A hyaluronic acid gel mixture, its preparation method and application

Abstract

The present invention discloses a hyaluronic acid gel mixture, a preparation method thereof, and an application, belonging to the technical field of prosthesis materials. The hyaluronic acid gel mixture includes a mixed disulfide-crosslinked sodium hyaluronate gel and crosslinked sodium hyaluronate gel particles. The crosslinked sodium hyaluronate gel particles are dispersed in the disulfide-crosslinked sodium hyaluronate gel. The hyaluronic acid gel mixture has high cohesion and high elasticity, and has good anti-enzymatic hydrolysis performance and stability, and is suitable for use in the preparation of medical products, especially medical products used as prosthesis materials. The preparation method of the hyaluronic acid gel mixture includes dispersing sodium hyaluronate gel particles in a sodium hyaluronate mercapto derivative solution, and then oxidizing the sodium hyaluronate mercapto derivative solution to form a disulfide-crosslinked sodium hyaluronate gel, thereby obtaining the hyaluronic acid gel mixture. The preparation method of the hyaluronic acid gel mixture of the present invention is simple and easy to operate, and is convenient for wide application.

Description

Technical Field

[0001] The present invention relates to the technical field of prosthetic materials, and particularly to a hyaluronic acid gel mixture, a preparation method thereof, and an application thereof. Background Art

[0002] Hyaluronic acid is a non-sulfated glycosaminoglycan composed of repeating disaccharide units (α-1,4-D-glucuronic acid and β-1,3-N-acetyl-D-glucosamine), which exists in all connective tissues and has good biocompatibility and unique physical and chemical properties. However, due to its fluidity and rapid degradation by hyaluronidase in vivo, its application in clinical practice is limited.

[0003] To solve this problem, it is usually necessary to chemically crosslink / modify sodium hyaluronate. Commonly used crosslinking agents include divinyl sulfoxide (DVS) and 1,4-butanediol diglycidyl ether (BDDE). The crosslinked hyaluronic acid gel prepared by the crosslinking reaction reduces the fluidity of hyaluronic acid and delays its degradation and absorption rate in vivo.

[0004] In the prior art, Chinese Patent CN101056891A provides a method for preparing a crosslinked hyaluronic acid gel. A mixture containing more than 10W / V% of hyaluronic acid, a crosslinking agent (divinyl sulfone, 1,4-butanediol diglycidyl ether, and ethylene glycol diglycidyl ether), and water is stirred and mixed under acidic (pH = 1 to 5) or alkaline (pH = 10 to 14) conditions. The gel prepared by this method has an excellent elastic modulus G'. US Patent US4582865 discloses a hyaluronic acid derivative crosslinked using divinyl sulfone (DVS) as a crosslinking agent, and its hydrogel has good viscoelastic properties.

[0005] Although the cross-linked hyaluronic acid gel prepared by the above-mentioned patented method reduces the fluidity of hyaluronic acid and delays its degradation and absorption rate in the body, in order to facilitate clinical use, these cross-linked hyaluronic acid gel products usually need to be crushed into fine particles. However, such an operation will lead to a decrease in the adhesion force between the gel particles, that is, a decrease in the cohesiveness of the gel. In some medical applications, materials are required to have good support properties. For example, in the case of prosthetic materials, the support properties of the gel are usually determined by cohesiveness and elasticity together. Cohesiveness represents the ability of the gel to maintain its original shape. The stronger the cohesiveness of the gel, the more resistant it is to external force extrusion and the less likely it is to spread; elasticity represents the hardness of the gel itself. The higher the elasticity, the less likely it is to deform. When used for medical aesthetic purposes, such as increasing the volume of the skin or tissue, especially when shaping the nasal dorsum and / or nasal root and the mandibular part, materials with strong bony support properties are required to achieve good plastic effects; when used for serious medical treatment of diseases, such as applying to sphincter tissue for the treatment of urinary incontinence, viscoelastic treatment of osteoarthritis, and soft tissue filling for metatarsalgia, materials are also required to have sufficient support properties. For another example, in eye surgeries such as plugging retinal holes for the treatment of rhegmatogenous retinal detachment and vitreous filling for the treatment of retinal detachment, materials are required to have overall high cohesiveness to prevent hyaluronic acid gel particles from blocking the tiny channels of the aqueous humor circulation between the anterior chamber and the posterior chamber. At the same time, the above clinical application scenarios all require materials to have high elasticity and high cohesiveness.

[0006] To solve this problem and achieve better injectability, one solution is to add a certain proportion of non-cross-linked hyaluronic acid to form a physically mixed biphasic gel, such as Restylane®, Durolane®, Pelane®, etc. They are prepared by mixing and homogenizing cross-linked hyaluronic acid and non-cross-linked hyaluronic acid and are used for facial filling or viscosifying treatment of arthritis. Such physically mixed biphasic gels improve the cohesiveness of the gel to a certain extent.

[0007] Another solution is to mix a cross-linked hyaluronic acid gel with better elasticity and a cross-linked hyaluronic acid gel with better viscosity. However, only a gel with compromised elasticity and viscosity can be prepared, and it is impossible to improve the high-viscosity performance of the gel without losing the high-elasticity performance of the gel.

[0008] However, the above technologies cannot improve the cohesiveness of the gel mixture without reducing the elastic modulus of the cross-linked hyaluronic acid gel, that is, they cannot have both high elasticity and high cohesiveness. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to provide a hyaluronic acid gel mixture, its preparation method and application. The hyaluronic acid gel mixture has high cohesion and high elasticity, and has good anti-enzymatic hydrolysis performance and stability, and is suitable for use in the preparation of medical products. The preparation method of the hyaluronic acid gel mixture is simple and easy to operate, facilitating wide application.

[0010] To solve the above technical problem, the first aspect of the present invention is to provide a hyaluronic acid gel mixture, comprising a mixed disulfide-crosslinked sodium hyaluronate gel and crosslinked sodium hyaluronate gel particles, wherein the crosslinked sodium hyaluronate gel particles are dispersed within the disulfide-crosslinked sodium hyaluronate gel.

[0011] In the linear viscoelastic region where the shear strain γ = 0.1% and the shear frequency is 1 Hz, the storage modulus G´ of the hyaluronic acid gel mixture is not less than 650 Pa, and the loss modulus is not less than 70 Pa.

[0012] In the present invention, the crosslinked sodium hyaluronate gel particles being dispersed within the disulfide-crosslinked sodium hyaluronate gel means that the crosslinked sodium hyaluronate gel particles within the disulfide-crosslinked sodium hyaluronate gel are wrapped by the disulfide-crosslinked sodium hyaluronate gel, and the crosslinked sodium hyaluronate gel particles are dispersed in the colloid of the disulfide-crosslinked sodium hyaluronate gel, and the two form an integral gel.

[0013] In the present invention, the crosslinked sodium hyaluronate gel particles provide high elastic support performance and anti-enzymatic hydrolysis ability for the mixture due to their high elastic modulus, and at the same time, also provide cohesion for the crosslinked sodium hyaluronate gel particles themselves; the disulfide-crosslinked sodium hyaluronate gel provides high cohesion for the mixture and further increases the anti-enzymatic hydrolysis ability of the mixture.

[0014] The hyaluronic acid gel mixture provided by the present invention forms an integral gel as a whole, and its elastic modulus G' tested in the linear viscoelastic region with a shear strain γ = 0.1% and a shear frequency of 1 Hz is not less than 650 Pa, and the viscous modulus is not less than 70 Pa. It has high cohesion and high elasticity, and at the same time has excellent anti-enzymatic hydrolysis ability. Compared with non-cohesive gels or gel compositions, the cohesive gel provided by the present invention can better maintain its shape and is not easily deformed under high shear force. Moreover, since no non-crosslinked sodium hyaluronate needs to be added, the problem of reduced viscosity stability is avoided. After testing, the hyaluronic acid gel mixture provided by the present invention can withstand high saturated steam for 10 - 20 minutes and can experience 9 months at 50°C or three years at 25°C, and the gel composition does not undergo significant degradation or become unstable. Specifically, after sterilization, the gel composition continues to be placed at 50°C for 274 days, and the retention value of its dynamic viscosity is above 90%. When the hyaluronic acid gel mixture provided by the present invention is used as a prosthetic material, its high cohesion and high elasticity endow it with excellent mechanical properties, and its excellent anti-enzymatic hydrolysis ability endows it with good biological stability.

[0015] In a specific embodiment, the particle size range of the crosslinked sodium hyaluronate gel particles is 50 μm - 2500 μm.

[0016] In a specific embodiment, in the crosslinked sodium hyaluronate gel particles, D50 is not greater than 800 μm and / or D90 is not greater than 2000 μm.

[0017] In a specific embodiment, the hyaluronic acid content in the crosslinked sodium hyaluronate gel particles is 12 - 50 mg / mL or 16 - 30 mg / mL; the hyaluronic acid content in the disulfide bond-crosslinked sodium hyaluronate gel is 0.2 - 10 mg / mL or 0.5 - 4 mg / mL.

[0018] In a specific embodiment, the crosslinking degree of the crosslinked sodium hyaluronate gel particles is 1.0% - 20%; the crosslinking degree of the disulfide bond-crosslinked sodium hyaluronate gel is 0.05% - 2.5% or 0.1% - 1.0%.

[0019] In a specific embodiment, the mass ratio of the crosslinked sodium hyaluronate gel particles to the disulfide bond-crosslinked sodium hyaluronate gel is 19:1 - 1:1.

[0020] To solve the above technical problems, the second aspect of the present invention is to provide the application of the aforementioned hyaluronic acid gel mixture in the preparation of medical products. Understandably, since the hyaluronic acid gel mixture provided by the present invention simultaneously has high cohesion, high elasticity and excellent anti-enzymatic ability, it is suitable for use in the preparation of medical products, especially prosthesis materials, etc. For example, it is applied to the preparation of products for blocking retinal holes to treat rhegmatogenous retinal detachment, products for vitreous filling, products for viscoelastic treatment of osteoarthritis, products for soft tissue filling of wrinkles, products for facial remodeling, and products for increasing the volume of sphincters.

[0021] To solve the above technical problems, the third aspect of the present invention is to provide a preparation method of the aforementioned hyaluronic acid gel mixture, comprising the following steps:

[0022] S1. Crush the cross-linked sodium hyaluronate gel into cross-linked sodium hyaluronate gel particles;

[0023] S2. Disperse the cross-linked sodium hyaluronate gel particles obtained in step S1 in a solution of sodium hyaluronate mercapto derivative;

[0024] S3. Gradually subject the solution of sodium hyaluronate mercapto derivative dispersed with cross-linked sodium hyaluronate gel particles obtained in step S2 to an oxidation process to form a disulfide-cross-linked sodium hyaluronate gel, thereby obtaining a hyaluronic acid gel mixture, and testing the elastic modulus G´ of the hyaluronic acid gel mixture in the linear viscoelastic region with a shear strain γ = 0.1% and a shear frequency of 1 Hz, which is not less than 650 Pa, and the viscous modulus is not less than 70 Pa.

[0025] In the present invention, the cross-linked sodium hyaluronate gel can be obtained by prior art methods. For example, dissolve non-cross-linked sodium hyaluronate in a sodium hydroxide solution, and then add a cross-linking agent such as 1,4-butanediol diglycidyl ether (BDDE). After the cross-linking reaction is completed, perform post-treatment on the reaction system to obtain the cross-linked sodium hyaluronate gel. It should be noted that the content of the cross-linked sodium hyaluronate gel refers to the weight (mg / mL) of the cross-linked sodium hyaluronate contained in a unit volume of the composition; the cross-linking degree can be calculated by the following formula:

[0026] Cross-linking degree = total molar number of cross-linking agent / total molar number of N-acetyl-D-glucosamine; it should be noted that the total molar number of the molecular cross-linking agent refers to the total molar number of the effectively grafted cross-linking agent, rather than the total molar number of the added cross-linking agent, because the grafting rate of the cross-linking agent is usually low and difficult to measure. Therefore, in general experimental processes, the cross-linking degree is directly measured using an instrument.

[0027] In the present invention, the sodium hyaluronate mercapto - modified derivative solution is a precursor of the disulfide - crosslinked sodium hyaluronate gel. After oxidation, it forms a disulfide - crosslinked sodium hyaluronate gel. The oxidation process is carried out in an aerobic environment. The general source of oxygen in the aerobic environment is the oxygen in the air contacted during the mixing process or the oxygen dissolved in the solution. The preparation method of the disulfide - crosslinked sodium hyaluronate gel can be referred to prior patents such as CN118406258A and prior literature such as Shu et al., Biomacromolecules 2002, 3: 1304 - 1311, etc. It should be noted that the molecular weight of the disaccharide structural unit of sodium hyaluronate is about 400, and each disaccharide unit contains a carboxyl group that can be mercapto - modified. That is, each gram of hyaluronic acid contains 2500 μmol of carboxyl groups that can be mercapto - modified. Further, two mercapto groups are oxidized and crosslinked to form a disulfide bond. For example, when the sodium hyaluronate mercapto - modified derivative solution with a modification degree of 1% is oxidized and crosslinked, the crosslinking degree of the crosslinked hyaluronic acid gel is 0.5%. In addition, it should also be noted that the mercapto content refers to the number of micromoles of mercapto groups contained in each gram of the sodium hyaluronate mercapto - modified derivative (μmol / g). The modification degree is the ratio of the mercapto content to the carboxyl content. The crosslinking degree of the disulfide - crosslinked sodium hyaluronate gel is the modification degree value divided by 2. The modification degree is the ratio of the mercapto content to the carboxyl content. The crosslinking degree of the disulfide - crosslinked sodium hyaluronate gel is the modification degree value divided by 2.

[0028] In the present invention, the hyaluronic acid gel mixture is prepared by first dispersing the crosslinked sodium hyaluronate gel particles in the sodium hyaluronate mercapto - modified derivative solution, and then the sodium hyaluronate mercapto - modified derivative solution undergoes an oxidation process to form a disulfide - crosslinked hyaluronic acid gel (due to disulfide crosslinking, the mass remains unchanged before and after crosslinking. The mass of the sodium hyaluronate mercapto - modified derivative solution before crosslinking is equal to the mass of the disulfide - crosslinked hyaluronic acid gel after crosslinking). Therefore, the hyaluronic acid gel mixture prepared by the method of the present invention is a monolithic gel with high cohesion. At the same time, due to the crosslinked sodium hyaluronate gel particles having a high elastic modulus, it provides high elastic support performance and anti - enzymatic degradation ability for the hyaluronic acid gel mixture; the hyaluronic acid gel mixture prepared by the method of the present invention also has better anti - enzymatic degradation ability.

[0029] In the present invention, after dispersing the crosslinked sodium hyaluronate gel particles in the sodium hyaluronate mercapto - modified derivative solution, sterilization and filling operations can be carried out. The oxidation crosslinking process occurs after filling. The size and shape of the final product hyaluronic acid gel mixture are the same as those of the filling container. It should be noted that sterilization means meeting the microbial standards for pharmaceutical or medical device products in the art (such as the Chinese Pharmacopoeia, the United States Pharmacopoeia, the European Pharmacopoeia or other national standards). The sterilization process can be carried out according to the GB18278.1 - 2015 standard. Specifically, sterilization means filling the mixture gel into a container and subjecting it to high - saturated steam at 121 °C for 10 - 20 minutes.

[0030] In a specific embodiment, the particle size range of the crosslinked sodium hyaluronate gel particles in step S1 is 50 μm to 2500 μm. Preferably, D50 is not greater than 800 μm and / or D90 is not greater than 2000 μm.

[0031] In a specific embodiment, the crosslinking degree of the crosslinked sodium hyaluronate gel particles in step S1 is 1.0% to 20%, and the elastic modulus G´ tested in the linear viscoelastic region with a shear strain γ = 0.1% and a shear frequency of 1 Hz is not less than 700 Pa.

[0032] In a specific embodiment, the hyaluronic acid content in the crosslinked sodium hyaluronate gel particles in step S1 is 12 to 50 mg / mL or 16 to 30 mg / mL.

[0033] In a specific embodiment, the hyaluronic acid content in the sodium hyaluronate thiolated derivative solution in step S2 is 0.2 to 10 mg / mL, and the pH value is 6.8 to 7.6. It should be noted that the adjustment and maintenance of the system pH are achieved, for example, by adding a phosphate buffer solution.

[0034] In a specific embodiment, the crosslinking degree of the disulfide bond-crosslinked sodium hyaluronate gel in the hyaluronic acid gel mixture obtained in step S3 is 0.05% to 2.5% or 0.1% to 1.0%.

[0035] In a specific embodiment, for the hyaluronic acid gel mixture obtained in step S3, the mass ratio of the crosslinked sodium hyaluronate gel particles to the disulfide bond-crosslinked sodium hyaluronate gel is 19:1 to 1:1. It should be noted that the mass ratio of the crosslinked sodium hyaluronate gel particles to the disulfide bond-crosslinked sodium hyaluronate gel in the present invention can be adjusted according to clinical needs. For example, when used for medical aesthetic purposes, when it is necessary to increase the volume of the skin or tissue for tissue shaping, especially for shaping the nasal dorsum and / or nasal root and the mandibular part, materials with strong bony support performance and high cohesiveness are required, and a combination with a relatively high proportion of crosslinked sodium hyaluronate gel particles can be selected, such as the mass ratio of the crosslinked sodium hyaluronate gel particles to the structural enhancement gel is 19:1 to 8:2, and such is usually injected with a 27G needle; again, when used for applications such as viscosifying treatment of osteoarthritis, filling wrinkles in soft tissues, filling treatment of gingival atrophy, and increasing the volume of sphincters and other prosthesis materials, the mass ratio of the crosslinked sodium hyaluronate gel particles to the disulfide bond-crosslinked sodium hyaluronate gel can be designed between 19:1 and 1:1 according to specific needs. Detailed Embodiments

[0036] The technical solutions in the present invention will be described clearly and completely. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0037] Example 1 Preparation of BDDE-crosslinked sodium hyaluronate gel:

[0038] Take 10 g of non-crosslinked sodium hyaluronate (1500 KDa), dissolve it in 100 g of sodium hydroxide solution (1%), add 0.5 g of crosslinking agent 1,4-butanediol diglycidyl ether (BDDE), heat it in a water bath at 50 °C, carry out crosslinking reaction for 2 h, after crosslinking is completed, dialyze it in neutral phosphate buffer for 48 - 72 hours, and obtain BDDE-crosslinked sodium hyaluronate gel with a content of 23 mg / ml by controlling the water absorption swelling rate, and measure the crosslinking degree to be 1.13%.

[0039] Example 2 Preparation of DVS-crosslinked sodium hyaluronate gel:

[0040] Take 10 g of non-crosslinked sodium hyaluronate (1500 KDa), dissolve it in 100 g of sodium hydroxide solution (1%), add 0.5 g of crosslinking agent divinyl sulfone (DVS), heat it in a water bath at 50 °C, carry out crosslinking reaction for 2 h, after crosslinking is completed, dialyze it in neutral phosphate buffer for 48 - 72 hours, and obtain DVS-crosslinked sodium hyaluronate gel with a content of 23 mg / ml by controlling the water absorption swelling rate, and measure the crosslinking degree to be 1.51%.

[0041] Example 3 Preparation of lysine-crosslinked sodium hyaluronate gel:

[0042] Take 10 g of non-crosslinked sodium hyaluronate (400 KDa) and 0.2 g of lysine, dissolve them in 150 g of purified water, stir at 25 °C until dissolved, adjust the pH to 4 with 0.1 mol / L hydrochloric acid, pre-dissolve 1.0 g of 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM) in 10 g of purified water, add it to the above sodium hyaluronate solution, and stir and react at 25 °C for 24 hours. Dialyze it in neutral phosphate buffer for 48 - 72 hours, and obtain lysine-crosslinked sodium hyaluronate gel with a content of 23 mg / ml by controlling the water absorption swelling rate, and measure the crosslinking degree to be 1.20%.

[0043] Example 4 Preparation of crosslinked sodium hyaluronate gel:

[0044] The cross-linked sodium hyaluronate gel in this embodiment is obtained by cross-linking with disulfide bonds and is used as the encapsulated cross-linked sodium hyaluronate particles. It is prepared by the method reported by Shu et al. (Shu et al., Biomacromolecules 2002, 3: 1304-1311). Specifically: Take 10.0 grams of sodium hyaluronate (MW 200 kDa) and dissolve it in 100 mL of distilled water at 40 °C. Add an appropriate amount of 0.1 N hydrochloric acid to adjust the pH value of the solution to 4.75. Then add 3.2 grams of Sulfo-NHS (N-hydroxysulfosuccinimide), 16.8 grams of cystamine dihydrochloride, and 1.2 grams of solid EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), stir to dissolve. Add an appropriate amount of acid or base (0.1 N hydrochloric acid or sodium hydroxide) to keep the pH value of the reaction solution stable at 4.75, and stir the reaction overnight. Add 1.0 N sodium hydroxide to adjust the pH of the reaction solution to 8.5 to terminate the reaction. Then add 5.0 grams of dithiothreitol, stir to dissolve, and react for 4 hours. Then add 1.0 N hydrochloric acid to adjust the pH value of the above reaction solution to 3.0, load it into a dialysis bag with a molecular weight cut-off of 10 kDa, and then dialyze and purify it with a mixed solution of 0.001 N hydrochloric acid and 0.2 N sodium chloride. Use gel permeation chromatography (GPC) to track the purification process of the final product until the impurity absorption peak is invisible, obtaining a solution of sodium hyaluronate thiolated derivative. After further freeze-drying, a freeze-dried product of sodium hyaluronate thiolated derivative is obtained. Take 5.0 g of the freeze-dried product of sodium hyaluronate thiolated derivative, add phosphate buffer to prepare a 16 mg / ml solution, adjust the pH to about 7.2, and place it at 40 °C for 7 days to obtain a 16 mg / ml disulfide bond-cross-linked sodium hyaluronate gel. Its thiol content is 51 μmol / g, and the calculated modification degree is 2.04%, and then the cross-linking degree is 1.02%. Cross-linked sodium hyaluronate gels with concentrations of 23 mg / ml and 30 mg / ml can be prepared in the same way, and the cross-linking degree is 1.02% for both. By adjusting the material ratio, cross-linked sodium hyaluronate gels with various thiol contents of 149 μmol / g (cross-linking degree of 2.98%) and 376 μmol / g (cross-linking degree of 7.52%) can be prepared by a similar method, a total of 9 groups, as shown in Table 1.

[0045] Table 1

[0046] 。

[0047] Preparation of sodium hyaluronate thiolated derivative solution in Example 5:

[0048] The sodium hyaluronate thiolated derivative solution was prepared from sodium hyaluronate with a molecular weight of 200KDa by the method reported by Shu et al. (Shu et al., Biomacromolecules 2002, 3: 1304 - 1311). Specifically, 10.0 g of sodium hyaluronate (MW 200kDa) was dissolved in 100 mL of distilled water at 40 °C. An appropriate amount of 0.1 N hydrochloric acid was added to adjust the pH of the solution to 4.75. 0.8 g of Sulfo-NHS, 8.4 g of cystamine dihydrochloride, and 0.3 g of solid EDCI were added and stirred until dissolved. An appropriate amount of acid or base (0.1 N hydrochloric acid or sodium hydroxide) was added to keep the pH of the reaction solution stable at the aforementioned specific value, and the reaction was stirred overnight. 1.0 N sodium hydroxide was added to adjust the pH of the reaction solution to 8.5 to terminate the reaction. Then, 5.0 g of dithiothreitol was added and stirred until dissolved, and the reaction was carried out for 4 hours. Then, 1.0 N hydrochloric acid was added to adjust the pH of the above reaction solution to 3.0. It was loaded into a dialysis bag with a molecular weight cut-off of 10 kDa, and then dialyzed and purified with a mixed solution of 0.001 N hydrochloric acid and 0.2 N sodium chloride. Gel permeation chromatography (GPC) was used to track the purification process of the final product until the impurity absorption peak was invisible, obtaining a sodium hyaluronate thiolated derivative solution with a thiol content of 5.1 μmol / g (the crosslinking degree after crosslinking was 0.1%). It was further formulated into solutions of 0.5 mg / ml, 2 mg / ml, and 4 mg / ml with phosphate buffer. By adjusting the material ratio, sodium hyaluronate thiolated derivative solutions with different thiol contents could be prepared in a similar method, including a polymer with a thiol content of 25.2 μmol / g (the crosslinking degree after crosslinking was 0.5%) and a polymer with a thiol content of 50.1 μmol / g (the crosslinking degree after crosslinking was 1.0%), a total of 9 groups, as shown in Table 2, for standby.

[0049] Table 2

[0050] 。

[0051] Example 6 Preparation of hyaluronic acid gel mixture:

[0052] S1. Take 18 g of the crosslinked sodium hyaluronate gel prepared in Example 1, crush it to obtain gel particles with a D50 of 400 μm and a D90 of 850 μm;

[0053] S2. Take the sodium hyaluronate thiolated derivative solution prepared in the 5th group of Example 5, adjust the pH to 7.2 with phosphate buffer, take 2 g of the solution after adjusting the pH, add it to the crosslinked sodium hyaluronate gel particles in step S1, and mix evenly;

[0054] S3. Fill the mixed system in S2 into a 1-ml prefilled syringe, and sterilize it by moist heat. After 2 days, the sodium hyaluronate mercapto derivative solution forms a disulfide-crosslinked sodium hyaluronate gel through an oxidation process, thus obtaining the hyaluronic acid gel mixture.

[0055] Example 7 Preparation of hyaluronic acid gel mixture:

[0056] S1. Take 18 g of the crosslinked sodium hyaluronate gel prepared in Example 2, crush it to obtain gel particles with D50 of 400 μm and D90 of 850 μm.

[0057] S2. Take the sodium hyaluronate mercapto derivative solution prepared in Group 5 of Example 5, adjust the pH to 7.2 with phosphate buffer solution. Take 2 g of the solution after pH adjustment, add the crosslinked sodium hyaluronate gel particles in Step S1 and mix evenly.

[0058] S3. Fill the mixed system in S2 into a 1-ml prefilled syringe, and sterilize it by moist heat. After 2 days, the sodium hyaluronate mercapto derivative solution forms a disulfide-crosslinked sodium hyaluronate gel through an oxidation process, thus obtaining the hyaluronic acid gel mixture.

[0059] Example 8 Preparation of hyaluronic acid gel mixture:

[0060] S1. Take 18 g of the crosslinked sodium hyaluronate gel prepared in Example 3, crush it to obtain gel particles with D50 of 400 μm and D90 of 850 μm.

[0061] S2. Take the sodium hyaluronate mercapto derivative solution prepared in Group 5 of Example 5, adjust the pH to 7.2 with phosphate buffer solution. Take 2 g of the solution after pH adjustment, add the crosslinked sodium hyaluronate gel particles in Step S1 and mix evenly.

[0062] S3. Fill the mixed system in S2 into a 1-ml prefilled syringe, and sterilize it by moist heat. After 2 days, the sodium hyaluronate mercapto derivative solution forms a disulfide-crosslinked sodium hyaluronate gel through an oxidation process, thus obtaining the hyaluronic acid gel mixture.

[0063] The information of the crosslinked sodium hyaluronate gel particles and the disulfide-crosslinked sodium hyaluronate gel in the hyaluronic acid gel mixtures in Examples 6 - 8 is shown in Table 3.

[0064] Table 3

[0065] .

[0066] Example 9 Preparation of hyaluronic acid gel mixture:

[0067] S1. Take 18 g of the cross-linked sodium hyaluronate gel prepared in Group 1 of Example 4, crush it to obtain gel particles with a D50 of 400 μm and a D90 of 850 μm;

[0068] S2. Take the sodium hyaluronate mercapto derivative solution prepared in Group 5 of Example 5, adjust the pH to 7.2 with phosphate buffer solution. Take 2 g of the solution after pH adjustment, add the cross-linked sodium hyaluronate gel particles in Step S1, and mix evenly;

[0069] S3. Fill the mixed system in S2 into a 1 ml pre-filled syringe, sterilize it by moist heat. After 2 days, the sodium hyaluronate mercapto derivative solution forms a disulfide bond-cross-linked sodium hyaluronate gel through an oxidation process, and thus a hyaluronic acid gel mixture is obtained.

[0070] Examples 10 - 17 Preparation of hyaluronic acid gel mixture:

[0071] Based on Example 9, in Step S1, the cross-linked sodium hyaluronate gels obtained from other groups in Example 4 are used respectively, and Examples 10 - 17 are obtained under the condition that other preparation conditions remain unchanged, as shown in Table 4.

[0072] Example 18 Preparation of hyaluronic acid gel mixture:

[0073] S1. Take 18 g of the cross-linked sodium hyaluronate gel prepared in Group 5 of Example 4, crush it to obtain gel particles with a D50 of 400 μm and a D90 of 850 μm;

[0074] S2. Take the sodium hyaluronate mercapto derivative solution prepared in Group 1 of Example 5, adjust the pH to 7.2 with phosphate buffer solution. Take 2 g of the solution after pH adjustment, add the cross-linked sodium hyaluronate gel particles in Step S1, and mix evenly;

[0075] S3. Fill the mixed system in S2 into a 1 ml pre-filled syringe, sterilize it by moist heat. After 2 days, the sodium hyaluronate mercapto derivative solution forms a disulfide bond-cross-linked sodium hyaluronate gel through an oxidation process, and thus a hyaluronic acid gel mixture is obtained.

[0076] Examples 19 - 25 Preparation of hyaluronic acid gel mixture:

[0077] Based on Example 18, in Step S2, the cross-linked sodium hyaluronate gels obtained from other groups in Example 5 are used respectively, and Examples 19 - 25 are obtained under the condition that other preparation conditions remain unchanged, as shown in Table 4.

[0078] The information of the cross-linked sodium hyaluronate gel particles and the disulfide bond-cross-linked sodium hyaluronate gel in the hyaluronic acid gel mixtures obtained in Examples 9 - 25 is shown in Table 4.

[0079] Table 4

[0080] 。

[0081] Example 26 Preparation of hyaluronic acid gel mixture:

[0082] S1. Take 19 g of the crosslinked sodium hyaluronate gel prepared in Group 5 of Example 4, crush it to obtain gel particles with D50 of 400 μm and D90 of 850 μm;

[0083] S2. Take the solution of sodium hyaluronate mercapto derivative prepared in Group 5 of Example 5, adjust the pH to 7.2 with phosphate buffer. Take 1 g of the solution after pH adjustment, add it to the crosslinked sodium hyaluronate gel particles in step S1 and mix evenly;

[0084] S3. Fill the mixed system in S2 into a 1 ml prefilled syringe, sterilize it by moist heat. After 2 days, the solution of sodium hyaluronate mercapto derivative forms a disulfide bond crosslinked sodium hyaluronate gel through an oxidation process, thus obtaining the hyaluronic acid gel mixture.

[0085] Examples 27 - 29 Preparation of hyaluronic acid gel mixture:

[0086] On the basis of Example 26, adjust the mass ratio of crosslinked sodium hyaluronate gel particles and disulfide bond crosslinked sodium hyaluronate gel. Under the condition that other preparation conditions remain unchanged, Examples 27 - 29 are obtained, as shown in Table 5.

[0087] The information of crosslinked sodium hyaluronate gel particles and disulfide bond crosslinked sodium hyaluronate gel in the hyaluronic acid gel mixture obtained in Examples 13, 26 - 29 is shown in Table 5.

[0088] Table 5

[0089]

[0090] Among them, the mass of the disulfide bond crosslinked sodium hyaluronate gel is the mass of the solution of sodium hyaluronate mercapto derivative with a pH value of 7.2 added in step S2.

[0091] Comparative Example 1:

[0092] S1. Take 18 g of the crosslinked sodium hyaluronate gel prepared in Example 1, crush it to obtain gel particles with D50 of 400 μm and D90 of 850 μm;

[0093] S2. Fill the gel in S1 into a 1 ml prefilled syringe, sterilize it by moist heat, and obtain the sample of Comparative Example 1.

[0094] Comparative Example 2:

[0095] S1. Take 18 g of the cross-linked sodium hyaluronate gel prepared in Example 2, crush it to obtain gel particles with a D50 of 400 μm and a D90 of 850 μm;

[0096] S2. Fill the gel from S1 into a 1-ml pre-filled syringe and sterilize it by moist heat to obtain the sample of Comparative Example 2.

[0097] Comparative Example 3:

[0098] S1. Take 18 g of the cross-linked sodium hyaluronate gel prepared in Example 3, crush it to obtain gel particles with a D50 of 400 μm and a D90 of 850 μm;

[0099] S2. Fill the gel from S1 into a 1-ml pre-filled syringe and sterilize it by moist heat to obtain the sample of Comparative Example 3.

[0100] Comparative Example 4:

[0101] S1. Take 18 g of the cross-linked sodium hyaluronate gel prepared in the 5th group of Example 4, crush it to obtain gel particles with a D50 of 400 μm and a D90 of 850 μm;

[0102] S2. Fill the gel from S1 into a 1-ml pre-filled syringe and sterilize it by moist heat to obtain the sample of Comparative Example 4.

[0103] Comparative Example 5:

[0104] S1. Take 18 g of the cross-linked sodium hyaluronate gel prepared in Example 1, crush it to obtain gel particles with a D50 of 600 μm and a D90 of 1500 μm;

[0105] S2. Dissolve non-cross-linked sodium hyaluronate (1500 KDa) in an isotonic phosphate buffer solution, adjust the content to 20 mg / ml, adjust the pH to 7.2, and take 2 g of the adjusted solution and mix it evenly with the cross-linked sodium hyaluronate gel particles obtained in S1;

[0106] S3. Fill the mixed system in S2 into a 1-ml pre-filled syringe and sterilize it by moist heat to obtain the sample of Comparative Example 5.

[0107] Comparative Example 6:

[0108] S1. Take 18 g of the cross-linked sodium hyaluronate gel prepared in Example 2, crush it to obtain gel particles with a D50 of 600 μm and a D90 of 1500 μm;

[0109] S2. Dissolve non-cross-linked sodium hyaluronate (1500 KDa) in an isotonic phosphate buffer solution, adjust the content to 20 mg / ml, adjust the pH to 7.2, and take 2 g of the adjusted solution and mix it evenly with the cross-linked sodium hyaluronate gel particles obtained in S1;

[0110] S3. Fill the mixed system in S2 into a 1-ml pre-filled syringe and sterilize it by moist heat to obtain the sample of Comparative Example 6.

[0111] Comparative Example 7:

[0112] S1. Take 18 g of the cross-linked sodium hyaluronate gel prepared in Example 3, crush it to obtain gel particles with D50 of 600 μm and D90 of 1500 μm.

[0113] S2. Dissolve non-cross-linked sodium hyaluronate (1500KDa) in an isotonic phosphate buffer solution, adjust the content to 20 mg / ml, adjust the pH to 7.2, and take 2 g of the adjusted solution and mix it evenly with the cross-linked sodium hyaluronate gel particles obtained in S1.

[0114] S3. Fill the mixed system in S2 into a 1-ml pre-filled syringe and sterilize it by moist heat to obtain the sample of Comparative Example 7.

[0115] Comparative Example 8:

[0116] S1. Take 18 g of the cross-linked sodium hyaluronate gel prepared in Group 5 of Example 4, crush it to obtain gel particles with D50 of 600 μm and D90 of 1500 μm.

[0117] S2. Dissolve non-cross-linked sodium hyaluronate (1500KDa) in an isotonic phosphate buffer solution, adjust the content to 20 mg / ml, adjust the pH to 7.2, and take 2 g of the adjusted solution and mix it evenly with the cross-linked sodium hyaluronate gel particles obtained in S1.

[0118] S3. Fill the mixed system in S2 into a 1-ml pre-filled syringe and sterilize it by moist heat to obtain the sample of Comparative Example 8.

[0119] Comparative Example 9:

[0120] S1. Take 10 g of non-cross-linked sodium hyaluronate (1500KDa), dissolve it in 100 g of sodium hydroxide solution (1%), add 0.5 g of cross-linking agent 1,4-butanediol diglycidyl ether (BDDE), heat it in a water bath at 50 °C for 2 h for cross-linking reaction. After cross-linking, dialyze it in a neutral phosphate buffer solution for 72 hours. By controlling the dialysis time, obtain the first portion of BDDE-cross-linked sodium hyaluronate gel with a content of 23 mg / ml.

[0121] Take 10 g of non-crosslinked sodium hyaluronate (1500 KDa), dissolve it in 100 g of sodium hydroxide solution (1%), add 0.05 g of crosslinking agent 1,4-butanediol diglycidyl ether (BDDE), heat it in a water bath at 50 °C, and carry out crosslinking reaction for 2 h. After the crosslinking is completed, dialyze it in neutral phosphate buffer for 72 hours. By controlling the dialysis time, the second portion of BDDE-crosslinked sodium hyaluronate gel is obtained, with a content of 23 mg / ml;

[0122] Respectively take the first and second portions of BDDE-crosslinked sodium hyaluronate gel, crush them to obtain gel particles, with D50 being 600 μm and D90 being 1500 μm;

[0123] S2: Take 18 g of the first portion of BDDE-crosslinked sodium hyaluronate gel particles and 2 g of the second portion of BEED-crosslinked sodium hyaluronate gel particles, stir mechanically, and mix evenly;

[0124] S3: Fill the mixed gel in S2 into a 1 ml pre-filled syringe, and sterilize it by moist heat to obtain the sample of Comparative Example 9.

[0125] Comparative Example 10:

[0126] S1. Take the crosslinked sodium hyaluronate gel prepared in the 5th group of Example 4, crush it to obtain gel particles, with D50 being 600 μm and D90 being 1500 μm;

[0127] S2. Take the thiolated hyaluronic acid solution obtained before lyophilization in the 2nd group of Example 4, adjust the pH to 7.2 with phosphate buffer, and then oxidize it to form a disulfide bond-crosslinked sodium hyaluronate gel, and crush it to obtain gel particles;

[0128] S3. Take 18 g of the gel particles in S1 and 2 g of the gel particles in S2, stir and mix them evenly, and fill the mixed gel particles into a 1 ml pre-filled syringe, and sterilize it by moist heat to obtain the sample of Comparative Example 10.

[0129] Comparative Example 11:

[0130] S1. Dissolve 2 g of non-crosslinked hyaluronic acid with a molecular weight of 1500 KDa in 200 mL of 2% sodium chloride solution, add 400 mL of ethanol while stirring, collect the flocculent precipitate, squeeze it dry, add 100 mL of ethanol, let it stand for 12 hours, extract the excess water, filter, remove the residual ethanol in the precipitate under reduced pressure, and crush the precipitate to obtain non-crosslinked sodium hyaluronate particles, with D50 being 600 μm and D90 being 1500 μm;

[0131] S2. Take the sodium hyaluronate thiolated derivative solution prepared in the 5th group of Example 5, adjust the pH to 7.2 with phosphate buffer, take 2 g of the solution after adjusting the pH, add the non-crosslinked sodium hyaluronate particles in step S1 and mix evenly;

[0132] S3. Fill the mixed system in S2 into a 1 ml pre-filled syringe, and sterilize it by moist heat. After 2 days, the sodium hyaluronate mercapto derivative solution forms a disulfide-crosslinked sodium hyaluronate gel through an oxidation process, and thus the sample of Comparative Example 11 is obtained.

[0133] Test Example 1 Gel viscoelasticity:

[0134] Viscoelasticity test method: Take 1 g of each sample of the examples and comparative examples, and use a rheometer (Anton Paar, MCR301) to test the samples. Select a flat plate with a diameter of 50 mm, set the plate spacing to 0.5 mm, the test temperature is 25 °C, the shear strain γ = 0.1%, 1 Hz, and test the elastic modulus (G´) and viscous modulus (G〞).

[0135] The elastic modulus G´ and viscous modulus G〞 of the samples prepared in Examples 6-29 and Comparative Examples 1-11 are shown in Table 6 below.

[0136] Table 6

[0137] .

[0138] As can be seen from Table 6, the gel compositions prepared in Examples 6-29 have the advantages of both high elastic modulus and high viscous modulus, making them have good support and anti-enzymatic hydrolysis properties. Comparative Examples 1-4 have a high elastic modulus, but their viscous modulus is low and the support is poor. Based on Comparative Examples 1-4, Comparative Examples 5-8 added non-crosslinked hyaluronic acid, which increased the viscous modulus to a certain extent, but their viscous modulus was still low and the support was still poor; for Comparative Examples 9-10, a crosslinked sodium hyaluronate gel with good elasticity and a crosslinked sodium hyaluronate gel with good viscosity were mixed to prepare a gel with balanced elasticity and viscosity, but its viscous modulus was still lower than that of each example; Comparative Example 11 adopted the form of encapsulating non-crosslinked sodium hyaluronate particles with a disulfide-crosslinked sodium hyaluronate gel, and both the elastic modulus and viscous modulus of the mixture were low. It is speculated that during the formation of the disulfide crosslinking of the sodium hyaluronate mercapto derivative solution, the non-crosslinked sodium hyaluronate particles partially absorbed the moisture in the sodium hyaluronate mercapto derivative solution, resulting in partial dissolution.

[0139] Test Example 2 Gel cohesion test:

[0140] According to the test method described in the reference (HEMA SUNDARAM et al., Plast Reconstr Surg, 2015, 136(4): 678 - 686), take 1 g of gel sample, use a syringe equipped with a slender needle to draw a small amount (about 0.2 ml) of 2% toluidine blue solution and insert it into the bottom of the sample. Slowly push the toluidine blue solution and gradually pull out the needle until all the samples are dyed. After dyeing, let it stand for 3 minutes to make the sample dye evenly; Push the gel (without connecting the injection needle) into a beaker containing 700 ml of purified water at a fixed speed of 400 mm / min at a distance of 2 cm from the water surface. Set the magnetic stirring speed to 160 r / min and the length of the stirring bar to 2.5 cm; Start video recording and taking pictures after the gel is extruded. The time points for taking pictures are 15, 70, and 95 s after the start. Take 3 pictures at each time point for each sample. Ten people independently score the cohesion of each picture according to five different states of the gel (1 point - the gel is completely dispersed; 2 points - the gel is partially dispersed; 3 points - the gel is partially dispersed and partially cohesive; 4 points - most of the gel is cohesive; 5 points - the gel is completely cohesive), and calculate the average value.

[0141] The test results of the cohesion of the samples prepared in Examples 6 - 29 and Comparative Examples 1 - 11 are shown in Table 7.

[0142] Table 7

[0143] 。

[0144] As can be seen from Table 7, the cohesion of the gel compositions prepared in Examples 6 - 29 is very high. Even when stirred for 95 s, it still maintains good cohesion, and the cohesion scores are all greater than 4 points. Comparative Examples 1 - 4 are single - crosslinked sodium hyaluronate gels, and their cohesion is very poor. They are completely dispersed when stirred for 15 s; After adding non - crosslinked sodium hyaluronate in Comparative Examples 5 - 8, the cohesion is improved. When stirred for 15 s, the gel is partially dispersed; After mixing with low - crosslinked crosslinked sodium hyaluronate gels in Comparative Examples 9 - 10, the cohesion is further improved. When stirred for 15 s, the gel is partially dispersed and partially cohesive. When continuously stirred to 95 s, the cohesion of the gel significantly decreases; Comparative Example 11 is encapsulated non - crosslinked sodium hyaluronate particles. When stirred to 15 s, its cohesion is good, but when further stirred to 70 s and 95 s, the cohesion becomes poor and it is completely dispersed.

[0145] Test Example 3 Stability Test:

[0146] Stability accelerated experiment conditions: 50 °C, humidity 75%.

[0147] Dynamic viscosity test method: Take an appropriate amount of gel particles from each group of samples and conduct the test according to the third method of the viscosity determination method in the General Rules of the Fourth Part of the Chinese Pharmacopoeia 2020 Edition, General Rule 0633, Rotating Viscometer Determination Method. The test conditions are a shear rate of not less than 0.25 Hz and (25 ± 0.1) °C.

[0148] The gels prepared in Example 10, Example 13, Example 16 and Comparative Example 1, Comparative Example 5, and Comparative Example 9 were placed in a temperature and humidity stability test chamber, and the dynamic viscosity of the samples was tested after 0 days, 137 days, and 274 days of accelerated stability. The test results are shown in Table 8 below.

[0149] Table 8

[0150] .

[0151] It can be seen from Table 8 that Comparative Example 1 is a cross-linked sodium hyaluronate gel, and its retention value of dynamic viscosity is 42.11% after 274 days of accelerated stability. After adding non-cross-linked sodium hyaluronate in Comparative Example 2, the stability decreased, and its retention value of dynamic viscosity was only 35.42% after 274 days of accelerated stability. After mixing with a low-cross-linking degree cross-linked sodium hyaluronate gel in Comparative Example 9, the stability was improved, and its retention value of dynamic viscosity was 46.57% after 274 days of accelerated stability. After 274 days of accelerated stability in Example 10, Example 13, and Example 16, the viscosity retention value reached more than 90% of that at 0 days, indicating that the gels prepared by the present invention have good viscosity performance stability.

[0152] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A hyaluronic acid gel mixture, characterized in that: Comprising a mixture of disulfide-crosslinked sodium hyaluronate gel and crosslinked sodium hyaluronate gel particles, wherein the crosslinked sodium hyaluronate gel particles are dispersed in the disulfide-crosslinked sodium hyaluronate gel; The hyaluronic acid gel mixture is obtained by the following method: S1. crushing the cross-linked sodium hyaluronate gel into cross-linked sodium hyaluronate gel particles; S2. dispersing the cross-linked sodium hyaluronate gel particles obtained in step S1 in a solution of a thiolated sodium hyaluronate derivative; S3, gradually subjecting the sodium hyaluronate thiolated derivative solution in which the cross-linked sodium hyaluronate gel particles are dispersed, obtained in step S2, to an oxidation process to form a disulfide-bonded cross-linked sodium hyaluronate gel, thereby obtaining a hyaluronic acid gel mixture; When the hyaluronic acid gel mixture is tested in a linear viscoelastic region with a shear strain γ=0.1% and a shear frequency of 1 Hz, the elastic modulus G' of the hyaluronic acid gel mixture is not less than 650 Pa, and the viscous modulus is not less than 70 Pa.

2. The hyaluronic acid gel mixture according to claim 1, wherein The particle size of the cross-linked sodium hyaluronate gel particles ranges from 50 μm to 2500 μm.

3. The hyaluronic acid gel mixture according to claim 2, wherein In the cross-linked sodium hyaluronate gel particles, D50 is not greater than 800 μm and / or D90 is not greater than 2000 μm.

4. The hyaluronic acid gel mixture according to claim 1, wherein The hyaluronic acid content in the cross-linked sodium hyaluronate gel particles is 12 to 50 mg / mL; The hyaluronic acid content in the disulfide bond cross-linked sodium hyaluronate gel is 0.2-10 mg / mL.

5. The hyaluronic acid gel mixture according to claim 4, characterized in that The hyaluronic acid content in the cross-linked sodium hyaluronate gel particles is 16 to 30 mg / mL; The hyaluronic acid content in the disulfide bond cross-linked sodium hyaluronate gel is 0.5-4 mg / mL.

6. The hyaluronic acid gel mixture according to claim 1, wherein The cross-linking degree of the cross-linked sodium hyaluronate gel particles is 1.0% to 20%; The cross-linking degree of the disulfide bond cross-linked sodium hyaluronate gel is 0.05% to 2.5%.

7. The hyaluronic acid gel mixture according to claim 6, wherein The cross-linking degree of the disulfide bond cross-linked sodium hyaluronate gel is 0.1% to 1.0%.

8. The hyaluronic acid gel mixture according to claim 1, wherein The mass ratio of the cross-linked sodium hyaluronate gel particles to the disulfide bond cross-linked sodium hyaluronate gel is 19:1 to 1:

1.

9. Use of the hyaluronic acid gel mixture according to any one of claims 1 to 8 in the preparation of medical products.

10. The use according to claim 9, characterized in that The medical products include products for sealing retinal holes to treat rhegmatogenous retinal detachment, products for vitreous filling, products for viscoelastic treatment of osteoarthritis, products for soft tissue wrinkle filling, products for facial reshaping, and products for increasing sphincter volume.

11. A method for preparing the hyaluronic acid gel mixture according to any one of claims 1 to 8, characterized in that: The steps include: S1. crushing the cross-linked sodium hyaluronate gel into cross-linked sodium hyaluronate gel particles; S2. dispersing the cross-linked sodium hyaluronate gel particles obtained in step S1 in a solution of a thiolated sodium hyaluronate derivative; S3. The sodium hyaluronate thiolated derivative solution in which the cross-linked sodium hyaluronate gel particles are dispersed, obtained in step S2, is gradually subjected to an oxidation process to form a disulfide-crosslinked sodium hyaluronate gel, thereby obtaining a hyaluronic acid gel mixture, wherein the elastic modulus G' of the hyaluronic acid gel mixture tested in the linear viscoelastic region with a shear strain γ = 0.1% and a shear frequency of 1 Hz is not less than 650 Pa, and the viscous modulus is not less than 70 Pa.

12. The preparation method according to claim 11, characterized in that The particle size of the cross-linked sodium hyaluronate gel particles in step S1 ranges from 50 μm to 2500 μm.

13. The preparation method according to claim 12, wherein In the cross-linked sodium hyaluronate gel particles, D50 is not greater than 800 μm and / or D90 is not greater than 2000 μm.

14. The preparation method according to claim 11, wherein The cross-linking degree of the cross-linked sodium hyaluronate gel particles in step S1 is 1.0% to 20%, and the elastic modulus G' tested in the linear viscoelastic region with a shear strain γ=0.1% and a shear frequency of 1 Hz is not less than 700 Pa.

15. The preparation method according to claim 11, wherein The hyaluronic acid content in the cross-linked sodium hyaluronate gel particles in step S1 is 12 to 50 mg / mL.

16. The preparation method according to claim 15, characterized in that The hyaluronic acid content in the cross-linked sodium hyaluronate gel particles in step S1 is 16 to 30 mg / mL.

17. The preparation method according to claim 11, wherein The hyaluronic acid content in the thiolated sodium hyaluronate derivative solution in step S2 is 0.2-10 mg / mL, and the pH value is 6.8-7.

6.

18. The preparation method according to claim 11, wherein The cross-linking degree of the disulfide-crosslinked sodium hyaluronate gel in the hyaluronic acid gel mixture obtained in step S3 is 0.05% to 2.5%.

19. The preparation method according to claim 18, characterized in that The cross-linking degree of disulfide-crosslinked sodium hyaluronate gel in the hyaluronic acid gel mixture obtained in step S3 is 0.1% to 1.0%.

20. The preparation method according to claim 11, wherein In the hyaluronic acid gel mixture obtained in step S3, the mass ratio of cross-linked sodium hyaluronate gel particles to disulfide bond cross-linked sodium hyaluronate gel is 19:1 to 1:1.

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