Method of preparing a composite injectable gel, composite injectable gel and use thereof

Abstract

The present application belongs to the technical field of biomaterials. The existing preparation process of composite injectable gel is complicated, and the prepared composite injectable gel has the problems of poor injectability, low swelling rate, low mechanical strength, low drug loading rate and high cytotoxicity. Based on this, the present application provides a method for preparing a composite injectable gel, which comprises: 1) dissolving hyaluronic acid and sodium chloride in an aqueous solution of NaOH or KOH; 2) adding a crosslinking agent 1,4-butanediol diglycidyl ether or genipin and mixing; 3) performing crosslinking reaction; 4) purifying the reaction product; 5) vacuum freeze-drying to obtain the final composite injectable gel. The method simplifies the preparation process, and the prepared composite injectable gel has significantly improved injectability, swelling rate, mechanical strength, drug loading rate, and lower cytotoxicity, and has good application prospect in the fields of drug delivery, tissue engineering, skin wound healing repair, medical cosmetology and the like.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to a method for preparing composite injectable gels, composite injectable gels and their uses. Background Technology

[0002] Biomaterials are a class of special functional materials, either natural or artificially synthesized, used to contact and interact with living systems, enabling the diagnosis, treatment, replacement, repair, or induced regeneration of cells, tissues, and organs. They are also known as biomedical materials. Based on material properties, they can be classified as medical metallic materials, medical inorganic materials, medical polymer materials, and medical composite materials; based on material function, they can be classified as hard tissue compatible materials, soft tissue compatible materials, blood compatible materials, biodegradable materials, and polymeric drugs; based on material source, they can be classified as autologous tissue, allogeneic organs and tissues, xenogeneic organs and tissues, natural biomaterials, and artificially synthesized materials; and based on the site of application, they can be classified as hard tissue materials, soft tissue materials, cardiovascular materials, blood substitute materials, and separation, filtration, and dialysis membrane materials.

[0003] In the field of biomaterials, current composite injectable gels require multiple steps to prepare, which is cumbersome. Furthermore, current composite injectable gels have poor injectability, low swelling rate, low mechanical strength, low drug loading rate, and high cytotoxicity.

[0004] Therefore, this invention urgently needs to develop a method for preparing composite injectable gels to obtain composite injectable gels with good injectability, high swelling ratio, high mechanical strength, high drug loading rate, low cytotoxicity, and excellent antioxidant properties. Summary of the Invention

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

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

[0007] In a second aspect, the present invention provides a composite injectable gel. According to an embodiment of the present invention, the composite injectable gel is prepared by the method described in the first aspect of the present invention. The composite injectable gel according to the embodiment of the present invention exhibits good injectability, increased swelling ratio, enhanced mechanical strength, improved drug loading capacity, reduced cytotoxicity, and excellent anti-free radical oxidation properties.

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

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

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

[0011] In a sixth aspect, the present invention proposes the use of the composite injectable gel described in the second aspect of the present invention in the healing and repair of skin wounds.

[0012] In a seventh aspect, the present invention proposes the use of the composite injectable gel described in the second aspect of the present invention in medical aesthetics.

[0013] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0014] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0015] Figure 1 This is a schematic diagram of the injectable gel obtained according to Examples 1-4 of the present invention.

[0016] Figure 2 The swelling rate results are shown in the figures for cross-linked gel samples with different compositions and reaction conditions according to Examples 1-3, Comparative Examples 1 and 3 of the present invention.

[0017] Figure 3 The graph shows the storage modulus and loss modulus of representative cross-linked gel samples prepared according to Examples 1-2 of the present invention.

[0018] Figure 4 The graph shows the drug loading rate of the gels prepared according to Examples 1-2 and Comparative Example 1 of the present invention.

[0019] Figure 5 The graph shows the survival rate of the gel samples prepared according to Examples 1 and 3 of the present invention.

[0020] Figure 6 This is a photographic comparison of the sample amounts obtained from two precipitations of the gel prepared according to Comparative Example 2 of the present invention.

[0021] Figure 7 Comparative Example 2 of the present invention shows a comparison of the supernatant of the gel prepared by adding NaCl before and after, and a comparison of the precipitate obtained by centrifuging the turbid liquid.

[0022] Figure 8 The graph shows the results of determining the scavenging rate of 1,1-diphenyl-2-trinitrophenylhydrazine radical (DPPH) in gels (6 mg / mL) prepared according to Examples 1, 2, and 3 of the present invention.

[0023] Figure 9 The images shown are scanning electron microscope (SEM) images of the gel samples prepared according to Examples 1, 2, and 3 of the present invention. Detailed Implementation

[0024] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0025] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0026] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0027] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this invention, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0028] In this invention, the terms "comprising" or "including" are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0029] In this invention, the term "hyaluronic acid" refers to 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 this invention (CAS 9004-61-9, West Asia Chemicals, catalog number A22873) contains a large number of carboxyl and hydroxyl groups on its molecular chain, giving it strong hydrophilicity. Aqueous solutions of hyaluronic acid exhibit high viscosity and good lubricity; its viscoelasticity is related to factors such as concentration and molecular weight. For example, hyaluronic acid solutions used in ophthalmic surgery utilize their 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 the vitreous humor of the eye. It can retain skin moisture, making the skin soft and smooth, and is therefore widely used in moisturizing products in the cosmetics industry. In the medical field, hyaluronic acid can be used to treat joint diseases such as osteoarthritis. Injecting hyaluronic acid into the joint cavity can increase the viscoelasticity of synovial fluid, reduce joint pain, and improve joint function.

[0030] In this invention, the term "1,4-butanediol diglycidyl ether" refers to a cross-linking agent. In tissue engineering, 1,4-butanediol diglycidyl ether (BDDE) is used to cross-link biomaterials to prepare scaffolds. For example, in cartilage tissue engineering, extracellular matrix components of chondrocytes, such as collagen and glycosaminoglycans (e.g., hyaluronic acid), are reacted with this cross-linking agent to prepare scaffolds with suitable porosity, mechanical strength, and biocompatibility. Such scaffolds can provide a favorable 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, encapsulating the drug within a biomaterial matrix cross-linked with 1,4-butanediol diglycidyl ether. The drug release rate can be controlled by adjusting factors such as the cross-linking density. For example, some drugs for treating arthritis are encapsulated in cross-linked hyaluronic acid materials. When the material is implanted at the joint site, the drug can be slowly released as the material degrades or interacts with the surrounding environment, thereby prolonging the duration of drug action and improving therapeutic efficacy.

[0031] In this invention, unless otherwise specified, the solvent of the solution is water. For example, a NaOH solution is obtained by dissolving NaOH in water.

[0032] This invention proposes a method for preparing a composite injectable gel, a composite injectable gel and its uses, which will be described in detail below.

[0033] Methods for preparing composite injectable gels

[0034] In a first aspect, the present invention provides a method for preparing a composite injectable gel. According to an embodiment of the invention, the method includes the following steps: 1) dissolving hyaluronic acid and sodium chloride in an aqueous solution of a strong alkali to obtain a mixed solution, wherein the strong alkali is at least one selected from NaOH and KOH; 2) mixing the mixed solution obtained in step 1) with a crosslinking agent, wherein the crosslinking agent is one or both selected from 1,4-butanediol diglycidyl ether and genipin; 3) subjecting the mixed product obtained in step 2) to a crosslinking reaction; 4) purifying the crosslinking reaction product obtained in step 3); and 5) subjecting the purified product obtained in step 4) to vacuum freeze-drying 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 regulating ionic strength, shielding charge, promoting collagen fibrillation, and stabilizing fiber structure. According to the method of the embodiments of the present invention, a composite injectable gel with good injectability, high swelling ratio, high mechanical strength, high drug loading rate, low cytotoxicity, and excellent antioxidant properties can be prepared.

[0035] According to an embodiment of the present invention, the purification process is achieved by performing alcohol precipitation and filtration:

[0036] i) The cross-linking reaction product obtained in step 3) is subjected to alcohol precipitation followed by filtration. The filtrate is then subjected to a first dialysis treatment in PBS solution. ii) The first dialysis product obtained in i) is subjected to a second dialysis treatment in deionized water. iii) The second dialysis product obtained in ii) is then subjected to vacuum freeze-drying. In step i), the PBS solution preferentially removes small molecule impurities (such as reactants, cross-linking agents, and alcohol solvents) from the product. By simulating a physiological environment, this avoids conformational changes or functional loss of collagen due to lack of ion balance in pure water, thus preserving the product's solubility and stability. In step ii), the purpose of pure water dialysis is to slowly desalt and further remove unreacted raw materials and cross-linking agents, reducing the risk of product inactivation due to rapid changes in salt concentration gradients. This provides a high-purity matrix for subsequent freeze-drying. The combination of steps i) and ii) forms a staged purification strategy that balances efficiency and product protection.

[0037] According to an embodiment of the present invention, the duration of the first dialysis treatment is 1-2 days, for example, 1 day or 2 days, and the duration of the second dialysis treatment is 3-5 days, for example, 3 days, 4 days or 5 days.

[0038] According to an embodiment of the present invention, step 1) further includes dissolving the collagen in the aqueous solution of the strong alkali.

[0039] According to embodiments of the present invention, the concentration of the strong alkali is 0.02-10 mg / mL, for example, it can be 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 between the two, such as 0.05-10 mg / mL or 0.1-10 mg / mL. According to embodiments of the present invention, if the concentration of the strong alkali is below 0.02 mg / mL, the HA will not dissolve sufficiently, and the cross-linking reaction will not proceed completely. If the concentration of the strong alkali is above 10 mg / mL, it will lead to excessive cross-linking, resulting in side reactions and yellowing of the reaction solution. The appropriate concentration of the strong alkali helps the components such as hyaluronic acid to be fully dispersed and dissolved in the solution, so that the components are evenly mixed, thereby preparing a uniform gel. This avoids differences in gel performance caused by uneven component distribution. The appropriate concentration of the strong alkali is also beneficial to controlling the rate of cross-linking reaction, ensuring that the degree of cross-linking and performance of the gel achieve the expected results.

[0040] According to an embodiment of the present invention, the weight ratio of the hyaluronic acid, the strong base, the sodium chloride, and the crosslinking agent is (0.5-1):(0.0004-0.05):(0.000001-1):(0.05-0.1). According to an embodiment of the present invention, the appropriate weight ratio of the hyaluronic acid, the strong base, the sodium chloride (NaCl), and the crosslinking agent is beneficial for preparing a composite injectable gel with good injectability, high swelling ratio, high mechanical strength, high drug loading rate, low cytotoxicity, and excellent antioxidant properties. It should be explained that hyaluronic acid plays a crucial matrix role in the composite injectable gel. An appropriate amount of hyaluronic acid ensures that the composite injectable gel has good elasticity and water retention. The strong base is used during the preparation process to adjust the pH value of the solution, making the solution reach a suitable alkaline environment to promote the full dissolution and mixing of hyaluronic acid with other components, as well as the subsequent crosslinking reaction. An appropriate amount of strong base ensures that the pH value of the solution remains stable within the optimal range, avoiding damage to the gel components and reducing the occurrence of side reactions. Sodium chloride helps hyaluronic acid and other components dissolve better in the solution, further promoting uniform mixing of the components and maintaining a certain ionic strength. An appropriate amount of cross-linking agent ensures that hyaluronic acid and other components are fully cross-linked, forming a uniform and dense cross-linked network. This gives the composite injectable gel good elasticity and strength, while avoiding the potential toxicity risks caused by excessive residue.

[0041] According to an embodiment of the present invention, the weight ratio of the hyaluronic acid, the strong alkali, the sodium chloride, the collagen, and the crosslinking 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 invention, the appropriate weight ratio of the hyaluronic acid, the strong alkali, the sodium chloride (NaCl), the collagen, and the crosslinking agent is beneficial for preparing a composite injectable gel with good injectability, high swelling ratio, high mechanical strength, high drug loading rate, low cytotoxicity, and excellent antioxidant properties. It should be explained that collagen is an important structural protein; an appropriate amount of collagen can form a composite network structure with hyaluronic acid, significantly enhancing the mechanical properties of the gel. Simultaneously, an appropriate amount of collagen can provide a favorable growth environment for cells, promoting cell adhesion, migration, and proliferation, accelerating tissue repair and regeneration, and improving the repair and therapeutic effect of the gel.

[0042] It should be noted that in this application, "gel" and "composite injectable gel" are synonymous.

[0043] According to an embodiment of the present invention, the collagen is selected from at least one of natural collagen and recombinant collagen.

[0044] It should be explained that, as those skilled in the art will know, the collagen mentioned includes, but is not limited to, natural collagen extracted from animal tissues (such as bovine Achilles tendon and pig skin), or recombinant humanized collagen expressed through genetic engineering (such as human-like collagen HLC).

[0045] According to an embodiment of the present invention, the collagen comprises natural collagen extracted from bovine Achilles tendon.

[0046] According to an embodiment of the present invention, the collagen comprises natural collagen extracted from pig skin.

[0047] According to an embodiment of the present invention, the collagen is selected from at least one of natural type I collagen, natural type II collagen, and natural type III collagen.

[0048] According to embodiments of the present invention, the crosslinking reaction is carried out at a temperature of 25-45°C for 3-24 hours, for example, at 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 between both, such as 26-45°C or 27-45°C; and for example, for 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 between both, such as 4-24 hours or 5-24 hours.

[0049] According to an embodiment of the present invention, the crosslinking reaction is carried out at a temperature of 37°C for 6 hours.

[0050] According to an embodiment of the present invention, the purification process is carried out in the presence of an alcohol precipitant, which includes 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% or 77%~100% ethanol.

[0051] According to an embodiment of the present invention, the alcohol precipitant is 90% ethanol.

[0052] Composite injectable gel

[0053] In a second aspect, the present invention provides a composite injectable gel. According to an embodiment of the present invention, the composite injectable gel is prepared by the method described in the first aspect of the present invention. The composite injectable gel according to the embodiment of the present invention exhibits good injectability, increased swelling ratio, enhanced mechanical strength, improved drug loading capacity, reduced cytotoxicity, and excellent anti-free radical oxidation properties.

[0054] use

[0055] In a third aspect, the present invention proposes the use of the composite injectable gel described in the second aspect of the present invention in tissue engineering repair.

[0056] According to an embodiment of the present invention, the tissue engineering repair includes the preparation of a tissue filler.

[0057] In a fourth aspect, the present invention proposes the use of the composite injectable gel described in the second aspect of the present invention in the preparation of a drug carrier.

[0058] According to an embodiment of the present invention, the drug carrier includes a drug controlled-release carrier.

[0059] In a fifth aspect, the present invention proposes the use of the composite injectable gel described in the second aspect of the invention in a bioactive scaffold.

[0060] In a sixth aspect, the present invention proposes the use of the composite injectable gel described in the second aspect of the present invention in the healing and repair of skin wounds.

[0061] In a seventh aspect, the present invention proposes the use of the composite injectable gel described in the second aspect of the present invention in medical aesthetics.

[0062] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0063] Example:

[0064] Example 1: Take 0.5g of hyaluronic acid powder and add 20 mL of 0.02 mg / mL NaOH solution under magnetic stirring, stirring thoroughly until completely dissolved. Then add 0.45g of NaCl powder, stir evenly, and then mix 0.05g of BDDE into the above HA solution. Incubate the mixture in a 45℃ water bath for 16h. Precipitate the product twice with 50mL of 90% ethanol, filter, and transfer the filtered material to a dialysis bag (Cutoff=12000-14000). Dialyze in 1×PBS solution for 24h, then dialyze in deionized water for 5 days, and freeze-dry under vacuum to obtain a gel (i.e., a composite injectable gel).

[0065] Example 2: Take 1g of hyaluronic acid powder, 0.5g of natural type II collagen, and 0.9g of NaCl particles, mix them, and add 20mL of 0.02mg / mL NaOH solution to dissolve them completely. The solution is a pale yellow viscous liquid. Add 0.1g of BDDE solution to the solution and mix well. Incubate in a 37℃ water bath for 3h. Add 50mL of 90% anhydrous ethanol to precipitate twice. Place the filtered and dried material in a dialysis bag (Cutoff=12000-14000), dialyze in PBS solution for 24h, then dialyze in deionized water for 3 days, and freeze-dry under vacuum to obtain a gel.

[0066] Example 3: 0.5g of hyaluronic acid powder, 0.3g of natural type III collagen, and 0.45g of NaCl granules were mixed and dissolved in 20mL of 0.1mg / mL NaOH solution until fully dissolved, resulting in a viscous solution. 0.05g of BDDE solution was added to the solution and mixed thoroughly. The mixture was then incubated in a 37℃ water bath for 6 hours. Precipitation was performed twice with 150mL of 90% anhydrous ethanol. The filtered material was placed in a pre-treated dialysis bag (Cutoff = 12000-14000) and dialyzed in PBS solution for 24 hours, followed by dialyzing in deionized water for 7 days. The gel was then lyophilized under vacuum to obtain a gel.

[0067] Example 4: 0.5g of hyaluronic acid powder, 0.25g of natural type II collagen, and 0.45g of NaCl granules were mixed and dissolved in 9mL of 0.02mg / mL NaOH solution, resulting in a pale yellow, viscous solution. An ethanol solution containing 0.055g of genipin (obtained by dissolving 0.055g of genipin in 1mL of ethanol) was added to the solution and mixed thoroughly. The mixture was then placed in a 37℃ water bath for 6 hours. Precipitation was achieved twice with 50mL of 90% anhydrous ethanol. The filtered material was placed in a pre-treated dialysis bag (Cutoff = 12000-14000) and dialyzed in PBS solution for 24 hours, followed by dialyzing in deionized water for 3 days. The gel was then lyophilized under vacuum to obtain a gel.

[0068] Comparative example:

[0069] Comparative Example 1:

[0070] The experimental procedures for Comparative Example 1 and Example 2 were basically the same, except that BDDE was not added to the solution. Other preparation procedures were the same as in Example 2. Because no cross-linking agent was added, most of the raw materials were lost after precipitation and dialysis, resulting in a low gel yield (<3%).

[0071] Comparative Example 2:

[0072] Comparative Example 2-1:

[0073] The experimental procedures for Comparative Example 2-1 and Example 2 were basically the same, except that NaCl was not added. Other preparation procedures were essentially the same as in Example 2. 1g of hyaluronic acid powder and 0.5g of natural type II collagen were added to 20mL of 0.02mg / mL NaOH solution to dissolve them completely. The solution was a pale yellow, viscous consistency. 0.1g of BDDE solution was added to the solution and mixed thoroughly. The mixture was then placed in a 37℃ water bath and reacted for 3 hours. 50mL of 90% anhydrous ethanol was added to precipitate the product. It was found that the yield of the recovered product (i.e., gel) was very low (<5%) during the second precipitation.

[0074] Comparative Example 2-2:

[0075] Comparative Example 2-2 and Example 2 followed essentially the same experimental procedure, except that NaCl was not added and the reaction time was extended to 6 hours. Other preparation procedures were essentially the same as in Example 2. 1 g of hyaluronic acid powder and 0.5 g of natural type II collagen were added to 20 mL of 0.02 mg / mL NaOH solution to dissolve them completely. The solution was a pale yellow, viscous consistency. 0.1 g of BDDE solution was added to the solution and mixed thoroughly. The mixture was then placed in a 37°C water bath and reacted for 6 hours. 50 mL of 90% anhydrous ethanol was added to precipitate the product. It was found that the yield of the recovered product was very low (<10%) during the second precipitation. For a detailed schematic diagram of the two precipitation processes, please refer to [link to schematic diagram]. Figure 6 Furthermore, the presence of NaCl affects the precipitation of collagen samples. Collecting the supernatant from the second precipitation step and adding 0.45g of NaCl, followed by shaking and mixing, revealed that the originally clear solution became cloudy. Centrifuging the cloudy solution showed deposits at the bottom of the tube, indicating the prepared sample. This demonstrates that NaCl plays a crucial role in sample precipitation. See details... Figure 7 .in, Figure 7 The bottle on the right in the first image from left to right illustrates the effect of adding NaCl to the supernatant of the gel.

[0076] Comparative Example 3:

[0077] The procedure was basically the same as in Example 1, except that the concentration of the NaOH solution was different and NaCl was not added. Other experimental procedures were essentially the same as in Example 1. 0.5 g of hyaluronic acid powder was added to 4.95 mL of 10 mg / mL NaOH solution under magnetic stirring and stirred thoroughly until completely dissolved. Then, 0.055 g of BDDE was added to the completely dissolved HA solution and mixed thoroughly. The mixture was then incubated in a 45°C water bath for 6 hours. The product was precipitated twice with 50 mL of 90% ethanol, filtered, and the filtered material was transferred to a dialysis bag (Cutoff = 12000-14000). Dialysis was performed in 1×PBS solution for 24 hours, followed by dialysis in deionized water for 5 days. The gel was then lyophilized under vacuum, resulting in a low gel yield.

[0078] Test Example 1:

[0079] Weigh 0.5 g of the lyophilized powder of the gels prepared in Examples 1-4 respectively, and dissolve them in 100 mL of pure water, 0.9% NaCl, or 1N PBS, stirring thoroughly to dissolve. Inject the resulting gel solution into the aqueous solution using an 18G-26G syringe. Add alizarin red to the gel solution for staining to better distinguish the imaging effect. After injection, the gel and aqueous solution show significant layering and can be freely written in, making it suitable for clinical medical injection scenarios. See the specific illustration. Figure 1 .

[0080] Test Example 2:

[0081] Test samples: gels prepared in Comparative Example 1, gels prepared in Comparative Example 3, and gels prepared in Examples 1-3. Each gel sample was freeze-dried to obtain porous solid dry gels. The mass of the freeze-dried gel was accurately weighed (unit: mg) and denoted as W. d 。 The dry gels were immersed in excess physiological saline as a swelling medium and allowed to stand at a constant temperature (25°C) for at least 12 hours until swelling equilibrium was reached. The swollen gels were then removed, and any residual liquid on the surface was gently wiped away with filter paper. The mass of the wet gel in its saturated swollen state was immediately measured and denoted as W. s The calculation formula is: Swelling ratio (SR) = [(W...] s- W d ) / W d ×100%, each group of samples was tested in parallel ≥3 times, and the average value was taken.

[0082] The swelling ratios of the gels prepared in Comparative Examples 1 and 3, as well as those prepared in Examples 1, 2, and 3, were measured. The swelling ratio of Comparative Example 1 was 1216%. After optimized crosslinking, the swelling ratios of the gels in Examples 1, 2, and 3 were 3511%, 2243%, and 2793%, respectively. The results show that compared to Comparative Examples 1 and 3, the swelling ratios of the gels prepared in Examples 1-3 of this invention were increased by 1.8 to 2.9 times. See the detailed schematic diagram below. Figure 2 .

[0083] Test Example 3:

[0084] The viscosity and rheological properties of the gels prepared in Examples 1 and 2 were tested using a rotational rheometer (MCR302) with a parallel plate (25 mm) at 25°C, 4% strain, and 2.5 Hz for 1000 s.

[0085] The results show that the gels prepared in Examples 1 and 2 of this invention exhibit higher viscosity (100~500 mPa•s) and more stable rheological properties. See the detailed schematic diagrams below. Figure 3 .

[0086] Test Example 4:

[0087] Standard solutions of Kartogenin (KGN) at different concentrations were prepared, and the absorbance at 277 nm was measured using UV-Vis absorption spectroscopy to plot a standard curve. Gel solutions (0.9% NaCl) of 10 mg / mL from Examples 1, 2, and Comparative Example 1 were prepared. A certain amount of KGN (final concentration 10 μM) was added to the gel solutions, mixed thoroughly, and stored overnight. The composite gels were filtered through a 0.22 μm filter membrane, and the absorbance of the filtrate was measured. The concentration of KGN in the solution was determined using the standard curve, and the drug loading rate of the gel was calculated. The results showed that, compared to the uncrosslinked sample (Comparative Example 1), the drug loading efficiency of the gels prepared in Examples 1 and 2 was effectively increased to over 80%. See the schematic diagram for details. Figure 4 .

[0088] Test Example 5:

[0089] HaCaT cells (a cell line derived from immortalized keratinocytes of adult humans; manufacturer: Shangen Biotechnology; catalog number: SNL-163) were co-cultured with cross-linked gel samples prepared in Examples 1 and 3 at a concentration of 10 mg / mL for 24 h. Cell viability was determined using a CCK-8 assay kit (Beyotime, C0048M). The results showed that the gels prepared in Examples 1 and 3 had low cytotoxicity, with cell viability exceeding 90%. See the detailed schematic diagram below. Figure 5 .

[0090] Test Example 6:

[0091] First, prepare 500 μL of each of the gel solutions prepared in Examples 1, 2, and 3, and 0.1 mmol / L DPPH solution (prepared with anhydrous ethanol and diluted to volume in a volumetric flask). Take 10 μL of each gel solution, add an equal volume of 0.1 mmol / L DPPH solution, mix well, and react at room temperature in the dark for 30 min. Then, measure the absorbance A1 at 517 nm using a UV spectrophotometer. The blank control group consists of 10 μL of sample solution with an equal volume of anhydrous ethanol, and the absorbance value A2 is measured. The model control group consists of 10 μL of 0.1 mmol / L DPPH solution with 10 μL of anhydrous ethanol, and the absorbance value A0 is measured. To avoid experimental error, each measurement should be performed in triplicate and the average value should be taken. The DPPH free radical scavenging rate of the sample is determined by the following formula:

[0092] DPPH radical scavenging rate (%) = [1 - (A1 - A2) / A0] × 100%.

[0093] The results showed that the gels prepared in Examples 2 and 3 had excellent DPPH free radical scavenging ability, with scavenging rates both exceeding 30%. See the detailed schematic diagrams below. Figure 8 ,

[0094] Test Example 7:

[0095] The gels prepared in Examples 1, 2, and 3 were scanned under a scanning electron microscope. The results showed that all three samples exhibited a distinct fibrous porous network structure. Specifically, the gel prepared in Example 1 exhibited a loose porous structure with the largest pore size; the gel prepared in Example 2 had a relatively dense network structure with smaller pore sizes, but was still predominantly a pore structure; while the gel prepared in Example 3 exhibited the finest structural features, with the smallest pore size and the highest degree of filamentation, forming a denser three-dimensional network composed of interwoven coarse and fine fibers. (See details...) Figure 9 .

[0096] In this specification, the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0097] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for preparing a composite injectable gel, characterized in that, Includes the following steps: 1) Hyaluronic acid and sodium chloride are dissolved in an aqueous solution of a strong alkali to obtain a mixed solution, wherein the strong alkali is at least one of NaOH and KOH; 2) The mixed solution described in 1) is mixed with a crosslinking agent, wherein the crosslinking agent is one or two of 1,4-butanediol diglycidyl ether and genipin; 3) The mixed product obtained in 2) is subjected to a cross-linking reaction; 4) The cross-linking reaction product obtained in 3) is purified; 5) The purified product obtained in 4) is subjected to vacuum freeze-drying to obtain the composite injectable gel.

2. The method according to claim 1, characterized in that, Step 1) further includes dissolving the collagen in the aqueous solution of the strong alkali.

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 according to claim 1, characterized in that, The weight ratio of the hyaluronic acid, the strong alkali, the sodium chloride, and the crosslinking agent is (0.5-1):(0.0004-0.05):(0.000001-1):(0.05-0.1).

5. The method according to claim 2, characterized in that, The weight ratio of the hyaluronic acid, the strong alkali, the sodium chloride, the collagen, and the crosslinking agent is (0.5-1):(0.0004-0.05):(0.000001-1):(0.000001-1):(0.05-0.1).

6. The method according to claim 2, characterized in that, 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 according to claim 1, characterized in that, The crosslinking reaction is carried out at a temperature of 25~45℃ for 3~24 hours.

8. The method according to claim 1, characterized in that, The crosslinking reaction was carried out at a temperature of 37°C for 6 hours.

9. The method according to claim 1, characterized in that, The purification process is carried out in the presence of an alcohol precipitant, which includes at least one of 75%-100% ethanol and methanol.

10. The method according to claim 9, characterized in that, The alcohol precipitant is 90% ethanol.

11. A composite injectable gel, characterized in that, It is prepared by the method described in any one of claims 1 to 10.

12. The use of the composite injectable gel of claim 11 in at least one of tissue engineering repair, preparation of drug carriers, bioactive scaffolds, skin wound healing and repair, and medical aesthetics.

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