A dense honeycomb structure cross-linked sodium hyaluronate gel and its preparation method

Abstract

This invention provides a dense honeycomb structure cross-linked sodium hyaluronate gel and its preparation method, comprising the following steps: S1. Preparing a chloride ion solution, adding a cross-linking agent, slowly adding sodium hyaluronate under stirring conditions, adjusting the pH to alkaline, and stirring evenly under vacuum conditions to obtain a mixture; S2. Maintaining vacuum conditions, heating the mixture in a water bath, cutting it to obtain a block gel, cross-linking it in a water bath to obtain a cross-linked sodium hyaluronate gel; S3. Adjusting the pH of the cross-linked sodium hyaluronate gel to neutral, performing a first vortex-shear composite grinding treatment to obtain a homogeneous paste-like gel, dialysis, then a second vortex-shear composite grinding treatment, adding non-cross-linked sodium hyaluronate and PBS solution, performing a third vortex-shear composite grinding treatment, filling, and sterilizing to obtain the dense honeycomb structure cross-linked sodium hyaluronate gel. The dense honeycomb structure cross-linked sodium hyaluronate gel obtained by this invention has smooth injection, strong support, long-lasting effect, and low adverse reaction rate.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a dense honeycomb structure cross-linked sodium hyaluronate gel and its preparation method. Background Technology

[0002] Hyaluronic acid is a biodegradable high-molecular-weight mucopolysaccharide, which typically exists in its sodium salt form under physiological conditions. Sodium hyaluronate is widely found in the matrix of many connective tissues, including skin, vitreous humor, cartilage, and synovial fluid, playing physiological roles such as moisturizing, nourishing, and repairing. It possesses excellent physicochemical properties and biocompatibility. By promoting epidermal cell proliferation and differentiation, and scavenging oxygen free radicals, it can promote skin regeneration at injured sites, thus having a regulatory effect on human skin. Cross-linked sodium hyaluronate gel, derived from sodium hyaluronate, has the advantages of good filling effect and high biocompatibility, and is therefore widely used in the medical and cosmetic fields as a filler injected into the body. However, with the widespread use of cross-linked sodium hyaluronate gel, some problems have emerged, such as short-lasting effects, poor support, high water absorption, high pushing resistance, and a high rate of adverse reactions after injection.

[0003] Currently, the conventional method for preparing cross-linked sodium hyaluronate gel mainly includes the following steps: cross-linking reaction - swelling - dialysis - filling - sterilization. A suitable cross-linking agent is used to react with the active groups on the sodium hyaluronate molecular chain, thereby connecting different sodium hyaluronate molecules to form a cross-linked network, resulting in cross-linked sodium hyaluronate gel. Gels obtained directly from cross-linking agents and sodium hyaluronate exhibit excellent mechanical properties and resistance to degradation, but they are relatively hard and difficult to inject. Therefore, non-cross-linked sodium hyaluronate is often 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 may exhibit uneven two-phase mixing, leading to a series of problems such as filling difficulties, differences in formulation extrusion force, insufficient support, and short duration of hold, which can seriously affect product quality and even cause adverse reactions in the human body.

[0004] Chinese patent CN108250457A, entitled "A Biphasic Crosslinked Sodium Hyaluronate Gel with Controllable Shear Viscosity and its Preparation Method and Formulation," discloses a biphasic gel consisting of crosslinked sodium hyaluronate particles and non-crosslinked sodium hyaluronate. The method involves adding non-crosslinked sodium hyaluronate after dialysis, granulating it using a colloid mill, and then mixing it with the non-crosslinked sodium hyaluronate. This allows the previously added non-crosslinked sodium hyaluronate to penetrate the interior of the crosslinked sodium hyaluronate gel particles, increasing the shear viscosity of the product and making the three-dimensional network structure of the finished gel more stable. However, the crosslinked sodium hyaluronate gel prepared by this method still suffers from uneven mixing, inconsistent formulation extrusion force, injection difficulties, and unstable quality control.

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

[0006] Given the problems of uneven biphasic mixing, inconsistent extrusion force, difficult injection, and unstable quality control in existing biphasic crosslinked sodium hyaluronate gels, this invention provides a dense honeycomb structure crosslinked sodium hyaluronate gel and its preparation method. By controlling the pH, temperature, ionic strength, time, and crosslinking medium during the crosslinking reaction process, the effective crosslinking efficiency of the product is improved, and a dense honeycomb structure is formed. This reduces the amount of free crosslinking agent residue, reduces the amount of crosslinking agent used, and improves mechanical properties. The resulting dense honeycomb structure crosslinked sodium hyaluronate gel is easy to inject, has strong support, long-lasting effect, and low adverse reaction rate.

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

[0008] A method for preparing a dense honeycomb structure cross-linked sodium hyaluronate gel includes the following steps:

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

[0010] S2. Under vacuum conditions, heat the mixture obtained in S1 in a water bath, cut it to obtain a block gel, crosslink it in a water bath to obtain a crosslinked sodium hyaluronate gel.

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

[0012] Furthermore, the three-stage vortex-shear composite grinding process described in S3 has a rotation speed gradient of 20-60-80 rpm, with each process lasting 30-60 minutes.

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

[0014] Furthermore, the crosslinking agent mentioned in S1 is one or more of 1,4-butanediol diglycidyl ether, divinyl sulfone, and polyethylene glycol diethylene oxide.

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

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

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

[0018] Furthermore, the water bath heating temperature described in S2 is 25-30℃, and the heating time is 4-6 hours.

[0019] Furthermore, the slicing described in S2 involves cutting the gel into uniform pieces of 0.5-1cm. 3 A block-shaped gel of various sizes.

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

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

[0022] Furthermore, in the dialysis described in S3, the dialysis bag is a composite cellulose dialysis bag with a molecular weight cutoff of 8000-14000 kDa, the dialysis solution is a flowing PBS solution at 2-8°C, the dialysis solution is changed every 6-8 hours, and the total dialysis time is 48-56 hours.

[0023] Furthermore, after adding non-crosslinked sodium hyaluronate and PBS solution as described in S3, the final content of non-crosslinked 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 dense honeycomb structure cross-linked sodium hyaluronate gel.

[0025] A dense honeycomb structure cross-linked sodium hyaluronate gel is prepared according to the method for preparing dense honeycomb structure cross-linked sodium hyaluronate gel described in any one of the preceding claims.

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

[0027] (1) This invention controls the ionic strength of the sodium hyaluronate dissolution medium during the crosslinking reaction, thereby opening the entangled structure of the hyaluronic acid macromolecular chain, exposing more reactive groups, and increasing the number of crosslinking sites; controls the pH value of the reaction so that the epoxy groups in the crosslinking agent react with the hydroxyl groups on the sodium hyaluronate molecular chain to generate stable ether bonds; controls the temperature and time to ensure the sufficiency of the crosslinking reaction, thereby improving the effective crosslinking efficiency of the product and forming a dense and uniform honeycomb structure; uses a semi-permeable membrane to perform long-term continuous dialysis on the crosslinked gel so that small molecule impurities in the gel (which can easily lead to adverse reactions after injection) are fully removed, improving the injection safety of the product; thereby reducing the free crosslinking agent residue, reducing the amount of crosslinking agent used, reducing the water absorption rate of the product, and improving the mechanical properties of the dense honeycomb structure crosslinked sodium hyaluronate gel.

[0028] (2) The present invention improves the uniformity and cohesion of the product through a multi-vortex-shear composite grinding process, so that the product always maintains a homogeneous gel state, improves injection smoothness and cohesion, and balances the elastic modulus, extrusion force and cohesion of the dense honeycomb cross-linked sodium hyaluronate gel, ensuring uniform mixing, smooth injection and stable quality control.

[0029] (3) This invention protects the activity of the crosslinking agent by using vacuum protection during the crosslinking process, thereby improving the effective crosslinking rate between the crosslinking agent and hyaluronic acid. By strictly controlling the sterilization heating and cooling time, the crosslinking degree, spatial mechanical structure and performance of the product are improved, and the physical properties of the product are significantly improved. This results in a dense honeycomb structure crosslinked sodium hyaluronate gel with excellent comprehensive physical properties, always maintaining a homogeneous gel state, excellent injection smoothness and cohesion, strong support, long maintenance effect and low adverse reaction rate. Attached Figure Description

[0030] The invention will be further illustrated with reference to the accompanying drawings, but the embodiments in the drawings do not constitute any limitation on the invention. For those skilled in the art, other drawings can be obtained based on the following drawings without any creative effort.

[0031] Figure 1 This is a SEM image of the dense honeycomb structure cross-linked sodium hyaluronate gel of Example 1 of the present invention.

[0032] Figure 2 This is a SEM image of the dense honeycomb structure cross-linked sodium hyaluronate gel in Example 2 of the present invention.

[0033] Figure 3 This is a SEM image of the dense honeycomb structure cross-linked sodium hyaluronate gel in Example 3 of the present invention.

[0034] Figure 4This is a SEM image of the cross-linked sodium hyaluronate gel of Comparative Example 1 of the present invention.

[0035] Figure 5 This is a SEM image of the cross-linked sodium hyaluronate gel of Comparative Example 2 of the present invention.

[0036] Figure 6 This is a SEM image of the cross-linked sodium hyaluronate gel of Comparative Example 3 of the present invention.

[0037] Figure 7 This is a SEM image of the cross-linked sodium hyaluronate gel of Comparative Example 4 of the present invention.

[0038] Figure 8 This is a SEM image of the cross-linked sodium hyaluronate gel of Comparative Example 5 of the present invention. Detailed Implementation

[0039] To better illustrate the objectives, technical solutions, and advantages of this invention, the following embodiments are provided. Obviously, the following embodiments are only a part of the embodiments of this invention, and not all of them; it should be understood that the embodiments of this invention are only used to illustrate the technical effects of this invention, and not to limit the scope of protection of this invention.

[0040] All raw materials used in the examples are commercially available; unless otherwise specified, the reagents, methods and equipment used in this invention are conventional reagents, methods and equipment in this 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. The mass ratio of 1,4-butanediol diglycidyl ether to sodium hyaluronate is 0.08:1. Adjust the pH to alkaline using a 1 mol / L sodium hydroxide solution. After adding hyaluronic acid, the total mass ratio is 9%, and after adding sodium hydroxide, the total mass ratio is 7%. Stir evenly under vacuum to obtain a mixture.

[0043] S2. Maintaining vacuum conditions, heat the mixture obtained in S1 in a 30°C water bath for 6 hours, then cut it into uniform pieces of 0.5-1cm. 3 The sized block gels were cross-linked in a water bath at 50°C for 3 hours to obtain cross-linked sodium hyaluronate gel.

[0044] S3. The cross-linked sodium hyaluronate gel obtained in S2 was adjusted to neutral pH using 1 mol / L hydrochloric acid solution and phosphate buffer solution. The amount of hydrochloric acid solution added was the same as the amount of sodium hydroxide solution added in step S1. Then, the same amount of phosphate buffer solution was added, the same as the amount of cross-linked sodium hyaluronate gel obtained in S2. A first vortex-shear composite grinding process was performed to obtain a homogeneous paste-like gel. After thoroughly removing air, the gel was dialyzed in 80 times its weight of dialysate. The dialysate bag was a composite cellulose dialysis bag with a molecular weight cutoff of 8000-14000 kDa, and the gel content was 2 / 3 of the dialysis bag's capacity. The dialysate was a 4°C flowing PBS solution. The dialysate was changed every 8 hours for a total of 48 hours. The gel was then removed from the bag. The gel was removed from the container and then subjected to a second vortex-shear composite grinding process. Non-crosslinked sodium hyaluronate and PBS solution were added, with the final content of non-crosslinked sodium hyaluronate being 1 mg / mL and the final total sodium hyaluronate content being 24 mg / mL. A third vortex-shear composite grinding process was then performed, with the rotation speed increasing at a gradient of 20-60-80 rpm for 30 minutes each time. After degassing in a filling machine, the gel was filled and then sterilized at 124°C for 6 minutes. The time from room temperature to sterilization temperature and the time from sterilization temperature to 70°C were both within 0-8 minutes during the sterilization process, thus obtaining the dense honeycomb structure crosslinked 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 while stirring. The mass ratio of 1,4-butanediol diglycidyl ether to sodium hyaluronate is 0.07:1. Adjust the pH to alkaline using a 1 mol / L sodium hydroxide solution. After adding hyaluronic acid, the proportion is 10.5%, and after adding sodium hydroxide, the proportion is 8%. Stir evenly under vacuum to obtain a mixture.

[0047] S2. Maintaining vacuum conditions, heat the mixture obtained in S1 in a water bath at 27°C for 5 hours, and then cut it into uniform pieces of 0.5-1cm. 3 The sized block gels were cross-linked in a water bath at 50°C for 2.5 hours to obtain cross-linked sodium hyaluronate gel.

[0048] S3. The cross-linked sodium hyaluronate gel obtained in S2 was adjusted to neutral pH using 1 mol / L hydrochloric acid solution and phosphate buffer solution. The amount of hydrochloric acid solution added was the same as the amount of sodium hydroxide solution added in step S1. Then, phosphate buffer solution was added, with the same amount as the cross-linked sodium hyaluronate gel obtained in S2. A first vortex-shear composite grinding process was performed to obtain a homogeneous paste-like gel. After thoroughly removing air, the gel was dialyzed in 90 times its weight of dialysate. The dialysate bag was a composite cellulose dialysis bag with a molecular weight cutoff of 8000-14000 kDa, and the gel content was half the bag's capacity. The dialysate was 6°C flowing PBS solution, and the dialysate was changed every 7 hours for a total of 52 hours. The gel was then removed from the bag. The gel was removed and then subjected to a second vortex-shear composite grinding process. Non-crosslinked sodium hyaluronate and PBS solution were added, resulting in a final non-crosslinked sodium hyaluronate content of 1.5 mg / mL and a final total sodium hyaluronate content of 20 mg / mL. A third vortex-shear composite grinding process was then performed, with the rotation speed increasing at a gradient of 20-60-80 rpm for 45 minutes each time. After degassing in a filling machine, the gel was filled and then sterilized at 126°C for 5 minutes. The time from room temperature to sterilization temperature and from sterilization temperature to 70°C was within 0-8 minutes during the sterilization process, resulting in the dense honeycomb structure crosslinked 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 while stirring. The mass ratio of 1,4-butanediol diglycidyl ether to sodium hyaluronate is 0.06:1. Adjust the pH to alkaline using a 1 mol / L sodium hydroxide solution. After adding hyaluronic acid, the proportion is 12%, and after adding sodium hydroxide, the proportion is 9%. Stir evenly under vacuum to obtain a mixture.

[0051] S2. Maintaining vacuum conditions, heat the mixture obtained in S1 in a water bath at 25°C for 4 hours, and then cut it into uniform pieces of 0.5-1cm. 3 The block-shaped gels of different sizes were cross-linked in a water bath at 50°C for 2 hours to obtain cross-linked sodium hyaluronate gel.

[0052] S3. The cross-linked sodium hyaluronate gel obtained in S2 was adjusted to neutral pH using 1 mol / L hydrochloric acid solution and phosphate buffer solution. The amount of hydrochloric acid solution added was the same as the amount of sodium hydroxide solution added in step S1. Then, the same amount of phosphate buffer solution was added, the same as the amount of cross-linked sodium hyaluronate gel obtained in S2. A first vortex-shear composite grinding process was performed to obtain a homogeneous paste-like gel. After thoroughly removing air, the gel was dialyzed in 100 times its weight of dialysate. The dialysate bag was a composite cellulose dialysate bag with a molecular weight cutoff of 8000-14000 kDa, and the gel content was 1 / 3 of the dialysate bag's capacity. The dialysate was 8°C flowing PBS solution, and the dialysate was changed every 6 hours for a total of 56 hours. The gel was then removed from the bag. The gel was removed and then subjected to a second vortex-shear composite grinding process. Non-crosslinked sodium hyaluronate and PBS solution were added, resulting in a final non-crosslinked sodium hyaluronate content of 2.0 mg / mL and a final total sodium hyaluronate content of 18 mg / mL. A third vortex-shear composite grinding process was then performed, with the rotation speed increasing at a gradient of 20-60-80 rpm for 60 minutes each time. After degassing in a filling machine, the gel was filled and then sterilized at 128°C for 4 minutes. The time from room temperature to sterilization temperature and from sterilization temperature to 70°C was within 0-8 minutes during the sterilization process, thus obtaining the dense honeycomb structure crosslinked sodium hyaluronate gel.

[0053] Comparative Example 1

[0054] A method for preparing cross-linked sodium hyaluronate gel includes the following steps:

[0055] S1. Add 1,4-butanediol diglycidyl ether to water, and slowly add sodium hyaluronate while stirring. 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. After adding hyaluronic acid, the proportion is 10.5%, and after adding sodium hydroxide, the proportion is 8%. Stir evenly under vacuum to obtain a mixture.

[0056] S2. Maintaining vacuum conditions, heat the mixture obtained in S1 in a water bath at 27°C for 5 hours, and then cut it into uniform pieces of 0.5-1cm. 3 The sized block gels were cross-linked in a water bath at 50°C for 2.5 hours to obtain cross-linked sodium hyaluronate gel.

[0057] S3. The cross-linked sodium hyaluronate gel obtained in S2 was adjusted to neutral pH using 1 mol / L hydrochloric acid solution and phosphate buffer solution. The amount of hydrochloric acid solution added was the same as the amount of sodium hydroxide solution added in step S1. Then, phosphate buffer solution was added, the same as the amount of cross-linked sodium hyaluronate gel obtained in S2. A first vortex-shear composite grinding process was performed to obtain a homogeneous paste-like gel. After thoroughly removing air, it was dialyzed in 90 times its weight of dialysate. The dialysate bag was a composite cellulose dialysate bag with a molecular weight cutoff of 8000-14000 kDa, and the gel content was half the bag's capacity. The dialysate was 6°C flowing PBS solution. The dialysate was changed every 7 hours for a total of 52 hours. The gel was then... The product is removed from the bag and then subjected to a second vortex-shear composite grinding process. Non-crosslinked sodium hyaluronate and PBS solution are added, resulting in a final non-crosslinked sodium hyaluronate content of 1.5 mg / mL and a final total sodium hyaluronate content of 20 mg / mL. A third vortex-shear composite grinding process is then performed, with the rotation speed increasing at 20-60-80 rpm for 45 minutes each time. After degassing in a filling machine, the product is filled and then sterilized at 126°C for 5 minutes. The time from room temperature to sterilization temperature and from sterilization temperature to 70°C is within 0-8 minutes during sterilization, resulting in the crosslinked sodium hyaluronate gel.

[0058] Compared with Example 2, the main difference in this comparative example is that the chloride ion solution is not prepared in step S1.

[0059] Comparative Example 2

[0060] A method for preparing cross-linked sodium hyaluronate gel includes 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 while stirring. The mass ratio of 1,4-butanediol diglycidyl ether to sodium hyaluronate is 0.07:1. Adjust the pH to alkaline using a 1 mol / L sodium hydroxide solution. After adding hyaluronic acid, the proportion is 10.5%, and after adding sodium hydroxide, the proportion is 8%. Stir evenly under vacuum to obtain a mixture.

[0062] S2. Maintaining vacuum conditions, heat the mixture obtained in S1 in a water bath at 27°C for 5 hours, and then cut it into uniform pieces of 0.5-1cm. 3 The sized block gels were cross-linked in a water bath at 50°C for 2.5 hours to obtain cross-linked sodium hyaluronate gel.

[0063] S3. The cross-linked sodium hyaluronate gel obtained in S2 was adjusted to neutral pH using 1 mol / L hydrochloric acid solution and phosphate buffer solution. The amount of hydrochloric acid solution added was the same as the amount of sodium hydroxide solution added in step S1. Then, phosphate buffer solution was added, with the same amount as the cross-linked sodium hyaluronate gel obtained in S2. A first vortex-shear composite grinding process was performed to obtain a homogeneous paste-like gel. After thorough removal of air, it was dialyzed in 90 times its weight of dialysate. The dialysate bag was a composite cellulose dialysate bag with a molecular weight cutoff of 8000-14000 kDa, and the gel content was half the bag's capacity. The dialysate was 6°C flowing PBS solution. The dialysate was changed every 7 hours for a total of [number missing] dialysis cycles. After 52 hours, the gel was removed from the bag and non-crosslinked sodium hyaluronate and PBS solution were added. The final content of non-crosslinked sodium hyaluronate was 1.5 mg / mL, and the final total sodium hyaluronate content was 20 mg / mL. A second vortex-shear composite grinding treatment was performed, with a rotation speed gradient of 20-60-80 rpm, each treatment lasting 45 minutes. After degassing in a filling machine, the gel was filled and then sterilized at 126°C for 5 minutes. During the sterilization process, the time from room temperature to sterilization temperature was within 0-8 minutes, and the time from sterilization temperature to 70°C was within 0-8 minutes, thus obtaining the crosslinked sodium hyaluronate gel.

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

[0065] Comparative Example 3

[0066] A method for preparing cross-linked sodium hyaluronate gel includes 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 while stirring. The mass ratio of 1,4-butanediol diglycidyl ether to sodium hyaluronate is 0.07:1. Adjust the pH to alkaline using a 1 mol / L sodium hydroxide solution. After adding hyaluronic acid, the proportion is 10.5%, and after adding sodium hydroxide, the proportion is 8%. Stir until homogeneous to obtain a mixture.

[0068] S2. Heat the mixture obtained in S1 in a water bath at 27°C for 5 hours, and cut it into uniform pieces of 0.5-1cm. 3 The sized block gels were cross-linked in a water bath at 50°C for 2.5 hours to obtain cross-linked sodium hyaluronate gel.

[0069] S3. The cross-linked sodium hyaluronate gel obtained in S2 was adjusted to neutral pH using 1 mol / L hydrochloric acid solution and phosphate buffer solution. The amount of hydrochloric acid solution added was the same as the amount of sodium hydroxide solution added in step S1. Then, phosphate buffer solution was added, the same as the amount of cross-linked sodium hyaluronate gel obtained in S2. A first vortex-shear composite grinding process was performed to obtain a homogeneous paste-like gel. After thoroughly removing air, it was dialyzed in 90 times its weight of dialysate. The dialysate bag was a composite cellulose dialysate bag with a molecular weight cutoff of 8000-14000 kDa, and the gel content was half the bag's capacity. The dialysate was 6°C flowing PBS solution. The dialysate was changed every 7 hours for a total of 52 hours. The gel was then... The product is removed from the bag and then subjected to a second vortex-shear composite grinding process. Non-crosslinked sodium hyaluronate and PBS solution are added, resulting in a final non-crosslinked sodium hyaluronate content of 1.5 mg / mL and a final total sodium hyaluronate content of 20 mg / mL. A third vortex-shear composite grinding process is then performed, with the rotation speed increasing at 20-60-80 rpm for 45 minutes each time. After degassing in a filling machine, the product is filled and then sterilized at 126°C for 5 minutes. The time from room temperature to sterilization temperature and from sterilization temperature to 70°C is within 0-8 minutes during sterilization, resulting in the crosslinked sodium hyaluronate gel.

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

[0071] Comparative Example 4

[0072] A method for preparing cross-linked sodium hyaluronate gel includes 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 while stirring. The mass ratio of 1,4-butanediol diglycidyl ether to sodium hyaluronate is 0.07:1. Adjust the pH to alkaline using a 1 mol / L sodium hydroxide solution. After adding hyaluronic acid, the proportion is 10.5%, and after adding sodium hydroxide, the proportion is 8%. Stir evenly under vacuum to obtain a mixture.

[0074] S2. Maintaining vacuum conditions, heat the mixture obtained in S1 in a water bath at 27°C for 5 hours, and then cut it into uniform pieces of 0.5-1cm. 3 The sized block gels were cross-linked in a water bath at 50°C for 2.5 hours to obtain cross-linked sodium hyaluronate gel.

[0075] S3. The cross-linked sodium hyaluronate gel obtained in S2 was adjusted to neutral pH using 1 mol / L hydrochloric acid solution and phosphate buffer solution. The amount of hydrochloric acid solution added was the same as the amount of sodium hydroxide solution added in step S1. Then, phosphate buffer solution was added, the same as the amount of cross-linked sodium hyaluronate gel obtained in S2. A first vortex-shear composite grinding process was performed to obtain a homogeneous paste-like gel. After thoroughly removing air, it was dialyzed in 90 times its weight of dialysate. The dialysate bag was a composite cellulose dialysate bag with a molecular weight cutoff of 8000-14000 kDa, and the gel content was half the bag's capacity. The dialysate was 6°C flowing PBS solution. The dialysate was changed every 7 hours for a total of 52 hours. The gel was removed from the bag and then subjected to a second vortex-shear composite grinding process. Non-crosslinked sodium hyaluronate and PBS solution were added, 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. A third vortex-shear composite grinding process was then performed, with the rotation speed increasing at a gradient of 20-60-80 rpm, each process lasting 45 minutes. After degassing in a filling machine, the gel was filled and then sterilized 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, thus obtaining the crosslinked sodium hyaluronate gel.

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

[0077] Comparative Example 5

[0078] A method for preparing cross-linked sodium hyaluronate gel includes the following steps:

[0079] S1. Add 1,4-butanediol diglycidyl ether to water, and slowly add sodium hyaluronate while stirring. 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. After adding hyaluronic acid, the proportion is 10.5%, and after adding sodium hydroxide, the proportion is 8%. Stir until homogeneous to obtain a mixture.

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

[0081] S3. The cross-linked sodium hyaluronate gel obtained in S2 was adjusted to neutral pH using 1 mol / L hydrochloric acid solution and phosphate buffer solution. The amount of hydrochloric acid solution added was the same as the amount of sodium hydroxide solution added in step S1. Then, phosphate buffer solution was added, the same as the amount of cross-linked sodium hyaluronate gel obtained in S2. A first vortex-shear composite grinding process was performed to obtain a homogeneous paste-like gel. After thorough removal of air, it was dialyzed in 90 times its weight of dialysate. The dialysate bag was a composite cellulose dialysate bag with a molecular weight cutoff of 8000-14000 kDa, and the gel content was half the bag's capacity. The dialysate was a 6°C flowing PBS solution, and the dialysate was changed every 7 hours. After 52 hours of analysis, the gel was removed from the bag and non-crosslinked sodium hyaluronate and PBS solution were added. The final content of non-crosslinked sodium hyaluronate was 1.5 mg / mL, and the final total sodium hyaluronate content was 20 mg / mL. A second vortex-shear composite grinding treatment was performed, with a rotation speed gradient of 20-60-80 rpm, each treatment lasting 45 minutes. After degassing in a filling machine, the gel was filled and then sterilized at 126℃ for 5 minutes. During the sterilization process, the time from room temperature to sterilization temperature was within 20 minutes, and the time from sterilization temperature to 70℃ was within 20 minutes, thus obtaining the crosslinked sodium hyaluronate gel.

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

[0083] SEM scans were performed on Examples 1-3 and Comparative Examples 1-5, and the SEM images are shown below. Figure 1-8 As shown.

[0084] Figure 1 , Figure 2 and Figure 3 The images show SEM images of the dense honeycomb cross-linked sodium hyaluronate gels from Examples 1-3. As can be seen from the images, the network of the dense honeycomb cross-linked sodium hyaluronate gels becomes denser with increasing cross-linking degree, but the overall structure exhibits a uniform dense honeycomb shape, demonstrating a stable cross-linked spatial structure.

[0085] Figure 4 The image shows a SEM image of the cross-linked sodium hyaluronate gel in Comparative Example 1. As can be seen from the image, compared with Example 2, the density of the sodium hyaluronate gel without controlled ion concentration is reduced, and the network structure is also more inconsistent.

[0086] Figure 5 The image shows a SEM image of the cross-linked sodium hyaluronate gel in Comparative Example 2. As can be seen from the image, compared with Example 2, the sodium hyaluronate gel that underwent only two vortex-shear composite milling treatments actually showed an increase in density.

[0087] Figure 6 , Figure 7 The image shows the SEM images of the cross-linked sodium hyaluronate gels of Comparative Example 3-4. As can be seen from the image, compared with Example 2, the network structure of the cross-linked sodium hyaluronate gels of Comparative Example 3-4 is significantly looser and more uneven, indicating that the vacuum environment cross-linking and rapid temperature-heating sterilization methods have a significant impact on the cross-linking effect and cross-linking spatial structure of the product.

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

[0089] Performance tests were conducted on the above embodiments and comparative examples. The experimental test methods are as follows:

[0090] Elastic modulus:

[0091] Using a rotational rheometer, the test temperature was set to 25℃, and an oscillation test was performed with a strain of 5%. The frequency was scanned from 0.08Hz to 5Hz, and the elastic modulus G' value corresponding to the 1Hz position was taken with the scanning frequency as the horizontal axis.

[0092] In vitro degradation:

[0093] The sample was enzymatically hydrolyzed using a 10 IU / mL hyaluronidase solution. After 12 hours of degradation, the hyaluronidase was inactivated using a 0.5 mol / L hydrochloric acid solution. The concentration of glucuronic acid in the degradation solution was then measured and converted into the concentration of sodium hyaluronate after degradation. The in vitro degradation rate after 12 hours was obtained by comparing the concentration with that of the sample.

[0094] Pushing force:

[0095] The sample was filled into a 1mL syringe and refrigerated at 2–10°C for 2 hours. It was then removed and allowed to cool to room temperature. The syringe was then mounted on a universal testing machine, and the pushing speed was set to 30 mm / min. The pushing force was measured using a 27G injection needle.

[0096] Swelling degree:

[0097] Take a 500-mesh sieve (an 8cm×8cm square, folded into a 4cm×4cm×2cm square groove) and place it in a drying oven. Heat it to constant weight at 80℃ and record it as m0.

[0098] Weigh out the cross-linked sodium hyaluronate gel and place it on a 500-mesh sieve. Place the sieve in an evaporating dish and add 0.9% sodium chloride solution to completely wet the sample. After the gel has fully swelled (at least 30 minutes), remove the sieve and sample together. Use filter paper to absorb the liquid from the bottom and sides of the sieve until there are no wet marks on the filter paper. Weigh the sample and record the weight as m1.

[0099] Place the sieve containing the swollen gel into a drying oven and dry it at 80°C until constant weight is achieved, which is recorded as m2.

[0100] Calculation formula: Cohesion test:

[0101] The product was uniformly dyed and placed on a universal testing machine. It was then pushed through an 18G cannula at a constant speed of 8 mm / min. The average weight (unit: mg) of the 10 drops of gel pushed out was recorded.

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

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

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

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

[0106] Compared to Example 2, Comparative Example 1, without controlling ionic strength, had a lower elastic modulus, higher swelling degree, and higher in vitro degradation rate, which was not conducive to maintaining the effect. Comparative Example 2, which only underwent two vortex-shear composite grinding processes, had a higher elastic modulus, higher extrusion force and cohesion, making injection difficult. Comparative Example 3, with crosslinking not performed under vacuum, had a lower elastic modulus, higher in vitro degradation rate, higher swelling degree, lower cohesion, and weaker support. Comparative Example 4, with a long sterilization and heating time, had a lower elastic modulus, lower cohesion, higher swelling degree, and poor support and maintenance effect. Comparative Example 5, prepared by conventional methods, showed the worst performance among all comparative examples. This indicates that controlling ionic strength, using vacuum protection for the crosslinking process, and controlling sterilization and heating time can improve the degree of crosslinking, spatial mechanical structure, and performance of the product, significantly improving the physical properties of the product. Performing a triple homogenization process can significantly improve the injection smoothness of the product.

[0107] In summary, this invention improves the effective cross-linking efficiency of the product and forms a dense honeycomb structure by controlling the pH, temperature, ionic strength, sterilization heating and cooling time, and cross-linking medium during the cross-linking reaction process. This reduces the amount of free cross-linking agent residue, lowers the amount of cross-linking agent used, and improves mechanical properties. Furthermore, the invention enhances the uniformity and cohesion of the product through a multi-stage stirring and grinding process, and fully removes residual small molecule impurities through semi-permeable membrane dialysis technology, reducing inflammatory reactions after product injection. This invention fully integrates the clinical application of the product with the cross-linking reaction mechanism. By controlling the ionic strength of the sodium hyaluronate dissolving medium, the entangled structure of the hyaluronic acid macromolecular chain is opened, thereby exposing more reactive groups and increasing the number of cross-linking sites. By controlling the pH value of the reaction, the epoxy groups in the cross-linking agent react with the hydroxyl groups on the sodium hyaluronate molecular chain to form stable ether bonds. By controlling the temperature and time, the sufficiency of the cross-linking reaction is ensured. Cross-linking in a vacuum environment protects the activity of the cross-linking agent and improves the effective cross-linking rate between the cross-linking agent and hyaluronic acid. By performing triple stirring and grinding treatment after cross-linking, after dialysis, and before the filling process, the product always maintains a homogeneous gel state, improving injection smoothness and cohesion. By strictly controlling the sterilization heating and cooling time and performing it rapidly, the degree of cross-linking, spatial mechanical structure and properties of the product are improved, significantly improving the physical properties of the product. The result is a dense honeycomb structure cross-linked sodium hyaluronate gel with excellent comprehensive physical properties, always maintaining a homogeneous gel state, excellent injection smoothness and cohesion, strong support, long-lasting effect, and low adverse reaction rate.

[0108] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention 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, 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, adjust the pH to alkaline, stir evenly under vacuum to obtain a mixture; S2. Under vacuum conditions, heat the mixture obtained in S1 in a water bath, cut it to obtain a block gel, crosslink it in a water bath to obtain a crosslinked sodium hyaluronate gel. S3. Adjust the pH of the cross-linked sodium hyaluronate gel obtained in S2 to neutral, perform a first vortex-shear composite grinding treatment to obtain a homogeneous paste-like gel, dialyze, then perform a second vortex-shear composite grinding treatment, add non-cross-linked sodium hyaluronate and PBS solution, perform a third vortex-shear composite grinding treatment, fill the package, and perform high-temperature steam sterilization treatment at a temperature of 124-128℃ for 4-6 minutes. During the sterilization process, the time from room temperature to the sterilization temperature should be within 0-8 minutes, and the time from the sterilization temperature to 70℃ should be within 0-8 minutes, thus obtaining the dense honeycomb structure cross-linked sodium hyaluronate gel. The crosslinking agent described in S1 is 1,4-butanediol diglycidyl ether; The mass ratio of the crosslinking agent to sodium hyaluronate in S1 is 0.07:1; S1 describes adjusting the pH to alkalinity by using a 1 mol / L sodium hydroxide solution, wherein the mass ratio of the sodium hydroxide solution to sodium hyaluronate is (15-20):(26-28). The water bath heating temperature described in S2 is 25-30℃, and the heating time is 4-6 hours; The water bath crosslinking temperature described in S2 is 50℃, and the time is 2-3 hours; The three-stage vortex-shear composite grinding process described in S3 involves a rotational speed gradient of 20-60-80 rpm, with each process lasting 30-60 minutes.

2. A method for preparing a dense honeycomb structure cross-linked sodium hyaluronate gel according to claim 1, characterized in that, S2 describes the slicing process, which involves cutting the gel into uniform pieces of 0.5-1cm. 3 A block-shaped gel of various sizes.

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

4. A dense honeycomb structure cross-linked sodium hyaluronate gel, characterized in that, The dense honeycomb structure crosslinked sodium hyaluronate gel according to any one of claims 1-3 is prepared by the method of preparation.

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