Sodium hyaluronate gel and its preparation method and application

Abstract

The present invention discloses a sodium hyaluronate gel, its preparation method, and its application, belonging to the field of medical materials technology. The process includes sodium hyaluronate purification, crosslinker pre-dispersion, room-temperature pre-activation, intermediate-temperature directional crosslinking, gradient temperature aging, neutralization and preliminary impurity removal, gradient dialysis purification, and molding and sterilization. By stably controlling the pH at a weakly alkaline level, the present invention suppresses excessive deacetylation of HA molecules under strong alkaline conditions. This balances the ring-opening reaction of BDDE with the degradation of HA, avoiding the imbalance between crosslinking efficiency and HA degradation in traditional strong alkaline processes.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and in particular to a sodium hyaluronate gel and a preparation method and application thereof. Background Art

[0002] Sodium hyaluronate gel is a polymer network structure material formed by cross-linking reaction of sodium hyaluronate. Due to its excellent biocompatibility, water retention and viscoelasticity, it is widely used in medicine, cosmetics and other fields.

[0003] Currently, some preparation processes use strong alkaline solutions to adjust the reaction system to an alkaline environment. For example, Chinese patent CN118126357B uses a 1-2% sodium hydroxide aqueous solution to adjust the alkalinity. While this can promote the ring-opening reaction of BDDE (1,4-butanediol diglycidyl ether), this strong alkaline condition (pH > 10) can severely damage the molecular structure of sodium hyaluronate (HA). The N-acetylglucosamine units in the HA chain are easily deacetylated under alkaline conditions, forming unsaturated double bonds. This leads to main chain breakage and a sharp decrease in molecular weight, which ultimately significantly affects the viscoelasticity of the sodium hyaluronate gel and reduces product quality.

[0004] Furthermore, commonly used crosslinkers, such as BDDE, contain potentially toxic groups, such as epoxy groups. However, existing technologies have significant shortcomings in product quality testing, with most methods only testing for free HA content without effectively monitoring crosslinker residues. Excessive crosslinker residues could trigger inflammatory reactions or cytotoxicity during long-term implantation of sodium hyaluronate gel in the human body, posing a threat to human health.

[0005] In view of the above-mentioned shortcomings of the prior art, the present invention provides a sodium hyaluronate gel and a preparation method and application thereof. Summary of the Invention

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions: A method for preparing sodium hyaluronate gel, comprising the following steps:

[0007] S1. Purification of sodium hyaluronate;

[0008] ① Dissolve the crude HA in phosphate buffer and stir until it is completely dissolved into a gel;

[0009] ② Pass through Protein A affinity chromatography column and collect the flow-through;

[0010] ③Ultrafilter the permeate to remove small molecule impurities and circulate ultrafiltration and concentration;

[0011] ④ Freeze-dry the concentrated solution to obtain purified HA powder;

[0012] S2. Crosslinker pre-dispersion;

[0013] ① Mix 1,4-butanediol diglycidyl ether and propylene glycol to form a uniform pre-dispersion liquid;

[0014] ② Add NaOH solution dropwise to the uniform pre-dispersion liquid. When the pH stabilizes at a weak alkaline state, add mercaptoethanol and heat to form a BDDE microemulsion.

[0015] S3. Pre-activation at room temperature;

[0016] ① Dissolve the purified HA powder in deionized water to form a HA solution;

[0017] ②Add pre-dispersed BDDE microemulsion and vitamin C at the same time;

[0018] ③Stir at low speed at room temperature;

[0019] S4. Medium temperature directional crosslinking;

[0020] ① Add NaOH solution dropwise to adjust the pH to weak alkaline;

[0021] ② Slowly increase the temperature;

[0022] S5. Gradient temperature rise and aging;

[0023] Slowly increase the temperature and then accelerate the temperature;

[0024] S6. Neutralization and preliminary impurity removal;

[0025] ①Add hydrochloric acid to adjust the pH to neutral;

[0026] ②Add pre-treated activated carbon and stir for adsorption;

[0027] ③ Centrifuge to remove activated carbon particles and collect the supernatant;

[0028] S7. Gradient dialysis purification;

[0029] ① Primary dialysis: the supernatant was placed in a dialysis bag and dialyzed in deionized water containing β-cyclodextrin;

[0030] ② Secondary dialysis: dialyze in PBS buffer containing bovine serum albumin;

[0031] ③Third dialysis: dialysis with deionized water;

[0032] S8. Molding and sterilization to obtain sodium hyaluronate gel.

[0033] Furthermore, S1 specifically comprises: ① dissolving crude HA with a molecular weight of 1800-2200 kDa in a phosphate buffer solution at pH 7.4-8.5 at a concentration of 20-30 mg / mL, and stirring until completely dissolved into a gel;

[0034] ② Pass the protein A affinity chromatography column at a flow rate of 4-6 mL / min and collect the flowthrough;

[0035] ③Ultrafiltrate the permeate to remove small molecule impurities, and repeat ultrafiltration 2-3 times until the volume is concentrated to 1 / 5-1 / 4 of the original volume;

[0036] ④ Freeze-dry the concentrated solution to obtain purified HA powder.

[0037] Furthermore, S2 is specifically as follows: ① mixing 1,4-butanediol diglycidyl ether and propylene glycol in a volume ratio of 1:5-8, and ultrasonically dispersing for 10-15 minutes to form a uniform pre-dispersion liquid;

[0038] ② Slowly add pre-cooled 1-1.5% NaOH aqueous solution to the uniform pre-dispersion liquid at a drop rate of 1-3 drops / second while stirring at a speed of 200-280 rpm. When the pH stabilizes at 8.2-9.6, add 0.05-0.1wt% of mercaptoethanol, stir for 5-8 minutes, and then heat to 45-50°C to form a BDDE microemulsion.

[0039] Furthermore, S3 is specifically as follows: ① dissolving the purified HA powder in deionized water and stirring until a 1-3% HA solution is formed;

[0040] ② Add pre-dispersed BDDE microemulsion, crosslinker and HA mass ratio of 1:150-200, and add 0.08-0.12% vitamin C;

[0041] ③ Stir at a low speed of 120-150 rpm for 30-40 minutes at room temperature to allow the HA molecular chains to fully absorb the crosslinker droplets.

[0042] Furthermore, S4 is specifically as follows: ① adding 1-3 M NaOH solution dropwise to adjust the pH to 9.0 ± 0.5;

[0043] ② Slowly increase the temperature to 42-45°C at 3-5°C / min, maintain the stirring rate at 260-300 rpm, and keep warm for 10-15 minutes.

[0044] Furthermore, S5 is specifically as follows: ① heating to 50-52°C at a rate of 5-8°C / h;

[0045] ②Raise the temperature to 55-57℃ at a rate of 1-2℃ / min.

[0046] Furthermore, S6 is specifically as follows: ① adding hydrochloric acid to adjust the pH to 6.8-7.2;

[0047] ② Add 0.5-1% pre-treated activated carbon and stir for 30-45 minutes to remove residual cross-linking agent and degradation products;

[0048] ③ Centrifuge to remove activated carbon particles and collect the supernatant.

[0049] Furthermore, S7 is specifically as follows: ① one dialysis: the supernatant is placed in a dialysis bag and dialyzed in deionized water containing 0.01-0.03M β-cyclodextrin;

[0050] ② Secondary dialysis: dialyze in PBS buffer containing 1-3% bovine serum albumin;

[0051] ③Third dialysis: Dialysis with deionized water to remove buffer salt ions.

[0052] A sodium hyaluronate gel prepared by the method for preparing sodium hyaluronate gel.

[0053] A use of the sodium hyaluronate gel in the preparation of joint cavity injections, ophthalmic viscoelastics, medical aesthetic fillers and / or moisturizing gels for skin care products.

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] 1. During the preparation and directional cross-linking of the BDDE microemulsion, the present invention maintains a stable pH at a weakly alkaline level, achieving the following effects: The weakly alkaline environment maintains the ring-opening activity of the BDDE epoxy group through nucleophilic attack, while simultaneously suppressing excessive deacetylation of the HA molecule under strong alkaline conditions. Strong alkaline conditions significantly increase the deacetylation rate of the N-acetylglucosamine units on the HA chain, while weak alkaline conditions reduce the degree of deacetylation. This regulation balances the ring-opening reaction of BDDE with the degradation of HA, avoiding the imbalance between cross-linking efficiency and HA degradation in traditional strong alkaline processes. Under weak alkaline conditions, the degradation of the HA molecular chain is suppressed, significantly increasing the number-average molecular weight while maintaining a stable weight-average molecular weight. This phenomenon is attributed to the reduction of short-chain fragments. The optimized molecular weight distribution directly improves the viscoelasticity of the gel and reduces the slip effect of short-chain fragments during dynamic shear. The weakly alkaline environment promotes uniform cross-linking of BDDE and HA, forming a more regular three-dimensional network. Both the elastic modulus and the viscous modulus are increased, indicating a significant enhancement of the elastic properties of the gel.

[0056] 2. The present invention utilizes the porous structure and surface chemical properties of activated carbon to remove free BDDE in the reaction system through physical adsorption and chemical action. The microporous structure of activated carbon matches the molecular size of BDDE, achieving preliminary enrichment and separation of residual cross-linkers and reducing the load of subsequent purification steps. The hydrophobic inner cavity of β-cyclodextrin forms a host-guest inclusion complex with the epoxy group of BDDE, which enhances the migration ability of BDDE through intermolecular interactions, making it easier to detach from the gel network, creating favorable conditions for subsequent dialysis steps. A gradient dialysis strategy of buffer solution and deionized water is adopted, while controlling the gel particle size to reduce the specific surface area, shorten the mass transfer path, improve the dialysis efficiency, and achieve deep removal of residual cross-linkers. Reduce potential cytotoxicity and inflammatory risks in the gel. The efficient removal of residual cross-linkers avoids adverse tissue reactions caused by chemical stimulation during long-term implantation and improves the biocompatibility of the material.

[0057] 3. The present invention achieves multi-scenario adaptation by precisely controlling pH and optimizing the cross-linking process:

[0058] Osteoarthritis treatment: By controlling the degree of cross-linking and molecular weight distribution in a weakly alkaline environment, the mechanical properties of the gel are made close to those of synovial fluid, achieving effective buffering and lubrication of joint friction;

[0059] Facial filler materials: By increasing the cross-linking density and the proportion of long-chain molecules, the support and structural stability of the gel are enhanced to meet the morphological maintenance requirements of areas with high support needs;

[0060] Skin moisturizing and sustained drug release: By reducing the cross-linking density and molecular weight, the gel is given low modulus properties, making it easy to penetrate into the superficial layer of the skin, achieving long-lasting moisturizing and controlled drug release. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0062] Figure 1 This is a physical picture of a sodium hyaluronate (crude product) of the present invention;

[0063] Figure 2 This is a physical picture of the sodium hyaluronate gel prepared in Example 1 of the present invention;

[0064] Figure 3 This is a physical picture of the sodium hyaluronate gel prepared in Example 2 of the present invention;

[0065] Figure 4This is a physical picture of the sodium hyaluronate gel prepared in Example 3 of the present invention. DETAILED DESCRIPTION

[0066] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0067] Example 1: This example provides a method for preparing sodium hyaluronate gel, comprising the following steps:

[0068] S1. Purification of sodium hyaluronate (HA);

[0069] ① Dissolve sodium hyaluronate (crude) (molecular weight 1800-2200 kDa) in pH 8.5 phosphate buffer (PBS) at a concentration of 30 mg / mL and stir until completely dissolved into a gel;

[0070] ② Pass the protein A affinity chromatography column (to remove residual proteins) at a flow rate of 6 mL / min and collect the flowthrough;

[0071] ③Ultrate the permeate (molecular weight cut-off 300,000) to remove small molecule impurities, and repeat the ultrafiltration three times until the volume is concentrated to 1 / 5 of the original volume;

[0072] ④ Freeze-dry the concentrate to obtain purified HA powder (protein residue <0.01%);

[0073] S2. Crosslinker pre-dispersion;

[0074] ① Mix 1,4-butanediol diglycidyl ether (BDDE) and propylene glycol in a volume ratio of 1:8 (propylene glycol serves as an inert dispersion medium to reduce the viscosity of BDDE) and ultrasonically disperse for 15 minutes (frequency 40kHz, power 200W) to form a uniform pre-dispersion liquid;

[0075] ② Slowly add 1.5% NaOH aqueous solution pre-cooled to 4°C to the uniform pre-dispersion liquid at a drop rate of 3 drops / second while stirring at 280 rpm. When the pH stabilizes at 8.2, add 0.1wt% mercaptoethanol, stir for 8 minutes, and then heat to 50°C to form a BDDE microemulsion.

[0076] The thiol group (-SH) of mercaptoethanol can undergo a ring-opening addition reaction with the epoxy group of BDDE, preferentially capturing free BDDE that does not participate in cross-linking to form a non-toxic thioether compound;

[0077] S3. Pre-activation at room temperature (promoting HA chain stretching);

[0078] ① Dissolve purified HA powder (3 g) in 100 mL of deionized water (preheated to 25°C) and stir until a 3% HA solution is formed;

[0079] ② Add pre-dispersed BDDE microemulsion (cross-linker: HA mass ratio 1:200) and 0.12% vitamin C (antioxidant, inhibiting HA degradation);

[0080] ③ Stir at low speed (150 rpm) for 40 min at room temperature to allow the HA molecular chains to fully absorb the crosslinker droplets;

[0081] S4. Medium temperature directional crosslinking (to improve the effective crosslinking rate);

[0082] ① Add 3M NaOH solution dropwise to adjust the pH to 9.0±0.5;

[0083] ② Slowly increase the temperature to 45°C at 5°C / min, maintain the stirring rate at 300 rpm (to promote the rupture of the BDDE microemulsion and release BDDE to react with the HA hydroxyl group), and keep warm for 15 minutes;

[0084] S5. Gradient temperature aging (homogenization of cross-linked network);

[0085] ① Raise the temperature to 52℃ at a rate of 8℃ / h;

[0086] ② Raise the temperature to 57°C at a rate of 2°C / min;

[0087] By increasing the temperature gradually, the distal HA molecular chains are gradually cross-linked, eliminating local excessive cross-linking and forming a gel network with uniform pore size distribution.

[0088] S6. Neutralization and preliminary impurity removal;

[0089] ① Add 0.1M hydrochloric acid to adjust the pH to 6.8-7.2 (to neutralize excess alkali solution and terminate the reaction);

[0090] ② Add 1% pretreated activated carbon (soak the activated carbon in 5% nitric acid for 24 hours, wash it with water until it is neutral, and then dry it at 120°C to generate more carboxyl and hydroxyl groups on the surface of the activated carbon, thereby improving the adsorption capacity of polar molecules containing epoxy groups). Stir and adsorb for 45 minutes to remove residual crosslinkers and degradation products.

[0091] ③ Centrifuge (3000 rpm, 10 min) to remove activated carbon particles and collect the supernatant;

[0092] S7. Gradient dialysis purification (double carryover control);

[0093] ① Primary dialysis: The supernatant was placed in a dialysis bag with a molecular weight cut-off of 100,000 and dialyzed in deionized water containing 0.03 M β-cyclodextrin for 12 h (the water was changed every 4 h);

[0094] ② Secondary dialysis: Replace the dialysis bag with a molecular weight cutoff of 50,000 and dialyze in PBS buffer containing 3% bovine serum albumin for 24 hours (through the binding of protein-drug interaction sites, the covalently bonded residual BDDE is separated from the HA chain and enters the dialysate);

[0095] ③ Three dialysis: Dialyze with deionized water for 8 hours to remove buffer salt ions. The free HA in the gel is less than 0.05 mg / mL and the BDDE residue is less than 5 ppm.

[0096] S8. Molding and sterilization to obtain sodium hyaluronate gel (denoted as HA Gel①);

[0097] ① The purified supernatant gel was extruded through a 200-mesh sieve to remove large particles of impurities and form a uniform paste;

[0098] ② Use gamma ray irradiation sterilization (dose 25kGy) to avoid the damage of the cross-linked network caused by high temperature sterilization;

[0099] ③ Aseptically fill into medical syringes, seal with nitrogen, and store at 4°C.

[0100] Example 2: This example provides a method for preparing sodium hyaluronate gel, comprising the following steps:

[0101] S1. Purification of sodium hyaluronate (HA);

[0102] ① Dissolve sodium hyaluronate (crude) (molecular weight 1800-2200 kDa) in pH 7.4 phosphate buffer at a concentration of 20 mg / mL and stir until completely dissolved into a gel;

[0103] ② Pass the protein A affinity chromatography column at a flow rate of 4 mL / min and collect the flowthrough;

[0104] ③Ultrate the permeate (molecular weight cut-off 300,000) to remove small molecule impurities, and repeat the ultrafiltration twice until the volume is concentrated to 1 / 4 of the original volume;

[0105] ④ Freeze-dry the concentrated solution to obtain purified HA powder;

[0106] S2. Crosslinker pre-dispersion;

[0107] ① Mix BDDE and propylene glycol in a volume ratio of 1:5 and ultrasonically disperse for 10 minutes (frequency 40kHz, power 200W) to form a uniform pre-dispersion liquid;

[0108] ② Slowly add 1% NaOH aqueous solution pre-cooled to 4°C to the uniform pre-dispersion liquid at a drop rate of 1 drop / second while stirring at 200 rpm. When the pH stabilizes at 9.6, add 0.05wt% mercaptoethanol, stir for 5 minutes, and then heat to 45°C to form a BDDE microemulsion;

[0109] S3. Pre-activation at room temperature;

[0110] ① Dissolve purified HA powder (1 g) in 100 mL of deionized water (preheated to 25°C) and stir until a 1% HA solution is formed;

[0111] ② Add pre-dispersed BDDE microemulsion (cross-linker: HA mass ratio 1:150) and 0.08% vitamin C;

[0112] ③ Stir at low speed (120 rpm) for 30 min at room temperature;

[0113] S4. Medium temperature directional crosslinking;

[0114] ① Add 1M NaOH solution dropwise to adjust the pH to 9.0±0.5;

[0115] ② Slowly increase the temperature to 42°C at 3°C / min, maintain the stirring rate at 260 rpm, and keep warm for 10 minutes;

[0116] S5. Gradient temperature rise and aging;

[0117] ①Heat to 50℃ at a rate of 5℃ / h;

[0118] ②Heat to 55℃ at a rate of 1℃ / min;

[0119] S6. Neutralization and preliminary impurity removal;

[0120] ①Add 0.1M hydrochloric acid to adjust the pH to 6.8-7.2;

[0121] ② Add 0.5% pretreated activated carbon (soak the activated carbon in 5% nitric acid for 24 hours, wash it with water until it is neutral, and then dry it at 120°C to generate more carboxyl and hydroxyl groups on the surface of the activated carbon, thereby improving the adsorption capacity of polar molecules containing epoxy groups). Stir and adsorb for 30 minutes to remove residual crosslinkers and degradation products.

[0122] ③ Centrifuge (3000 rpm, 10 min) to remove activated carbon particles and collect the supernatant;

[0123] S7. Gradient dialysis purification;

[0124] ① Primary dialysis: The supernatant was placed in a dialysis bag with a molecular weight cut-off of 100,000 and dialyzed in deionized water containing 0.01 M β-cyclodextrin for 12 h (the water was changed every 4 h);

[0125] ② Secondary dialysis: Replace the dialysis bag with a molecular weight cutoff of 50,000 and dialyze in PBS buffer containing 1% bovine serum albumin for 24 hours;

[0126] ③Three dialysis: dialysis with deionized water for 8 hours to remove buffer salt ions;

[0127] S8. Molding and sterilization to obtain sodium hyaluronate gel (denoted as HA Gel②);

[0128] ① The purified supernatant gel was extruded through a 200-mesh sieve to remove large particles of impurities and form a uniform paste;

[0129] ② Use gamma ray irradiation sterilization (dose 25kGy) to avoid the damage of the cross-linked network caused by high temperature sterilization;

[0130] ③ Aseptically fill into medical syringes, seal with nitrogen, and store at 4°C.

[0131] Example 3: This example provides a method for preparing sodium hyaluronate gel, comprising the following steps:

[0132] S1. Purification of sodium hyaluronate (HA);

[0133] ① Dissolve sodium hyaluronate (crude) (molecular weight 1800-2200 kDa) in pH 8.2 phosphate buffer at a concentration of 28 mg / mL and stir until completely dissolved into a gel;

[0134] ② Pass the protein A affinity chromatography column at a flow rate of 5 mL / min and collect the flow-through;

[0135] ③Ultrate the permeate (molecular weight cut-off 300,000) to remove small molecule impurities, and repeat the ultrafiltration three times until the volume is concentrated to 1 / 5 of the original volume;

[0136] ④ Freeze-dry the concentrated solution to obtain purified HA powder;

[0137] S2. Crosslinker pre-dispersion;

[0138] ① Mix BDDE and propylene glycol in a volume ratio of 1:6 and ultrasonically disperse for 12 minutes (frequency 40kHz, power 200W) to form a uniform pre-dispersion liquid;

[0139] ② Slowly add 1.3% NaOH aqueous solution pre-cooled to 4°C to the uniform pre-dispersion liquid at a drop rate of 2 drops / second while stirring at 220 rpm. When the pH stabilizes at 8.8, add 0.08 wt% mercaptoethanol, stir for 6 minutes, and then heat to 48°C to form a BDDE microemulsion.

[0140] S3. Pre-activation at room temperature;

[0141] ① Dissolve purified HA powder (1.8 g) in 100 mL of deionized water (preheated to 25°C) and stir until a 1.8% HA solution is formed;

[0142] ② Add pre-dispersed BDDE microemulsion (cross-linker: HA mass ratio 1:170) and 0.1% vitamin C;

[0143] ③ Stir at low speed (140 rpm) for 38 min at room temperature;

[0144] S4. Medium temperature directional crosslinking;

[0145] ① Add 2M NaOH solution dropwise to adjust the pH to 9.0±0.5;

[0146] ② Slowly increase the temperature to 43°C at 4°C / min, maintain the stirring rate at 280 rpm, and keep warm for 12 minutes;

[0147] S5. Gradient temperature rise and aging;

[0148] ① Raise the temperature to 51℃ at a rate of 6℃ / h;

[0149] ② Raise the temperature to 56°C at a rate of 1°C / min;

[0150] S6. Neutralization and preliminary impurity removal;

[0151] ①Add 0.1M hydrochloric acid to adjust the pH to 6.8-7.2;

[0152] ② Add 0.6% pretreated activated carbon (soak the activated carbon in 5% nitric acid for 24 hours, wash it with water until it is neutral, and then dry it at 120°C to generate more carboxyl and hydroxyl groups on the surface of the activated carbon, thereby improving the adsorption capacity of polar molecules containing epoxy groups). Stir and adsorb for 42 minutes to remove residual crosslinkers and degradation products.

[0153] ③ Centrifuge (3000 rpm, 10 min) to remove activated carbon particles and collect the supernatant;

[0154] S7. Gradient dialysis purification;

[0155] ① Primary dialysis: The supernatant was placed in a dialysis bag with a molecular weight cut-off of 100,000 and dialyzed in deionized water containing 0.02M β-cyclodextrin for 12 hours (the water was changed every 4 hours);

[0156] ② Secondary dialysis: Replace the dialysis bag with a molecular weight cutoff of 50,000 and dialyze in PBS buffer containing 2% bovine serum albumin for 24 hours;

[0157] ③Three dialysis: dialysis with deionized water for 8 hours to remove buffer salt ions;

[0158] S8. Molding and sterilization to obtain sodium hyaluronate gel (denoted as HA Gel③);

[0159] ① The purified supernatant gel was extruded through a 200-mesh sieve to remove large particles of impurities and form a uniform paste;

[0160] ② Use gamma ray irradiation sterilization (dose 25kGy) to avoid the damage of the cross-linked network caused by high temperature sterilization;

[0161] ③ Aseptically fill into medical syringes, seal with nitrogen, and store at 4°C.

[0162] Comparative Example 1: This comparative example differs from Example 3 in that S6 and S7 are not performed.

[0163] Comparative Example 2: This comparative example differs from Example 3 in that mercaptoethanol is added only when the pH in S2 is stabilized at 11; and 2M NaOH solution is added dropwise in S4 to adjust the pH to 11.

[0164] Comparative Example 3: This comparative example differs from Example 3 in that S6 and S7 are not performed, mercaptoethanol is added to S2 only when the pH is stabilized at 11; and 2M NaOH solution is added dropwise to S4 to adjust the pH to 11.

[0165] Experimental example: 1. Detection of molecular weight changes;

[0166] Detection method: gel permeation chromatography (GPC).

[0167] Principle: GPC is used to separate HA fragments of different molecular weights, and the molecular weight distribution is quantitatively analyzed using a refractive index detector (RI) or a multi-angle laser light scattering detector (MALLS).

[0168] Sample preparation: Sodium hyaluronate gel was dissolved in 0.1 M NaCl solution at a concentration of approximately 1-2 mg / mL and sonicated until completely dissolved.

[0169] Chromatographic column: TSKgelG6000PWXL (300 mm × 7.8 mm).

[0170] Mobile phase: 0.1 M NaCl solution, flow rate 0.5 mL / min.

[0171] Column temperature: 30℃.

[0172] Standard curve: A standard curve was established using HA standards of known molecular weight.

[0173] Detection: Inject sample, record chromatogram, calculate number average molecular weight (Mn) and weight average molecular weight (Mw).

[0174] Among them, the number average molecular weight (Mn) reflects the lower limit of the average chain length of the HA molecular chain and is more sensitive to low molecular weight components (a small amount of short chain molecules can lower Mn). If HA undergoes β-elimination degradation (such as main chain breakage under alkaline conditions), the number of short chain fragments increases and Mn will decrease significantly.

[0175] The weight-average molecular weight (Mw) reflects the upper limit of the average chain length of the HA molecular chain and is more sensitive to high molecular weight components (a small amount of long-chain molecules can increase Mw). In cross-linked gels, Mw is related to the cross-linking density and network structure of the molecular chain. High Mw usually corresponds to more complex entanglement or cross-linking.

[0176] Therefore, molecular weight often refers to the weight average molecular weight (Mw).

[0177] 2. Viscoelasticity test;

[0178] Detection method: rotational rheometer.

[0179] Principle: The viscoelasticity of gels is evaluated by measuring the storage modulus (G') and loss modulus (G").

[0180] Sample preparation: Spread the gel evenly on parallel plates (25 mm diameter, 1 mm gap).

[0181] Frequency sweep: 0.1-10 Hz, strain 1%.

[0182] Temperature: 25℃.

[0183] Data analysis: Start the frequency sweep program and record the calculated storage modulus (G', elastic modulus, unit: Pa) and loss modulus (G", viscous modulus, unit: Pa) at each frequency point. Test each sample at least three times in parallel and take the average value of G' and G".

[0184] 3. DDE residue detection;

[0185] Detection method: High performance liquid chromatography-mass spectrometry (HPLC-MS / MS).

[0186] Principle: BDDE is separated by HPLC and detected with high sensitivity using the multiple reaction monitoring (MRM) mode of mass spectrometry.

[0187] Sample preparation:

[0188] Weigh 0.1 g of gel sample, add 1 mL of acetonitrile, vortex for 10 minutes, centrifuge (10,000 rpm, 10 minutes), and collect the supernatant.

[0189] The extraction was repeated twice, and the supernatants were combined, dried under nitrogen, and reconstituted to 1 mL with 0.1% formic acid aqueous solution.

[0190] Chromatographic column: C18 column (150 mm × 2.1 mm, 3.5 μm).

[0191] Mobile phase: Phase A is 0.1% formic acid aqueous solution, phase B is acetonitrile, gradient elution (0-5 min, 5%-95% B; 5-7 min, 95% B; 7-7.1 min, 5% B; 7.1-10 min, 5% B).

[0192] Flow rate: 0.3 mL / min.

[0193] Column temperature: 30℃.

[0194] Ion source: electrospray ionization (ESI+).

[0195] Monitored ion pairs: m / z 165.1→123.1 (quantification ion), m / z 165.1→87.1 (qualification ion).

[0196] Fragmentation voltage: 35 V, collision energy: 15 eV.

[0197] Standard curve: A standard curve was established using BDDE standards.

[0198] Calculation: Calculate the BDDE content (ppm) in the sample based on the peak area.

[0199] The results are shown in the following table:

[0200] project Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Weight average molecular weight Mw (kDa) 2108 1813 2005 2094 1617 1578 Number average molecular weight Mn (kDa) 2014 416 821 2003 697 347 Elastic modulus G' (Pa) 360 87 120 117 45 37 Viscous modulus G" (Pa) 152 74 89 85 21 18 BDDE content (ppm) 0.23 0.25 0.24 1.05 0.38 1.34

[0201] As can be seen from the above table, the present invention achieves the removal of residual cross-linking agent BDDE through pre-treatment with activated carbon for impurity removal and gradient dialysis purification using β-cyclodextrin + PBS buffer + deionized water; the above impurity removal step is combined with the stable control of pH at a weak alkaline level during the preparation of the BDDE microemulsion and the directional cross-linking process, which can achieve a synergistic effect in removing the residual cross-linking agent BDDE.

[0202] By stably controlling the pH at a weak alkaline level during the preparation of the BDDE microemulsion and the directional crosslinking process, although there is no obvious effect on the commonly used molecular weight indicator, the weight-average molecular weight, it can significantly increase the number-average molecular weight, that is, significantly reduce the number of short-chain fragments; thereby increasing the elastic modulus and viscous modulus.

[0203] In addition, the present invention can also control the pH and the number of short-chain fragments, thereby adjusting the elastic modulus and viscous modulus of the sodium hyaluronate gel to adapt to different application scenarios;

[0204] For example, hyaluronic acid injections used to treat osteoarthritis usually have an elastic modulus of 50-150 Pa and a viscous modulus of 30-100 Pa;

[0205] Cross-linked hyaluronic acid products used for areas with high support requirements, such as the nasal dorsum and brow arch, typically have an elastic modulus of 350-400 Pa and a viscous modulus of 50-150 Pa.

[0206] Products used for superficial skin moisturizing or drug sustained release, with elastic modulus and viscosity modulus as low as 1-10Pa.

[0207] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements will not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for preparing sodium hyaluronate gel, characterized in that: The following steps are involved: S1. Purification of sodium hyaluronate; Dissolve the crude HA in phosphate buffer and stir until it is completely dissolved into a gel; Pass through Protein A affinity chromatography column and collect the flow-through; Ultrafiltration is performed on the permeate to remove small molecule impurities, and the ultrafiltration is circulated and concentrated; The concentrated solution was freeze-dried to obtain purified HA powder; S2. Crosslinker pre-dispersion; Mixing 1,4-butanediol diglycidyl ether and propylene glycol to form a uniform pre-dispersion liquid; NaOH solution was added dropwise to the uniform pre-dispersion solution. When the pH was stable at 8.2-9.6, mercaptoethanol was added and the temperature was raised to form a BDDE microemulsion. S3. Pre-activation at room temperature; S3: dissolving the purified HA powder in deionized water and stirring until a 1-3% HA solution is formed; Add pre-dispersed BDDE microemulsion, crosslinker and HA mass ratio of 1:150-200, and add 0.08-0.12% vitamin C; Stir at a low speed of 120-150 rpm for 30-40 minutes at room temperature to allow the HA molecular chains to fully absorb the crosslinker droplets; S4. Medium temperature directional crosslinking; Add NaOH solution dropwise to adjust the pH to 9.0 ± 0.5; Slowly increase the temperature; S5. Gradient temperature rise and aging; Slowly increase the temperature and then accelerate the temperature; S6. Neutralization and preliminary impurity removal; Add hydrochloric acid to adjust the pH to neutral; Add pretreated activated carbon and stir for adsorption; The activated carbon particles were removed by centrifugation and the supernatant was collected; S7. Gradient dialysis purification; Primary dialysis: the supernatant was placed in a dialysis bag and dialyzed in deionized water containing β-cyclodextrin; Secondary dialysis: dialyzed in PBS buffer containing bovine serum albumin; Three dialysis: dialysis with deionized water; S8. Molding and sterilization to obtain sodium hyaluronate gel.

2. The method for preparing sodium hyaluronate gel according to claim 1, wherein S1 specifically comprises: dissolving crude HA with a molecular weight of 1800-2200 kDa in a phosphate buffer solution at pH 7.4-8.5 at a concentration of 20-30 mg / mL, and stirring until completely dissolved into a gel; Pass the protein A affinity chromatography column at a flow rate of 4-6 mL / min and collect the flowthrough; Ultrafiltration is performed on the permeate to remove small molecule impurities, and the ultrafiltration cycle is repeated 2-3 times until the volume is concentrated to 1 / 5-1 / 4 of the original volume; The concentrated solution was freeze-dried to obtain purified HA powder.

3. The method for preparing sodium hyaluronate gel according to claim 1, wherein S2 is specifically as follows: 1,4-butanediol diglycidyl ether and propylene glycol are mixed in a volume ratio of 1:5-8, and ultrasonically dispersed for 10-15 minutes to form a uniform pre-dispersion liquid; Slowly add pre-cooled 1-1.5% NaOH aqueous solution to the uniform pre-dispersion liquid at a dropping rate of 1-3 drops / second while stirring at a speed of 200-280 rpm. When the pH stabilizes at 8.2-9.6, add 0.05-0.1wt% of mercaptoethanol, stir for 5-8 minutes, and then heat to 45-50°C to form a BDDE microemulsion.

4. The method for preparing sodium hyaluronate gel according to claim 1, wherein S4 specifically comprises: adding 1-3 M NaOH solution dropwise to adjust the pH to 9.0 ± 0.5; Slowly increase the temperature to 42-45°C at 3-5°C / min, maintain the stirring rate at 260-300 rpm, and keep warm for 10-15 minutes.

5. The method for preparing sodium hyaluronate gel according to claim 1, wherein S5 is specifically as follows: heating to 50-52°C at a rate of 5-8°C / h; The temperature was raised to 55-57°C at a rate of 1-2°C / min.

6. The method for preparing sodium hyaluronate gel according to claim 1, wherein S6 specifically includes: adding hydrochloric acid to adjust the pH to 6.8-7.2; Add 0.5-1% pre-treated activated carbon and stir for 30-45 minutes to remove residual cross-linking agent and degradation products; The activated carbon particles were removed by centrifugation and the supernatant was collected.

7. The method for preparing sodium hyaluronate gel according to claim 2, wherein: S7 specifically includes: primary dialysis: placing the supernatant into a dialysis bag and dialyzing it in deionized water containing 0.01-0.03 M β-cyclodextrin; Secondary dialysis: dialyzed in PBS buffer containing 1-3% bovine serum albumin; Three dialysis: Dialysis with deionized water to remove buffer salt ions.

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