A method for preparing an amino acid cross-linked sodium hyaluronate gel for injection

Abstract

This invention belongs to the field of medical aesthetics and biomedical materials technology, specifically relating to a method for preparing injectable amino acid crosslinked sodium hyaluronate gel. This method uses amino acids as crosslinking agents to modify two types of sodium hyaluronate with different molecular weights, reacting to form an amide bond structure. A high-low temperature alternating crosslinking process enhances the gel's cohesive energy, constructing a dense and stable three-dimensional network structure, effectively improving the problems of insufficient viscoelasticity and rapid in vivo degradation of pure sodium hyaluronate. Amino acids are endogenous active substances with excellent biocompatibility and safety, avoiding the safety hazards caused by residues of traditional chemical crosslinking agents. The crosslinked sodium hyaluronate gel prepared by this process has properties suitable for clinical applications such as soft tissue filling and medical aesthetic repair.

Description

Technical Field

[0001] This invention relates to the fields of medical aesthetics and biomedical materials technology, specifically to a method for preparing injectable amino acid crosslinked sodium hyaluronate gel. Background Technology

[0002] Sodium hyaluronate is the sodium salt of hyaluronic acid, widely found in human skin, synovial fluid, and cartilage. It is a natural polysaccharide macromolecule composed of glucuronic acid and N-acetylglucosamine structural units linked by repeated β-1,3 and β-1,4 glycosidic bonds. Due to the large number of hydroxyl and carboxyl groups in its molecular chain and its negative charge, sodium hyaluronate possesses unique water-absorbing and moisturizing properties. Because of these unique properties and good biocompatibility, sodium hyaluronate is widely used for soft tissue defect filling and synovial fluid replenishment. However, once inside the human body, sodium hyaluronate is degraded by hyaluronidase, resulting in a short retention time and affecting the therapeutic and repair effects.

[0003] Crosslinking sodium hyaluronate using chemical and physical methods to form a rigid spatial network structure can prolong its retention time in vivo. Physical crosslinking forms a network structure through intermolecular hydrogen bonds or ionic bonds, but the bonding is weak and the crosslinked structure is unstable. Therefore, the mainstream technology currently on the market mainly uses chemical crosslinking agents to enhance the structure of sodium hyaluronate. With the development of technology and the market, the crosslinking agents currently used for crosslinking sodium hyaluronate are mainly divided into two categories: 1,4-butanediol diglycidyl ether (BDDE) and divinyl sulfone (DVS). Both improve the physical properties of sodium hyaluronate by crosslinking the hydroxyl groups on sodium hyaluronate. However, both have significant biotoxicity. BDDE contains epoxy groups and has potential toxicity, and DVS itself has potential toxicity; residual unreacted DVS can cause adverse reactions such as allergies. Therefore, finding and using crosslinking agents with high biosafety is an important way to develop crosslinked sodium hyaluronate.

[0004] Amino acids are the basic building blocks of proteins, biologically functional macromolecules, participating in various life activities in the human body and exhibiting extremely high biocompatibility. Furthermore, structural analysis reveals that amino acids possess bifunctional groups (amino and carboxyl groups), enabling them to undergo various reactions such as amidation and esterification. They can react with the carboxyl groups in the structural units of sodium hyaluronate to form amide bonds. Therefore, amino acids are a relatively ideal chemical cross-linking agent for cross-linking sodium hyaluronate.

[0005] When amino acids are used as chemical crosslinking agents for sodium hyaluronate, the resulting crosslinked sodium hyaluronate exhibits lower mechanical support properties, such as strength and elastic modulus, compared to traditional chemical crosslinking agents like BDDE. Therefore, further modification of the amino acid-crosslinked sodium hyaluronate gel is necessary to improve its mechanical properties. Summary of the Invention

[0006] To address the aforementioned shortcomings, the present invention aims to provide a method for preparing injectable amino acid-crosslinked sodium hyaluronate gel. The amino acid-crosslinked sodium hyaluronate gel prepared by this invention overcomes the drawbacks of current technologies, such as the short in vivo retention time of sodium hyaluronate, the potential biotoxicity of crosslinking agents like BDDE and DVS, and the poor mechanical properties of amino acid crosslinking. This invention uses amino acids as crosslinking agents to prepare crosslinked sodium hyaluronate gel. By modifying two sodium hyaluronate molecules with different molecular weights using amino acids, amide bonds are formed, and hydrogen bonds are formed through temperature control, constructing a robust dual-dimensional network structure. This reduces the use of small-molecule crosslinking agents and overcomes the defects of poor viscoelasticity and rapid degradation of amino acid-crosslinked sodium hyaluronate. Furthermore, amino acids, as substances involved in human life activities, possess ideal biocompatibility and safety, mitigating the potential risks associated with crosslinking agent residues. The crosslinked sodium hyaluronate material obtained through the preparation method of amino acid-crosslinked sodium hyaluronate gel can well meet the needs of medical filling and repair.

[0007] This invention provides the following technical solution: A method for preparing injectable amino acid crosslinked sodium hyaluronate gel includes the following steps: Step 1: Mix high molecular weight sodium hyaluronate in water for injection to obtain a sodium hyaluronate system, and then add a coupling agent to activate it; Step 2: After adding the cross-linking agent amino acid and mixing well, carry out a high-temperature cross-linking reaction; Step 3: Add low molecular weight sodium hyaluronate and carry out a low-temperature cross-linking reaction; Step 4: The cross-linked sodium hyaluronate after low-temperature cross-linking undergoes dialysis treatment; Step 5: After dialysis until the mass of cross-linked sodium hyaluronate no longer increases, granulation is performed; Step 6: The granulated cross-linked sodium hyaluronate is dehydrated using a dehydrating agent; Step 7: The dehydrated cross-linked sodium hyaluronate is then dried. Step 8: The dried cross-linked sodium hyaluronate is reconstituted in phosphate buffer to obtain amino acid cross-linked sodium hyaluronate gel.

[0008] As a preferred technical solution for the preparation method of injectable amino acid crosslinked sodium hyaluronate gel, the high molecular weight sodium hyaluronate in step one has a weight average molecular weight of 1000 kDa to 3000 kDa, and the low molecular weight sodium hyaluronate in step three has a weight average molecular weight of 100 kDa to 5000 kDa.

[0009] As a preferred technical solution for the preparation method of injectable amino acid crosslinked sodium hyaluronate gel, the mass fraction of high molecular weight sodium hyaluronate in the sodium hyaluronate system in step one is 3%-5%, and the mass fraction of low molecular weight sodium hyaluronate in the sodium hyaluronate system in step three is 1%-3%.

[0010] In a preferred embodiment of a method for preparing injectable amino acid-crosslinked sodium hyaluronate gel, the coupling agent in step one is one or more selected from succinimidyl glutarate (SG group), N-hydroxysuccinimidyl ester (NSH ester), carbodiimide (such as EDC), and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM). Further, the molar amount of the coupling agent is 50%-100% of the molar amount of the repeating unit of sodium hyaluronate.

[0011] In a preferred embodiment of the preparation method of injectable amino acid crosslinked sodium hyaluronate gel, the crosslinking agent amino acid in step two is a basic amino acid and its derivatives: one or more of lysine, arginine, histidine, L-lysine hydrochloride, N'-tert-butoxycarbonyl-D-lysine tert-butyl ester hydrochloride, L-lysine diisocyanate, phosphate-L-arginine, and L-arginine hydrochloride.

[0012] In a preferred embodiment of the preparation method of injectable amino acid crosslinked sodium hyaluronate gel, the molar amount of the crosslinking agent amino acid in step two is 5%-30% of the molar amount of the repeating unit of hyaluronic acid.

[0013] As a preferred technical solution for the preparation method of injectable amino acid crosslinked sodium hyaluronate gel, the high-temperature crosslinking reaction in step two has a temperature range of 40℃-60℃ and a reaction time range of 5-24 hours, and the low-temperature crosslinking reaction in step three has a temperature range of 0℃-10℃ and a reaction time range of 12-48 hours.

[0014] As a preferred technical solution for the preparation of injectable amino acid crosslinked sodium hyaluronate gel, the granulation method in step five is one of screen extrusion granulation and high-pressure homogenization granulation.

[0015] In a preferred embodiment of the preparation method of injectable amino acid crosslinked sodium hyaluronate gel, the dehydrating agent in step six is ​​one or more of ethanol, acetone, and isopropanol, and the mass of the dehydrating agent is 3-5 times the mass of the granulated crosslinked sodium hyaluronate.

[0016] As a preferred technical solution for the preparation of injectable amino acid crosslinked sodium hyaluronate gel, the drying method in step seven is either vacuum drying or freeze drying.

[0017] In a preferred embodiment of the preparation method of injectable amino acid crosslinked sodium hyaluronate gel, the phosphate buffer solution in step eight consists of disodium hydrogen phosphate, sodium dihydrogen phosphate, and sodium chloride, and the pH range of the phosphate buffer solution is 6.5-7.5.

[0018] Beneficial effects of the technology: 1. The injectable amino acid crosslinked sodium hyaluronate gel provided by this invention uses amino acids as a chemical crosslinking agent. Through a two-step crosslinking process at high and low temperatures, the crosslinking of amino acids and sodium hyaluronate is achieved. While constructing an amidation crosslinking mechanism, hydrogen bonds are used to strengthen the structure, forming a double three-dimensional network structure, which improves the mechanical properties of the sodium hyaluronate gel and enhances the filling effect. The formation of amide bonds and hydrogen bonds delays the collapse of the gel structure caused by hyaluronidase and improves the long-lasting effect of the gel filling.

[0019] 2. The injectable amino acid crosslinked sodium hyaluronate gel provided by the present invention uses natural and non-toxic amino acids to replace traditional chemical crosslinking agents, avoiding the potential toxicity of traditional crosslinking agents, and the metabolites have no chemical residues and can be excreted with human metabolism, significantly improving product safety.

[0020] 3. The injectable amino acid cross-linked sodium hyaluronate gel provided by the present invention uses amino acids that can stimulate collagen regeneration, exhibiting physiological synergy and enhancing the filling effect.

[0021] 4. The method for preparing injectable amino acid crosslinked sodium hyaluronate gel provided by the present invention has mild crosslinking reaction conditions, does not require high pressure, and is convenient for standardized mass production in the workshop. Attached Figure Description

[0022] Figure 1 a is a macroscopic photograph of the gel before granulation after dialysis in Example 5; Figure 1 b is a macroscopic photograph of the gel before granulation after dialysis in Example 6; Figure 1 c is a macroscopic photograph of the gel before granulation at the end of dialysis in Control Example 3; Figure 2 The 1H NMR spectrum of the amino acid crosslinked sodium hyaluronate gel prepared in Example 4; Figure 3 The 1H NMR spectrum of the amino acid crosslinked sodium hyaluronate gel prepared in Example 5; Figure 4 The 1H NMR spectrum of the amino acid crosslinked sodium hyaluronate gel prepared in Example 6; Figure 5 Frequency-modulus diagram of the amino acid crosslinked sodium hyaluronate gel prepared in Example 5; Detailed Implementation

[0023] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the following description is provided in conjunction with the accompanying drawings. Figure 1 The embodiments in the specification provide a detailed description of the specific implementation methods of the present invention.

[0024] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.

[0025] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0026] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0027] Test method: 1. The pushing force of the sample shall be tested using a universal testing machine in accordance with the method specified in Appendix A of YY / T0962-2021 "Cross-linked Sodium Hyaluronate Gel for Plastic Surgery"; 2. The dynamic viscosity of the samples was determined using a rotational rheometer at a shear rate of 0.25 s⁻¹. -1 The test was conducted at (25±2)℃, and the average value within 0-30s was taken. 3. The elastic modulus and viscous modulus of the samples were measured using a rotational rheometer in oscillating shear mode at (25±2)℃ with 1% shear deformation. The modulus change was measured from 0.1 to 10 Hz. The elastic modulus and viscous modulus of the samples at 1 Hz were taken. 4. The 1H NMR spectrum of the sample was obtained by testing the dehydrated gel sample after it was degraded with a deuterium aqueous solution of hyaluronidase. The chemical crosslinking degree of the gel can be calculated by taking the integral values ​​of the peaks at f1=2.0ppm and f1=3.0ppm.

[0028] Example 1 Step 1. Mix and activate Weigh 3.0 g of high molecular weight sodium hyaluronate (1500 kDa) and add water for injection until the mass fraction of sodium hyaluronate is 3%. Stir at 25°C. After stirring evenly, a sodium hyaluronate system is obtained. Add 2.0 g of DMTMM and stir to activate for 15 min.

[0029] Step 2. High-temperature crosslinking After activation, add 0.3g of arginine, stir and mix well, stir for 30 minutes, and then let stand at 50℃ for 5 hours to carry out high-temperature cross-linking reaction.

[0030] Step 3. Low-temperature crosslinking After the high-temperature crosslinking reaction, 1g of low molecular weight sodium hyaluronate (500kDa) was added to the sodium hyaluronate system and stirred evenly at 25°C. Then, it was moved to a 4°C environment and allowed to stand for 24 hours to carry out the low-temperature crosslinking reaction.

[0031] Step 4. Dialysis After a two-step crosslinking reaction at high and low temperatures, sodium hyaluronate gel was obtained. The gel was cut into 1cm×1cm×1cm cubes and dialyzed with 1L of water for injection. The dialysate was changed every 4 hours. Dialysis was stopped when the gel mass no longer increased, and the gel cubes were removed.

[0032] Step 5. Granulation The gel block obtained in step 4 is sequentially passed through 600μm, 300μm and 200μm sieves for sieve extrusion granulation to obtain gel particles.

[0033] Step 6. Dehydration treatment The gel particles obtained in step 5 were added to 300g of anhydrous ethanol for dehydration. After standing for 10 minutes, the supernatant was removed, and then another 300g of anhydrous ethanol was added. This process was repeated 3 times to obtain dehydrated gel particles.

[0034] Step 7. Drying treatment The dehydrated gel particles obtained in step 6 were dried in a vacuum drying oven at -0.09 MPa and 40°C for 24 hours.

[0035] Step 8. Gel Reconstitution The dried gel particles obtained in step 7 are added to 50 times their mass of phosphate buffer solution at pH 7.1 and mixed thoroughly to dissolve, thus obtaining amino acid crosslinked sodium hyaluronate gel.

[0036] The results of the extrusion force test and macroscopic state test of amino acid crosslinked sodium hyaluronate gel are shown in Table 1, and the results of the dynamic viscosity and modulus test are shown in Table 2.

[0037] Example 2 Step 1. Mix and activate Weigh 3.0 g of high molecular weight sodium hyaluronate (2000 kDa) and add water for injection until the mass fraction of sodium hyaluronate is 3%. Stir at 25°C. After stirring evenly, a sodium hyaluronate system is obtained. Add 2.0 g of DMTMM and stir to activate for 15 min.

[0038] Step 2. High-temperature crosslinking After activation, add 0.3g of arginine, stir and mix well, stir for 30 minutes, and then let stand at 50℃ for 5 hours to carry out high-temperature cross-linking reaction.

[0039] Step 3. Low-temperature crosslinking After the high-temperature crosslinking reaction, 1g of low molecular weight sodium hyaluronate (500kDa) was added to the sodium hyaluronate system and stirred evenly at 25°C. Then, it was moved to a 4°C environment and allowed to stand for 24 hours to carry out the low-temperature crosslinking reaction.

[0040] Step 4. Dialysis After a two-step crosslinking reaction at high and low temperatures, sodium hyaluronate gel was obtained. The gel was cut into 1cm×1cm×1cm cubes and dialyzed with 1L of water for injection. The dialysate was changed every 4 hours. Dialysis was stopped when the gel mass no longer increased, and the gel cubes were removed.

[0041] Step 5. Granulation The gel block obtained in step 4 is sequentially passed through 600μm, 300μm and 200μm sieves for sieve extrusion granulation to obtain gel particles.

[0042] Step 6. Dehydration treatment The gel particles obtained in step 5 were added to 300g of anhydrous ethanol for dehydration. After standing for 10 minutes, the supernatant was removed, and then another 300g of anhydrous ethanol was added. This process was repeated 3 times to obtain dehydrated gel particles.

[0043] Step 7. Drying treatment The dehydrated gel particles obtained in step 6 were dried in a vacuum drying oven at -0.09 MPa and 40°C for 24 hours.

[0044] Step 8. Gel Reconstitution The dried gel particles obtained in step 7 are added to 50 times their mass of phosphate buffer solution at pH 7.1 and mixed thoroughly to dissolve, thus obtaining amino acid crosslinked sodium hyaluronate gel.

[0045] The results of the extrusion force test and macroscopic state test of amino acid crosslinked sodium hyaluronate gel are shown in Table 1, and the results of the dynamic viscosity and modulus test are shown in Table 2.

[0046] Example 3 Step 1. Mix and activate Weigh 3.0 g of high molecular weight sodium hyaluronate (1500 kDa) and add water for injection until the mass fraction of sodium hyaluronate is 3%. Stir at 25°C. After stirring evenly, a sodium hyaluronate system is obtained. Add 2.0 g of DMTMM and stir to activate for 15 min.

[0047] Step 2. High-temperature crosslinking After activation, add 0.3g of arginine, stir and mix well, stir for 30 minutes, and then let stand at 50℃ for 5 hours to carry out high-temperature cross-linking reaction.

[0048] Step 3. Low-temperature crosslinking After the high-temperature crosslinking reaction, 1g of low molecular weight sodium hyaluronate (1000KDa) was added to the sodium hyaluronate system and stirred evenly at 25°C. Then, it was moved to a 4°C environment and allowed to stand for 24 hours to carry out the low-temperature crosslinking reaction.

[0049] Step 4. Dialysis After a two-step crosslinking reaction at high and low temperatures, sodium hyaluronate gel was obtained. The gel was cut into 1cm×1cm×1cm cubes and dialyzed with 1L of water for injection. The dialysate was changed every 4 hours. Dialysis was stopped when the gel mass no longer increased, and the gel cubes were removed.

[0050] Step 5. Granulation The gel block obtained in step 4 is sequentially passed through 600μm, 300μm and 200μm sieves for sieve extrusion granulation to obtain gel particles.

[0051] Step 6. Dehydration treatment The gel particles obtained in step 5 were added to 300g of anhydrous ethanol for dehydration. After standing for 10 minutes, the supernatant was removed, and then another 300g of anhydrous ethanol was added. This process was repeated 3 times to obtain dehydrated gel particles.

[0052] Step 7. Drying treatment The dehydrated gel particles obtained in step 6 were dried in a vacuum drying oven at -0.09 MPa and 40°C for 24 hours.

[0053] Step 8. Gel Reconstitution The dried gel particles obtained in step 7 are added to 50 times their mass of phosphate buffer solution at pH 7.1 and mixed thoroughly to dissolve, thus obtaining amino acid crosslinked sodium hyaluronate gel.

[0054] The results of the extrusion force test and macroscopic state test of amino acid crosslinked sodium hyaluronate gel are shown in Table 1, and the results of the dynamic viscosity and modulus test are shown in Table 2.

[0055] Example 4 Step 1. Mix and activate Weigh 3.0 g of high molecular weight sodium hyaluronate (1500 kDa) and add water for injection until the mass fraction of sodium hyaluronate is 3%. Stir at 25°C. After stirring evenly, a sodium hyaluronate system is obtained. Add 2.0 g of DMTMM and stir to activate for 15 min.

[0056] Step 2. High-temperature crosslinking After activation, add 0.3g of lysine, stir and mix well, stir for 30 minutes, and then let stand at 50℃ for 5 hours to carry out high-temperature cross-linking reaction.

[0057] Step 3. Low-temperature crosslinking After the high-temperature crosslinking reaction, 1g of low molecular weight sodium hyaluronate (500kDa) was added to the sodium hyaluronate system and stirred evenly at 25°C. Then, it was moved to a 4°C environment and allowed to stand for 24 hours to carry out the low-temperature crosslinking reaction.

[0058] Step 4. Dialysis After a two-step crosslinking reaction at high and low temperatures, sodium hyaluronate gel was obtained. The gel was cut into 1cm×1cm×1cm cubes and dialyzed with 1L of water for injection. The dialysate was changed every 4 hours. Dialysis was stopped when the gel mass no longer increased, and the gel cubes were removed.

[0059] Step 5. Granulation The gel block obtained in step 4 is sequentially passed through 600μm, 300μm and 200μm sieves for sieve extrusion granulation to obtain gel particles.

[0060] Step 6. Dehydration treatment The gel particles obtained in step 5 were added to 300g of anhydrous ethanol for dehydration. After standing for 10 minutes, the supernatant was removed, and then another 300g of anhydrous ethanol was added. This process was repeated 3 times to obtain dehydrated gel particles.

[0061] Step 7. Drying treatment The dehydrated gel particles obtained in step 6 were dried in a vacuum drying oven at -0.09 MPa and 40°C for 24 hours.

[0062] Step 8. Gel Reconstitution The dried gel particles obtained in step 7 are added to 50 times their mass of phosphate buffer solution at pH 7.1 and mixed thoroughly to dissolve, thus obtaining amino acid crosslinked sodium hyaluronate gel.

[0063] The results of the extrusion force test and macroscopic state test of amino acid crosslinked sodium hyaluronate gel are shown in Table 1, and the results of the dynamic viscosity and modulus test are shown in Table 2.

[0064] Example 5 Step 1. Mix and activate Weigh 3.0 g of high molecular weight sodium hyaluronate (2000 kDa) and add water for injection until the mass fraction of sodium hyaluronate is 3%. Stir at 25°C. After stirring evenly, a sodium hyaluronate system is obtained. Add 2.0 g of DMTMM and stir to activate for 15 min.

[0065] Step 2. High-temperature crosslinking After activation, add 0.5g of lysine, stir and mix well, stir for 30min, and then let stand at 50℃ for 3h to carry out high-temperature cross-linking reaction.

[0066] Step 3. Low-temperature crosslinking After the high-temperature crosslinking reaction, 1g of low molecular weight sodium hyaluronate (500kDa) was added to the sodium hyaluronate system and stirred evenly at 25°C. Then, it was moved to a 4°C environment and allowed to stand for 24 hours to carry out the low-temperature crosslinking reaction.

[0067] Step 4. Dialysis After a two-step crosslinking reaction at high and low temperatures, sodium hyaluronate gel was obtained. The gel was cut into 1cm×1cm×1cm cubes and dialyzed with 1L of water for injection. The dialysate was changed every 4 hours. Dialysis was stopped when the gel mass no longer increased, and the gel cubes were removed.

[0068] Step 5. Granulation The gel block obtained in step 4 is sequentially passed through 600μm, 300μm and 200μm sieves for sieve extrusion granulation to obtain gel particles.

[0069] Step 6. Dehydration treatment The gel particles obtained in step 5 were added to 300g of anhydrous ethanol for dehydration. After standing for 10 minutes, the supernatant was removed, and then another 300g of anhydrous ethanol was added. This process was repeated 3 times to obtain dehydrated gel particles.

[0070] Step 7. Drying treatment The dehydrated gel particles obtained in step 6 were dried in a vacuum drying oven at -0.09 MPa and 40°C for 24 hours.

[0071] Step 8. Gel Reconstitution The dried gel particles obtained in step 7 are added to 50 times their mass of phosphate buffer solution at pH 7.1 and mixed thoroughly to dissolve, thus obtaining amino acid crosslinked sodium hyaluronate gel.

[0072] The results of the extrusion force test and macroscopic state test of amino acid crosslinked sodium hyaluronate gel are shown in Table 1, and the results of the dynamic viscosity and modulus test are shown in Table 2.

[0073] Example 6 Step 1. Mix and activate Weigh 3.0 g of high molecular weight sodium hyaluronate (1500 kDa) and add water for injection until the mass fraction of sodium hyaluronate is 3%. Stir at 25°C. After stirring evenly, a sodium hyaluronate system is obtained. Add 2.0 g of DMTMM and stir to activate for 15 min.

[0074] Step 2. High-temperature crosslinking After activation, add 0.3g of lysine, stir and mix well, stir for 30 minutes, and then let stand at 50℃ for 5 hours to carry out high-temperature cross-linking reaction.

[0075] Step 3. Low-temperature crosslinking After the high-temperature crosslinking reaction, 1g of low molecular weight sodium hyaluronate (500kDa) was added to the sodium hyaluronate system and stirred evenly at 25°C. Then, it was transferred to a 4°C environment and allowed to stand for 48 hours to carry out a low-temperature crosslinking reaction.

[0076] Step 4. Dialysis After a two-step crosslinking reaction at high and low temperatures, sodium hyaluronate gel was obtained. The gel was cut into 1cm×1cm×1cm cubes and dialyzed with 1L of water for injection. The dialysate was changed every 4 hours. Dialysis was stopped when the gel mass no longer increased, and the gel cubes were removed.

[0077] Step 5. Granulation The gel block obtained in step 4 is sequentially passed through 600μm, 300μm and 200μm sieves for sieve extrusion granulation to obtain gel particles.

[0078] Step 6. Dehydration treatment The gel particles obtained in step 5 were added to 300g of anhydrous ethanol for dehydration. After standing for 10 minutes, the supernatant was removed, and then another 300g of anhydrous ethanol was added. This process was repeated 3 times to obtain dehydrated gel particles.

[0079] Step 7. Drying treatment The dehydrated gel particles obtained in step 6 were dried in a vacuum drying oven at -0.09 MPa and 40°C for 24 hours.

[0080] Step 8. Gel Reconstitution The dried gel particles obtained in step 7 are added to 50 times their mass of phosphate buffer solution at pH 7.1 and mixed thoroughly to dissolve, thus obtaining amino acid crosslinked sodium hyaluronate gel.

[0081] The results of the extrusion force test and macroscopic state test of amino acid crosslinked sodium hyaluronate gel are shown in Table 1, and the results of the dynamic viscosity and modulus test are shown in Table 2.

[0082] Compare with Example 1 Step 1. Mix and activate Weigh 2g of NaOH and add it to water for injection to prepare 100mL of a 20g / L NaOH solution. Add 0.08g of 1,4-butanediol diglycidyl ether (BDDE) to the NaOH solution and mix to activate.

[0083] Step 2. High-temperature crosslinking Weigh 3.0 g of high molecular weight sodium hyaluronate (1500 kDa), add NaOH solution containing BDDE until the mass fraction of sodium hyaluronate is 3%, and stir at 25 °C for 30 min. After 30 min, transfer to 50 °C and let stand for 3 h to carry out high-temperature cross-linking reaction.

[0084] Step 3. Low-temperature crosslinking After the high-temperature crosslinking reaction, 1g of low molecular weight sodium hyaluronate (500kDa) was added to the sodium hyaluronate system and stirred evenly at 25°C. Then, it was transferred to a 4°C environment and allowed to stand for 48 hours to carry out a low-temperature crosslinking reaction.

[0085] Step 4. Dialysis After a low-temperature cross-linking reaction, sodium hyaluronate gel was obtained. The gel was cut into 1cm×1cm×1cm cubes and dialyzed with 1L of water for injection. The dialysate was changed every 4 hours. Dialysis was stopped when the gel mass no longer increased, and the gel cubes were removed.

[0086] Step 5. Granulation The gel block obtained in step 3 was sequentially passed through 600μm, 300μm and 200μm sieves for sieve extrusion granulation to obtain gel particles.

[0087] Step 6. Dehydration treatment The gel particles obtained in step 4 were added to 300g of anhydrous ethanol for dehydration. After standing for 10 minutes, the supernatant was removed, and then another 300g of anhydrous ethanol was added. This process was repeated 3 times to obtain dehydrated gel particles.

[0088] Step 7. Drying treatment The dehydrated gel particles obtained in step 5 were dried in a vacuum drying oven at -0.09 MPa and 40°C for 24 hours.

[0089] Step 8. Gel Reconstitution The dried gel particles obtained in step 7 are added to 50 times their mass of phosphate buffer solution at pH 7.1 and mixed thoroughly to dissolve, thus obtaining BDDE crosslinked sodium hyaluronate gel.

[0090] The results of the extrusion force test and macroscopic state test of BDDE crosslinked sodium hyaluronate gel are shown in Table 1, and the results of the dynamic viscosity and modulus test are shown in Table 2.

[0091] Compare with Example 2 Step 1. Mix and activate Weigh 2g of NaOH and add it to water for injection to prepare 100mL of a 20g / L NaOH solution. Add 0.08g of 1,4-butanediol diglycidyl ether (BDDE) to the NaOH solution and mix to activate.

[0092] Step 2. High-temperature crosslinking Weigh 4.0 g of high molecular weight sodium hyaluronate (1500 kDa), add NaOH solution containing BDDE until the mass fraction of sodium hyaluronate is 3%, and stir at 25 °C for 30 min. After 30 min, transfer to 35 °C and let stand for 3 h to carry out high-temperature cross-linking reaction.

[0093] Step 3. Dialysis After undergoing a high-temperature cross-linking reaction, sodium hyaluronate gel was obtained. The gel was cut into 1cm×1cm×1cm cubes and dialyzed with 1L of water for injection. The dialysate was changed every 4 hours. Dialysis was stopped when the gel mass no longer increased, and the gel cubes were removed.

[0094] Step 4. Granulation The gel block obtained in step 3 was sequentially passed through 600μm, 300μm and 200μm sieves for sieve extrusion granulation to obtain gel particles.

[0095] Step 5. Dehydration treatment The gel particles obtained in step 4 were added to 300g of anhydrous ethanol for dehydration. After standing for 10 minutes, the supernatant was removed, and then another 300g of anhydrous ethanol was added. This process was repeated 3 times to obtain dehydrated gel particles.

[0096] Step 6. Drying treatment The dehydrated gel particles obtained in step 5 were dried in a vacuum drying oven at -0.09 MPa and 40°C for 24 hours.

[0097] Step 7. Gel Reconstitution The dried gel particles obtained in step 6 are added to 50 times their mass of phosphate buffer solution at pH 7.1 and mixed thoroughly to dissolve, thus obtaining BDDE crosslinked sodium hyaluronate gel.

[0098] The results of the extrusion force test and macroscopic state test of BDDE crosslinked sodium hyaluronate gel are shown in Table 1, and the results of the dynamic viscosity and modulus test are shown in Table 2.

[0099] Compare with Example 3 Step 1. Mix and activate Weigh 4.0g of low molecular weight sodium hyaluronate (500kDa) and add water for injection until the mass fraction of sodium hyaluronate is 3%. Stir at 25°C. After stirring until homogeneous, a sodium hyaluronate system is obtained. Add 2.0g of DMTMM and stir to activate for 15min.

[0100] Step 2. High-temperature crosslinking After activation, 0.4g of lysine was added and stirred until homogeneous. After stirring for 30 minutes, the mixture was allowed to stand at 4°C for 48 hours to carry out a low-temperature cross-linking reaction.

[0101] Step 3. Dialysis After a low-temperature cross-linking reaction, sodium hyaluronate gel was obtained. The gel was cut into 1cm×1cm×1cm cubes and dialyzed with 1L of water for injection. The dialysate was changed every 4 hours. Dialysis was stopped when the gel mass no longer increased, and the gel cubes were removed.

[0102] Step 4. Granulation The gel block obtained in step 3 was sequentially passed through 600μm, 300μm and 200μm sieves for sieve extrusion granulation to obtain gel particles.

[0103] Step 5. Dehydration treatment The gel particles obtained in step 4 were added to 300g of anhydrous ethanol for dehydration. After standing for 10 minutes, the supernatant was removed, and then another 300g of anhydrous ethanol was added. This process was repeated 3 times to obtain dehydrated gel particles.

[0104] Step 6. Drying treatment The dehydrated gel particles obtained in step 5 were dried in a vacuum drying oven at -0.09 MPa and 40°C for 24 hours.

[0105] Step 7. Reconstitute the gel.

[0106] The dried gel particles obtained in step 6 are added to 50 times their mass of phosphate buffer solution at pH 7.1 and mixed thoroughly to dissolve, thus obtaining amino acid crosslinked sodium hyaluronate gel.

[0107] The results of the extrusion force test and macroscopic state test of amino acid crosslinked sodium hyaluronate gel are shown in Table 1, and the results of the dynamic viscosity and modulus test are shown in Table 2.

[0108] Experimental data: The gel was placed in a 1mL glass syringe, and a 27G needle was attached to perform a pushing force test. Table 1. Results of extrusion force and macroscopic state testing of cross-linked sodium hyaluronate gel.

[0109] Table 2. Results of dynamic viscosity, elastic modulus, and viscous modulus tests on cross-linked sodium hyaluronate gel.

[0110] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing an injectable amino acid-crosslinked sodium hyaluronate gel, characterized in that, Includes the following steps: Step 1: Mix high molecular weight sodium hyaluronate in water for injection to obtain a sodium hyaluronate system, and then add a coupling agent to activate it; Step 2: After adding the cross-linking agent amino acid and mixing well, carry out a high-temperature cross-linking reaction; Step 3: Add low molecular weight sodium hyaluronate and carry out a low-temperature cross-linking reaction; Step 4: The cross-linked sodium hyaluronate after low-temperature cross-linking undergoes dialysis treatment; Step 5: After dialysis until the mass of cross-linked sodium hyaluronate no longer increases, granulation is performed; Step Six: The granulated cross-linked sodium hyaluronate is dehydrated using a dehydrating agent; Step 7: The dehydrated cross-linked sodium hyaluronate is then dried. Step 8: The dried cross-linked sodium hyaluronate is reconstituted in phosphate buffer to obtain amino acid cross-linked sodium hyaluronate gel.

2. The method for preparing an injectable amino acid crosslinked sodium hyaluronate gel according to claim 1, characterized in that, The high molecular weight sodium hyaluronate mentioned in step one has a weight-average molecular weight of 1000 kDa to 3000 kDa, and the low molecular weight sodium hyaluronate mentioned in step three has a weight-average molecular weight of 100 kDa to 500 kDa.

3. The method for preparing an injectable amino acid crosslinked sodium hyaluronate gel according to claim 1, characterized in that, The mass fraction of high molecular weight sodium hyaluronate in the sodium hyaluronate system in step one is 3%-5%, and the mass fraction of low molecular weight sodium hyaluronate in the sodium hyaluronate system in step three is 1%-3%.

4. The method for preparing an injectable amino acid crosslinked sodium hyaluronate gel according to claim 1, characterized in that, The coupling agent mentioned in step one is one or more of succinimide glutarate, N-hydroxysuccinimide esters (NSH esters), carbodiimides (such as EDC), and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM); the molar amount of the coupling agent is 50%-100% of the molar amount of the repeating unit of sodium hyaluronate.

5. The method for preparing an injectable amino acid crosslinked sodium hyaluronate gel according to claim 1, characterized in that, The crosslinking agent amino acid mentioned in step two is a basic amino acid and its derivatives: one or more of lysine, arginine, histidine, L-lysine hydrochloride, N'-tert-butoxycarbonyl-D-lysine tert-butyl ester hydrochloride, L-lysine diisocyanate, phosphate-L-arginine, and L-arginine hydrochloride.

6. The method for preparing an injectable amino acid crosslinked sodium hyaluronate gel according to claim 5, characterized in that, The molar amount of the cross-linking agent amino acid in step two is 5%-30% of the molar amount of the repeating unit of hyaluronic acid.

7. The method for preparing an injectable amino acid crosslinked sodium hyaluronate gel according to claim 1, characterized in that, The high-temperature crosslinking reaction in step two has a temperature range of 40℃-60℃ and a reaction time range of 5-24 hours. The low-temperature crosslinking reaction in step three has a temperature range of 0℃-10℃ and a reaction time range of 12-48 hours.

8. The method for preparing an injectable amino acid crosslinked sodium hyaluronate gel according to claim 1, characterized in that, The granulation process described in step five is one of screen extrusion granulation and high-pressure homogenization granulation.

9. The method for preparing an injectable amino acid crosslinked sodium hyaluronate gel according to claim 1, characterized in that, The dehydrating agent mentioned in step six is ​​one or more of ethanol, acetone, and isopropanol, and the mass of the dehydrating agent is 3-5 times the mass of the cross-linked sodium hyaluronate after granulation.

10. The method for preparing an injectable amino acid crosslinked sodium hyaluronate gel according to claim 1, characterized in that, The drying method described in step seven is either vacuum drying or freeze drying; the pH range of the phosphate buffer solution described in step eight is 6.5-7.5.

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