Heterogeneous hyaluronic acid gel particles, their application, preparation and products containing same

Abstract

The present invention discloses a non-uniform hyaluronic acid gel particle, its application, preparation and products containing the same. The non-uniform hyaluronic acid gel particle has an average content of cross-linked hyaluronic acid of not less than 25 mg / mL, and the content of cross-linked hyaluronic acid in the inner core is higher than that on the surface, and is suitable for application in cosmetics, joint cavity injection, tissue filler and other products. The present invention prepares the non-uniform hyaluronic acid gel particles by sequentially immersing first hyaluronic acid gel particles having uniformly distributed cross-linked hyaluronic acid in a gradient ethanol aqueous solution with gradually increasing ethanol concentration, in a reducing compound aqueous solution and in an isotonic buffer solution. The non-uniform hyaluronic acid gel particles of the present invention have a large elastic modulus and high resistance to enzymolysis, can meet the shaping requirements of medical aesthetics and the sufficient retention time and supporting performance required for serious medical treatment, and the pushing force meets the injection requirements.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a kind of non-uniform hyaluronic acid gel particles, the application and preparation thereof and products containing the same. Background Art

[0002] Hyaluronic acid is a non-sulfonated 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 mechanical properties. Injections made of hyaluronic acid can be used in ophthalmology, pelvic and abdominal surgery to prevent postoperative adhesions, to increase viscosity in osteoarthritis and rheumatoid arthritis, and in facial injection beauty.

[0003] Since hyaluronic acid is rapidly degraded by hyaluronidase in the body, its clinical application is limited. The cross-linking reaction can effectively reduce the fluidity of hyaluronic acid and delay its degradation and absorption in the body. Generally, the higher the content of cross-linked hyaluronic acid, the higher the cross-linking rate, the greater the elastic modulus G' of the material, and the stronger its resistance to enzymatic hydrolysis and the support performance of the material. When used for medical and cosmetic purposes, it is necessary to increase the volume of the skin or tissue, especially when shaping the dorsum and / or nasal root and the mandibular area, the material is required to have strong bony support properties to achieve a good plastic effect; when used for serious medical purposes, such as application to sphincter tissue to treat urinary incontinence, filling the volume of damaged vocal cords, and soft tissue filling for metatarsalgia, the material is required to have sufficient retention time and support performance.

[0004] Patent publication number CN101056891A provides a method for preparing a cross-linked hyaluronic acid gel, providing a new method for preparing a cross-linked hyaluronic acid gel with a low cross-linking agent content and excellent viscoelasticity: a mixture containing more than 10 W / V% of hyaluronic acid, a cross-linking agent and water is stirred and mixed under acidic or alkaline conditions. The sodium hyaluronate gel prepared by this method has a high elastic modulus, but its cross-linking rate is low, and the comprehensive anti-enzymatic ability is still insufficient.

[0005] Products already on the market include Hyaluronic acid content can reach 20mg / mL and is used for facial filling or viscosity enhancement treatment of arthritis. They can provide longer-lasting results compared with bovine collagen or hylan B. Some products (such as Extreme and Elegant) increase the hyaluronic acid content to 24mg / mL, but their support performance and maintenance effect still have room for improvement. In addition, the hyaluronic acid gel in this type of product has consistent content inside and outside (that is, in the hyaluronic acid gel system, the hyaluronic acid is evenly dispersed, and the inner core content is consistent with the surface content). In order to facilitate clinical injection, a certain proportion (5-10%) of non-cross-linked hyaluronic acid needs to be added. Although this type of product reduces the pushing force of the gel to a certain extent, due to the poor thermal stability of non-cross-linked hyaluronic acid, the gel viscosity stability is also reduced.

[0006] Therefore, there is an urgent need in the art for a new hyaluronic acid gel that has both a high content and an uneven content, and whose enzymatic hydrolysis ability is better than the currently marketed products and technologies, and does not require the addition of non-cross-linked hyaluronic acid. This new hyaluronic acid gel has both better maintenance time and stability, which is of positive significance for the preparation and application of hyaluronic acid gel. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a kind of heterogeneous hyaluronic acid gel particles, their application, preparation and products containing the same. The heterogeneous hyaluronic acid gel particles prepared by the preparation method of heterogeneous hyaluronic acid gel particles have high anti-enzymatic performance, cross-linked hyaluronic acid content and viscosity stability. Without adding non-cross-linked hyaluronic acid, the pushing force can meet the clinical use requirements.

[0008] In order to solve the above technical problems, the first aspect of the present invention is to provide a heterogeneous hyaluronic acid gel particle, wherein the average content of cross-linked hyaluronic acid is not less than 25 mg / mL, and the content of cross-linked hyaluronic acid in the inner core is higher than that on the surface; preferably, in the heterogeneous hyaluronic acid gel particle, the average content of cross-linked hyaluronic acid is not less than 28 mg / mL, and the content of cross-linked hyaluronic acid in the inner core is 2.5 times or more of the content on the surface.

[0009] Specifically, the elastic modulus G' of the heterogeneous hyaluronic acid gel particles is greater than 1400 Pa (shear rate 5 Hz); preferably, the elastic modulus G' of the heterogeneous hyaluronic acid gel particles is greater than 2000 Pa (shear rate 5 Hz).

[0010] Specifically, the particle size of the non-uniform hyaluronic acid gel particles is 20-3000μm; preferably, the particle size of the non-uniform hyaluronic acid gel particles is 50-1600μm; more preferably, the particle size of the non-uniform hyaluronic acid gel particles is 50μm, 100μm, 150μm, 200μm, 300μm, 400μm, 500μm, 600μm, 700μm, 800μm, 900μm, 1000μm, 1100μm, 1200μm, 1300μm, 1400μm, 1500μm or 1600μm.

[0011] In the present invention, non-uniform hyaluronic acid gel particles refer to that in a granular gel system, hyaluronic acid is not uniformly distributed in the gel system, especially cross-linked (disulfide cross-linked) hyaluronic acid is non-uniformly distributed in the gel system; in particular, the non-uniform hyaluronic acid gel particles provided by the present invention are characterized in that the concentration of cross-linked hyaluronic acid gradually increases from the surface to the center, and preferably the content of its kernel is at least 2.5 times the surface content, so that the cross-linked hyaluronic acid content on its surface is low, and the pushing force required for clinical needs can be met without adding non-cross-linked hyaluronic acid, and since non-cross-linked hyaluronic acid does not need to be added, its viscosity stability is not affected. It should be noted that the kernel described in the present invention refers to the central part of the non-uniform hyaluronic acid gel particles, and the volume occupied by the kernel is 10%-30% of the total volume of the non-uniform hyaluronic acid gel particles. At the same time, since the non-uniform hyaluronic acid gel particles of the present invention have a high content of cross-linked hyaluronic acid and a large elastic modulus G', they have a high resistance to enzymolysis.

[0012] In order to solve the above technical problems, the second aspect of the present invention is to provide a pharmaceutical product, which is obtained by the aforementioned non-uniform hyaluronic acid gel particles through filling, sterilization and other steps, without the need to compound non-cross-linked hyaluronic acid. Since the pharmaceutical product of the present invention is prepared from the aforementioned non-uniform hyaluronic acid gel particles of the present invention, it at least has all the beneficial effects of the aforementioned non-uniform hyaluronic acid gel particles.

[0013] In order to solve the above technical problems, the third aspect of the present invention is to provide the use of the aforementioned heterogeneous hyaluronic acid gel particles in cosmetics, joint cavity injections, and tissue fillers. In particular, the heterogeneous hyaluronic acid gel particles of the present invention have good resistance to enzymatic hydrolysis, high elastic modulus and viscosity stability, can meet the sufficient retention time and support performance required for medical aesthetics and serious medical treatment, and the pushing force meets the injection requirements.

[0014] In order to solve the above technical problems, the fourth aspect of the present invention is to provide a method for preparing the above-mentioned heterogeneous hyaluronic acid gel particles, comprising the following steps:

[0015] Step S1, soaking the first hyaluronic acid gel particles with uniform cross-linked hyaluronic acid content in gradient ethanol aqueous solutions with gradually increasing ethanol concentrations, each soaking time is 16-48 hours, and filtering after the soaking to obtain second hyaluronic acid gel particles;

[0016] Step S2, soaking the second hyaluronic acid gel particles obtained in step S1 in an aqueous solution of a reducing compound for 0.02-2 hours, and filtering after the soaking to obtain third hyaluronic acid gel particles;

[0017] Step S3, soaking the third hyaluronic acid gel particles obtained in step S2 in an isotonic buffer solution for 6-24 hours, and filtering after soaking to obtain non-uniform hyaluronic acid gel particles.

[0018] In the present invention, the first hyaluronic acid gel particles with uniform cross-linked hyaluronic acid content can be prepared by using thiolated hyaluronic acid derivatives. Specifically, a certain proportion of sustained-release solution is added to the thiolated hyaluronic acid derivatives and the pH is adjusted to 7.2-7.4. Under the oxidation of oxygen, disulfide bonds are cross-linked to obtain the first hyaluronic acid gel particles with uniform cross-linked hyaluronic acid content; wherein the thiol content of the thiolated hyaluronic acid derivatives is preferably 50-500 μmol / g, more preferably 100-250 μmol / g, and the sustained-release solution can be, for example, phosphate buffer. It should be noted that the thiolated hyaluronic acid derivative refers to a hyaluronic acid derivative containing a thiol group, which can be prepared by thiolation modification of hyaluronic acid, and also includes thiolated derivatives prepared by further thiolation modification of various hyaluronic acid derivatives (such as carboxymethyl hyaluronic acid, acetylated hyaluronic acid and its sodium salt, potassium salt, etc.). For example, the various methods for preparing thiolated hyaluronic acid derivatives disclosed in previous documents such as Shu et al., Biomacromolecules 2002, 3:1304-1311 can be used to prepare the thiolated hyaluronic acid derivatives described in the present invention.

[0019] In the present invention, first hyaluronic acid gel particles with uniform cross-linked hyaluronic acid content are sequentially immersed in a gradient ethanol aqueous solution with gradually increasing ethanol concentration, in a reducing compound aqueous solution, and in an isotonic buffer solution to finally form non-uniform hyaluronic acid gel particles, and the cross-linked hyaluronic acid content in the inner core is greater than the content on the surface; the first hyaluronic acid gel particles are sequentially immersed in a gradient ethanol aqueous solution with gradually increasing ethanol concentration, so that the volume of the first hyaluronic acid gel particles is gradually shrunk, thereby increasing the content of cross-linked hyaluronic acid, and forming second hyaluronic acid gel particles, wherein the cross-linked hyaluronic acid is uniformly distributed and the cross-linked structure is more compact. It should be noted that the second hyaluronic acid gel particles may also turn white, which may be caused by the precipitation of hyaluronic acid, which makes the second hyaluronic acid gel particles The hyaluronic acid gel particles turn white; the second hyaluronic acid gel particles are continuously immersed in the aqueous solution of the reducing compound in order to partially open the disulfide bonds on the surface, so that the dense surface structure becomes loose, and a third hyaluronic acid gel particle is formed; the third hyaluronic acid gel particle is continuously immersed in the isotonic buffer solution in order to remove the residual ethanol and the reducing compound. At the same time, the surface of the third hyaluronic acid gel particle absorbs water, so that the content of cross-linked hyaluronic acid on the surface is reduced, but the inner core still maintains a high content, and finally non-uniform hyaluronic acid gel particles are formed. For the whitish second hyaluronic acid gel particles, after this step, the transparent state is restored; the content of the inner core (the central area accounting for 10%-30% of the volume) of the third hyaluronic acid gel particle is at least 2.5 times the surface content.

[0020] Specifically, in step S1, the content of cross-linked hyaluronic acid in the first hyaluronic acid gel particles is 15-25 mg / mL;

[0021] In the various ethanol aqueous solutions, the volume concentration of ethanol is not less than 30% and not more than 80%, and the volume concentration difference between adjacent ethanol aqueous solutions is not less than 15%.

[0022] In the first hyaluronic acid gel particles, the content of cross-linked hyaluronic acid is preferably not more than 25 mg / mL. In order to further increase its content, the first hyaluronic acid gel particles are sequentially immersed in gradient ethanol aqueous solutions with gradually increasing concentrations. It should be noted that if the concentration of the ethanol aqueous solution is too low (for example, less than 30%), the volume shrinkage of the first hyaluronic acid gel particles is limited, and the degree of increase in the content of cross-linked hyaluronic acid is limited. However, if the concentration of the ethanol aqueous solution is too high (for example, higher than 80%), the surface of the gel particles will shrink too quickly, affecting the internal water loss, thereby affecting the further shrinkage of the gel particles, so that the overall cross-linked hyaluronic acid content of the second hyaluronic acid gel particles finally formed is lower than expected, and the volume shrinkage rate cannot reach the expected level. At the same time, it is also found that when the concentration of the ethanol aqueous solution is too high, the formation The second hyaluronic acid gel particles turn white, and the transparency cannot be restored by soaking in an isotonic buffer solution later; therefore, in the present invention, the first hyaluronic acid gel particles need to be soaked in an ethanol aqueous solution with a concentration between 30% and 80% and a concentration gradient in sequence, and the concentration difference of adjacent gradient concentration ethanol solutions is not less than 15%, and finally second hyaluronic acid gel particles with a higher cross-linked hyaluronic acid content are formed. For the second hyaluronic acid gel particles with a white surface, they can restore their transparency after being soaked in a subsequent isotonic buffer solution; compared with the first hyaluronic acid gel particles, the elastic modulus of the second hyaluronic acid with a higher cross-linked hyaluronic acid content is effectively improved, and its anti-enzymatic ability is also improved, and its support is also stronger, but it cannot yet meet the injection requirements.

[0023] Specifically, in step S2, the mass concentration of the reducing compound in the reducing compound aqueous solution is 0.001%-0.2%; preferably, the reducing compound is dithiothreitol and / or tris(2-carboxyethyl)phosphine hydrochloride.

[0024] During the immersion of the second hyaluronic acid gel particles in the reducing compound aqueous solution, the disulfide bonds on their surface are partially opened, and the dense surface structure becomes loose, forming third hyaluronic acid gel particles; it should be noted that if the disulfide bonds are excessively opened, the subsequent gel will absorb too much water and reduce the content of cross-linked hyaluronic acid. Therefore, the concentration and immersion time of the reducing compound aqueous solution need to be reasonably controlled. For example, it can be 0.001% dithiothreitol solution and / or tris(2-carboxyethyl)phosphine hydrochloride solution for 2 hours, 0.01% dithiothreitol solution and / or tris(2-carboxyethyl)phosphine hydrochloride solution for 0.2 hours, 0.1% dithiothreitol solution and / or tris(2-carboxyethyl)phosphine hydrochloride solution for 0.02 hours, etc.

[0025] Specifically, in step S3, the isotonic buffer is a phosphate buffer.

[0026] The third hyaluronic acid is immersed in an isotonic buffer solution to remove impurities such as residual ethanol and reducing compounds, and its surface absorbs water, thereby reducing the content of surface cross-linked hyaluronic acid, but the inner core still maintains a high content of cross-linked hyaluronic acid, forming non-uniform hyaluronic acid gel particles. At the same time, for the whitish second hyaluronic acid gel particles, they gradually recover their transparency during the immersion process. It should be noted that the isotonic buffer solution is preferably replaced at an interval of, for example, 2 hours. In addition, the immersion in the isotonic buffer solution is terminated when the ethanol content in the non-uniform hyaluronic acid gel particles is less than 40 ppm and the content of dithiothreitol and / or tri(2-carboxyethyl)phosphine hydrochloride is less than 2 ppm. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solution of the present invention, the following briefly introduces the drawings required for use in the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 These are the in vitro enzymatic hydrolysis curves of the hyaluronic acid gel particles prepared in Example 2, Example 4, Example 7, and Comparative Examples 1-2. DETAILED DESCRIPTION

[0029] The following will be combined with the accompanying drawings to clearly and completely describe the technical solutions in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Example 1

[0031] Preparation of thiolated derivatives of hyaluronic acid: Sodium hyaluronate with a molecular weight of 1200 KDa was used as raw material and prepared by the method reported by Shu et al. (Shu et al., Biomacromolecules 2002, 3:1304-1311). By adjusting the material ratio, thiolated modified derivatives of hyaluronic acid were synthesized, and the thiol contents of the derivatives were 101.2 μmol / g polymer, 152.3 μmol / g polymer and 253.4 μmol / g polymer, respectively.

[0032] Preparation of the first hyaluronic acid gel particles: add phosphate buffer solution to the thiolated hyaluronic acid derivative prepared above to adjust the pH to 7.2-7.4, and obtain hyaluronic acid gel after oxidative cross-linking and homogenization under the action of oxygen, which is granulated to obtain the first hyaluronic acid gel particles.

[0033] In this embodiment, nine groups of first hyaluronic acid gel particles were prepared, as shown in Table 1.

[0034] Table 1

[0035]

[0036]

[0037] Preparation of the second hyaluronic acid gel particles: 6 g of the first hyaluronic acid gel particles-1 were soaked in 50 ml of 40% ethanol aqueous solution for 16 hours and in 50 ml of 80% ethanol aqueous solution for 16 hours, and filtered to obtain the second hyaluronic acid gel particles;

[0038] Preparation of the third hyaluronic acid gel particles: The second hyaluronic acid gel particles were immersed in 20 ml of 0.01% dithiothreitol solution (pH=8.0) for 0.2 hours, and filtered to obtain the third hyaluronic acid gel particles.

[0039] Preparation of non-uniform hyaluronic acid gel particles: The third hyaluronic acid gel particles are immersed in 50 ml of isotonic buffer (phosphate buffer) (the isotonic buffer is replaced every 2 hours). When the ethanol content is lower than 40 ppm and the dithiothreitol content is lower than 2 ppm, the non-uniform hyaluronic acid gel particles are obtained by filtration.

[0040] Example 2

[0041] Preparation of the second hyaluronic acid gel particles: Based on the first hyaluronic acid gel particles prepared in Example 1, 6 g of the first hyaluronic acid gel particles-1 were sequentially immersed in 50 ml of 40% ethanol aqueous solution for 16 hours, 50 ml of 65% ethanol aqueous solution for 8 hours, and 50 ml of 80% ethanol aqueous solution for 16 hours, and filtered to obtain the second hyaluronic acid gel particles;

[0042] Preparation of the third hyaluronic acid gel particles: soak the second hyaluronic acid gel particles in 20 ml of 0.01% dithiothreitol solution (pH=8.0) for 0.2 hours, and filter to obtain the third hyaluronic acid gel particles;

[0043] Preparation of non-uniform hyaluronic acid gel particles: The third hyaluronic acid gel particles are immersed in 50 ml of isotonic buffer (phosphate buffer) (the isotonic buffer is replaced every 2 hours). When the ethanol content is lower than 40 ppm and the dithiothreitol content is lower than 2 ppm, the non-uniform hyaluronic acid gel particles are obtained by filtration.

[0044] Example 3

[0045] Preparation of the second hyaluronic acid gel particles: Based on the first hyaluronic acid gel particles prepared in Example 1, 6 g of the first hyaluronic acid gel particles-1 were soaked in 50 ml of 30% ethanol aqueous solution for 16 hours and in 50 ml of 80% ethanol aqueous solution for 16 hours, and filtered to obtain the second hyaluronic acid gel particles;

[0046] Preparation of the third hyaluronic acid gel particles: soak the second hyaluronic acid gel particles in 20 ml of 0.01% dithiothreitol solution (pH=8.0) for 0.2 hours, and filter to obtain the third hyaluronic acid gel particles;

[0047] Preparation of non-uniform hyaluronic acid gel particles: The third hyaluronic acid gel particles are immersed in 50 ml of isotonic buffer (phosphate buffer) (the isotonic buffer is replaced every 2 hours). When the ethanol content is lower than 40 ppm and the dithiothreitol content is lower than 2 ppm, the non-uniform hyaluronic acid gel particles are obtained by filtration.

[0048] Example 4

[0049] Preparation of the second hyaluronic acid gel particles: Based on the first hyaluronic acid gel particles prepared in Example 1, 6 g of the first hyaluronic acid gel particles-1 were sequentially placed in 50 ml of 30% ethanol aqueous solution for 16 hours, 50 ml of 65% ethanol aqueous solution for 8 hours, and 50 ml of 80% ethanol aqueous solution for 16 hours, and filtered to obtain the second hyaluronic acid gel particles;

[0050] Preparation of the third hyaluronic acid gel particles: soak the second hyaluronic acid gel particles in 20 ml of 0.01% dithiothreitol solution (pH=8.0) for 0.2 hours, and filter to obtain the third hyaluronic acid gel particles;

[0051] Preparation of non-uniform hyaluronic acid gel particles: The third hyaluronic acid gel particles are immersed in 50 ml of isotonic buffer (phosphate buffer) (the isotonic buffer is replaced every 2 hours). When the ethanol content is lower than 40 ppm and the dithiothreitol content is lower than 2 ppm, the non-uniform hyaluronic acid gel particles are obtained by filtration.

[0052] Example 5

[0053] Preparation of the second hyaluronic acid gel particles: Based on the first hyaluronic acid gel particles prepared in Example 1, 6 g of the first hyaluronic acid gel particles-1 were sequentially immersed in 50 ml of 40% ethanol aqueous solution for 16 hours, 50 ml of 65% ethanol aqueous solution for 8 hours, and 50 ml of 80% ethanol aqueous solution for 16 hours, and filtered to obtain the second hyaluronic acid gel particles;

[0054] Preparation of the third hyaluronic acid gel particles: soak the second hyaluronic acid gel particles in 20 ml of 0.1% dithiothreitol solution (pH=8.0) for 0.02 hours, and filter to obtain the third hyaluronic acid gel particles;

[0055] Preparation of non-uniform hyaluronic acid gel particles: The third hyaluronic acid gel particles are immersed in 50 ml of isotonic buffer (phosphate buffer) (the isotonic buffer is replaced every 2 hours). When the ethanol content is lower than 40 ppm and the dithiothreitol content is lower than 2 ppm, the non-uniform hyaluronic acid gel particles are obtained by filtration.

[0056] Example 6

[0057] Preparation of the second hyaluronic acid gel particles: Based on the first hyaluronic acid gel particles prepared in Example 1, 6 g of the first hyaluronic acid gel particles-1 were sequentially immersed in 50 ml of 40% ethanol aqueous solution for 16 hours, 50 ml of 65% ethanol aqueous solution for 8 hours, and 50 ml of 80% ethanol aqueous solution for 16 hours, and filtered to obtain the second hyaluronic acid gel particles;

[0058] Preparation of the third hyaluronic acid gel particles: soak the second hyaluronic acid gel particles in 20 ml of 0.001% dithiothreitol solution (pH=8.0) for 2 hours, and filter to obtain the third hyaluronic acid gel particles;

[0059] Preparation of non-uniform hyaluronic acid gel particles: The third hyaluronic acid gel particles are immersed in 50 ml of isotonic buffer (phosphate buffer) (the isotonic buffer is replaced every 2 hours). When the ethanol content is lower than 40 ppm and the dithiothreitol content is lower than 2 ppm, the non-uniform hyaluronic acid gel particles are obtained by filtration.

[0060] Example 7

[0061] Preparation of the second hyaluronic acid gel particles: Based on the first hyaluronic acid gel particles prepared in Example 1, 6 g of the first hyaluronic acid gel particles-1 were sequentially immersed in 50 ml of 40% ethanol aqueous solution for 16 hours, 50 ml of 60% ethanol aqueous solution for 8 hours, and 50 ml of 80% ethanol aqueous solution for 16 hours, and filtered to obtain the second hyaluronic acid gel particles;

[0062] Preparation of the third hyaluronic acid gel particles: soak the second hyaluronic acid gel particles in 20 ml of 0.01% dithiothreitol solution (pH=8.0) for 0.2 hours, and filter to obtain the third hyaluronic acid gel particles;

[0063] Preparation of non-uniform hyaluronic acid gel particles: The third hyaluronic acid gel particles are immersed in 50 ml of isotonic buffer (phosphate buffer) (the isotonic buffer is replaced every 2 hours). When the ethanol content is lower than 40 ppm and the dithiothreitol content is lower than 2 ppm, the non-uniform hyaluronic acid gel particles are obtained by filtration.

[0064] Example 8

[0065] Preparation of the second hyaluronic acid gel particles: Based on the first hyaluronic acid gel particles prepared in Example 1, 6 g of the first hyaluronic acid gel particles-2 were sequentially placed in 50 ml of 40% ethanol aqueous solution for 16 hours, 50 ml of 60% ethanol aqueous solution for 8 hours, and 50 ml of 80% ethanol aqueous solution for 16 hours, and filtered to obtain the second hyaluronic acid gel particles;

[0066] Preparation of the third hyaluronic acid gel particles: soak the second hyaluronic acid gel particles in 20 ml of 0.01% dithiothreitol solution (pH=8.0) for 0.2 hours, and filter to obtain the third hyaluronic acid gel particles;

[0067] Preparation of non-uniform hyaluronic acid gel particles: The third hyaluronic acid gel particles are immersed in 50 ml of isotonic buffer (phosphate buffer) (the isotonic buffer is replaced every 2 hours). When the ethanol content is lower than 40 ppm and the dithiothreitol content is lower than 2 ppm, the non-uniform hyaluronic acid gel particles are obtained by filtration.

[0068] Example 9

[0069] Preparation of the second hyaluronic acid gel particles: Based on the first hyaluronic acid gel particles prepared in Example 1, 6 g of the first hyaluronic acid gel particles-3 were sequentially placed in 50 ml of 40% ethanol aqueous solution for 16 hours, 50 ml of 60% ethanol aqueous solution for 8 hours, and 50 ml of 80% ethanol aqueous solution for 16 hours, and filtered to obtain the second hyaluronic acid gel particles;

[0070] Preparation of the third hyaluronic acid gel particles: soak the second hyaluronic acid gel particles in 20 ml of 0.01% dithiothreitol solution (pH=8.0) for 0.2 hours, and filter to obtain the third hyaluronic acid gel particles;

[0071] Preparation of non-uniform hyaluronic acid gel particles: The third hyaluronic acid gel particles are immersed in 50 ml of isotonic buffer (phosphate buffer) (the isotonic buffer is replaced every 2 hours). When the ethanol content is lower than 40 ppm and the dithiothreitol content is lower than 2 ppm, the non-uniform hyaluronic acid gel particles are obtained by filtration.

[0072] Example 10

[0073] Preparation of the second hyaluronic acid gel particles: Based on the first hyaluronic acid gel particles prepared in Example 1, 6 g of the first hyaluronic acid gel particles-4 were sequentially immersed in 50 ml of 40% ethanol aqueous solution for 16 hours, 50 ml of 60% ethanol aqueous solution for 8 hours, and 50 ml of 80% ethanol aqueous solution for 16 hours, and filtered to obtain the second hyaluronic acid gel particles;

[0074] Preparation of the third hyaluronic acid gel particles: soak the second hyaluronic acid gel particles in 20 ml of 0.01% dithiothreitol solution (pH=8.0) for 0.2 hours, and filter to obtain the third hyaluronic acid gel particles;

[0075] Preparation of non-uniform hyaluronic acid gel particles: The third hyaluronic acid gel particles are immersed in 50 ml of isotonic buffer (phosphate buffer) (the isotonic buffer is replaced every 2 hours). When the ethanol content is lower than 40 ppm and the dithiothreitol content is lower than 2 ppm, the non-uniform hyaluronic acid gel particles are obtained by filtration.

[0076] Embodiment 11

[0077] Preparation of the second hyaluronic acid gel particles: Based on the first hyaluronic acid gel particles prepared in Example 1, 6 g of the first hyaluronic acid gel particles-5 were sequentially placed in 50 ml of 40% ethanol aqueous solution for 16 hours, 50 ml of 60% ethanol aqueous solution for 8 hours, and 50 ml of 80% ethanol aqueous solution for 16 hours, and filtered to obtain the second hyaluronic acid gel particles;

[0078] Preparation of the third hyaluronic acid gel particles: soak the second hyaluronic acid gel particles in 20 ml of 0.01% dithiothreitol solution (pH=8.0) for 0.2 hours, and filter to obtain the third hyaluronic acid gel particles;

[0079] Preparation of non-uniform hyaluronic acid gel particles: The third hyaluronic acid gel particles are immersed in 50 ml of isotonic buffer (phosphate buffer) (the isotonic buffer is replaced every 2 hours). When the ethanol content is lower than 40 ppm and the dithiothreitol content is lower than 2 ppm, the non-uniform hyaluronic acid gel particles are obtained by filtration.

[0080] Example 12

[0081] Preparation of the second hyaluronic acid gel particles: Based on the first hyaluronic acid gel particles prepared in Example 1, 6 g of the first hyaluronic acid gel particles-6 were sequentially placed in 50 ml of 40% ethanol aqueous solution for 16 hours, 50 ml of 60% ethanol aqueous solution for 8 hours, and 50 ml of 80% ethanol aqueous solution for 16 hours, and filtered to obtain the second hyaluronic acid gel particles;

[0082] Preparation of the third hyaluronic acid gel particles: soak the second hyaluronic acid gel particles in 20 ml of 0.01% dithiothreitol solution (pH=8.0) for 0.2 hours, and filter to obtain the third hyaluronic acid gel particles;

[0083] Preparation of non-uniform hyaluronic acid gel particles: The third hyaluronic acid gel particles are immersed in 50 ml of isotonic buffer (phosphate buffer) (the isotonic buffer is replaced every 2 hours). When the ethanol content is lower than 40 ppm and the dithiothreitol content is lower than 2 ppm, the non-uniform hyaluronic acid gel particles are obtained by filtration.

[0084] Example 13

[0085] Preparation of the second hyaluronic acid gel particles: Based on the first hyaluronic acid gel particles prepared in Example 1, 6 g of the first hyaluronic acid gel particles-7 were sequentially placed in 50 ml of 40% ethanol aqueous solution for 16 hours, 50 ml of 60% ethanol aqueous solution for 8 hours, and 50 ml of 80% ethanol aqueous solution for 16 hours, and filtered to obtain the second hyaluronic acid gel particles;

[0086] Preparation of the third hyaluronic acid gel particles: soak the second hyaluronic acid gel particles in 20 ml of 0.01% dithiothreitol solution (pH=8.0) for 0.2 hours, and filter to obtain the third hyaluronic acid gel particles;

[0087] Preparation of non-uniform hyaluronic acid gel particles: The third hyaluronic acid gel particles are immersed in 50 ml of isotonic buffer (phosphate buffer) (the isotonic buffer is replaced every 2 hours). When the ethanol content is lower than 40 ppm and the dithiothreitol content is lower than 2 ppm, the non-uniform hyaluronic acid gel particles are obtained by filtration.

[0088] Embodiment 14

[0089] Preparation of the second hyaluronic acid gel particles: Based on the first hyaluronic acid gel particles prepared in Example 1, 6 g of the first hyaluronic acid gel particles-8 were sequentially placed in 50 ml of 40% ethanol aqueous solution for 16 hours, 50 ml of 60% ethanol aqueous solution for 8 hours, and 50 ml of 80% ethanol aqueous solution for 16 hours, and filtered to obtain the second hyaluronic acid gel particles;

[0090] Preparation of the third hyaluronic acid gel particles: soak the second hyaluronic acid gel particles in 20 ml of 0.01% dithiothreitol solution (pH=8.0) for 0.2 hours, and filter to obtain the third hyaluronic acid gel particles;

[0091] Preparation of non-uniform hyaluronic acid gel particles: The third hyaluronic acid gel particles are immersed in 50 ml of isotonic buffer (phosphate buffer) (the isotonic buffer is replaced every 2 hours). When the ethanol content is lower than 40 ppm and the dithiothreitol content is lower than 2 ppm, the non-uniform hyaluronic acid gel particles are obtained by filtration.

[0092] Embodiment 15

[0093] Preparation of the second hyaluronic acid gel particles: Based on the first hyaluronic acid gel particles prepared in Example 1, 6 g of the first hyaluronic acid gel particles-9 were sequentially placed in 50 ml of 40% ethanol aqueous solution for 16 hours, 50 ml of 60% ethanol aqueous solution for 8 hours, and 50 ml of 80% ethanol aqueous solution for 16 hours, and filtered to obtain the second hyaluronic acid gel particles;

[0094] Preparation of the third hyaluronic acid gel particles: soak the second hyaluronic acid gel particles in 20 ml of 0.01% dithiothreitol solution (pH=8.0) for 0.2 hours, and filter to obtain the third hyaluronic acid gel particles;

[0095] Preparation of non-uniform hyaluronic acid gel particles: The third hyaluronic acid gel particles are immersed in 50 ml of isotonic buffer (phosphate buffer) (the isotonic buffer is replaced every 2 hours). When the ethanol content is lower than 40 ppm and the dithiothreitol content is lower than 2 ppm, the non-uniform hyaluronic acid gel particles are obtained by filtration.

[0096] Comparative Example 1

[0097] Preparation of the second hyaluronic acid gel particles: Based on the first hyaluronic acid gel particles prepared in Example 1, 6 g

[0098] The first hyaluronic acid gel particle-1 is immersed in 50 ml of 40% ethanol aqueous solution for 32 hours, and filtered to obtain the second hyaluronic acid gel particle; the second hyaluronic acid gel particle is immersed in 50 ml of isotonic buffer solution (the isotonic buffer solution is replaced every 2 hours), and when the ethanol content is lower than 40 ppm, the final product is filtered to obtain transparent hyaluronic acid gel particles. Since it has not been immersed in an aqueous solution of a reducing compound, no disulfide bonds are opened, and the hyaluronic acid gel particles absorb water uniformly in each part of the gel in the isotonic buffer solution. Therefore, the cross-linked hyaluronic acid is uniformly distributed in the hyaluronic acid gel particles of this comparative example. According to Appendix C of YY / T 0962-2021 (Cross-linked sodium hyaluronate gel for plastic surgery), the cross-linked hyaluronic acid content is detected, and the content of cross-linked hyaluronic acid is 14.5 mg / mL.

[0099] Comparative Example 2

[0100] Preparation of the second hyaluronic acid gel particles: Based on the first hyaluronic acid gel particles prepared in Example 1, 6 g

[0101] The first hyaluronic acid gel particle-1 is placed in 50ml 80% concentration of ethanol aqueous solution and soaked for 32 hours, and the second hyaluronic acid gel particle is filtered to obtain the second hyaluronic acid gel particle; The second hyaluronic acid gel particle is placed in 50ml isotonic buffer and soaked (isotonic buffer is replaced at intervals of 2 hours), when the ethanol content is less than 40ppm, the hyaluronic acid gel particles with whitish surface are filtered, and continue to be placed in 50ml isotonic buffer and soak for 72 hours, and the surface of the hyaluronic acid gel particles is still whitish. In this comparative example, due to the first hyaluronic acid gel particle in 80% volume concentration of ethanol aqueous solution, its surface loses water rapidly, forms a dense structure, hinders further internal water loss, and the volume shrinkage is limited, and it is not possible to reach expectations. The content of cross-linked hyaluronic acid in the particle is lower than expected, and subsequent soaking in isotonic buffer, its surface absorbs water, and further reduces the content of cross-linked hyaluronic acid; In addition, the second hyaluronic acid obtained is directly soaked in isotonic solution, and the surface is still whitish, which may be due to its limited water absorption capacity, and the precipitated hyaluronic acid can not be dissolved, causing, affecting its application. In this comparative example, the cross-linked hyaluronic acid content was tested according to Appendix C of YY / T 0962-2021 (Cross-linked sodium hyaluronate gel for plastic surgery), and the average content of cross-linked hyaluronic acid was 10.5 mg / mL.

[0102] Comparative Example 3

[0103] Preparation of the second hyaluronic acid gel particles: Based on the first hyaluronic acid gel particles prepared in Example 1, 6 g

[0104] The first hyaluronic acid gel particle-1 was sequentially immersed in 50 ml of 40% ethanol aqueous solution for 16 hours, 50 ml of 65% ethanol aqueous solution for 8 hours, and 50 ml of 80% ethanol aqueous solution for 16 hours, and filtered to obtain the second hyaluronic acid gel particles; the second hyaluronic acid was immersed in 50 ml of isotonic buffer solution (the isotonic buffer solution was replaced every 2 hours), and when the ethanol content was lower than 40 ppm, transparent hyaluronic acid gel particles were obtained by filtration. Since the hyaluronic acid gel particles were not immersed in an aqueous solution of a reducing compound, no disulfide bonds were opened, and the hyaluronic acid gel particles absorbed water uniformly in each part of the gel in the isotonic buffer solution. Therefore, the cross-linked hyaluronic acid was uniformly distributed in the hyaluronic acid gel particles of this comparative example. According to Appendix C of YY / T 0962-2021 (Cross-linked sodium hyaluronate gel for plastic surgery), the cross-linked hyaluronic acid content was detected, and the content of cross-linked hyaluronic acid was 30.4 mg / mL.

[0105] Comparative Example 4

[0106] Preparation of the second hyaluronic acid gel particles: Based on the first hyaluronic acid gel particles prepared in Example 1, 6 g

[0107] The first hyaluronic acid gel particles-4 were sequentially immersed in 50 ml of 40% ethanol aqueous solution for 16 hours, 50 ml of 65% ethanol aqueous solution for 8 hours, and 50 ml of 80% ethanol aqueous solution for 16 hours, and filtered to obtain the second hyaluronic acid gel particles; the second hyaluronic acid gel particles were immersed in 50 ml of isotonic buffer solution (the isotonic buffer solution was replaced every 2 hours), and when the ethanol content was lower than 40 ppm, transparent hyaluronic acid gel particles were obtained by filtration. Since the hyaluronic acid gel particles were not immersed in an aqueous solution of a reducing compound, no disulfide bonds were opened, and the hyaluronic acid gel particles absorbed water uniformly in each part of the gel in the isotonic buffer solution. Therefore, the cross-linked hyaluronic acid was uniformly distributed in the hyaluronic acid gel particles of this comparative example. According to Appendix C of YY / T0962-2021 (Cross-linked sodium hyaluronate gel for plastic surgery), the cross-linked hyaluronic acid content was detected, and the cross-linked hyaluronic acid content was 39.2 mg / mL.

[0108] Comparative Example 5

[0109] Preparation of the second hyaluronic acid gel particles: On the basis of the first hyaluronic acid gel particles prepared in Example 1, 6 g of the first hyaluronic acid gel particles-7 were sequentially placed in 50 ml of 40% ethanol aqueous solution for 16 hours, 50 ml of 65% ethanol aqueous solution for 8 hours, and 50 ml of 80% ethanol aqueous solution for 16 hours, and filtered to obtain the second hyaluronic acid gel particles; the second hyaluronic acid gel particles were placed in 50 ml of isotonic buffer solution for immersion (the isotonic buffer solution was replaced every 2 hours), and when the ethanol content was lower than 40 ppm, transparent hyaluronic acid gel particles were obtained by filtration. Since the hyaluronic acid gel particles were not soaked in an aqueous solution of a reducing compound, no disulfide bonds were opened, and the hyaluronic acid gel particles absorbed water uniformly in each part of the gel in the isotonic buffer solution. Therefore, the cross-linked hyaluronic acid was uniformly distributed in the hyaluronic acid gel particles of this comparative example. According to Appendix C of YY / T0962-2021 (Cross-linked sodium hyaluronate gel for plastic surgery), the cross-linked hyaluronic acid content was detected, and the cross-linked hyaluronic acid content was 44.7 mg / mL.

[0110] Test Example 1 Determination of the content of cross-linked hyaluronic acid in non-uniform hyaluronic acid gel particles of Examples 1-15

[0111] Test method for internal content and surface content of non-uniform hyaluronic acid gel particles:

[0112] 1) Take 3 g of sample and place it on the surface of a 200-mesh sieve. Use a rubber scraper to squeeze the gel particles and collect the gel particles on the back of the sieve for later use.

[0113] 2) Screening of gel separation liquid: Take the gel particles after passing through the sieve and soak them in 34%, 35%, 36%, 37%, and 38% ethanol aqueous solutions for 1 hour. After filtering, dry the residual solution on the surface of the gel particles and weigh them. The mass of the gel particles after soaking is 98%-102% of the mass of the gel particles before soaking, which is used as the separation liquid for this group of gels.

[0114] 3) Take another 0.5 g of the gel particles after the sieve and place them in a 15 ml centrifuge tube, add 10 ml of separation solution, and centrifuge at 100-200 rpm for 1 minute. The part with lower hyaluronic acid content will be suspended / floated on the upper layer of the separation solution, and the part with higher hyaluronic acid content will gather at the bottom of the centrifuge tube. Use a pipette to separate the two and set aside.

[0115] 4) Test the hyaluronic acid content according to Appendix C of YY / T 0962-2021 (Cross-linked sodium hyaluronate gel for plastic surgery);

[0116] The test results of the internal content and surface content of the non-uniform hyaluronic acid gel particles of Examples 1-15 are shown in Table 2.

[0117] Table 2

[0118]

[0119] As can be seen from the above table, the content of the cross-linked hyaluronic acid in the inner core of the non-uniform hyaluronic acid gel particles prepared in each group of Examples 1-15 is more than 2.5 times the content in the outer core.

[0120] Test Example 2 Elastic modulus G' and push force test

[0121] Elastic modulus G′ test method: Take 1 g of sample and use a rheometer (Anton Paar, MCR 301) to test the sample. The test uses a flat plate with a diameter of 50 mm, the plate spacing is set to 0.5 mm, the test temperature is 25° C., and the shear strain is 0.1%.

[0122] Pushing force test method: 1ml glass tube syringe, equipped with a 27G needle, is pushed at a speed of 30mm / min, and the average pushing force of the syringe handle is recorded.

[0123] The elastic modulus G′ of the gels prepared in Examples 1-15 and Comparative Examples 1 and 3-5 was tested, and the test results are shown in Table 3 below:

[0124] Table 3

[0125]

[0126]

[0127] As shown in the table above, the elastic modulus G' of each group of samples in Examples 1-15 is greater than 2000Pa, and the average pushing force is appropriate; although the gel prepared in Comparative Example 1 has a smaller pushing force, the cross-linked hyaluronic acid content is low, and the elastic modulus is very low, which does not meet the application requirements; although the elastic modulus G' of the gel prepared in Comparative Examples 3-5 is higher, the average pushing force is greater than 80N, and pushing is very difficult. Each group of samples in Examples 1-15 has both a higher elastic modulus and a more appropriate pushing force.

[0128] Test Example 3: Stability Test

[0129] The stability test item tests the viscosity and pushing force of the sample.

[0130] Viscosity test method: Take an appropriate amount of sample gel particles from each group and test them according to the third rotational viscometer method of viscosity determination method 0633 in the fourth general rule of the 2020 edition of the Pharmacopoeia of the People's Republic of China. The test conditions are a shear rate of not less than 0.25 Hz and (25±0.1)°C.

[0131] Pushing force test method: Use a 1ml glass tube syringe, install a 27G needle, push at a speed of 30mm / min, and record the average pushing force of the syringe handle.

[0132] The non-uniform hyaluronic acid gel particles obtained in Examples 1, 10 and 13 were taken and filled with a 1 mL glass tube syringe, and then the hyaluronic acid gel particles prepared in Comparative Examples 3-5 were mixed with 20 mg / mL non-cross-linked sodium hyaluronate solution (Mw=1200 KDa) at a mass ratio of 9:1 and filled with a 1 mL glass tube syringe. The glass tube syringe filled with Examples 1, 10, 13 and Comparative Examples 3-5 was placed in a 50°C, 75% humidity stability test box for accelerated testing, and the test was performed after 0 days, 68 days, and 137 days. The test items included adhesion and pushing force, and the test results are shown in Table 4 below.

[0133] Table 4

[0134]

[0135]

[0136] According to Table 4, after adding non-cross-linked sodium hyaluronate, the pushing force of the gel was reduced in Comparative Examples 3 to 5, however, the viscosity stability thereof was greatly reduced, and after 137 days of accelerated stability, the viscosity retention values ​​of Comparative Examples 3 to 5 were only about 33% of that of day 0. However, after 137 days of accelerated stability, the viscosity retention values ​​of Example 1, Example 10, and Example 13 reached about 89% of that of day 0, indicating that the gel prepared by the present invention has good viscosity performance stability.

[0137] Test Example 4 In vitro anti-enzymatic performance test

[0138] In vitro anti-enzymatic performance test method: Accurately weigh 0.5g of the hyaluronic acid gel particles prepared in Examples 2, 4, 7 and Comparative Examples 1 and 2, add 1.5ml of hyaluronidase solution (100U / mL), place the sample in a constant temperature shaker (37°C, 20rmp / min), take out within the specified time, boil at 100°C for 10min to inactivate, centrifuge the inactivated liquid, filter with a 0.22um microporous filter membrane, take 1.0mL of the filtrate, and use the glucuronic acid method (YY / T 0962-2021, Appendix C, Determination of sodium hyaluronate content) for content test.

[0139] Calculation method for in vitro enzymatic hydrolysis of hyaluronic acid gel particles:

[0140] Enzyme hydrolysis rate / %=4a / b*100%; wherein a is the content of sodium hyaluronate in the inactivation solution after centrifugation; b is the content of sodium hyaluronate in the sample.

[0141] The lower the enzymatic degradation rate, the better the gel's resistance to enzymatic degradation and the longer it can be stored in the body.

[0142] like Figure 1 As shown, the in vitro enzymatic hydrolysis curves of the hyaluronic acid gel particles prepared in Example 2, Example 4, Example 7, Comparative Example 1 and Comparative Example 2.

[0143] It can be seen that compared with Comparative Example 1, at the same time point, Examples 2, 4, and 7 have lower enzymatic degradation percentages, better anti-enzymatic degradation performance of the gel, and longer storage time in the body; and although the anti-enzymatic degradation performance of Comparative Example 2 in the figure seems to be better, after 24 hours, about 35% of the gel is still not enzymatically degraded, indicating that its biocompatibility is poor.

[0144] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A non-uniform hyaluronic acid gel particle, characterized in that: The non-uniform hyaluronic acid gel particles are obtained by the following steps: Step S1, soaking the first hyaluronic acid gel particles with uniform cross-linked hyaluronic acid content in gradient ethanol aqueous solutions with gradually increasing ethanol concentrations, each soaking time is 16-48 hours, and filtering after the soaking to obtain second hyaluronic acid gel particles; Step S2, soaking the second hyaluronic acid gel particles obtained in step S1 in an aqueous solution of a reducing compound for 0.02-2 hours, and filtering after the soaking to obtain third hyaluronic acid gel particles; Step S3, placing the third hyaluronic acid gel particles obtained in step S2 in an isotonic buffer solution and soaking for 6-24 hours, and filtering after soaking to obtain non-uniform hyaluronic acid gel particles; In step S1, in the first hyaluronic acid gel particles, the content of cross-linked hyaluronic acid is 15-25 mg / mL; in the multiple ethanol aqueous solutions, the volume concentration of ethanol is not less than 30% and not more than 80%, and the volume concentration difference of adjacent concentrations of ethanol aqueous solutions is not less than 15%; In step S2, the mass concentration of the reducing compound in the reducing compound aqueous solution is 0.001%-0.2%; The reducing compound is dithiothreitol and / or tris(2-carboxyethyl)phosphine hydrochloride; In the non-uniform hyaluronic acid gel particles, the average content of cross-linked hyaluronic acid is not less than 25 mg / mL, and the content of cross-linked hyaluronic acid in the inner core is 2.5 times or more of the content on the surface.

2. The heterogeneous hyaluronic acid gel particles according to claim 1, characterized in that: The elastic modulus G' of the non-uniform hyaluronic acid gel particles is greater than 1400Pa.

3. The heterogeneous hyaluronic acid gel particles according to claim 1, characterized in that: The particle size of the non-uniform hyaluronic acid gel particles is 20-3000 μm.

4. The heterogeneous hyaluronic acid gel particles according to claim 1, characterized in that: In step S3, the isotonic buffer is a phosphate buffer.

5. A pharmaceutical product obtained by filling and sterilizing the heterogeneous hyaluronic acid gel particles according to any one of claims 1 to 4.

6. Use of the heterogeneous hyaluronic acid gel particles according to any one of claims 1 to 4 in the preparation of cosmetics, joint cavity injections, and tissue filler products.

7. The method for preparing the heterogeneous hyaluronic acid gel particles according to any one of claims 1 to 4, characterized in that: The steps include: Step S1, soaking the first hyaluronic acid gel particles with uniform cross-linked hyaluronic acid content in gradient ethanol aqueous solutions with gradually increasing ethanol concentrations, each soaking time is 16-48 hours, and filtering after the soaking to obtain second hyaluronic acid gel particles; Step S2, soaking the second hyaluronic acid gel particles obtained in step S1 in an aqueous solution of a reducing compound for 0.02-2 hours, and filtering after the soaking to obtain third hyaluronic acid gel particles; Step S3, placing the third hyaluronic acid gel particles obtained in step S2 in an isotonic buffer solution and soaking for 6-24 hours, and filtering after soaking to obtain non-uniform hyaluronic acid gel particles; In step S1, in the first hyaluronic acid gel particles, the content of cross-linked hyaluronic acid is 15-25 mg / mL; in the multiple ethanol aqueous solutions, the volume concentration of ethanol is not less than 30% and not more than 80%, and the volume concentration difference of adjacent concentrations of ethanol aqueous solutions is not less than 15%; In step S2, the mass concentration of the reducing compound in the reducing compound aqueous solution is 0.001%-0.2%; The reducing compound is dithiothreitol and / or tris(2-carboxyethyl)phosphine hydrochloride.

8. The preparation method according to claim 7, characterized in that: In step S3, the isotonic buffer is a phosphate buffer.

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