tissues, natural biomaterials, artificially synthesized materials, etc.; according to use sites, biomaterials can be classified into hard tissue materials, soft tissue materials, cardiovascular materials, blood substitute materials, and separation, filtration, dialysis membrane materials, etc.

[0003] In the field of biomaterials, the current composite injectable gel needs to be prepared in multiple steps, which is cumbersome. The current composite injectable gel has poor injectability, low swelling rate, low mechanical strength, low drug loading rate, and high cytotoxicity.

[0004] Therefore, there is an urgent need in the art to develop a method for preparing a composite injectable gel to obtain a composite injectable gel with good injectability, high swelling rate, high mechanical strength, high drug loading rate, low cytotoxicity, and excellent antioxidant performance. SUMMARY

[0005] The present application aims to at least partially solve at least one of the technical problems existing in the prior art.

[0006] In a first aspect, the present application provides a method for preparing a composite injectable gel. According to an embodiment of the present application, the method comprises the following steps: 1) dissolving hyaluronic acid and sodium chloride in an aqueous solution of a strong base to obtain a mixed solution, the strong base being at least one of NaOH and KOH; 2) mixing the mixed solution of 1) with a crosslinking agent, the crosslinking agent being one or both of 1,4-butanediol diglycidyl ether and genipin; 3) performing a crosslinking reaction on the mixed product obtained in 2); 4) performing a purification treatment on the crosslinking reaction product obtained in 3); and 5) performing a vacuum freeze-drying treatment on the purification product obtained in 4) to obtain the composite injectable gel. According to the method of the present application, a composite injectable gel with good injectability, improved swelling rate, enhanced mechanical strength, increased drug loading rate, reduced cytotoxicity, and excellent antioxidant performance can be prepared.

[0007] In a second aspect, the present application provides a composite injectable gel. According to embodiments of the present application, the composite injectable gel is prepared by the method of the first aspect of the present application. According to embodiments of the present application, the composite injectable gel has good injectability, improved swelling ratio, enhanced mechanical strength, improved drug loading, reduced cytotoxicity, and excellent anti-oxidation performance.

[0008] In a third aspect, the present application provides use of the composite injectable gel of the second aspect of the present application in tissue engineering repair.

[0009] In a fourth aspect, the present application provides use of the composite injectable gel of the second aspect of the present application in preparation of a drug carrier.

[0010] In a fifth aspect, the present application provides use of the composite injectable gel of the second aspect of the present application in a bioactive scaffold.

[0011] In a sixth aspect, the present application provides use of the composite injectable gel of the second aspect of the present application in skin wound healing repair.

[0012] In a seventh aspect, the present application provides use of the composite injectable gel of the second aspect of the present application in medical cosmetology.

[0013] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0014] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the appended drawings. Figure 1 Schematic diagram of injectability of the gels obtained according to embodiments 1-4 of the present application.

[0015] Figure 2 Swelling ratio results plot of the crosslinked gel samples of different compositions and different reaction conditions according to embodiments 1-3, comparative example 1, comparative example 3 of the present application.

[0016] Figure 3 Storage modulus and loss modulus results plot of representative crosslinked gel samples prepared according to embodiments 1-2 of the present application.

[0017] Figure 4 Drug loading results plot of the gels prepared according to embodiments 1-2, comparative example 1 of the present application.

[0018] Figure 5A graph of the survival rate of the gel sample prepared according to Example 1, Example 3 of the present application.

[0019] Figure 6 A graph of the comparison of the amount of the precipitate of the gel prepared according to Comparative Example 2 of the present application.

[0020] Figure 7 A graph of the comparison of the supernatant of the gel prepared according to Comparative Example 2 of the present application before and after the addition of NaCl and a graph of the comparison of the precipitate obtained by centrifugation of the turbid solution.

[0021] Figure 8 A graph of the results of the determination of the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) radical scavenging rate of the gel (6 mg / mL) prepared according to Example 1, 2, 3 of the present application.

[0022] Figure 9 A scanning electron microscope graph of the gel sample prepared according to Example 1, 2, 3 of the present application. DETAILED DESCRIPTION

[0023] Hereinafter, embodiments of the present application will be described in detail. The embodiments described hereinafter are exemplary only, and are not to be construed as limiting the present application.

[0024] It is to be noted that the terms "first", "second", etc. are used only for the purpose of description, and are not to be construed as indicating or implying relative importance or an indicated number of technical features. Thus, features defined with "first", "second", etc. can explicitly or implicitly include one or more of the features. Further, in the description of the present application, the meaning of "a plurality of" is two or more, unless otherwise specified.

[0025] The endpoints of the ranges and any values described herein are not limited to the precise values stated. The endpoints of the ranges and any values are provided as approximations only, and are understood to include values approximately around the endpoints. Any numerical range recited herein is intended to include all sub-ranges of the same numbers (i.e., every subset of numbers within the indicated range). For numerical ranges expressed in lower and upper limits, the range is intended to include all numbers and sub-ranges between the lower and upper limits. For example, a range of 1 to 10 is intended to include all numbers between 1 and 10, for example, 1, 1.1, 1.2,..., 9.9, 10, etc.

[0026] In order to facilitate the understanding of the present application, certain technical and scientific terms are specifically defined below. Unless otherwise explicitly provided herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which the present application pertains.

[0027] In the present application, the term "comprising" or "including" is an open expression that includes the indicated content of the present application, but does not exclude other aspects. In the present application, the term "comprising" or "including" is an open expression that includes the indicated content of the present application, but does not exclude other aspects.

[0028] In the present application, the term "hyaluronic acid" is an acidic mucopolysaccharide composed of D-glucuronic acid and N-acetylglucosamine disaccharide units with a unit molecular weight of 403.31. The hyaluronic acid used in the present application (CAS 9004-61-9, Sigma-Aldrich, Catalog No. A22873) contains a large number of carboxyl and hydroxyl groups in its molecular chain, which makes it have strong hydrophilicity. The aqueous solution of hyaluronic acid has high viscosity and good lubricity, and its viscoelasticity is related to factors such as concentration and molecular weight. For example, the hyaluronic acid solution used in ophthalmic surgery uses its high viscosity to protect the cornea and intraocular tissues. In the human body, hyaluronic acid is widely present in tissues such as skin, synovial fluid, and vitreous body of the eye. It can keep the skin moist, making the skin soft and smooth, so it is widely used in the cosmetics industry for moisturizing products. In the medical field, hyaluronic acid can be used for the treatment of joint diseases such as osteoarthritis. Injecting hyaluronic acid into the joint cavity can increase the viscoelasticity of the synovial fluid, reduce joint pain, and improve joint function.

[0029] In the present application, the term "1,4-butanediol diglycidyl ether" is a crosslinking agent. In tissue engineering, 1,4-butanediol diglycidyl ether (BDDE) is used to crosslink biomaterials to prepare scaffolds. For example, for cartilage tissue engineering, cartilage extracellular matrix components such as collagen and glycosaminoglycans (such as hyaluronic acid) are reacted with this crosslinking agent to prepare scaffolds with appropriate porosity, mechanical strength, and biocompatibility. This scaffold can provide a good environment for the growth, proliferation, and differentiation of chondrocytes, promoting the repair and regeneration of cartilage tissue. 1,4-Butanediol diglycidyl ether can also be used to prepare drug release materials. The drug is wrapped in a biomaterial matrix crosslinked by 1,4-butanediol diglycidyl ether, and the release rate of the drug is controlled by adjusting factors such as crosslinking density. For example, some drugs for treating arthritis are wrapped in crosslinked hyaluronic acid materials. When the material is implanted in the joint area, the drug can be slowly released as the material degrades or interacts with the surrounding environment, thereby prolonging the action time of the drug and improving the therapeutic effect.

[0030] In the present application, unless otherwise specified, the solvent of the solution is water. For example, the NaOH solution is obtained by dissolving NaOH in water.

[0031] The present application proposes a method for preparing a composite injectable gel, a composite injectable gel, and the use thereof, which will be described in detail below.

[0032] Method for preparing a composite injectable gel In a first aspect, the present application provides a method for preparing a composite injectable gel. According to an embodiment of the present application, the method comprises the following steps: 1) dissolving hyaluronic acid and sodium chloride in an aqueous solution of a strong base to obtain a mixed solution, the strong base being at least one of NaOH and KOH; 2) mixing the mixed solution of 1) with a crosslinking agent, the crosslinking agent being one or both of 1,4-butanediol diglycidyl ether and genipin; 3) performing a crosslinking reaction on the mixed product obtained in 2); 4) performing a purification treatment on the crosslinking reaction product obtained in 3); and 5) performing vacuum freeze-drying treatment on the purified product obtained in 4) to obtain the composite injectable gel. Sodium chloride (NaCl) plays a key role in inhibiting PICs and optimizing the performance of the composite hydrogel by adjusting the ionic strength, shielding the charge, promoting collagen fiberization, and stabilizing the fiber structure. According to the method of the embodiment of the present application, a composite injectable gel with good injectability, high swelling rate, high mechanical strength, high drug loading rate, low cytotoxicity, and excellent antioxidant performance can be prepared.

[0033] According to an embodiment of the present application, the purification treatment is achieved by performing alcohol precipitation treatment and filtration treatment. i) performing alcohol precipitation treatment on the crosslinking reaction product obtained in 3) and then performing filtration treatment, and then performing first dialysis treatment on the filtrate obtained after the filtration treatment, the first dialysis treatment being performed in a PBS solution; ii) performing second dialysis treatment on the first dialysis product obtained in i), the second dialysis treatment being performed in deionized water; and iii) performing vacuum freeze-drying treatment on the second dialysis treatment product obtained in ii). In step i), the PBS solution preferentially removes small molecular impurities (such as reactants, crosslinking agents, and alcohol solvents) in the product. By simulating the physiological environment, the solubility and stability of the product are preserved, and the collagen protein conformation change or functional loss caused by the lack of ion balance in pure water is avoided. In step ii), the purpose of pure water dialysis is to slowly desalt and further remove unreacted raw materials and crosslinking agents, thereby reducing the risk of product inactivation caused by rapid changes in salt concentration gradients. A high-purity matrix is provided for subsequent freeze-drying. The combination of steps i) and ii) forms a staged purification strategy that takes into account both efficiency and product protection.

[0034] According to an embodiment of the present application, the first dialysis treatment is performed for 1-2 days, for example, 1 day or 2 days, and the second dialysis treatment is performed for 3-5 days, for example, 3 days, 4 days, or 5 days.

[0035] According to an embodiment of the present application, in step 1), the collagen is also subjected to the dissolving treatment in the aqueous solution of the strong base.

[0036] According to an embodiment of the present application, the concentration of the strong base is 0.02-10 mg / mL, for example, 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 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 a range value between any two of the above values, for example, 0.05-10 mg / mL, 0.1-10 mg / mL. According to an embodiment of the present application, when the concentration of the strong base is less than 0.02 mg / mL, the dissolution of HA is not sufficient, and the cross-linking reaction is not complete. When the concentration of the strong base is greater than 10 mg / mL, the cross-linking reaction is excessive, side reactions occur, and the reaction solution is yellow. The moderate concentration of the strong base helps the components such as HA to be fully dispersed and dissolved in the solution, and the components are uniformly mixed, so that a uniform gel is prepared, the performance difference of the gel caused by uneven distribution of the components is avoided, and the moderate concentration of the strong base helps to control the rate of the cross-linking reaction, and ensures that the cross-linking degree and performance of the gel achieve the expected effect.

[0037] According to an embodiment of the present application, the weight ratio of the HA, the strong base, the sodium chloride, and the cross-linking agent is (0.5-1):(0.0004-0.05):(0.000001-1):(0.05-0.1). According to an embodiment of the present application, the moderate weight ratio of the HA, the strong base, the sodium chloride (NaCl), and the cross-linking agent helps to prepare a composite injectable gel with good injectability, high swelling rate, high mechanical strength, high drug loading rate, low cytotoxicity, and excellent antioxidant performance. It should be noted that the HA plays a key role as a matrix in the composite injectable gel. The appropriate amount of HA can ensure that the composite injectable gel has good elasticity and water retention. The strong base is used to adjust the pH value of the solution during preparation, so that the solution reaches an appropriate alkaline environment to promote the sufficient dissolution and mixing of the HA and other components, and the subsequent cross-linking reaction. The appropriate amount of the strong base can ensure that the pH value of the solution is stable within the optimal range, avoid the damage to the components of the gel, and reduce the occurrence of side reactions. The sodium chloride can help the components such as HA to be better dissolved in the solution, further promote the uniform mixing of the components, and play a role in maintaining a certain ionic strength. The appropriate amount of the cross-linking agent can ensure that the components such as HA are fully cross-linked to form a uniform and dense cross-linking network, so that the composite injectable gel has good elasticity and strength, and at the same time avoids the potential risk of excessive residual.

[0038] According to an embodiment of the present application, the weight ratio of the hyaluronic acid, the strong alkali, the sodium chloride, the collagen and the cross-linking agent is (0.5-1):(0.0004-0.05):(0.000001-1):(0.000001-1):(0.05-0.1). According to an embodiment of the present application, the weight ratio of the hyaluronic acid, the strong alkali, the sodium chloride (NaCl), the collagen and the cross-linking agent is moderate, which is conducive to preparing a composite injectable gel with good injectability, high swelling rate, high mechanical strength, high drug loading rate, low cytotoxicity and excellent antioxidant performance. It should be explained that collagen is an important structural protein, and an appropriate amount of collagen can form a composite network structure with the hyaluronic acid, thereby significantly enhancing the mechanical properties of the gel. Meanwhile, an appropriate amount of collagen can provide a good growth environment for cells, promote the adhesion, migration and proliferation of cells, accelerate the repair and regeneration of tissues, and improve the repair and treatment effect of the gel.

[0039] It should be explained that the "gel" and the "composite injectable gel" in the present application are synonymous.

[0040] According to an embodiment of the present application, the collagen is selected from at least one of natural collagen and recombinant collagen.

[0041] It should be explained that the collagen includes but is not limited to natural collagen extracted from animal tissues (such as bovine tendons and pig skin) or recombinant humanized collagen (such as human-like collagen HLC) expressed by genetic engineering, which can be known by those skilled in the art.

[0042] According to an embodiment of the present application, the collagen includes natural collagen extracted from bovine tendons.

[0043] According to an embodiment of the present application, the collagen includes natural collagen extracted from pig skin.

[0044] According to an embodiment of the present application, the collagen is selected from at least one of natural type I collagen, natural type II collagen and natural type III collagen.

[0045] According to an embodiment of the present application, the cross-linking reaction is performed at a temperature of 25-45°C for 3-24 hours, for example, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, or a range value between any two of them, 26-45°C, 27-45°C; and for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, or a range value between any two of them, 4-24, 5-24 hours.

[0046] According to an embodiment of the present application, the cross-linking reaction is performed at a temperature of 37°C for 6 hours.

[0047] According to an embodiment of the present application, the purification treatment is performed in the presence of an alcohol precipitant, which comprises at least one of 75%-100% ethanol and methanol, for example, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% ethanol, or 76%-100%, 77%-100% ethanol.

[0048] According to an embodiment of the present application, the alcohol precipitant is 90% ethanol.

[0049] Composite injectable gel According to a second aspect of the present application, the present application provides a composite injectable gel. According to an embodiment of the present application, the composite injectable gel is prepared by the method of the first aspect of the present application. The composite injectable gel according to an embodiment of the present application has good injectability, improved swelling rate, enhanced mechanical strength, increased drug loading rate, reduced cytotoxicity, and excellent anti-free radical oxidation performance.

[0050] Use According to a third aspect of the present application, the present application provides a use of the composite injectable gel of the second aspect of the present application in tissue engineering repair.

[0051] According to an embodiment of the present application, the tissue engineering repair comprises preparing a tissue filler.

[0052] According to a fourth aspect of the present application, the present application provides a use of the composite injectable gel of the second aspect of the present application in preparing a drug carrier. According to an embodiment of the present application, the cross-linking reaction is performed at a temperature of 25-45°C for 3-24 hours, for example, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, or a range value between any two of them, 26-45°C, 27-45°C; and for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, or a range value between any two of them, 4-24, 5-24 hours.

[0053] According to an embodiment of the present application, the drug carrier comprises a drug controlled release carrier.

[0054] In a fifth aspect of the present application, there is provided use of the composite injectable gel according to the second aspect of the present application in a bioactive scaffold.

[0055] In a sixth aspect of the present application, there is provided use of the composite injectable gel according to the second aspect of the present application in skin wound healing repair.

[0056] In a seventh aspect of the present application, there is provided use of the composite injectable gel according to the second aspect of the present application in medical cosmetology.

[0057] The schemes of the present application will be explained below with reference to examples. Those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. If a specific technique or condition is not specified in the examples, it is performed according to the technique or condition described in the literature in the art or according to the product manual. If the manufacturer of a reagent or instrument is not specified, it is a conventional product that can be obtained from the market.

[0058] Examples: Example 1: Take 0.5 g of hyaluronic acid powder, add 20 mL of 0.02 mg / mL NaOH solution under the action of a magnetic stirrer, stir thoroughly until completely dissolved. Then add 0.45 g of NaCl powder, stir uniformly, then mix 0.05 g of BDDE in the above HA solution, place it in a 45℃ constant temperature water bath for incubation for 16 h. The product is precipitated with 50 mL of 90% ethanol for 2 times, filtered, and the filtered material is transferred to a dialysis bag (Cutoff = 12000-14000), dialyzed in 1×PBS solution for 24 h, then dialyzed in deionized water for 5 days, vacuum freeze-dried to obtain a gel (i.e. composite injectable gel).

[0059] Example 2: Take 1 g of hyaluronic acid powder, 0.5 g of natural type II collagen, and 0.9 g of NaCl particles, mix, add 20 mL of 0.02 mg / mL NaOH solution to make it dissolve thoroughly, the solution is light yellow and viscous, add 0.1 g of BDDE solution to it and mix uniformly, place it in a 37℃ water bath for incubation for 3 h. Precipitate with 50 mL of 90% anhydrous ethanol for 2 times, place the filtered and dried material in a dialysis bag (Cutoff = 12000-14000), dialyze in PBS solution for 24 h, then dialyze in deionized water for 3 days, vacuum freeze-dried to obtain a gel.

[0060] Example 3: Take 0.5 g of hyaluronic acid powder, 0.3 g of natural type III collagen, 0.45 g of NaCl particles, mix them, add 20 mL of 0.1 mg / mL NaOH solution to fully dissolve them, the solution is viscous, add 0.05 g of BDDE solution to it and mix it evenly, place it in a 37°C water bath for 6 h of incubation. Add 150 mL of 90% anhydrous ethanol to precipitate twice. Place the filtered material in a prepared dialysis bag (Cutoff = 12000-14000), dialyze it in PBS solution for 24 h, then dialyze it in deionized water for 7 days, vacuum freeze-dry it, and obtain the gel.

[0061] Example 4: Take 0.5 g of hyaluronic acid powder, 0.25 g of natural type II collagen, 0.45 g of NaCl particles, mix them, add 9 mL of 0.02 mg / mL NaOH solution to fully dissolve them, the solution is light yellow and viscous. Add 0.055 g of genipin in ethanol solution (0.055 g of genipin is dissolved in 1 mL of ethanol) to it and mix it evenly, place it in a 37°C water bath for 6 h of reaction. Add 50 mL of 90% anhydrous ethanol to precipitate twice. Place the filtered material in a prepared dialysis bag (Cutoff = 12000-14000), dialyze it in PBS solution for 24 h, then dialyze it in deionized water for 3 days, vacuum freeze-dry it, and obtain the gel.

[0062] Comparative Example: Comparative Example 1: Comparative Example 1 is basically the same as Example 2 in experimental process, the difference is that no BDDE is added to the solution, and the other preparation process is the same as Example 2. Since no crosslinking agent is added, most of the raw materials are lost after precipitation and dialysis, and the yield of the obtained gel is low (<3%).

[0063] Comparative Example 2: Comparative Example 2-1: Comparative Example 2-1 is basically the same as Example 2 in experimental process, the difference is that no NaCl is added, and the other preparation process is basically the same as Example 2. Take 1 g of hyaluronic acid powder, 0.5 g of natural type II collagen, add 20 mL of 0.02 mg / mL NaOH solution to fully dissolve them, the solution is light yellow and viscous, add 0.1 g of BDDE solution to it and mix it evenly, place it in a 37°C water bath for 3 h of reaction. Add 50 mL of 90% anhydrous ethanol to precipitate. It is found that the yield of the recovered product (i.e. the gel) is very low (<5%) in the second precipitation.

[0064] Comparative Example 2-2: The experimental process of Comparative Example 2-2 is basically the same as that of Example 2, except that no NaCl is added, the reaction time is extended to 6 h, and the other preparation processes are basically the same as those of Example 2. Take 1 g of hyaluronic acid powder, 0.5 g of natural type II collagen, and add 20 mL of 0.02 mg / mL NaOH solution to fully dissolve it. The solution is light yellow and viscous. Add 0.1 g of BDDE solution to the solution and mix evenly. Place it in a 37°C water bath for 6 h. Add 50 mL of 90% anhydrous ethanol to precipitate. It is found that the recovery rate of the product is very low (<10%) in the second precipitation. The schematic diagram of the gel after two times of precipitation is shown in Figure 6 . In addition, the presence of NaCl affects the precipitation of the collagen sample. Collect the supernatant of the second precipitation operation and add a corresponding amount of 0.45 g of NaCl and shake to mix evenly. The originally clear solution becomes turbid, and the turbid liquid is collected and centrifuged. It is found that there are attachments at the bottom of the tube wall, which are the prepared samples, thereby proving that NaCl plays a crucial role in the precipitation of the sample. See Figure 7 . Among them, Figure 7 The right bottle in the first picture from left to right is the effect of adding NaCl to the supernatant of the gel.

[0065] Comparative Example 3: The process is basically the same as that of Example 1, except that the concentration of the NaOH solution is different, no NaCl is added, and the other experimental processes are basically the same as those of Example 1. Take 0.5 g of hyaluronic acid powder and add 4.95 mL of 10 mg / mL NaOH solution under the action of a magnetic stirrer to fully dissolve it. Then add 0.055 g of BDDE to the fully dissolved HA solution and mix evenly. Place it in a 45°C constant temperature water bath for 6 h. The product is precipitated twice with 50 mL of 90% ethanol, filtered, and the filtered material is transferred to a dialysis bag (Cutoff = 12000-14000). Dialyze in 1×PBS solution for 24 h, then dialyze in deionized water for 5 days, and vacuum freeze-dry. The yield of the obtained gel is low.

[0066] Test Example 1: Take 0.5 g of the freeze-dried powder of the gel prepared in Examples 1-4, respectively, and dissolve it in 100 mL of pure water, 0.9% NaCl or 1N PBS, and fully stir to dissolve. Use an 18G-26G needle syringe to inject the gel solution in the aqueous solution. The gel solution is dyed with alizarin red to better present the differentiation and shooting effect. After injection, the gel and the aqueous solution show a significant layering phenomenon, and can be freely written in shape, which is suitable for clinical medical injection scenes. See Figure 1 .

[0067] Test Example 2: Test samples: the gel prepared in Comparative Example 1, the gel prepared in Comparative Example 3, and the gels prepared in Examples 1-3. Each gel sample was freeze-dried to obtain a porous solid-state dry gel. The mass (unit: mg) of the dry gel after freeze-drying was accurately weighed and recorded as W d 。 Each dry gel was immersed in excess swelling medium (normal saline) and placed in a constant temperature condition (25℃) for no less than 12 hours until reaching swelling equilibrium. The swollen gel was taken out and the surface residual liquid was wiped with filter paper. The mass of the wet gel in the saturated swelling state was immediately weighed and recorded as W s . The calculation formula was: Swelling rate (SR) = [(W s- W d ) / W d ] x 100%, and each group of samples was tested in parallel for ≥3 times, and the average value was taken.

[0068] The swelling rates of the gels prepared in Comparative Example 1, Comparative Example 3, and Examples 1, 2, and 3 were measured. The swelling rate of Comparative Example 1 was 1216%. After optimized crosslinking, the swelling rates of the gels of Example 1, Example 2, and Example 3 were 3511%, 2243%, and 2793%, respectively. The results showed that the swelling rates of the gels prepared in Examples 1-3 were 1.8 to 2.9 times higher than those of Comparative Examples 1 and 3, and specific diagrams are shown in Figure 2 .

[0069] Test Example 3: A rotational rheometer instrument (MCR302) was used to test the viscosity and rheological properties of the gels prepared in Examples 1 and 2 under the conditions of a parallel plate (25 mm), 25℃, 4% strain, and 2.5 Hz frequency, and the test time was 1000 s.

[0070] The results showed that the gels prepared in Examples 1 and 2 exhibited high viscosity (100-500 mPa•s) and more stable rheological properties, and specific diagrams are shown in Figure 3 .

[0071] Test Example 4: Prepare standard solutions of different concentrations of Kartogenin (KGN), and use ultraviolet-visible absorption spectroscopy to determine the absorbance at 277 nm to draw a standard curve. Prepare a 10 mg / mL gel solution (0.9% NaCl) of the gels prepared in Examples 1, 2, and Comparative Example 1, and add a certain amount of KGN (final concentration 10 μM) to the gel solution, mix well, and store overnight. Filter the above composite gel with a 0.22 μm filter membrane, take the filtrate to determine the ultraviolet absorbance, and determine the concentration of KGN in the solution by the standard curve to calculate the drug loading rate of the gel. The results show that, compared with the uncrosslinked sample of Comparative Example 1, the drug loading efficiency of the gels prepared in Examples 1 and 2 is effectively improved to more than 80%, and the specific schematic diagram is shown in Figure 4 .

[0072] Test Example 5: Use HaCaT cells (a cell line of immortalized keratinocytes from adult humans; manufacturer: Shangnian Biological; item number: SNL-163), and co-culture HaCaT cells with the crosslinked gel samples prepared in Examples 1 and 3 at a concentration of 10 mg / mL for 24 h. Use a CCK-8 kit (Bi Yun Tian, C0048M) to determine the cell survival rate. The results show that the gels prepared in Examples 1 and 3 have low cytotoxicity, and the cell viability is more than 90%, and the specific schematic diagram is shown in Figure 5 .

[0073] Test Example 6: First, prepare 500 μL of gel solutions prepared in Examples 1, 2, and 3 at a certain concentration and 0.1 mmol / L DPPH solution (prepared with anhydrous ethanol, and the volumetric flask is filled with solvent). Take 10 μL of the above gel solution, add an equal volume of 0.1 mmol / L DPPH solution, mix well, and react at room temperature for 30 min in the dark, and then use an ultraviolet spectrophotometer to determine the absorbance A1 at 517 nm. The blank control group is 10 μL of sample solution added with an equal volume of anhydrous ethanol, and the absorbance A2 is determined. The model control group is set to 10 μL of 0.1 mmol / L DPPH solution added with 10 μL of anhydrous ethanol, and the absorbance A0 is determined. At the same time, in order to avoid test errors, each group should be measured in triplicate and then averaged. The DPPH free radical scavenging rate of the sample is determined by the following calculation formula: DPPH free radical scavenging rate (%) = [1 - (A1 - A2) / A0] x 100%.

[0074] The results show that the gels prepared in Examples 2 and 3 have excellent DPPH free radical scavenging ability, and the scavenging rate is more than 30%, and the specific schematic diagram is shown in Figure 8 , Test Example 7: The gels prepared in Examples 1, 2 and 3 were scanned under a scanning electron microscope. The results showed that the three groups of samples all presented obvious fibrous porous network structure. Among them, the gel prepared in Example 1 presented loose porous structure with the largest pore size; the network structure of the gel prepared in Example 2 was relatively dense, and the pore size was relatively small, but it was still mainly in the form of hole structure; while the gel prepared in Example 3 presented the most subtle structural characteristics, with the smallest pore size and the highest degree of filamentous, forming a more dense three-dimensional network composed of interwoven and intertwined coarse and fine fibers, see Figure 9 .

[0075] In the description of the present specification, the description referring to the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, different embodiments or examples described in the present specification and the features of different embodiments or examples can be combined and modified by those skilled in the art without contradiction, within the scope of the present application.

[0076] Although the embodiments of the present application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.

Claims

1. A method of preparing a composite injectable gel, characterized in that, The method comprises the following steps: 1) dissolving hyaluronic acid and sodium chloride in an aqueous solution of a strong base, the strong base being at least one of NaOH and KOH, to obtain a mixed solution; 2) mixing the mixed solution of step 1) with a cross-linking agent, the cross-linking agent being one or both of 1,4-butanediol diglycidyl ether and genipin; 3) subjecting the mixed product of step 2) to a cross-linking reaction; 4) purifying the cross-linking reaction product of step 3); 5) subjecting the purified product of step 4) to vacuum freeze-drying to obtain the composite injectable gel.

2. The method of claim 1, wherein, In step 1), collagen is also subjected to the dissolving treatment in the aqueous solution of the strong base.

3. The method according to claim 1 or 2, characterized in that, The concentration of the strong base is 0.02-10 mg / mL.

4. The method of claim 1, wherein, The weight ratio of the hyaluronic acid, the strong base, the sodium chloride and the cross-linking agent is (0.5-1):(0.0004-0.05):(0.000001-1):(0.05-0.1).

5. The method of claim 2, wherein, The weight ratio of the hyaluronic acid, the strong base, the sodium chloride, the collagen and the cross-linking agent is (0.5-1):(0.0004-0.05):(0.000001-1):(0.000001-1):(0.05-0.1).

6. The method of claim 2, wherein, The collagen is selected from at least one of natural type I collagen, natural type II collagen and natural type III collagen.

7. The method of claim 1, wherein, The cross-linking reaction is performed at a temperature of 25-45℃ for 3-24 hours. Preferably, the cross-linking reaction is performed at a temperature of 37℃ for 6 hours.

8. The method of claim 1, wherein, The purification treatment is performed in the presence of an alcoholic precipitant, the alcoholic precipitant comprising at least one of 75%-100% ethanol and methanol. Preferably, the alcoholic precipitant is 90% ethanol.

9. A composite injectable gel, characterized in that, Prepared by the method of any one of claims 1-10.

10. Use of the composite injectable gel of claim 9 in at least one of tissue engineering repair, preparation of a drug carrier, a bioactive scaffold, skin wound healing repair, medical cosmetology.

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