Cross-linked sodium hyaluronate gel with compact honeycomb structure and preparation method of cross-linked sodium hyaluronate gel

Abstract

The invention provides cross-linked sodium hyaluronate gel with a compact honeycomb structure and a preparation method of the cross-linked sodium hyaluronate gel. The preparation method comprises the following steps: S1, preparing a chloride ion solution, adding a cross-linking agent, slowly adding sodium hyaluronate under a stirring condition, adjusting the pH value to be alkaline, and uniformly stirring under a vacuum condition to obtain a mixture; s2, keeping a vacuum condition, heating the mixture in a water bath, cutting to obtain blocky gel, and cross-linking in the water bath to obtain cross-linked sodium hyaluronate gel; s3, adjusting the pH value of the cross-linked sodium hyaluronate gel to be neutral, carrying out first vortex-shear composite grinding treatment to obtain homogeneous pasty gel, dialyzing, then carrying out second vortex-shear composite grinding treatment, adding non-cross-linked sodium hyaluronate and a PBS solution, carrying out third vortex-shear composite grinding treatment, filling, and sterilizing to obtain the hydrogel. The cross-linked sodium hyaluronate gel with the compact honeycomb structure is obtained. The cross-linked sodium hyaluronate gel with the compact honeycomb structure is smooth to inject, strong in supporting force, long in maintaining effect and low in adverse reaction rate.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and particularly relates to a dense honeycomb structure cross-linked sodium hyaluronate gel and a preparation method thereof. Background Art

[0002] Hyaluronic acid is a biodegradable high molecular mucopolysaccharide. Under physiological conditions, hyaluronic acid usually exists in the form of sodium salt. Sodium hyaluronate widely exists in the matrix of many connective tissues such as skin, eye vitreous body, cartilage and joint synovial fluid, playing physiological roles such as moisturizing, nourishing and repairing, and having good physical and chemical properties and biocompatibility. By promoting the proliferation and differentiation of epidermal cells and scavenging oxygen free radicals, it can promote the regeneration of the skin at the injured site and has a regulatory effect on human skin. The cross-linked sodium hyaluronate gel prepared from sodium hyaluronate has the advantages of good filling effect and high biocompatibility, so it is widely used in the medical and cosmetic fields and injected into the body as a filler. However, with the popularization and use of cross-linked sodium hyaluronate gel, some problems have emerged one after another, such as short maintenance effect, poor support force, high water absorption rate, large extrusion resistance, and high adverse reaction rate after injection.

[0003] At present, the conventional preparation method of cross-linked sodium hyaluronate gel mainly includes the following steps: cross-linking reaction - swelling - dialysis - filling - sterilization. A corresponding cross-linking agent is used to react with the active groups on the molecular chain of sodium hyaluronate to connect different sodium hyaluronate molecules with each other to form a cross-linked network, and a cross-linked sodium hyaluronate gel is obtained. The gel directly obtained by the cross-linking agent and sodium hyaluronate has excellent mechanical properties and anti-degradation ability, but the texture is relatively hard and it is not easy to inject and fill. Often, non-cross-linked sodium hyaluronate needs to be added as a lubricant to meet the requirements of subsequent filling and clinical use. However, due to the presence of non-cross-linked sodium hyaluronate, the product itself will have the situation of uneven biphasic mixing. A series of problems caused thereby, such as difficult filling, different extrusion forces of the preparation, insufficient support force, short maintenance time, etc., will seriously affect the product quality and even cause adverse reactions to the human body.

[0004] Chinese Patent CN108250457A, "A Biphasic Cross-Linked Sodium Hyaluronate Gel with Controllable Shear Viscosity, Its Preparation Method and Preparation", discloses a biphasic gel of cross-linked sodium hyaluronate particles and non-cross-linked sodium hyaluronate. By adding non-cross-linked sodium hyaluronate after dialysis, then using a colloid mill to granulate, and then mixing with non-cross-linked sodium hyaluronate, the previously added non-cross-linked sodium hyaluronate enters the inside of the cross-linked sodium hyaluronate gel particles, improving the shear viscosity of the product and making the three-dimensional network structure of the gel finished product more stable. However, the cross-linked sodium hyaluronate gel prepared by this method still has the problem of uneven mixing, different extrusion forces of the preparation, difficult injection, and unstable quality control.

[0005] Therefore, it is of great significance to provide a dense honeycomb structure cross-linked sodium hyaluronate gel with uniform texture, smooth injection, strong support force and stable quality, as well as a preparation method thereof. Summary of the Invention

[0006] In view of the problems existing in the existing dual-phase cross-linked sodium hyaluronate gel, such as non-uniform dual-phase mixing, different formulation extrusion forces, difficult injection, and unstable quality control, the present invention provides a dense honeycomb structure cross-linked sodium hyaluronate gel and a preparation method thereof. By controlling the pH, temperature, ionic strength, time and cross-linking medium during the cross-linking reaction, the effective cross-linking efficiency of the product is improved, and a dense honeycomb structure is formed, thereby reducing the residual free cross-linking agent, reducing the usage amount of the cross-linking agent, improving the mechanical properties. The obtained dense honeycomb structure cross-linked sodium hyaluronate gel has smooth injection, strong support force, long-lasting effect and low adverse reaction rate.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] A preparation method of a dense honeycomb structure cross-linked sodium hyaluronate gel, comprising the following steps:

[0009] S1. Prepare a 0.1-0.2 mol / L chloride ion solution, add a cross-linking agent, slowly add sodium hyaluronate under stirring conditions, adjust the pH to alkaline, and stir evenly under vacuum conditions to obtain a mixture;

[0010] S2. Maintain the vacuum condition, water bath heat the mixture obtained in S1, cut it into pieces to obtain block gels, and perform water bath cross-linking to obtain cross-linked sodium hyaluronate gels;

[0011] S3. Adjust the pH of the cross-linked sodium hyaluronate gel obtained in S2 to neutral, perform the first vortex-shear composite grinding treatment to obtain a homogeneous paste-like gel, dialyze it, then perform the second vortex-shear composite grinding treatment, add non-cross-linked sodium hyaluronate and PBS solution, perform the third vortex-shear composite grinding treatment, fill and sterilize to obtain the dense honeycomb structure cross-linked sodium hyaluronate gel.

[0012] Further, for the three vortex-shear composite grinding treatments described in S3, the rotation speed gradient increases in increments of 20-60-80 rpm, and each treatment is carried out for 30-60 minutes.

[0013] Further, the chloride ion solution described in S1 is one or more of sodium chloride, potassium chloride, and calcium chloride.

[0014] Further, the cross-linking agent described in S1 is one or more of 1,4-butanediol diglycidyl ether, divinyl sulfone, and polyethylene glycol diepoxide.

[0015] Furthermore, the cross-linking agent in S1 is 1,4-butanediol diglycidyl ether.

[0016] Further, the mass ratio of the cross-linking agent in S1 to sodium hyaluronate is (0.06 - 0.08):1.

[0017] Further, the adjustment of pH to alkaline in S1 is achieved by using 1 mol / L sodium hydroxide solution, and the mass ratio of the sodium hydroxide solution to sodium hyaluronate is (15 - 20):(26 - 28).

[0018] Further, the water bath heating temperature in S2 is 25 - 30 °C and the time is 4 - 6 hours.

[0019] Further, the slicing in S2 means slicing the gel into uniform gel blocks with a size of 0.5 - 1 cm 3 in size.

[0020] Further, the water bath cross-linking temperature in S2 is 50 °C and the time is 2 - 3 hours.

[0021] Further, the adjustment of pH to neutral in S3 is achieved by using 1 mol / L hydrochloric acid solution and phosphate buffer solution. The mass ratio of the hydrochloric acid solution to sodium hyaluronate is (15 - 20):(26 - 28), and the phosphate buffer solution has the same mass as the cross-linked sodium hyaluronate gel obtained in S2.

[0022] Further, for the dialysis in S3, the dialysis bag is a composite cellulose dialysis bag with a molecular weight cut-off of 8000 - 14000 KDa, the dialysis fluid is a flowing PBS solution at 2 - 8 °C, and the dialysis fluid is changed every 6 - 8 hours for a total of 48 - 56 hours.

[0023] Further, after adding non-cross-linked sodium hyaluronate and PBS solution in S3, the final content of non-cross-linked sodium hyaluronate is 1 - 2 mg / mL, and the final total sodium hyaluronate content is 18 - 24 mg / mL.

[0024] Another object of the present invention is to provide a cross-linked sodium hyaluronate gel with a dense honeycomb structure.

[0025] A cross-linked sodium hyaluronate gel with a dense honeycomb structure, which is prepared by the preparation method of the cross-linked sodium hyaluronate gel with a dense honeycomb structure according to any one of the foregoing.

[0026] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0027] (1) By controlling the ionic strength of the dissolution medium of sodium hyaluronate during the crosslinking reaction, the entanglement structure of the large hyaluronic acid molecular chains is opened, thereby exposing more reactive groups, increasing the number of crosslinking sites; by controlling the pH value of the reaction, the epoxy groups in the crosslinking agent react with the hydroxyl groups on the sodium hyaluronate molecular chains to form stable ether bonds; by controlling the temperature and time, the sufficiency of the crosslinking reaction is ensured, the effective crosslinking efficiency of the product is increased, and a dense and uniform honeycomb structure is formed; by using a semi-permeable membrane to continuously dialyze the crosslinked gel for a long time, small molecular impurities (which are likely to cause adverse reactions after injection) in the gel are fully removed, improving the injection safety of the product; thus reducing the residual free crosslinking agent, reducing the usage amount of the crosslinking agent, reducing the water absorption rate of the product, and improving the mechanical properties of the sodium hyaluronate gel with a dense honeycomb structure crosslinked

[0028] (2) By means of a multiple vortex-shear composite grinding treatment process, the uniformity and cohesiveness of the product are improved, so that the product always maintains a homogeneous gel state, improving the injection smoothness and cohesiveness, and evenly controlling the elastic modulus, pushing force and cohesiveness of the sodium hyaluronate gel with a dense honeycomb structure crosslinked, ensuring its uniform mixing, smooth injection and stable quality control.

[0029] (3) By using a vacuum to protect the crosslinking process, the activity of the crosslinking agent is protected, and the effective crosslinking rate of the crosslinking agent and hyaluronic acid is increased; by strictly controlling the rapid rise and fall of the temperature during sterilization, the crosslinking degree, spatial mechanical structure and performance of the product are improved, significantly improving the physical properties of the product, obtaining a sodium hyaluronate gel with a dense honeycomb structure crosslinked having excellent comprehensive physical properties, always maintaining a homogeneous gel state, excellent injection smoothness and cohesiveness, strong supporting force, long-lasting maintenance effect and low adverse reaction rate. Description of the Drawings

[0030] The invention is further described with reference to the drawings, but the embodiments in the drawings do not constitute any limitation to the present invention. For those of ordinary skill in the art, other drawings can also be obtained according to the following drawings without creative work.

[0031] Figure 1 It is the SEM image of the sodium hyaluronate gel with a dense honeycomb structure crosslinked in Example 1 of the present invention.

[0032] Figure 2 It is the SEM image of the sodium hyaluronate gel with a dense honeycomb structure crosslinked in Example 2 of the present invention.

[0033] Figure 3 It is the SEM image of the sodium hyaluronate gel with a dense honeycomb structure crosslinked in Example 3 of the present invention.

[0034] Figure 4SEM image of the crosslinked sodium hyaluronate gel of Comparative Example 1 of the present invention.

[0035] Figure 5 SEM image of the crosslinked sodium hyaluronate gel of Comparative Example 2 of the present invention.

[0036] Figure 6 SEM image of the crosslinked sodium hyaluronate gel of Comparative Example 3 of the present invention.

[0037] Figure 7 SEM image of the crosslinked sodium hyaluronate gel of Comparative Example 4 of the present invention.

[0038] Figure 8 SEM image of the crosslinked sodium hyaluronate gel of Comparative Example 5 of the present invention. Detailed implementation mode

[0039] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention is further illustrated by the following examples. Obviously, the following examples are only a part of the examples of the present invention, rather than all the examples; it should be understood that the embodiments of the present invention are only used to illustrate the technical effects of the present invention, rather than to limit the protection scope of the present invention.

[0040] The raw materials in the examples can all be obtained commercially; unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0041] Example 1:

[0042] S1. Prepare a 0.2 mol / L sodium chloride solution, add 1,4-butanediol diglycidyl ether, and slowly add sodium hyaluronate under stirring conditions. The mass ratio of 1,4-butanediol diglycidyl ether to sodium hyaluronate is 0.08:1. Adjust the pH to alkaline with 1 mol / L sodium hydroxide solution, where the proportion of sodium hyaluronic acid after addition accounts for 9% of the total mass, and the proportion of sodium hydroxide after addition accounts for 7%. Stir evenly under vacuum conditions to obtain a mixture;

[0043] S2. Maintain the vacuum condition, water bath heat the mixture obtained in S1 at 30 °C for 6 hours, cut it into uniform block gels with a size of 0.5 - 1 cm 3 to obtain block gels, and crosslink them in a water bath at 50 °C for 3 hours to obtain crosslinked sodium hyaluronate gels;

[0044] S3. Adjust the pH of the cross-linked sodium hyaluronate gel obtained in S2 to neutral with 1 mol / L hydrochloric acid solution and phosphate buffer solution. The addition amount of the hydrochloric acid solution is equal to the mass of the sodium hydroxide solution added in step S1, and then add the phosphate buffer solution with an addition amount equal to the mass of the cross-linked sodium hyaluronate gel obtained in S2. Conduct the first vortex-shear composite grinding treatment to obtain a homogeneous paste-like gel. After fully removing air, dialyze it in a dialysis solution with a mass 80 times that of the gel. The dialysis bag is a composite cellulose dialysis bag with a molecular weight cut-off of 8000 - 14000 KDa, and the gel loading amount is 2 / 3 of the dialysis bag capacity. The dialysis solution is a flowing PBS solution at 4°C, and the dialysis solution is changed every 8 hours for a total of 48 hours. Take out the gel from the bag, and then conduct the second vortex-shear composite grinding treatment. Add non-cross-linked sodium hyaluronate and PBS solution, with the final content of non-cross-linked sodium hyaluronate being 1 mg / mL and the final total sodium hyaluronate content being 24 mg / mL. Conduct the third vortex-shear composite grinding treatment. The three vortex-shear composite grinding treatments have a rotational speed gradient increasing from 20 - 60 - 80 rpm, with each treatment lasting for 30 minutes. After degassing treatment by a filling machine, conduct filling and then perform high-temperature steam sterilization treatment at 124°C for 6 minutes. The time for increasing from room temperature to the sterilization temperature during the sterilization process is within 0 - 8 minutes, and the time for cooling from the sterilization temperature to 70°C is within 0 - 8 minutes, thus obtaining the dense honeycomb structure cross-linked sodium hyaluronate gel.

[0045] Example 2:

[0046] S1. Prepare a 0.15 mol / L sodium chloride solution, add 1,4-butanediol diglycidyl ether, and slowly add sodium hyaluronate under stirring conditions. The mass ratio of 1,4-butanediol diglycidyl ether to sodium hyaluronate is 0.07:1. Adjust the pH to alkaline with 1 mol / L sodium hydroxide solution, where the proportion of hyaluronic acid after addition is 10.5% and the proportion of sodium hydroxide after addition is 8%. Stir evenly under vacuum conditions to obtain a mixture.

[0047] S2. Maintain the vacuum condition, water-bath heat the mixture obtained in S1 at 27°C for 5 hours, cut it into uniform block gels with a size of 0.5 - 1 cm 3 to obtain block gels, and cross-link them in a water bath at 50°C for 2.5 hours to obtain cross-linked sodium hyaluronate gels.

[0048] S3. Adjust the pH of the cross-linked sodium hyaluronate gel obtained in S2 to neutral with 1 mol / L hydrochloric acid solution and phosphate buffer solution. The addition amount of the hydrochloric acid solution is equal to the mass of the sodium hydroxide solution added in step S1. Then add the phosphate buffer solution, and the addition amount is equal to the mass of the cross-linked sodium hyaluronate gel obtained in S2. Perform the first vortex-shear composite grinding treatment to obtain a homogeneous paste-like gel. After fully removing air, dialyze it in a dialysis solution with a mass 90 times that of the gel. The dialysis bag is a composite cellulose dialysis bag with a molecular weight cut-off of 8000 - 14000 KDa. The gel loading amount is 1 / 2 of the dialysis bag loading amount. The dialysis solution is a flowing PBS solution at 6°C, and the dialysis solution is changed every 7 hours for a total of 52 hours. Take the gel out of the bag, and then perform the second vortex-shear composite grinding treatment. Add non-cross-linked sodium hyaluronate and PBS solution. The final content of non-cross-linked sodium hyaluronate is 1.5 mg / mL, and the final total sodium hyaluronate content is 20 mg / mL. Perform the third vortex-shear composite grinding treatment. For the three vortex-shear composite grinding treatments, the rotation speed gradient increases in increments of 20 - 60 - 80 rpm, and each treatment lasts for 45 minutes. After degassing treatment by a filling machine, perform filling, and perform high-temperature steam sterilization treatment at 126°C for 5 minutes. The time from room temperature to the sterilization temperature during the sterilization process is within 0 - 8 minutes, and the time from the sterilization temperature to 70°C is within 0 - 8 minutes, thus obtaining the dense honeycomb structure cross-linked sodium hyaluronate gel.

[0049] Example 3:

[0050] S1. Prepare a 0.1 mol / L sodium chloride solution, add 1,4-butanediol diglycidyl ether, and slowly add sodium hyaluronate under stirring conditions. The mass ratio of 1,4-butanediol diglycidyl ether to sodium hyaluronate is 0.06:1. Adjust the pH to alkaline with 1 mol / L sodium hydroxide solution, where the proportion of sodium hyaluronic acid after addition is 12%, and the proportion of sodium hydroxide after addition is 9%. Stir evenly under vacuum conditions to obtain a mixture.

[0051] S2. Maintain the vacuum condition, heat the mixture obtained in S1 in a water bath at 25°C for 4 hours, cut it into uniform block gels with a size of 0.5 - 1 cm 3 to obtain block gels, and cross-link them in a water bath at 50°C for 2 hours to obtain cross-linked sodium hyaluronate gels.

[0052] S3. Adjust the pH of the crosslinked sodium hyaluronate gel obtained in S2 to neutral with 1 mol / L hydrochloric acid solution and phosphate buffer solution. The addition amount of the hydrochloric acid solution is equal in mass to the sodium hydroxide solution added in step S1. Then add the phosphate buffer solution with an addition amount equal in mass to the crosslinked sodium hyaluronate gel obtained in S2. Conduct the first vortex-shear composite grinding treatment to obtain a homogeneous pasty gel. After thoroughly removing air, dialyze it in a dialysis solution with a mass 100 times that of the gel. The dialysis bag is a composite cellulose dialysis bag with a molecular weight cut-off of 8000 - 14000 KDa. The gel loading amount is 1 / 3 of the dialysis bag loading amount. The dialysis solution is a flowing PBS solution at 8°C, and the dialysis solution is changed every 6 hours for a total of 56 hours. Take out the gel from the bag, then conduct the second vortex-shear composite grinding treatment, add non-crosslinked sodium hyaluronate and PBS solution, with the final content of non-crosslinked sodium hyaluronate being 2.0 mg / mL and the final total sodium hyaluronate content being 18 mg / mL. Conduct the third vortex-shear composite grinding treatment. For the three vortex-shear composite grinding treatments, the rotational speed gradient increases in increments of 20 - 60 - 80 rpm, with each treatment lasting for 60 minutes. After degassing treatment by a filling machine, conduct filling, and perform high-temperature steam sterilization treatment at 128°C for 4 minutes. During the sterilization process, the time for increasing the temperature from room temperature to the sterilization temperature is within 0 - 8 minutes, and the time for cooling from the sterilization temperature to 70°C is within 0 - 8 minutes, thus obtaining the dense honeycomb structure crosslinked sodium hyaluronate gel.

[0053] Comparative Example 1

[0054] A preparation method of a crosslinked sodium hyaluronate gel, comprising the following steps:

[0055] S1. Add 1,4-butanediol diglycidyl ether to water, and slowly add sodium hyaluronate under stirring conditions. The mass ratio of 1,4-butanediol diglycidyl ether to sodium hyaluronate is 0.07:1. Adjust the pH to alkaline with 1 mol / L sodium hydroxide solution, where the proportion of sodium hyaluronic acid after addition is 10.5%, and the proportion of sodium hydroxide after addition is 8%. Stir evenly under vacuum conditions to obtain a mixture;

[0056] S2. Maintain the vacuum condition, water bath heat the mixture obtained in S1 at 27°C for 5 hours, cut it into uniform block gels with a size of 0.5 - 1 cm 3 to obtain block gels, and crosslink them in a water bath at 50°C for 2.5 hours to obtain a crosslinked sodium hyaluronate gel;

[0057] S3. Adjust the pH of the crosslinked sodium hyaluronate gel obtained in S2 to neutral with 1 mol / L hydrochloric acid solution and phosphate buffer solution. The addition amount of the hydrochloric acid solution is equal in mass to the sodium hydroxide solution added in step S1. Then add the phosphate buffer solution, with the addition amount equal in mass to the crosslinked sodium hyaluronate gel obtained in S2. Conduct the first vortex-shear composite grinding treatment to obtain a homogeneous paste-like gel. After fully removing air, dialyze it in a dialysis solution with a mass 90 times that of the gel. The dialysis bag is a composite cellulose dialysis bag with a molecular weight cut-off of 8000 - 14000KDa. The gel loading amount is 1 / 2 of the dialysis bag loading capacity. The dialysis solution is a flowing PBS solution at 6°C, and the dialysis solution is changed every 7 hours for a total of 52 hours. Take the gel out of the bag, then conduct the second vortex-shear composite grinding treatment, add non-crosslinked sodium hyaluronate and PBS solution, with the final content of non-crosslinked sodium hyaluronate being 1.5 mg / mL and the final total sodium hyaluronate content being 20 mg / mL. Conduct the third vortex-shear composite grinding treatment. For the three vortex-shear composite grinding treatments, the rotation speed gradient increases in increments of 20 - 60 - 80 rpm, and each treatment lasts for 45 minutes. After degassing treatment by a filling machine, conduct filling and then perform high-temperature steam sterilization treatment at 126°C for 5 minutes. During the sterilization process, the time for increasing the temperature from room temperature to the sterilization temperature is within 0 - 8 minutes, and the time for cooling from the sterilization temperature to 70°C is within 0 - 8 minutes, thus obtaining the crosslinked sodium hyaluronate gel.

[0058] Compared with Example 2, the main difference in this comparative example is that no chloride ion solution is prepared in step S1.

[0059] Comparative Example 2

[0060] A preparation method of a crosslinked sodium hyaluronate gel, comprising the following steps:

[0061] S1. Prepare a 0.15 mol / L sodium chloride solution, add 1,4-butanediol diglycidyl ether, and slowly add sodium hyaluronate under stirring conditions. The mass ratio of 1,4-butanediol diglycidyl ether to sodium hyaluronate is 0.07:1. Adjust the pH to alkaline with 1 mol / L sodium hydroxide solution, where the proportion of sodium hyaluronic acid after addition is 10.5%, and the proportion of sodium hydroxide after addition is 8%. Stir evenly under vacuum conditions to obtain a mixture;

[0062] S2. Maintain the vacuum condition, heat the mixture obtained in S1 in a water bath at 27°C for 5 hours, cut it into uniform block gels with a size of 0.5 - 1 cm 3 to obtain block gels, and crosslink them in a water bath at 50°C for 2.5 hours to obtain a crosslinked sodium hyaluronate gel;

[0063] S3. Adjust the pH of the crosslinked sodium hyaluronate gel obtained in S2 to neutral with 1 mol / L hydrochloric acid solution and phosphate buffer solution. The addition amount of the hydrochloric acid solution is equal in mass to the sodium hydroxide solution added in step S1, and then add the phosphate buffer solution with an addition amount equal in mass to the crosslinked sodium hyaluronate gel obtained in S2. Conduct the first vortex-shear composite grinding treatment to obtain a homogeneous paste-like gel. After fully removing air, dialyze it in a dialysis solution with a mass 90 times that of the gel. The dialysis bag is a composite cellulose dialysis bag with a molecular weight cut-off of 8000 - 14000KDa. The gel loading amount is 1 / 2 of the dialysis bag loading capacity. The dialysis solution is a flowing PBS solution at 6°C, and the dialysis solution is changed every 7 hours for a total of 52 hours. Take the gel out of the bag, add non-crosslinked sodium hyaluronate and PBS solution, with the final content of non-crosslinked sodium hyaluronate being 1.5 mg / mL and the final total sodium hyaluronate content being 20 mg / mL. Conduct the second vortex-shear composite grinding treatment, and for the secondary vortex-shear composite grinding treatment, the rotation speed gradient increases in increments of 20 - 60 - 80 rpm, with each treatment lasting for 45 minutes. After degassing treatment by a filling machine, conduct filling and perform high-temperature steam sterilization treatment at 126°C for 5 min. The time for increasing from room temperature to the sterilization temperature during the sterilization process is within 0 - 8 min, and the time for cooling from the sterilization temperature to 70°C is within 0 - 8 min, thus obtaining the crosslinked sodium hyaluronate gel.

[0064] Compared with Example 2, the main difference in this comparative example is that only two vortex-shear composite grinding treatments are conducted in step S3.

[0065] Comparative Example 3

[0066] A preparation method of a crosslinked sodium hyaluronate gel, comprising the following steps:

[0067] S1. Prepare a 0.15 mol / L sodium chloride solution, add 1,4-butanediol diglycidyl ether, and slowly add sodium hyaluronate under stirring conditions. The mass ratio of 1,4-butanediol diglycidyl ether to sodium hyaluronate is 0.07:1. Adjust the pH to alkaline with 1 mol / L sodium hydroxide solution, where the proportion of sodium hyaluronic acid after addition is 10.5% and the proportion of sodium hydroxide after addition is 8%. Stir evenly to obtain a mixture;

[0068] S2. Heat the mixture obtained in S1 in a water bath at 27°C for 5 hours, cut it into uniform block gels with a size of 0.5 - 1 cm 3 to obtain block gels, and crosslink them in a water bath at 50°C for 2.5 hours to obtain a crosslinked sodium hyaluronate gel;

[0069] S3. Adjust the pH of the cross-linked sodium hyaluronate gel obtained in S2 to neutral with 1 mol / L hydrochloric acid solution and phosphate buffer solution. The addition amount of the hydrochloric acid solution is equal to the mass of the sodium hydroxide solution added in step S1. Then add the phosphate buffer solution, and the addition amount is equal to the mass of the cross-linked sodium hyaluronate gel obtained in S2. Perform the first vortex-shear composite grinding treatment to obtain a homogeneous paste-like gel. After fully removing air, dialyze it in a dialysis solution with a mass 90 times that of the gel. The dialysis bag is a composite cellulose dialysis bag with a molecular weight cut-off of 8000 - 14000KDa. The gel loading amount is 1 / 2 of the dialysis bag loading capacity. The dialysis solution is a flowing PBS solution at 6°C, and the dialysis solution is changed every 7 hours for a total of 52 hours. Take the gel out of the bag, and then perform the second vortex-shear composite grinding treatment. Add non-cross-linked sodium hyaluronate and PBS solution. The final content of non-cross-linked sodium hyaluronate is 1.5 mg / mL, and the final total sodium hyaluronate content is 20 mg / mL. Perform the third vortex-shear composite grinding treatment. For the three vortex-shear composite grinding treatments, the rotation speed gradient increases in increments of 20 - 60 - 80 rpm, and each treatment lasts for 45 minutes. After degassing treatment by a filling machine, perform filling, and perform high-temperature steam sterilization treatment at 126°C for 5 minutes. The time for increasing from room temperature to the sterilization temperature during the sterilization process is within 0 - 8 minutes, and the time for cooling from the sterilization temperature to 70°C is within 0 - 8 minutes, thus obtaining the cross-linked sodium hyaluronate gel.

[0070] Compared with Example 2, the main difference in this comparative example is that the cross-linking in step S2 is not carried out under vacuum.

[0071] Comparative Example 4

[0072] A preparation method of a cross-linked sodium hyaluronate gel, comprising the following steps:

[0073] S1. Prepare a 0.15 mol / L sodium chloride solution, add 1,4-butanediol diglycidyl ether, and slowly add sodium hyaluronate under stirring conditions. The mass ratio of 1,4-butanediol diglycidyl ether to sodium hyaluronate is 0.07:1. Adjust the pH to alkaline with 1 mol / L sodium hydroxide solution, where the proportion of sodium hyaluronic acid after addition is 10.5%, and the proportion of sodium hydroxide after addition is 8%. Stir evenly under vacuum conditions to obtain a mixture;

[0074] S2. Maintain the vacuum condition, water-bath heat the mixture obtained in S1 at 27°C for 5 hours, cut it into uniform block gels with a size of 0.5 - 1 cm 3 to obtain block gels, and cross-link them in a water bath at 50°C for 2.5 hours to obtain a cross-linked sodium hyaluronate gel;

[0075] S3. Adjust the pH of the cross-linked sodium hyaluronate gel obtained in S2 to neutral with 1 mol / L hydrochloric acid solution and phosphate buffer solution. The addition amount of the hydrochloric acid solution is equal to the mass of the sodium hydroxide solution added in step S1. Then add the phosphate buffer solution, and the addition amount is equal to the mass of the cross-linked sodium hyaluronate gel obtained in S2. Perform the first vortex-shear composite grinding treatment to obtain a homogeneous paste-like gel. After fully removing air, dialyze it in a dialysis solution with a mass 90 times that of the gel. The dialysis bag is a composite cellulose dialysis bag with a molecular weight cut-off of 8000 - 14000 KDa, and the gel loading amount is 1 / 2 of the dialysis bag capacity. The dialysis solution is a flowing PBS solution at 6°C, and the dialysis solution is changed every 7 hours for a total of 52 hours. Take out the gel from the bag, then perform the second vortex-shear composite grinding treatment, add non-cross-linked sodium hyaluronate and PBS solution, and the final content of non-cross-linked sodium hyaluronate is 1.5 mg / mL, and the final total sodium hyaluronate content is 20 mg / mL. Perform the third vortex-shear composite grinding treatment. The three vortex-shear composite grinding treatments have a rotational speed gradient increasing from 20 - 60 - 80 rpm, and each treatment lasts for 45 minutes. After degassing treatment by a filling machine, perform filling and then perform high-temperature steam sterilization treatment at 126°C for 5 minutes. The time from room temperature to the sterilization temperature during the sterilization process is within 20 minutes, and the time from the sterilization temperature to 70°C is within 20 minutes, thus obtaining the cross-linked sodium hyaluronate gel.

[0076] Compared with Example 2, the main difference in this comparative example is that the temperature increase and decrease time during sterilization is controlled within 20 minutes.

[0077] Comparative Example 5

[0078] A preparation method of a cross-linked sodium hyaluronate gel, comprising the following steps:

[0079] S1. Add 1,4-butanediol diglycidyl ether to water, and slowly add sodium hyaluronate under stirring conditions. The mass ratio of 1,4-butanediol diglycidyl ether to sodium hyaluronate is 0.07:1. Adjust the pH to alkaline with 1 mol / L sodium hydroxide solution, where the proportion of sodium hyaluronic acid after addition is 10.5%, and the proportion of sodium hydroxide after addition is 8%. Stir evenly to obtain a mixture;

[0080] S2. Heat the mixture obtained in S1 in a water bath at 50°C for 2.5 hours to obtain a cross-linked sodium hyaluronate gel;

[0081] S3. The pH of the cross-linked sodium hyaluronate gel obtained in S2 is adjusted to neutral by 1 mol / L hydrochloric acid solution and phosphate buffer solution, the amount of hydrochloric acid solution added is equal to the mass of the sodium hydroxide solution added in step S1, and then a phosphate buffer solution is added, the amount of which is equal to the mass of the cross-linked sodium hyaluronate gel obtained in S2, and a first vortex-shear composite grinding treatment is performed to obtain a homogeneous paste gel, which is dialyzed in a 90-fold mass of dialysate after fully removing air, the dialysis bag is a composite cellulose dialysis bag with a molecular weight cutoff of 8000-14000KDa, the gel loading is 1 / 2 of the dialysis bag loading, the dialysis fluid is a 6°C running PBS solution, and the dialysis fluid is replaced every 7 hours, for a total of 10 minutes. After 52 hours of analysis, the gel was taken out of the bag, and non-cross-linked sodium hyaluronate and PBS solution were added, the final content of non-cross-linked sodium hyaluronate was 1.5 mg / mL, and the final total sodium hyaluronate content was 20 mg / mL. The second vortex-shear composite grinding treatment was carried out, and the second vortex-shear composite grinding treatment was carried out. The speed gradient was increased by 20-60-80 rpm, and each treatment was 45 minutes. After degassing treatment by a filling machine, filling was carried out, and high-temperature steam sterilization was carried out at 126°C for 5 minutes. During the sterilization process, the time from room temperature to the sterilization temperature was within 20 minutes, and the time from the sterilization temperature to 70°C was within 20 minutes. The cross-linked sodium hyaluronate gel was obtained.

[0082] Compared with Example 2, the main difference of this comparative example is that the cross-linked sodium hyaluronate gel is prepared by a conventional method.

[0083] SEM scanning tests were performed on Examples 1-3 and Comparative Examples 1-5, and the SEM images thereof are shown as follows: Figure 1-8 shown.

[0084] Figure 1 , Figure 2 and Figure 3 The SEM image of the dense honeycomb structure cross-linked sodium hyaluronate gel of Example 1-3. As can be seen from the figure, the network of the dense honeycomb structure cross-linked sodium hyaluronate gel becomes denser with the increase of the cross-linking degree, but the whole presents a uniform dense honeycomb shape, showing a stable cross-linked spatial structure.

[0085] Figure 4 This is a SEM image of the cross-linked sodium hyaluronate gel of comparative example 1. As can be seen from the figure, compared with Example 2, the density of the sodium hyaluronate gel without controlling the ion concentration is reduced, and the network result is also relatively sparse and uneven.

[0086] Figure 5 This is a SEM image of the cross-linked sodium hyaluronate gel of comparative example 2. As can be seen from the figure, compared with Example 2, the density of the sodium hyaluronate gel subjected to only two vortex-shear composite grinding processes is increased.

[0087] Figure 6 ,Figure 7 SEM image of the cross-linked sodium hyaluronate gel of Comparative Example 3-4. As can be seen from the figure, compared with Example 2, the network structure of the cross-linked sodium hyaluronate gel of Comparative Example 3-4 is significantly looser and more uneven, indicating that the cross-linking effect and cross-linked spatial structure of the product are significantly affected by the cross-linking in a vacuum environment and the rapid heating and cooling sterilization method.

[0088] Figure 8 SEM image of the cross-linked sodium hyaluronate gel of Comparative Example 5. As can be seen from the figure, compared with Example 2, the cross-linked sodium hyaluronate gel prepared by the conventional method not only has a loose and uneven network, but also shows a relatively rough cross-linked spatial structure and poor texture.

[0089] Performance tests were carried out on the above-mentioned examples and comparative examples, and the experimental test methods are as follows:

[0090] Elastic modulus:

[0091] Using a rotational rheometer, set the test temperature at 25 °C, perform an oscillation test with a strain of 5%, scan from 0.08 Hz to 5 Hz, take the elastic modulus G' value corresponding to the position of 1 Hz with the scanning frequency as the abscissa.

[0092] In vitro degradation:

[0093] Use a hyaluronidase solution of 10 IU / mL to enzymatically hydrolyze the sample. After 12 h of degradation, use a hydrochloric acid solution of 0.5 mol / L to inactivate the hyaluronidase in it. Then detect the concentration of glucuronic acid in the degradation solution, convert it to the concentration of degraded sodium hyaluronate, and then compare it with the labeled concentration of the sample to obtain the in vitro degradation rate at 12 h.

[0094] Pushing force:

[0095] Fill the sample into a 1 mL syringe, place it in a refrigerator at 2-10 °C for 2 h, take it out and let it cool to room temperature. Then install the syringe on a universal material testing machine, set the pushing speed at 30 mm / min, and measure the pushing force by using a 27G injection needle in cooperation.

[0096] Swelling degree:

[0097] Put a 500-mesh sieve (a square of 8 cm × 8 cm, folded into a square groove of 4 cm × 4 cm × 2 cm) into the drying oven, and dry it to a constant weight at 80 °C, denoted as m0.

[0098] Weigh the cross-linked sodium hyaluronate gel and place it on a 500-mesh sieve. Place the sieve in an evaporating dish, add 0.9% sodium chloride solution to completely soak the sample. After the gel has fully swollen (at least 30 min), take out the sieve and the sample together, and use filter paper to absorb the liquid at the bottom and around the sieve until there is no wet mark on the filter paper, and weigh it, denoted as m1.

[0099] Put the sieve with the swollen gel into an oven and dry it at 80 °C until constant weight, denoted as m2.

[0100] Calculation formula: Cohesion test:

[0101] Evenly dye the product, place it on a universal material testing machine, and push it through an 18G trocar at a constant speed of 8 mm / min, and record the average weight of 10 drops of gel pushed out (unit: mg)

[0102] The specific test data are shown in Table 1 below:

[0103] Table 1 Test data of Examples 1-3 and Comparative Examples 1-5

[0104] sample G' value in vitro degradation rate pushing force swelling degree cohesion Example 1 485 Pa 58％ 21N 35 85 mg Example 2 370 Pa 67％ 14N 40 78 mg Example 3 290 Pa 80％ 10N 51 69 mg Comparative Example 1 316 Pa 76％ 16N 48 72 mg Comparative Example 2 391 Pa 65％ 18N 43 75 mg Comparative Example 3 287 Pa 78％ 17N 54 68 mg Comparative Example 4 236 Pa 81％ 16N 58 66 mg Comparative Example 5 195 Pa 100％ 19N 68 42 mg

[0105] As can be seen from Table 1, the dense honeycomb structure cross-linked sodium hyaluronate gels obtained in Examples 1-3 of the present invention have excellent comprehensive physical properties at different cross-linking degrees, always maintain a homogeneous gel state, have excellent injection smoothness and cohesion, strong support force, long-lasting maintenance effect, and low adverse reaction rate.

[0106] Compared with Example 2, in Comparative Example 1, the ionic strength is not controlled, the elastic modulus is lower, the swelling degree is higher, and the in vitro degradation rate is higher, which is not conducive to the maintenance effect; in Comparative Example 2, only two vortex-shear composite grinding treatments are carried out, the elastic modulus is high, the pushing force and cohesion are high, and injection is difficult; in Comparative Example 3, the cross-linking is not carried out under vacuum, the elastic modulus is low, the in vitro degradation rate is high, the swelling degree is high, the cohesion is lower, and the support force is weak; in Comparative Example 4, the sterilization temperature rise and fall time is long, the elastic modulus is low, the cohesion is low, the swelling degree is high, and the support force and maintenance effect are poor; in Comparative Example 5, it is prepared by a conventional method, and the obtained cross-linked sodium hyaluronate gel performs the worst among the comparative examples, indicating that the control of ionic strength, the use of vacuum protection for the cross-linking process, and the control of the sterilization temperature rise and fall time can improve the cross-linking degree and spatial mechanical structure and performance of the product, significantly improve the physical properties of the product, and the triple homogenization process can significantly improve the injection smoothness of the product.

[0107] In summary, the present invention improves the effective crosslinking efficiency of the product and forms a dense honeycomb structure by controlling the pH, temperature, ionic strength, sterilization heating and cooling time, and crosslinking medium during the crosslinking reaction, thereby reducing the residual free crosslinking agent, reducing the usage amount of the crosslinking agent, and improving the mechanical properties; the uniformity and cohesion of the product are improved through a multiple stirring and grinding process; the residual small molecule impurities are fully removed through a semipermeable membrane dialysis technology, reducing the inflammatory reaction after the product is injected. The present invention fully combines the clinical use of the product and the crosslinking reaction mechanism. By controlling the ionic strength of the sodium hyaluronate dissolution medium, the entanglement structure of the hyaluronic acid macromolecular chain is opened, thereby exposing more reactive groups and increasing the number of crosslinking sites; by controlling the pH value of the reaction, the epoxy group in the crosslinking agent reacts with the hydroxyl group on the sodium hyaluronate molecular chain to form a stable ether bond; by controlling the temperature and time, the sufficiency of the crosslinking reaction is ensured; by crosslinking in a vacuum environment, the activity of the crosslinking agent is protected and the effective crosslinking rate of the crosslinking agent and hyaluronic acid is increased; by performing triple stirring and grinding treatments after the crosslinking ends, after the dialysis ends, and before the filling process, the product always remains in a homogeneous gel state, improving the injection smoothness and cohesion; by strictly controlling the rapid sterilization heating and cooling time, the crosslinking degree, spatial mechanical structure and performance of the product are improved, the physical properties of the product are significantly improved, and a dense honeycomb structure crosslinked sodium hyaluronate gel with excellent comprehensive physical properties, always remaining in a homogeneous gel state, excellent injection smoothness and cohesion, strong support force, long-lasting effect, and low adverse reaction rate is obtained.

[0108] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a dense honeycomb structure cross-linked sodium hyaluronate gel, characterized in that, It includes the following steps: S1. Prepare a 0.1 - 0.2 mol / L chloride ion solution, add a crosslinking agent, slowly add sodium hyaluronate under stirring conditions, adjust the pH to alkaline, and stir evenly under vacuum conditions to obtain a mixture; S2. Maintain the vacuum condition, water bath heat the mixture obtained in S1, cut it, obtain block gels, and carry out water bath crosslinking to obtain crosslinked sodium hyaluronate gels; S3. Adjust the pH of the crosslinked sodium hyaluronate gel obtained in S2 to neutral, carry out the first vortex - shear composite grinding treatment to obtain a homogeneous paste - like gel, dialyze, then carry out the second vortex - shear composite grinding treatment, add non - crosslinked sodium hyaluronate and PBS solution, carry out the third vortex - shear composite grinding treatment, fill, and carry out high - temperature steam sterilization treatment at a temperature of 124 - 128 °C for 4 - 6 min. The time for raising the temperature from room temperature to the sterilization temperature during the sterilization process is within 0 - 8 min, and the time for cooling from the sterilization temperature to 70 °C is within 0 - 8 min, thus obtaining the dense honeycomb - structured crosslinked sodium hyaluronate gel.

2. A method for preparing a dense honeycomb structure crosslinked sodium hyaluronate gel according to claim 1, characterized in that, For the three - time vortex - shear composite grinding treatment described in S3, the rotation speed gradient increases in increments of 20 - 60 - 80 rpm, and each treatment lasts for 30 - 60 minutes.

3. A method for preparing a dense honeycomb structure cross-linked sodium hyaluronate gel according to claim 1, characterized in that, The crosslinking agent described in S1 is one or more of 1,4 - butanediol diglycidyl ether, divinyl sulfone, and polyethylene glycol diepoxide.

4. A method for preparing a dense honeycomb structure cross-linked sodium hyaluronate gel according to claim 1, characterized in that, The mass ratio of the crosslinking agent to sodium hyaluronate described in S1 is (0.06 - 0.08):

1.

5. A method for preparing a dense honeycomb structure crosslinked sodium hyaluronate gel according to claim 1, characterized in that, Adjusting the pH to alkaline in S1 is carried out by using a 1 mol / L sodium hydroxide solution, and the mass ratio of the sodium hydroxide solution to sodium hyaluronate is (15 - 20):(26 - 28).

6. A method for preparing a dense honeycomb structure cross-linked sodium hyaluronate gel according to claim 1, characterized in that, The water bath heating temperature in S2 is 25 - 30 °C, and the time is 4 - 6 hours.

7. A method for preparing a dense honeycomb structure cross-linked sodium hyaluronate gel according to claim 1, characterized in that, The slitting described in S2 is to slit the gel into uniform block gels with a size of 0.5 - 1 cm. 3 ​ 8. A method for preparing a dense honeycomb structure cross-linked sodium hyaluronate gel according to claim 1, characterized in that, The water bath crosslinking temperature in S2 is 50 °C, and the time is 2 - 3 hours.

9. A method for preparing a dense honeycomb structure cross-linked sodium hyaluronate gel according to claim 1, characterized in that, For the dialysis described in S3, the dialysis bag is a composite cellulose dialysis bag with a molecular weight cut - off of 8000 - 14000 KDa, the dialysis solution is a flowing PBS solution at 2 - 8 °C, and the dialysis solution is changed every 6 - 8 hours, with a total dialysis time of 48 - 56 hours.

10. A crosslinked sodium hyaluronate gel with a dense honeycomb structure, characterized in that, It is prepared by the preparation method of the dense honeycomb - structured crosslinked sodium hyaluronate gel according to any one of claims 1 - 9.

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