Preparation method and application of double-modified cross-linked hyaluronic acid gel

Abstract

The invention relates to the technical field of polymer materials, and in particular to a preparation method and application of a double-modified cross-linked hyaluronic acid gel. The method adopts natural active substances to perform double modification on hyaluronic acid. First, fucoidan is used to perform single cross-linking with hyaluronic acid and its salt under suitable conditions to form a stable inner core structure; then, calycosin isoflavone is used to perform double cross-linking with the single-cross-linked fucoidan hyaluronic acid gel to form a hydrophobic outer layer; and non-polar groups on the calycosin isoflavone are introduced to form a cross-linked fucoidan-hyaluronic acid-calycosin isoflavone composite gel, which has a hydrophilic / hydrophobic vesicle structure. The invention does not need to use any chemical cross-linking agent, thus avoiding adverse reactions caused by cross-linking agent residues. Meanwhile, the elastic modulus and mechanical strength of the gel are optimized by combining high temperature and low temperature step-by-step cross-linking reactions, and the gel can be used for medical filling materials, sustained-release carriers or preparation of skin care products.

Description

Technical Field

[0001] The invention belongs to the technical field of polymer material preparation, and specifically relates to a preparation method and application of a double-modified cross-linked hyaluronic acid gel. Background Art

[0002] As a natural polysaccharide, hyaluronic acid (HA) has important application value in the biomedical field due to its excellent moisturizing, lubricating and tissue repairing properties. However, the unmodified natural HA molecular chain has a linear structure and is easily degraded by hyaluronidase in the body. Its half-life is usually less than 24-72 hours, resulting in significant defects such as poor stability and short action period in practical applications.

[0003] In order to overcome the above limitations, cross-linking technology has been introduced into the field of HA modification: chemical cross-linking agents (such as BDDE, DVS) are covalently bonded to the hydroxyl groups on the HA molecular chain to form a three-dimensional network cross-linking structure, which can significantly improve the mechanical strength and resistance to enzymatic hydrolysis of the material, and extend its retention time in the body to 6-12 months. At present, cross-linked hyaluronic acid gel has become the mainstream subcutaneous filling material in the field of medical cosmetology, and is widely used in wrinkle improvement, contour shaping and soft tissue augmentation. Despite this, the existing technology still faces the following key bottlenecks:

[0004] 1. Safety hazards caused by cross-linking agent residues

[0005] Traditional processes usually use chemical crosslinkers (such as 1,4-butanediol diglycidyl ether (BDDE) and divinyl sulfone (DVS)) to prepare crosslinked hyaluronic acid gels. However, due to the characteristics of crosslinked hyaluronic acid gels, the subsequent purification process of chemical crosslinkers is relatively complicated and it is difficult to completely remove the residues. The chemical crosslinkers themselves or their degradation products have certain cytotoxicity. If the purification is not thorough, the residual crosslinkers may be toxic to skin cells and cause adverse reactions.

[0006] Current evidence-based medicine also shows that adverse reactions such as skin allergies, redness, swelling, and infection that occur after using cross-linked hyaluronic acid gel may be related to the metabolites of the cross-linking agent in the body.

[0007] 2. Limitations of crosslinker selection and performance

[0008] Currently, there are limited types of crosslinking agents used to crosslink hyaluronic acid (HA) gels, including BDDE (1,4-butanediol diglycidyl ether) and DVS (divinyl sulfone), but they have limitations in terms of biocompatibility and crosslinking controllability:

[0009] Lack of biocompatibility

[0010] The sulfone group of DVS can induce inflammatory response. Therefore, DVS cross-linked HA gel tends to be gradually eliminated.

[0011] Excessive cross-linking: The high activity of the diepoxy groups of BDDE can easily lead to excessive cross-linking, forming a rigid insoluble gel, increasing the risk of granulomas, and affecting biodegradability. It can also lead to uneven cross-linking: Due to the high reactivity of BDDE, it may lead to an uneven cross-linking network, affecting the overall mechanical properties and stability.

[0012] The development of new cross-linking agents is still lagging behind: green technologies such as photocross-linking and enzyme cross-linking are still in the laboratory stage. Due to high costs or low reaction efficiency, it is difficult to meet the needs of large-scale production.

[0013] 3. Difficulty in controlling the uniformity of gel structure

[0014] Existing conventional synthesis methods have certain limitations. Some processes use static cross-linking reactions, and there are also differences in temperature control. These factors work together to make it difficult to achieve good uniformity in the cross-linking reaction.

[0015] Specifically, during the static cross-linking process, there are differences in the diffusion of reactants in the system, making it difficult to ensure that the degree of cross-linking in each part is consistent. At the same time, the temperature control is not precise enough, and temperature fluctuations in different areas will affect the rate and degree of the cross-linking reaction, further exacerbating the difference in uniformity of the reaction. This difference in uniformity of the cross-linking reaction causes the spatial structure of the cross-linked hyaluronic acid gel to show a state of poor uniformity. Under such circumstances, it becomes very difficult to prepare gel particles with good uniformity and smooth surface by subsequent screening.

[0016] Poorly uniform gel structures can lead to a series of adverse consequences. The most obvious one is that the mechanical properties and biocompatibility of the gel will be inconsistent. This will not only affect the performance stability of the gel in practical applications, but may also have an adverse effect on its effect and safety in vivo.

[0017] 4. Balance between mechanical properties and water absorption expansion

[0018] The ideal filling material needs to have both an appropriate elastic modulus and excellent support performance, while maintaining low water absorption, so as to meet long-term filling needs while minimizing the probability of filling deformation and the "bread face" phenomenon caused by water absorption. However, the hyaluronic acid gel currently on the market faces challenges in coordinating mechanical properties and water absorption, and it is difficult to achieve an ideal balance.

[0019] 5. Issues of degradation kinetics and biosafety

[0020] Most of the injectable cross-linked hyaluronic acids on the market now have a short metabolic degradation time in the body, and most of them can maintain the filling effect for 6 months, and multiple injections are required to maintain the filling effect. In order to improve its resistance to enzymatic hydrolysis, the conventional strategy is to increase the amount of cross-linking agent to enhance the cross-linking density. Commonly used cross-linking agents include 1,4-butanediol diglycidyl ether (BDDE) and divinyl sulfone (DVS). However, increasing the amount of cross-linking agent may lead to excessive cross-linking, making HA unable to be effectively hydrolyzed by hyaluronidase, and residual gel may induce chronic inflammation; high cross-linking agent dosage makes purification more difficult exponentially, and trace impurities (such as unreacted monomers) may produce cytotoxicity.

[0021] 6. Limitations of existing technology improvement solutions

[0022] In recent years, some studies have tried to optimize performance through the following approaches, but there are still shortcomings:

[0023] In the prior art, it has been reported that a single-phase hyaluronic acid gel with uniform and smooth particles has been prepared, but the gel prepared with uncross-linked hyaluronic acid as raw material inevitably has the problem of short degradation cycle; a study provides a method for preparing a cosmetic injection gel, dissolving sodium alginate in water, and preparing a cosmetic injection gel as one of the components and mixing other substances for improving wrinkles, skin filling, etc. However, in this method, alginate is not the main component, but only one of the components, and the prepared gel is multi-component, which needs to be mixed in a relatively complicated step before use before injection. This method is prone to uneven mixing or its product is prone to contamination, and cannot guarantee the best effect and sterile state that the product can achieve to the greatest extent; a document provides a hyaluronic acid and biodegradable polymer modified material and preparation method. In this method, the polymer material is grafted with hyaluronic acid to form a polymer, so that the cross-linked structure is more stable, but this preparation method is cumbersome and complicated, and the residual reagents in the gel are difficult to remove, which may cause some inflammation and infection after injection. There is also the use of microreactor continuous production to improve uniformity, but the equipment cost is high and it is difficult to promote industrialization.

[0024] Based on the above problems, the development of new cross-linked hyaluronic acid gels with low cytotoxicity, high cross-linking efficiency, and better structural uniformity is of urgent significance to promote the upgrading of long-lasting and safe medical beauty fillers. Summary of the invention

[0025] The present invention provides a method for preparing a cross-linked hyaluronic acid gel doubly modified with natural active molecules, and obtains a cross-linked hyaluronic acid gel with good biocompatibility, high elastic modulus, strong supporting performance and excellent resistance to enzymatic hydrolysis through double cross-linking, which has wide application value in the biomedical field.

[0026] The technical solution of the present invention is as follows:

[0027] A method for preparing a double-modified cross-linked hyaluronic acid gel, comprising the following steps:

[0028] (1) Dissolution: Fucoidan is swelled in an acidic solution to form a phase A solution; hyaluronic acid and its salts are dissolved in purified water to form a phase B solution;

[0029] (2) Single cross-linking: adding the phase A solution to the phase B solution to obtain a phase AB mixed solution, performing a thermal cross-linking reaction at a reaction temperature of 20-50° C. for 2-24 hours, and after the reaction, adjusting the pH to 9.2-11.4, placing the solution in an environment of 2-10° C. for a low-temperature cross-linking reaction for 2-48 hours to obtain a single cross-linked hyaluronic acid gel;

[0030] (3) Double cross-linking: dissolve calycosin in a polar solvent to form a phase C solution; add the phase C solution to a single cross-linked hyaluronic acid gel and stir to form a heterogeneous system. Gradually increase the temperature to 20-50°C for 2-24 hours. After reaching the final temperature, maintain the temperature and continue the reaction for 2-6 hours. Stir continuously during the reaction. After the reaction is completed, reduce the system temperature to 2-10°C and continue the low-temperature cross-linking reaction for 2-4 hours to finally form a double cross-linked hyaluronic acid gel.

[0031] Preferably, the specific steps of preparing the phase A solution in step (1) are as follows: the pH of the acidic solution used for the phase A solution is 2.1-4.1, the acidic solution is a hydrochloric acid solution or a phosphoric acid solution, fucoidan is swelled in the acidic solution so that the mass fraction of fucoidan in the phase A solution is 1-20%, the temperature is 15-35°C, the stirring speed is 50-150 rpm, and the stirring is performed for 2-6 hours to obtain the phase A solution.

[0032] Preferably, the specific steps for preparing the phase B solution in step (1) are as follows: hyaluronic acid and its salt are dissolved in purified water, the mass fraction of hyaluronic acid and its salt in the phase B solution is 1-30%, and the molecular weight of hyaluronic acid and its salt is 1 million-2.6 million Daltons.

[0033] Preferably, in step (2), the process of adding the phase A solution to the phase B solution is speed-controlled, and the adding time is controlled within 30 minutes; the pH of the AB phase mixed solution is 2.1-4.1, and the stirring speed of the AB phase mixed solution during the mixing process is 50-150 rpm; the mass fraction of hyaluronic acid and its salts in the AB phase mixed solution is 1-15%.

[0034] Preferably, in step (3), the polar solvent is obtained by mixing an organic solvent, water, ethanol and methanol in a volume ratio of 1:0-10:0-20:0-20, and the organic solvent is one or more of dichloromethane, chloroform, acetone and acetonitrile; the mass fraction of calycosin in the phase C solution is 1-15%;

[0035] In step (3), the pH of the heterogeneous system is 1.5-4.0, and the stirring speed is 50-100 rpm.

[0036] Preferably, the method further comprises (4) washing: washing the prepared double cross-linked hyaluronic acid gel with purified water to obtain a double cross-linked hyaluronic acid gel after impurities are removed, wherein the specific steps are as follows: washing the prepared double cross-linked hyaluronic acid gel with purified water and then standing for 2-12 hours to obtain a washed double cross-linked hyaluronic acid gel, dividing the washed double cross-linked hyaluronic acid gel and placing it in PBS buffer for swelling, wherein the volume ratio of the washed double cross-linked hyaluronic acid gel to the PBS buffer during swelling is 1:5-20, and the PBS buffer is replaced every 2-5 hours until the gel pH is 6.8-7.5 and the osmotic pressure is 280-350 mOsmol / L, thereby obtaining a double cross-linked hyaluronic acid gel with a swelling multiple of 5-20 times.

[0037] Preferably, the method further comprises step (5) wet heat sterilization, which specifically comprises placing the double cross-linked hyaluronic acid gel in a wet heat sterilizer, controlling the temperature at 100-130° C. in a saturated steam state, and wet heat sterilizing for 10-40 minutes.

[0038] An application of a doubly modified cross-linked hyaluronic acid gel, and an application of the doubly cross-linked hyaluronic acid gel prepared by a doubly modified cross-linked hyaluronic acid gel preparation method in the preparation of medical filling materials; or in the preparation of injections; or in the preparation of cross-linked hyaluronic acid gel containing drugs.

[0039] The invention discloses an application of a double-modified cross-linked hyaluronic acid gel, and the specific steps for preparing an injection are as follows: the double-cross-linked hyaluronic acid gel is filled into a pre-filled syringe, and sterilized by wet heat at 100-130° C. for 10-40 minutes in a saturated steam state.

[0040] The invention discloses an application of a double-modified cross-linked hyaluronic acid gel, and the specific steps for preparing the double-cross-linked hyaluronic acid gel containing a drug carrier are as follows: adding the bioactive components of the required concentration according to the actual application requirements during the preparation of the double-cross-linked hyaluronic acid gel, fully mixing and stirring at 20-100 rpm for 0.5-24 hours, so as to form double-cross-linked hyaluronic acid gels containing bioactive components with different encapsulation rates; the bioactive components include small molecule peptides and natural active substances, but are not limited to one or more of the above.

[0041] Beneficial effects of the present invention:

[0042] The present invention provides a preparation method of a cross-linked hyaluronic acid gel double-modified with natural active molecules, wherein a multi-level step-by-step cross-linking method is used to cross-link fucoidan polysaccharide, and at the same time, the fucoidan polysaccharide is cross-linked with hyaluronic acid and its salt to form a composite gel, thereby obtaining a structurally stable composite gel, and then, calycosin isoflavone is introduced, and it is cross-linked with the composite gel to undergo a secondary cross-linking reaction and introduce a non-polar group, so that while forming a primary cross-linked core of fucoidan hyaluronic acid, a fucoidan-hyaluronic acid-calycosin isoflavone structure is formed, and the cross-linked hyaluronic acid gel is hydrophobically modified, and the non-polar group of the calycosin isoflavone is used as a hydrophobic outer layer to form a novel composite cross-linked hyaluronic acid (fucoidan-hyaluronic acid-calycosin isoflavone) gel with a vesicle structure, and the multi-layer cross-linking method can enhance the mechanical properties and stability of the gel. The present invention is cross-linked by natural materials, has excellent biocompatibility, no toxic residue, and obtains a cross-linked hyaluronic acid gel with good biocompatibility, high elastic modulus, strong supporting performance, low water absorption, and excellent resistance to enzymatic hydrolysis through double cross-linking. At the same time, the fucoidan-hyaluronic acid-calycosin structure can form a hydrophilic-hydrophobic configuration to form a vesicle structure, so that the multi-layer cross-linked hyaluronic acid gel can also encapsulate different active products, thereby having a hyaluronic acid gel with multiple biological activities such as anti-oxidation, anti-inflammatory, and anti-fibrosis. The gel has a wide range of application value in the biomedical field.

[0043] In the prior art, the cross-linking temperature is usually fixed within a range (such as high temperature cross-linking or low temperature cross-linking), resulting in the inability to balance the mechanical properties and elasticity of the gel. The gel prepared by high temperature cross-linking (30-60°C) has high hardness but poor elasticity, and the gel prepared by low temperature cross-linking (2-10°C) has good elasticity but low hardness. Unlike traditional hyaluronic acid gel, the present invention does not require the use of any traditional chemical cross-linking agent, avoiding adverse reactions caused by cross-linking agent residues. At the same time, the patent optimizes the elastic modulus and mechanical strength of the gel by combining high temperature cross-linking (20-50°C) with low temperature step-by-step cross-linking (2-10°C) and controlling the cross-linking temperature by gradient temperature rise and fall. High temperature cross-linking increases the hardness of the gel, while low temperature cross-linking enhances the elasticity and density of the gel, and finally obtains a gel with high elasticity, support performance and low water absorption. In terms of biological activity, the vesicle structure can also encapsulate different bioactive small molecules such as antioxidant small molecules, giving the gel antioxidant properties or other biological activities; the fucoidan and calycosin in the gel have natural anti-inflammatory effects; the hydrophobic outer layer of the gel can inhibit fibrosis. In terms of application areas, it can be used as a medical filling material for facial anti-wrinkle, shaping and soft tissue repair; as a sustained-release carrier, it can be used as a drug carrier for anti-inflammatory, antioxidant, anti-fibrosis and other treatments; it can be used in skin care products for the preparation of moisturizing, freckle removal and other skin care products.

[0044] The cross-linked hyaluronic acid gel of the present invention can encapsulate these different bioactive substances by virtue of its vesicle structure, providing an ideal protective barrier for their transport and release in the body. In the field of anti-infection treatment, it can carry active ingredients with anti-inflammatory effects, act on the site of inflammation, slowly release drugs, continuously inhibit inflammatory reactions, and relieve the discomfort and damage caused by inflammation; in terms of antioxidant treatment, by encapsulating antioxidant active small molecules or peptides, it can effectively remove excessive free radicals in the body, protect cells from oxidative stress damage, and delay the aging process of cells; in the field of anti-fibrosis treatment, sustained-release drug carriers can deliver related drugs, inhibit excessive proliferation of fibrous tissue, improve fibrotic lesions of tissues and organs, and restore their normal structure and function.

[0045] The innovative features of the present invention are as follows:

[0046] The present invention discloses a preparation method and application of a hyaluronic acid gel doubly modified with natural active molecules. The method adopts a multi-level double cross-linking method to perform a single cross-linking and cross-linking reaction on brown algae polysaccharide and hyaluronic acid and its salt under a suitable reaction ratio and conditions, and at the same time, the cross-linked hyaluronic acid gel complex after the single cross-linking is subjected to a secondary cross-linking with calycosin under suitable conditions to form a hyaluronic acid cross-linked gel with a multi-layer composite structure.

[0047] 1. Double modified cross-linking structure:

[0048] The present invention constructs a multi-level cross-linked hyaluronic acid gel through dual modification of fucoidan and calycosin. Fucoidan is cross-linked with hyaluronic acid and its salt to form a stable inner core structure, while calycosin is cross-linked with the modified hyaluronic acid after the reaction to form a hydrophobic outer layer, thereby constructing a fucoidan-hyaluronic acid-calycosin cross-linked hyaluronic acid gel with a hydrophilic / hydrophobic vesicle structure.

[0049] This double modification not only enhances the mechanical properties and stability of the gel, but also gives the gel a variety of physical properties and biological activities such as sustained release, antioxidant, anti-inflammatory and anti-fibrosis by introducing new chemical groups. For example, cross-linked hyaluronic acid gel can encapsulate these different bioactive substances by virtue of its vesicle structure, providing an ideal protective barrier for their transportation and release in the body. In the field of anti-inflammatory treatment, it can carry active ingredients with anti-inflammatory effects, act on the site of inflammation, slowly release drugs, continuously inhibit inflammatory responses, and relieve the discomfort and damage caused by inflammation; in terms of antioxidant therapy, by encapsulating antioxidant active small molecules or peptides, it can effectively remove excessive free radicals in the body, protect cells from oxidative stress damage, and delay the process of cell aging; in the field of anti-fibrosis treatment, sustained-release drug carriers can deliver related drugs, inhibit excessive proliferation of fibrous tissue, improve fibrotic lesions of tissues and organs, and restore their normal structure and function.

[0050] 2. No chemical cross-linking agent:

[0051] The present invention does not require the use of chemical cross-linking agents (such as BDDE or DVS). Through the cross-linking reaction of natural materials (fucoidan and calycosin), a high cross-linking degree and excellent biocompatibility of the gel are achieved, avoiding skin allergies and adverse reactions caused by cross-linking agent residues.

[0052] 3. Multi-level cross-linking method:

[0053] The present invention adopts a method combining high temperature (20-50°C) and low temperature (2-10°C) cross-linking, and optimizes the elastic modulus and mechanical strength of the gel by controlling the gradient change process of the cross-linking temperature. Under high temperature conditions, the cross-linking reaction is accelerated, resulting in an increase in the hardness of the gel; while at low temperatures, the cross-linking network is more uniform, enhancing the elasticity and density of the gel. The gel finally prepared exhibits excellent high elasticity, support performance and low water absorption. This method can be widely used in the development of gel materials that require specific mechanical properties.

[0054] 4. Formation of vesicle structure:

[0055] Through the different polarities of fucoidan and calycosin, active groups were introduced into hyaluronic acid in stages, forming a vesicle structure with hydrophilic and hydrophobic properties. This structure enables the gel to effectively encapsulate and protect a variety of active small molecules or bioactive peptides, thus significantly expanding its application potential in biomedical fields such as drug delivery, tissue engineering and regenerative medicine.

[0056] The following table is used to illustrate the comparison with the prior art, see Table 1 for details.

[0057] Table 1 Differences between the prior art and the present invention

[0058] BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is the H&E staining picture of skin tissue in the rat skin irritation experiment of the double-modified cross-linked hyaluronic acid gel of the present invention. DETAILED DESCRIPTION

[0060] A method for preparing a double-modified cross-linked hyaluronic acid gel, comprising the following steps:

[0061] Dissolution: Dissolve fucoidan in an acidic solution with a pH of 2.1-4.1, so that the mass fraction of fucoidan in the acidic solution is 1-20%, the temperature is 15-35°C, and the stirring speed is controlled at 50-150 rpm for 2-6 hours until it is uniformly stirred to form phase A; hyaluronic acid and its salts (1 million-2.6 million Daltons) are dissolved in purified water for use as phase B, and the mass fraction of hyaluronic acid and its salts is 1-30%.

[0062] Single cross-linking: slowly add phase A to the fully swollen hyaluronic acid phase B solution and mix it with the mixture, so that the mass fraction of hyaluronic acid and its salt in the mixed solution is 1-15%; the pH of the reaction system is 2.1-4.1, the stirring speed is controlled at 50-150 rpm, and the mixture is fully stirred and evenly cross-linked for 2-24 hours. The reaction temperature is 20-50°C in an environment with a gradient temperature increase and a thermal cross-linking reaction. After the reaction is completed, the pH is adjusted to 9.2-11.4, and the mixture is placed in an environment of 2-10°C for a secondary low-temperature cross-linking reaction, which is cross-linked for 2-48 hours to obtain a single cross-linked gel.

[0063] Double cross-linking: dissolve calycosin in a polar solvent with a mass fraction of 1-15% to form a C phase solution, slowly add the C phase solution into a single cross-linked gel, the pH of the reaction system is 1.5-4.0, to form a heterogeneous phase system, control the stirring speed at 50-100 rpm, stir thoroughly and evenly, adopt a gradient heating thermal cross-linking reaction, the final reaction temperature is 20-50°C, the gradient heating time is 2-24 hours, after reaching the terminal temperature, continue to react for 2-6 hours, cool to 2-10°C, continue to cross-link for 2-4 hours to form a double cross-linked hyaluronic acid gel.

[0064] Cleaning: Wash the prepared double cross-linked hyaluronic acid with purified water, let it stand for 2-12 hours after washing, divide the gel into small pieces, and put it into PBS buffer again to swell, and replace the PBS buffer every 2-5 hours until the gel pH is 6.8-7.5 and the osmotic pressure is 280-350mOsmol / L, to obtain a cross-linked hyaluronic acid gel with a swelling multiple of 5-20 times.

[0065] Moist heat sterilization: Place the cleaned gel in a moist heat sterilizer, control the temperature at 100-130°C, and sterilize for 10-40 minutes. Based on the above technical solution, the volume ratio of the gel to the PBS buffer during swelling is 1:5-20, and the PBS buffer is replaced every 2-5 hours until the gel pH is 6.8-7.5 and the osmotic pressure is 280-350 mOsmol / L, and a hyaluronic acid gel with a swelling multiple of 5-20 times is obtained.

[0066] The purpose of replacing the PBS buffer is to make the gel absorb water and swell, and to adjust the pH and osmotic pressure of the gel. The volume ratio of the gel to the PBS buffer used in Examples 1-5 of the present invention and the comparative example during swelling is 1:10, and the PBS buffer is replaced every 3 hours until the pH of the gel is 6.8-7.5 and the osmotic pressure is 280-350 mOsmol / L. The embodiments can also use a volume ratio of the gel to the PBS buffer during swelling of 1:20, 1:5, 1:8; the interval time for replacing the PBS buffer is 2, 2.5, 3.5, 4, 4.5, 5 hours, etc., and is not limited to the above ratios and values.

[0067] The PBS buffer used in Examples 1-5 and Comparative Examples of the present invention is a buffer salt system with a pH value of 7.2 (in 1 L of deionized water: sodium chloride (NaCl), 8.0 g; potassium dihydrogen phosphate ( ), 0.24 g; disodium hydrogen phosphate ( ), 1.44 g; potassium chloride (KCl), 0.2 g)

[0068] The polar solvent used in Examples 1-5 of the present invention and the comparative example is a mixed solution of dichloromethane and ethanol (dichloromethane solution) with a volume ratio of 3: 0.5. The polar solvent that can also be used in the embodiment is a mixed solution of dichloromethane, water, ethanol, and methanol with a volume ratio of 1:10:20:20; the polar solvent that can also be used in the embodiment is a mixed solution of dichloromethane and chloroform, water, ethanol, and methanol with a volume ratio of 1:10:20:20; the polar solvent that can also be used in the embodiment is only an acetone solution, and is not limited to the above ratios, values, and components.

[0069] Embodiment 1:

[0070] 5.4g of fucoidan was swollen in 55g of HCl solution with a pH of 2.1, the temperature was 15°C, the speed was controlled and stirred at 150 rpm, and stirred for 3 hours. 10g of hyaluronic acid and its salt were dissolved in 35g of purified water. Fucoidan was slowly added to hyaluronic acid and mixed, the pH of the reaction system was 2.1, the speed was controlled and stirred at 50 rpm, and the mixture was fully stirred and cross-linked for 24 hours. The reaction temperature was 50°C, and the temperature was raised within 2 hours to perform a thermal cross-linking reaction. After the thermal cross-linking reaction was completed, the pH was adjusted to 9.2, and the mixture was placed in a 2°C environment for 5 minutes for a secondary low-temperature cross-linking reaction. The reaction was cross-linked for 36 hours, 4.8g of calycosin was dissolved in 48g of dichloromethane solution, and the calycosin solution was slowly added. The pH of the reaction system was 1.5, the speed was controlled and stirred at 50 rpm, the temperature was raised to 50°C within 3 hours, and the reaction was continued for 2 hours, and the temperature was lowered to 2°C within 10 minutes, and the reaction was continued for 2 hours. The prepared double cross-linked hyaluronic acid gel was washed with purified water and allowed to stand for 12 hours. The gel was divided into small pieces and placed in PBS buffer again for swelling.

[0071] Embodiment 2:

[0072] 8.4g of fucoidan was swollen in 42g of HCl solution with a pH of 4.1, the temperature was 15°C, the speed was controlled and stirred at 50 rpm, and stirred for 3 hours. 10g of hyaluronic acid and its salt were dissolved in 35g of purified water. Fucoidan was slowly added to hyaluronic acid and mixed, the pH of the reaction system was 4.1, the speed was controlled and stirred at 50 rpm, and the mixture was fully stirred. The cross-linking reaction took 2 hours, the reaction temperature was 20°C, and the heat cross-linking reaction was carried out in an environment where the temperature was raised within 1.5 hours. After the reaction, the pH was adjusted to 11.4, and the secondary low-temperature cross-linking reaction was carried out in a 10°C environment for 30 minutes. The cross-linking reaction lasted for 36 hours. 8.9g of calycosin was dissolved in 62.3g of dichloromethane solution, and the calycosin solution was slowly added. The pH of the reaction system was 4.0, the speed was controlled and stirred at 75 rpm, the temperature was raised to 20°C within 3 hours, and then the reaction was carried out for 6 hours, and the temperature was lowered to 10°C within 10 minutes, and then the reaction was carried out for 4 hours. The prepared double cross-linked hyaluronic acid gel was washed with purified water and allowed to stand for 12 hours. The gel was divided into small pieces and placed in PBS buffer again for swelling.

[0073] Embodiment 3:

[0074] 6.7g of fucoidan was swollen in 65.23g of HCl solution with a pH of 4.1, the temperature was 25°C, the speed was controlled and stirred at 75 rpm, and stirred for 3 hours. 9g of hyaluronic acid and its salt were dissolved in 46.77g of purified water. Fucoidan was slowly added to hyaluronic acid and mixed, the pH of the reaction system was 3.2, the speed was controlled and stirred at 50 rpm, and the mixture was fully stirred. The cross-linking reaction lasted for 24 hours, the reaction temperature was 45°C, and the heat cross-linking reaction was carried out in an environment where the temperature was raised within 1.5 hours. After the reaction, the pH was adjusted to 10.0, and the solution was placed in a 6°C environment for a secondary low-temperature cross-linking reaction within 2 hours. The cross-linking reaction lasted for 48 hours. 6.4g of calycosin was dissolved in 43.12g of dichloromethane solution, and the calycosin solution was slowly added. The pH of the reaction system was 3.0, the speed was controlled and stirred at 100 rpm, the temperature was raised to 50°C within 24 hours, and then the reaction was carried out for 2 hours, and the temperature was lowered to 10°C within 1 hour, and then the reaction was carried out for 4 hours. The prepared double cross-linked hyaluronic acid gel was washed with purified water and allowed to stand for 12 hours. The gel was divided into small pieces and placed in PBS buffer again for swelling.

[0075] Embodiment 4:

[0076] 10g of fucoidan was swollen in 61.23g of HCl solution with a pH of 2.1, the temperature was 20°C, the speed was controlled and stirred at 100 rpm, and stirred for 3 hours. 8g of hyaluronic acid and its salt were dissolved in 32g of purified water. Fucoidan was slowly added to hyaluronic acid and mixed, the pH of the reaction system was 2.5, the speed was controlled and stirred at 50 rpm, and the mixture was fully stirred and cross-linked for 24 hours. The reaction temperature was 30°C, and the temperature was raised within 3 hours to perform a thermal cross-linking reaction. After the thermal cross-linking reaction was completed, the pH was adjusted to 9.2, and the mixture was placed in a 2°C environment for 10 minutes for a secondary low-temperature cross-linking reaction. The reaction was cross-linked for 36 hours, 5g of calycosin was dissolved in 50g of dichloromethane solution, and the calycosin solution was slowly added. The pH of the reaction system was 3.3, the speed was controlled and stirred at 60 rpm, the temperature was raised to 40°C within 2 hours, and then the reaction was performed for 3 hours. The temperature was lowered to 5°C within 20 minutes, and then the reaction was performed for 3 hours. The prepared double cross-linked hyaluronic acid gel was washed with purified water and allowed to stand for 12 hours. The gel was divided into small pieces and placed in PBS buffer again for swelling.

[0077] Embodiment 5:

[0078] 1g of fucoidan was swollen in 18.3g of HCl solution with a pH of 4.0, the temperature was 15°C, the speed was controlled and stirred at 75 rpm, and stirred for 3 hours. 7g of hyaluronic acid and its salt were dissolved in 30.43g of purified water. Fucoidan was slowly added to hyaluronic acid and mixed, the pH of the reaction system was 4.0, the speed was controlled and stirred at 50 rpm, and the mixture was fully stirred. The cross-linking reaction lasted for 24 hours, the reaction temperature was 25°C, and the temperature was raised within 1 hour, and a thermal cross-linking reaction was performed. After the thermal cross-linking reaction was completed, the pH was adjusted to 11.0, and the mixture was placed in a 10°C environment for 15 minutes for a secondary low-temperature cross-linking reaction, and the cross-linking reaction lasted for 24 hours. 9g of calycosin was dissolved in 81.8g of dichloromethane solution, and the calycosin solution was slowly added. The pH of the reaction system was 2.8, the speed was controlled and stirred at 100 rpm, the temperature was raised to 30°C within 4 hours, the reaction was performed for 2 hours, the temperature was lowered to 8°C within 30 minutes, and the reaction was performed for 3 hours. The prepared double cross-linked hyaluronic acid gel was washed with purified water and allowed to stand for 12 hours. The gel was divided into small pieces and placed in PBS buffer again for swelling.

[0079] Comparative Example 1:

[0080] Accurately weigh 5g of hyaluronic acid and dissolve it in 200ml of purified water. To ensure the stability of the swelling process, set the temperature to 25℃ and control the stirring speed at 100 rpm through the speed-controlled stirring device. Allow the hyaluronic acid to swell fully to reach the state required for the subsequent reaction.

[0081] After the swelling is completed, adjust the pH value of the reaction system to 9.1. At this time, adjust the stirring speed to 50 rpm and slowly add 10 ml of BDDE (1,4-butanediol diglycidyl ether). Allow the reaction to continue for 12 hours under room temperature. During this process, BDDE, as a cross-linking agent, undergoes a cross-linking reaction with hyaluronic acid to form a cross-linked hyaluronic acid gel. After the reaction, in order to remove unreacted substances and impurities, the prepared cross-linked hyaluronic acid gel is carefully washed with purified water. After washing, let it stand for 12 hours to allow the system to reach a relatively stable state. Subsequently, the gel is divided into small pieces, and these small pieces of gel are again placed in PBS buffer for swelling, so as to further observe and analyze its performance characteristics.

[0082] The cross-linked hyaluronic acid gel was synthesized using the traditional cross-linking agent BDDE, and the performance was compared with the samples of the examples of the present application to investigate the effects of different preparation methods and conditions on the performance of the cross-linked hyaluronic acid gel.

[0083] Comparative Example 2:

[0084] This comparative example uses a commercially available cross-linked hyaluronic acid gel injection finished product as the research object. The content of the finished injection is 20 mg / ml, and its main components are cross-linked hyaluronic acid gel, lidocaine and purified water. Among them, the cross-linked hyaluronic acid gel is the key component that gives the product specific properties, lidocaine plays a role in local anesthesia, and purified water as a solvent ensures the uniform dispersion of each component.

[0085] The commercially available sample is a single cross-linked hyaluronic acid gel, and the cross-linking agent used is butanediol diglycidyl ether (BDDE). It provides us with a reference standard for existing products on the market, which helps us to more clearly evaluate the effects of different preparation methods and conditions on the properties of cross-linked hyaluronic acid gel.

[0086] Comparative Example 3:

[0087] In this comparative example, Example 1 was also used as a comparison basis, the material ratio and pH value conditions were kept unchanged, and only the cross-linking temperature and gradient temperature control were investigated, and the relevant performance indicators of the gel were determined. In this comparative example, 5.4 g of fucoidan was swollen in 55 g of HCl solution with a pH of 2.1, the temperature was 15°C, the speed was controlled and stirred at 150 rpm, and stirred for 3 hours, and 10 g of hyaluronic acid and its salt were dissolved in 35 g of purified water. Slowly add fucoidan to hyaluronic acid and mix, the pH of the reaction system is 2.1, control the stirring speed at 50 rpm, stir thoroughly and evenly, directly crosslink for 24 hours at a reaction temperature of 50°C, and after the thermal crosslinking reaction is completed, no cooling process is performed, the pH is adjusted to 9.2, and a secondary crosslinking reaction is directly performed for 36 hours. Dissolve 4.8g of calycosin in 48g of dichloromethane solution, slowly add the calycosin solution, the pH of the reaction system is 1.5, control the stirring speed at 50 rpm, and directly react for 2 hours at 50°C. Wash the prepared double crosslinked hyaluronic acid gel with purified water, let it stand for 12 hours after washing, divide the gel into small pieces, and put it into PBS buffer again for swelling.

[0088] Used for comparative study with the examples, to examine the effects of cross-linking temperature and non-gradient temperature change on the properties of cross-linked hyaluronic acid gel under specific high and low temperature conditions.

[0089] Example Sample Quality Control and Standardization:

[0090] Batch consistency test: The consistency test is performed on different batches of gels. See Table 2 to ensure that the physicochemical properties and biological characteristics of each batch of products meet the standards.

[0091] Table 2 Quality evaluation results of different batches of double-modified cross-linked hyaluronic acid

[0092]

[0093] Results: The samples of the embodiments were tested according to international standards. The results showed that the samples of the embodiments met the international quality inspection standards or product nominal content in terms of pH value, viscosity, particle size distribution, heavy metal content, bacterial endotoxin limit and other testing items.

[0094] Performance and efficacy evaluation experiments of comparative examples and example samples:

[0095] 1. Evaluation of elastic modulus in mechanical strength of different samples

[0096] The elastic modulus was measured by using a rheometer at a detection temperature of 24° C. and a setting frequency of 10 rads / sec to measure the elastic modulus of the gel.

[0097] Experimental results:

[0098] The experimental results (see Table 3) show that compared with Comparative Examples 1 and 2, the storage modulus of the embodiment is significantly improved, and the complex modulus is also higher than that of Comparative Examples 1 and 2. At the same time, compared with Comparative Example 3, the storage modulus and complex modulus of the embodiment implementing high and low temperature and gradient temperature control are also higher than those of Comparative Example 3. Through the rheometer test, the double cross-linked hyaluronic acid gel of the present invention exhibits excellent elastic modulus, indicating that it has high mechanical strength and elasticity, and is suitable as a medical filling material.

[0099] Table 3. Differences in elastic modulus of different samples

[0100]

[0101] In general, the example samples are better than the comparative example samples in terms of storage modulus, loss modulus and complex modulus. Compared with the comparative example samples, the example samples have better elasticity and ability to resist deformation.

[0102] , Evaluation of enzymatic hydrolysis time of different samples

[0103] To evaluate the enzymatic degradation performance of the gel, hyaluronidase was used to carry out the degradation experiment. 5 g of cross-linked hyaluronic acid gel was accurately weighed and placed in a specific reaction system. 30 mL of hyaluronidase gel solution with a concentration of 200 U / mL was added to the system, and 20 mL of PBS buffer with a pH of 7.0 was added at the same time. After that, the reaction system was placed in a constant temperature environment of 37°C for enzymatic hydrolysis, and the enzymatic hydrolysis time was set to 12 hours.

[0104] After the 12h enzymatic hydrolysis reaction was completed, the reaction mixture was taken out for centrifugation and the supernatant was taken. 30mL PBS buffer was added to the supernatant and the content of glucuronic acid therein was determined by the carbazole method. The content of degraded hyaluronic acid gel was calculated by the formula, and the ratio of the content of degraded hyaluronic acid gel to the content of undegraded hyaluronic acid gel was used as the degradation rate. The specific experimental data are shown in the following table:

[0105] Experimental results:

[0106] The results (see Table 4) show that under the same degradation conditions, the degradation rates of the gels in Examples 1-5 are significantly lower than those in Comparative Examples 1 and 2. This data difference fully demonstrates that the dual modification of fucoidan and calycosin effectively prolongs the degradation time of the gel and significantly improves the stability of the gel in an enzymatic hydrolysis environment. At the same time, compared with Comparative Example 3, the embodiments implementing high and low temperature and gradient temperature control have better degradation resistance than the comparative example.

[0107] Table 4. Differences in degradation rates of different samples

[0108]

[0109] The data in Table 4 show that the degradation rates of the comparative samples are generally higher than those of the example samples, among which the degradation rates of comparative examples 1 and 2 are significantly higher than those of all samples, while the degradation rates of examples 4 and 5 are significantly lower than those of all samples. The example samples may have more stable components or structures, thereby reducing the degradation rates.

[0110] , Evaluation of swelling results of different samples

[0111] The specific test method of swelling degree is carried out in accordance with the industry standard YY / T 0962-2014 "Cross-linked Sodium Hyaluronate Gel for Plastic Surgery". Accurately weigh about 0.2g of cross-linked hyaluronic acid gel and place it on two clean culture dishes respectively. Then, put the two culture dishes together in a drying oven, set the drying oven temperature to 80℃, and accurately weigh the gel after the gel reaches a constant weight state at this temperature. The weight obtained is recorded as M1. Then, add water drop by drop to the gel until the gel is fully expanded. After the expansion is completed, carefully remove the excess water and weigh the gel again. The weight at this time is recorded as M2. Finally, calculate the swelling degree according to the following swelling degree formula: [Swelling degree calculation formula] = Swelling rate (%) = (M2-M1) / M1x100%.

[0112] Experimental results:

[0113] The experimental results (see Table 5) show that: It can be seen from the above experimental results that: under the same experimental conditions, compared with Comparative Examples 1 and 2, the swelling degrees of the gels in Examples 1-5 are significantly lower than those in the Comparative Examples. The swelling test results further show that the swelling degree of the gel prepared by the present invention is low. This characteristic shows that the gel has good stability and excellent anti-swelling properties, and can maintain the stability of its own structure and performance for a long time, so it is suitable for use as a long-term implant material, providing a reliable product choice for related medical applications. At the same time, compared with Comparative Example 3, the embodiment implementing high and low temperature and gradient temperature control has better anti-swelling properties than the comparative example.

[0114] Table 5. Differences in swelling degree of different samples

[0115]

[0116] Performance comparison study results analysis:

[0117] The comparative samples are respectively the traditional cross-linked hyaluronic acid gel prepared by using the traditional chemical cross-linking agent BDDE (Comparative Example 1); the commercially available finished cross-linked hyaluronic acid gel (Comparative Example 2); the cross-linked hyaluronic acid gel prepared without using the gradient temperature control reaction and deleting some patent synthesis steps (Comparative Example 3); and the double cross-linked hyaluronic acid gel prepared strictly in accordance with the scope of patent rights (Examples 1-5); the detection and evaluation indicators include the following elastic properties, swelling degree, and enzymatic degradation rate. The following items are used to provide a comparative benchmark for the cross-linked hyaluronic acid gel prepared by the traditional process and commercially available samples for the present application, and verify the effectiveness, feasibility and superiority of the present application method. Through comparative analysis, it is evaluated whether the present application method meets the market standards, and the process is optimized to improve product performance.

[0118]

[0119] Result analysis:

[0120] 1. Viscoelastic properties:

[0121] Elastic modulus and storage modulus are one of the important indicators for evaluating the elastic deformation and viscosity of cross-linked hyaluronic acid gel. This study shows that the self-assembled novel double cross-linked hyaluronic acid gel using fucoidan, calycosin and hyaluronic acid has a significant improvement in elastic modulus-related indicators compared with commercially available cross-linked hyaluronic acid gels. Compared with Comparative Examples 1 and 2, the storage modulus of the embodiment is significantly improved, and the complex modulus is also higher than that of Comparative Examples 1 and 2. The results show that the double cross-linked hyaluronic acid gel of the present invention has good mechanical strength and elasticity, and is suitable as a medical filling material.

[0122] Precise temperature control plays an important role in the present invention. Temperature control directly affects the arrangement of molecular chains and the change of cross-linking state, and also affects viscosity and elasticity. Under high temperature conditions, the cross-linking reaction is accelerated, resulting in rapid generation of molecular networks and increased gel hardness; while at low temperatures, the cross-linked network is more uniform, which enhances the elasticity and density of the gel. The reaction temperature of Example 3 is non-gradient temperature change, and there is no low-temperature cross-linking reaction process, which causes its gel internal structure to be different from that of Example 1. Under the same test conditions, the viscoelasticity of Example 3 is significantly different from that of the present application. Compared with Example 3, the cross-linked hyaluronic acid gel prepared in Example 1, which implements high and low temperature and gradient temperature control, has a significantly higher storage modulus and complex modulus than that of Example 3, and its loss modulus is significantly lower than that of Example 3. Cross-linking temperature and non-gradient temperature change have a significant effect on the physicochemical properties and biocompatibility of the gel. Although Example 3 uses fucoidan and calycosin to cross-link hyaluronic acid, the elastic modulus of the synthetic sample is quite different from that of the embodiment. The experimental results provide an important reference for further studying the relationship between gel properties and cross-linking temperature, and help optimize the gel preparation process to obtain products with more ideal performance.

[0123] Swelling degree:

[0124] The swelling degree of cross-linked sodium hyaluronate gel is related to the three-dimensional grid of cross-linked sodium hyaluronate gel and the cross-linking degree of cross-linked hyaluronic acid gel. Sodium hyaluronate gels with different cross-linking degrees will have different degrees of volume increase after absorbing liquid. Sodium hyaluronate gels with higher cross-linking degrees have a tighter network structure and may have a lower swelling degree because liquid molecules are more difficult to penetrate into their tight structure. On the contrary, sodium hyaluronate gels with lower cross-linking degrees have a looser network structure and may have a higher swelling degree because liquid molecules are more likely to penetrate and fill the gaps in their structure. This study shows that the self-assembled new cross-linked hyaluronic acid using fucoidan, calycosin and hyaluronic acid has better low swelling than commercially available cross-linked hyaluronic acid gels, indicating that the degree of cross-linking is better and the spatial molecules are tighter. At the same time, temperature control is very important for this application. Through gradient temperature control, the swelling degree of the synthesized embodiments is lower than that of Comparative Example 3 in which gradient temperature control is not implemented. It can be seen that the cross-linking temperature and non-gradient temperature change have a significant effect on the hydrophilicity of the gel. The special temperature history of Comparative Example 3 changed the chemical structure of the surface and interior of the gel, thereby affecting its ability to interact with water.

[0125] , enzyme degradation

[0126] Enzymatic hydrolysis of cross-linked hyaluronic acid gel is a process of decomposing cross-linked hyaluronic acid gel into small molecular fragments through the action of specific enzymes. Appropriate enzymatic hydrolysis ability is an important parameter of cross-linked hyaluronic acid. Enzymatic hydrolysis has a significant impact on the quality of cross-linked hyaluronic acid gel, mainly reflected in molecular weight, mechanical properties, biological activity and degradation rate. Enzymatic hydrolysis weakens or destroys the cross-linked network, reducing the mechanical strength and elasticity of the material. Therefore, having a certain degree of resistance to enzymatic hydrolysis is a key parameter of cross-linked hyaluronic acid gel.

[0127] This study shows that the self-assembled novel double cross-linked hyaluronic acid gel using fucoidan, calycosin and hyaluronic acid has similar results with the commercially available cross-linked hyaluronic acid gel in terms of elastic modulus and storage modulus. At the same time, compared with Comparative Examples 1 and 2, under the same enzymatic conditions, the enzymatic resistance of the embodiment is higher than that of Comparative Examples 1 and 2. The double cross-linked hyaluronic acid gel of the present invention has good enzymatic resistance, and its ability to resist the decline in mechanical properties caused by hyaluronidase and its action time to maintain its filling capacity in vivo are more durable, and it is suitable as a medical filling material. At the same time, through gradient temperature control, the enzymatic ability of the synthesized embodiment is also higher than that of Comparative Example 3, which does not implement gradient temperature control and high and low temperature synthesis. It can be seen that the cross-linking temperature and non-gradient temperature change also have a significant effect on the gel's enzymatic resistance.

[0128] Application Examples

[0129] Application Example 1 Application of hyaluronic acid gel for preparing injections

[0130] Hyaluronic acid gel is of great significance in the preparation of injections, providing a convenient and effective dosage form for clinical treatment and beauty. The specific steps are: First, carefully fill the hyaluronic acid gel that has undergone strict quality inspection into a prefilled syringe. Prefilled syringes have many advantages. They can minimize the contact between the drug and the external environment, reduce the risk of contamination, and provide great convenience for clinical use. The filling process needs to be carried out in a strict sterile environment to ensure the quality and safety of the product.

[0131] After filling, the prefilled syringe containing the cross-linked hyaluronic acid gel is placed in a specific sterilization device and treated with moist heat sterilization at a temperature of 100-130°C. Moist heat sterilization is an efficient and reliable sterilization method that uses the latent heat and moisture of high-temperature steam to quickly kill microorganisms and ensure the sterility of the product. The sterilization time is controlled at 10-40 minutes. This time period has been verified by a large number of experiments. It can not only effectively kill various possible microorganisms, but also maintain the chemical stability and biological activity of the hyaluronic acid gel to the greatest extent, thereby ensuring that the final prepared injection is of stable quality, safe and effective. After such a series of rigorous operating procedures, a cross-linked hyaluronic acid gel injection that meets the quality standards is finally obtained.

[0132] Application Example 2 Application of hyaluronic acid gel in the preparation of medical filling materials

[0133] In the field of medical cosmetology and repair, cross-linked hyaluronic acid gel has shown great application potential in the preparation of medical filling materials due to its remarkable properties. The cross-linked hyaluronic acid in this application showed excellent viscoelasticity and moderate hydrophilicity in basic physical and chemical property tests. Theoretically, excellent viscoelasticity can enable the filling material to better maintain its shape after being injected into the human body, and it is not easy to deform and shift, thereby ensuring the durability of the filling effect; moderate hydrophilicity helps to achieve good integration with surrounding tissues, reduce the occurrence of rejection reactions, and ensure the biocompatibility of the material in filling applications.

[0134] Cross-linked hyaluronic acid gel can provide cells with a suitable growth environment and promote normal cell growth and metabolism, which indicates that it will not have obvious toxic effects on cells. In animal experiments, no obvious inflammatory response or tissue damage was observed after the cross-linked hyaluronic acid gel was implanted into the animal body, further verifying its safety.

[0135] Therefore, in actual application, the new double cross-linked hyaluronic acid gel synthesized in this application can be used for the repair, filling and shaping of human tissues under the conventional properties, and is expected to improve tissue defects or appearance problems caused by diseases, trauma or congenital factors. Through specific process and formula adjustments, cross-linked hyaluronic acid gel can be processed into medical filling materials suitable for different parts and needs. For example, in facial beauty, it can be used for fine filling of facial contours, such as lip augmentation, rhinoplasty, filling of nasolabial folds, etc., to help shape a more three-dimensional and youthful facial appearance; in soft tissue repair, it can be used to repair soft tissue defects caused by trauma or surgery, and promote tissue healing and regeneration. Based on the above characteristics and experimental basis, cross-linked hyaluronic acid gel has broad application prospects as a medical filling material.

[0136] Application Example 3: Application of cross-linked hyaluronic acid gel containing drug

[0137] In the fields of drug delivery and tissue engineering, the preparation of cross-linked hyaluronic acid gel containing drugs has important research and application value. Small molecule peptides are short-chain compounds composed of 2-20 amino acids connected by peptide bonds, and their molecular weight is usually less than 1000 Da. They have the characteristics of high biological activity, easy absorption, and low immunogenicity. They are widely used in the fields of medicine, cosmetics, food, etc. Combining with hyaluronic acid can give the cross-linked product better performance.

[0138] The following is an example of protein peptides. Preparation method of drug-loaded double cross-linked hyaluronic acid gel:

[0139] The protein polypeptide (molecular weight 500-1000 Da) was dissolved in deionized water to prepare an aqueous phase solution with a mass fraction of 2.5%, and stirred at 4°C for 2 hours until completely dissolved. According to the method described in Example 1 of the patent, calycosin isoflavone was dissolved in the polar solution to form a stable dispersed phase; in a constant temperature reactor at 25°C, the protein polypeptide aqueous phase and the calycosin isoflavone dispersed phase were mixed with a pre-cross-linked hyaluronic acid gel at a volume ratio of 1:0.2; after physical blending was completed by stirring at 150 rpm for 4 hours. Continue to implement according to Example 1. After the synthesis is completed, the gel is transferred to a dialysis bag with a molecular weight cutoff of 3.5 kDa, and dialyzed with ultrapure water for 48 hours (liquid change every 12 hours) to remove the unencapsulated protein polypeptide. The double cross-linked hyaluronic acid gel not only retains the original excellent properties of the hyaluronic acid gel, but also has more unique functions due to the introduction of functional protein polypeptides, including new biological activities and drug loading capacity, which lays a solid foundation for its wide application in the biomedical field.

[0140] This application example investigates the encapsulation effect of the gel on active substances (protein peptides) through encapsulation experiments. Using the dynamic dialysis method, 1.00 g of gel was accurately weighed and placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa. The bag was oscillated at 37°C (100 rpm) and 1 mL was sampled every 24 hours to determine the protein peptide concentration in the dialysate and draw a cumulative release curve.

[0141] According to the concentration of free protein polypeptides and the ratio of total protein polypeptides, the encapsulation rate can be calculated. The formula for calculating the encapsulation rate is: Encapsulation rate (%) = (1-free active ingredient concentration / total active ingredient concentration) × 100%. By this formula, the encapsulation effect of the gel on the active substance is evaluated. The protein polypeptide content is detected by GB 5009.5-2016 Kjeldahl nitrogen determination method. The experimental results show that: from the above experimental results, it can be seen that under the same experimental conditions, compared with Examples 1-5, the encapsulation rates in Comparative Examples 1 and 2 are only 12.4% and 8.6%. The encapsulation rate test results further show that the gel prepared by the present invention can wrap the protein polypeptide well. This characteristic shows that the gel can slowly release the protein polypeptide in the body through enzymatic hydrolysis in the body, achieving a sustained release effect, thereby slowly releasing the protein polypeptide in the skin tissue to exert its biological activity. At the same time, since Comparative Example 3 does not adopt steps such as temperature control reaction, its encapsulation rate is lower than that of Examples 1-5.

[0142] Table 6. Differences in encapsulation efficiency of different samples

[0143]

[0144] Application Example 4 Biocompatibility and Safety Evaluation

[0145] Biocompatibility and safety are key indicators for measuring whether medical materials can be successfully applied to the human body. For the hyaluronic acid gel and related products involved in the above application examples, skin irritation tests and sensitization tests were carried out. According to the standard experimental method, a certain amount of hyaluronic acid gel of Example 1 was injected into the skin tissue of rats. The results of skin tissue staining showed that there was no obvious inflammatory infiltration reaction in the skin tissue, the tissue structure was intact and the stratum corneum was intact, indicating that the hyaluronic acid gel did not damage the barrier function of the skin; the granular layer and the germinal layer cells were clearly arranged, without edema and arranged tightly, indicating that the normal physiological function of the cells was not disturbed, and the structure and function between cells remained good; the structural unit morphology in the dermis was clear, the structure was continuous, and there was no inflammatory cell aggregation and edema reaction. After evaluation and data analysis, the results showed that the hyaluronic acid gel of the present invention was non-irritating to the skin, indicating that the hyaluronic acid gel did not induce a local inflammatory reaction and had no adverse effects on the tissue structure and cell environment of the dermis.

[0146] The experimental results show that the hyaluronic acid gel of the present invention does not cause any obvious inflammatory reaction or damage when implanted in experimental animals, thus providing a solid safety foundation for its application in the medical field.

[0147] like Figure 1 As shown in the results of the rat skin irritation test and sensitization test, the results of H&E staining of the skin tissue show that: normal skin tissue is closely arranged, the structural unit morphology in the dermis is clear, the structure is continuous, and there is no inflammatory cell aggregation and edema reaction; after 2 weeks and 8 weeks of injection of the cross-linked hyaluronic acid gel of the embodiment of this patent, there is no edema in the rat skin tissue, the structural unit morphology in the dermis is clear, and there is no obvious inflammatory cell aggregation and edema reaction, indicating that the hyaluronic acid gel of the present invention and its related products have good biocompatibility and safety, making the application of the product in the medical field more reliable and safe, and providing a strong basis for clinicians to choose suitable medical materials. With the continuous improvement of the requirements for material safety and effectiveness in the biomedical field, the good performance of the hyaluronic acid gel of the present invention and its related products is expected to bring new development opportunities to this field and promote the development and application of more innovative medical technologies.

[0148] Application prospects of the present invention

[0149] 1. Medical filling materials:

[0150] The hyaluronic acid gel of the present invention has excellent mechanical properties, low water absorption, sustained release and biocompatibility, and is suitable as a subcutaneous injection filling material for facial anti-wrinkle, body shaping and soft tissue repair. Its characteristic of no chemical cross-linking agent reduces the risk of allergies and adverse reactions and has broad market prospects.

[0151] 2. Drug carrier:

[0152] Because the gel has a vesicle structure, it can encapsulate a variety of active small molecules or bioactive peptides and can be used to prepare sustained-release drug carriers for use in anti-inflammatory, antioxidant, anti-fibrosis and other therapeutic fields.

[0153] 3. Skin care products:

[0154] The gel of the present invention can be used in combination with additives such as moisturizing and freckle removal to prepare various skin care products (such as lotions, creams, gels, etc.) for improving skin texture, moisturizing and repairing.

[0155] 4. Cosmetic injections:

[0156] The gel of the present invention can be directly used for cosmetic injection in the form of a prefilled syringe, has the characteristics of simple operation and lasting effect, and is expected to become a new generation of cosmetic injection material.

[0157] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for preparing a double-modified cross-linked hyaluronic acid gel, characterized in that: Here are the steps: (1) Dissolution: Fucoidan is swelled in an acidic solution to form a phase A solution; uncross-linked hyaluronic acid and its salts are dissolved in purified water to form a phase B solution; (2) Single cross-linking: adding the phase A solution to the phase B solution to obtain a phase AB mixed solution, and performing a thermal self-crosslinking reaction at a reaction temperature of 20-50° C. for 2-24 hours. After the reaction, adjusting the pH to 9.2-11.4, and continuing to place in an environment of 2-10° C. for low-temperature self-crosslinking reaction for 2-48 hours to obtain a single self-crosslinked hyaluronic acid gel; (3) Double cross-linking: dissolve calycosin in a polar solvent to form a phase C solution; add the phase C solution to a single self-cross-linked hyaluronic acid gel, stir to form a heterogeneous system, heat to 20-50°C, and control the temperature for 2-24 hours. After reaching the final temperature, maintain the temperature and continue to react for 2-6 hours, stirring continuously during the reaction; after the reaction is completed, reduce the system temperature to 2-10°C, and continue the low-temperature cross-linking reaction for 2-4 hours to finally form a double cross-linked hyaluronic acid gel.

2. The method for preparing a double-modified cross-linked hyaluronic acid gel according to claim 1, characterized in that: The specific steps for preparing the phase A solution in step (1) are as follows: the pH of the acidic solution used for the phase A solution is 2.1-4.1, the acidic solution is a hydrochloric acid solution or a phosphoric acid solution, fucoidan is swelled in the acidic solution so that the mass fraction of fucoidan in the phase A solution is 1-20%, the temperature is 15-35° C., the stirring speed is 50-150 rpm, and the stirring is performed for 2-6 hours to obtain the phase A solution.

3. The method for preparing a double-modified cross-linked hyaluronic acid gel according to claim 1, characterized in that: The specific steps for preparing the phase B solution in step (1) are as follows: uncross-linked hyaluronic acid and its salt are dissolved in purified water, the mass fraction of uncross-linked hyaluronic acid and its salt in the phase B solution is 1-30%, and the molecular weight is 1 million-2.6 million Daltons.

4. The method for preparing a double-modified cross-linked hyaluronic acid gel according to claim 1, characterized in that: In step (2), the process of adding the phase A solution to the phase B solution is speed-controlled, and the adding time is controlled within 30 minutes; the pH of the AB phase mixed solution is 2.1-4.1, and the stirring speed of the AB phase mixed solution during the mixing process is 50-150 rpm; The mass fraction of hyaluronic acid and its salt in the AB phase mixed solution is 1-15%.

5. The method for preparing a double-modified cross-linked hyaluronic acid gel according to claim 1, characterized in that: In step (3), the polar solvent is obtained by mixing an organic solvent, water, ethanol, and methanol in a volume ratio of 1:0-10:0-20:0-20, and the organic solvent is one or more of dichloromethane, acetone, and acetonitrile; the mass fraction of calycosin in the phase C solution is 1-15%; In step (3), the pH of the heterogeneous system is 1.5-4.0, and the stirring speed is 50-100 rpm.

6. The method for preparing a double-modified cross-linked hyaluronic acid gel according to claim 1, characterized in that: The method further comprises (4) washing: washing the prepared double cross-linked hyaluronic acid gel with purified water to obtain the double cross-linked hyaluronic acid gel after impurities are removed, wherein the specific steps are as follows: washing the prepared double cross-linked hyaluronic acid gel with purified water and then standing for 2-12 hours to obtain the washed double cross-linked hyaluronic acid gel, dividing the washed double cross-linked hyaluronic acid gel and placing it in PBS buffer for swelling, wherein the volume ratio of the washed double cross-linked hyaluronic acid gel to the PBS buffer during swelling is 1:5-20, and the PBS buffer is replaced every 2-5 hours until the pH of the gel is 6.8-7.5 and the osmotic pressure is 280-350 mOsmol / L, thereby obtaining the double cross-linked hyaluronic acid gel with a swelling multiple of 5-20 times.

7. The method for preparing a double-modified cross-linked hyaluronic acid gel according to claim 1, characterized in that: The method further includes step (5) wet heat sterilization, which specifically comprises placing the double cross-linked hyaluronic acid gel in a wet heat sterilizer, controlling the temperature at 100-130° C. in a saturated steam state, and wet heat sterilizing for 10-40 minutes.

8. An application of a double-modified cross-linked hyaluronic acid gel, the double-cross-linked hyaluronic acid gel prepared by the method for preparing a double-modified cross-linked hyaluronic acid gel according to any one of claims 1 to 7, characterized in that: Application in the preparation of medical filling materials; or in the preparation of injections; or in the preparation of cross-linked hyaluronic acid gel containing drugs.

9. The use of a double-modified cross-linked hyaluronic acid gel according to claim 8, characterized in that: The specific steps for preparing the injection are: filling the double-crosslinked hyaluronic acid gel into a prefilled syringe, and sterilizing it by wet heat at 100-130° C. for 10-40 minutes in a saturated steam state.

10. The use of a double-modified cross-linked hyaluronic acid gel according to claim 8, characterized in that: The specific steps for preparing the double cross-linked hyaluronic acid gel containing drug carriers are: adding the bioactive ingredients of the required concentration according to the actual application needs during the preparation of the double cross-linked hyaluronic acid gel, maintaining stirring at 20-100 rpm for 0.5-24 hours, and fully mixing to form double cross-linked hyaluronic acid gels containing bioactive ingredients with different encapsulation rates; the bioactive ingredients include one or more of small molecule peptides and natural active substances.

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