Sodium hyaluronate gel as well as preparation method and application thereof

Abstract

The invention discloses sodium hyaluronate gel as well as a preparation method and application thereof, and belongs to the technical field of medical materials, and the preparation method comprises the steps of sodium hyaluronate purification, cross-linking agent pre-dispersion, normal-temperature pre-activation, medium-temperature directional cross-linking, gradient heating curing, neutralization and preliminary impurity removal, gradient dialysis purification and forming and sterilization. According to the method, the pH is stably controlled to be alkalescent, so that excessive deacetylation of HA molecules under a strong alkali condition is inhibited. The ring-opening reaction of the BDDE and the degradation of the HA are balanced, and the unbalance between the crosslinking efficiency and the degradation of the HA in the traditional strong alkali process is avoided. The microporous structure of the activated carbon is matched with the molecular size of the BDDE, preliminary enrichment and separation of the residual cross-linking agent are realized, a hydrophobic inner cavity of the beta-cyclodextrin and an epoxy group of the BDDE form a host-guest inclusion compound, and the migration ability of the BDDE is improved through intermolecular interaction, so that the BDDE can be separated from a gel network more easily. The deep removal of the residual cross-linking agent is realized. And potential cytotoxicity and inflammation risks in the gel are reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and specifically relates to a sodium hyaluronate gel, a preparation method thereof, and an 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 the fields of medicine, cosmetics and the like.

[0003] At present, some preparation processes use strong alkali solutions to adjust the alkaline environment of the reaction system. For example, in Chinese Patent CN118126357B, 1-2% sodium hydroxide aqueous solution is used to adjust the alkalinity. Although it can promote the ring-opening reaction of BDDE (1,4-butanediol diglycidyl ether), this strong alkali condition (pH>10) will cause serious damage to the molecular structure of sodium hyaluronate (HA). The N-acetylglucosamine unit on the HA chain is extremely prone to deacetylation under alkaline conditions, generating unsaturated double bonds, resulting in the breakage of the main chain, a sharp drop in molecular weight, and ultimately significantly affecting the viscoelasticity of sodium hyaluronate gel and reducing the product quality.

[0004] In addition, common cross-linking agents such as BDDE contain potential toxic groups such as epoxy groups. However, there are obvious deficiencies in the product quality detection link of the existing technology. Most only detect the content of free HA, but do not effectively monitor the residual amount of the cross-linking agent. If the residual amount of the cross-linking agent exceeds the standard, during the long-term implantation of sodium hyaluronate gel into the human body, it may cause inflammatory reactions or cytotoxicity, threatening human health.

[0005] In view of the above-mentioned disadvantages of the existing technology, the present invention provides a sodium hyaluronate gel, a preparation method thereof, and an application thereof. Summary of the Invention

[0006] To achieve the above object, the present invention is realized through the following technical solutions: A preparation method of a sodium hyaluronate gel, comprising the following steps: S1. Purification of sodium hyaluronate; ① Dissolve the crude HA in phosphate buffer solution and stir until it is completely dissolved into a gel; ② Pass through a ProteinA affinity chromatography column and collect the breakthrough solution; ③ Ultrafilter the breakthrough solution to remove small molecule impurities, and cycle ultrafiltration for concentration; ④ Lyophilize the concentrated solution to obtain purified HA powder; S2. Predispersion of the cross-linking agent; ① Mix 1,4-butanediol diglycidyl ether with propylene glycol to form a uniform predispersion solution; ② Add NaOH solution dropwise to the uniformly pre-dispersed solution. When the pH is stabilized at weakly alkaline, add mercaptoethanol and raise the temperature to form a BDDE microemulsion; S3. Room temperature pre-activation; ① Dissolve the purified HA powder in deionized water to form an HA solution; ② Add the pre-dispersed BDDE microemulsion and simultaneously add vitamin C; ③ Stir at low speed at room temperature; S4. Medium temperature directional cross-linking; ① Add NaOH solution dropwise to adjust the pH to weakly alkaline; ② Slowly raise the temperature; S5. Gradient temperature rise for curing; After slowly raising the temperature, accelerate the temperature rise; S6. Neutralization and preliminary impurity removal; ① Add hydrochloric acid to adjust the pH to neutral; ② Add pretreated activated carbon and stir for adsorption; ③ Centrifuge to remove activated carbon particles and collect the supernatant; S7. Gradient dialysis purification; ① First dialysis: Load the supernatant into a dialysis bag and dialyze it in deionized water containing β-cyclodextrin; ② Second dialysis: Dialyze it in PBS buffer containing bovine serum albumin; ③ Third dialysis: Dialyze it with deionized water; S8. Molding and sterilization to obtain sodium hyaluronate gel.

[0007] Further, S1 is specifically: ① Dissolve the HA crude product with a molecular weight of 1800 - 2200 kDa in a phosphate buffer with a pH of 7.4 - 8.5 and a concentration of 20 - 30 mg / mL, and stir until it is completely dissolved into a gel; ② Pass through a Protein A affinity chromatography column at a flow rate of 4 - 6 mL / min and collect the breakthrough solution; ③ Ultrafilter the breakthrough solution to remove small molecule impurities, and ultrafilter 2 - 3 times in a cycle until the volume is concentrated to 1 / 5 - 1 / 4 of the original volume; ④ Lyophilize the concentrated solution to obtain the purified HA powder.

[0008] Further, S2 is specifically: ① Mix 1,4-butanediol diglycidyl ether and propylene glycol in a volume ratio of 1:5 - 8, and ultrasonically disperse for 10 - 15 min to form a uniformly pre-dispersed solution; ② Slowly add the pre-cooled 1 - 1.5% NaOH aqueous solution to the homogeneous pre-dispersion solution at a dropping rate of 1 - 3 drops per second, and stir at a speed of 200 - 280 rpm while dropping. When the pH stabilizes at 8.2 - 9.6, add 0.05 - 0.1 wt% of mercaptoethanol, stir for 5 - 8 min, and then heat up to 45 - 50 °C to form a BDDE microemulsion.

[0009] Furthermore, S3 is specifically as follows: ① Dissolve the purified HA powder in deionized water and stir until a 1 - 3% HA solution is formed; ② Add the pre-dispersed BDDE microemulsion, with the mass ratio of the cross-linking agent to HA being 1:150 - 200, and simultaneously add 0.08 - 0.12% of vitamin C; ③ Stir at a low speed of 120 - 150 rpm at room temperature for 30 - 40 min to allow the HA molecular chains to fully adsorb the cross-linking agent microdroplets.

[0010] Furthermore, S4 is specifically as follows: ① Dropwise add 1 - 3 M NaOH solution to adjust the pH to 9.0 ± 0.5; ② Slowly heat up to 42 - 45 °C at a rate of 3 - 5 °C / min, maintain the stirring rate at 260 - 300 rpm, and keep warm for 10 - 15 min.

[0011] Furthermore, S5 is specifically as follows: ① Heat up to 50 - 52 °C at a rate of 5 - 8 °C / h; ② Heat up to 55 - 57 °C at a rate of 1 - 2 °C / min.

[0012] Furthermore, S6 is specifically as follows: ① Add hydrochloric acid to adjust the pH to 6.8 - 7.2; ② Add 0.5 - 1% of pretreated activated carbon, stir and adsorb for 30 - 45 min to remove residual cross-linking agent and degradation products; ③ Centrifuge to remove the activated carbon particles and collect the supernatant.

[0013] Furthermore, S7 is specifically as follows: ① First dialysis: Load the supernatant into a dialysis bag and dialyze it in deionized water containing 0.01 - 0.03 M β-cyclodextrin; ② Second dialysis: Dialyze it in PBS buffer containing 1 - 3% bovine serum albumin; ③ Third dialysis: Dialyze it with deionized water to remove buffer salt ions.

[0014] Sodium hyaluronate gel prepared by the preparation method of the sodium hyaluronate gel described above.

[0015] Use of the sodium hyaluronate gel described above in the preparation of intra-articular injection agents, ophthalmic viscoelastic agents, aesthetic filling agents and / or skin care product moisturizing gels.

[0016] The beneficial effects of the present invention compared with the prior art are as follows: 1. During the preparation and directional crosslinking of the BDDE microemulsion in the present invention, the pH is stably controlled in the weakly alkaline range, achieving the following effects: The weakly alkaline environment maintains the ring-opening activity of the BDDE epoxy group through nucleophilic attack, while inhibiting the excessive deacetylation of HA molecules under strong alkaline conditions. Strong alkaline conditions can lead to a significant increase in the deacetylation rate of N-acetylglucosamine units on the HA chain, while the weakly alkaline condition can reduce the degree of deacetylation. This regulation balances the ring-opening reaction of BDDE and the degradation of HA, avoiding the imbalance between crosslinking efficiency and HA degradation in the traditional strong alkaline process. Under weakly alkaline conditions, the degradation of HA molecular chains is inhibited, and the number-average molecular weight is significantly increased, while the weight-average molecular weight remains stable. This phenomenon is due to the reduction of short-chain fragments. The optimization of the molecular weight distribution directly improves the viscoelasticity of the gel and reduces the slip effect of short-chain fragments in dynamic shear. The weakly alkaline environment promotes the uniform crosslinking 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 characteristics of the gel.

[0017] 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, realizing the preliminary enrichment and separation of the residual crosslinking agent 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, enhancing the migration ability of BDDE through intermolecular interactions, making it easier to detach from the gel network and creating favorable conditions for the subsequent dialysis step. A gradient dialysis strategy of buffer solution and deionized water is adopted, while controlling the reduction of the gel particle size and specific surface area, shortening the mass transfer path, improving the dialysis efficiency, and achieving the deep removal of the residual crosslinking agent. Reducing the potential cytotoxicity and inflammation risk in the gel. The efficient removal of the residual crosslinking agent avoids tissue adverse reactions caused by chemical stimulation during long-term implantation and improves the biocompatibility of the material.

[0018] 3. The present invention realizes multi-scenario adaptation by precisely regulating the pH and optimizing the crosslinking process: Treatment of osteoarthritis: By controlling the crosslinking degree and molecular weight distribution under weakly alkaline conditions, the mechanical properties of the gel are close to those of joint synovial fluid, effectively buffering and lubricating joint friction; Facial filling material: By increasing the crosslinking density and the proportion of long-chain molecules, the supporting force and structural stability of the gel are enhanced, meeting the morphological maintenance requirements of high-support-demand sites; Skin moisturization and drug sustained release: By reducing the crosslinking density and molecular weight, the gel is given low modulus characteristics, making it easy to penetrate into the shallow layer of the skin, achieving long-term moisturization and controlled release of drugs. Description of the Drawings

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0020] Figure 1 It is a physical picture of a crude sodium hyaluronate of the present invention; Figure 2 It is a physical picture of the sodium hyaluronate gel prepared in Example 1 of the present invention; Figure 3 It is a physical picture of the sodium hyaluronate gel prepared in Example 2 of the present invention; Figure 4 It is a physical picture of the sodium hyaluronate gel prepared in Example 3 of the present invention. Detailed implementation manners

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0022] Example 1: This example provides a preparation method for a sodium hyaluronate gel, including the following steps: S1. Purification of sodium hyaluronate (HA); ① Dissolve crude sodium hyaluronate (molecular weight 1800 - 2200 kDa) in a pH 8.5 phosphate buffer solution (PBS) at a concentration of 30 mg / mL, and stir until completely dissolved into a gel; ② Pass through a Protein A affinity chromatography column (to remove residual proteins) at a flow rate of 6 mL / min, and collect the breakthrough solution; ③ Ultrafilter the breakthrough solution (molecular weight cut-off 300,000) to remove small molecule impurities, and circulate the ultrafiltration 3 times until the volume is concentrated to 1 / 5 of the original volume; ④ Lyophilize the concentrated solution to obtain purified HA powder (protein residue < 0.01%); S2. Predispersion of the cross-linking agent; ① Mix 1,4-butanediol diglycidyl ether (BDDE) and propylene glycol at a volume ratio of 1:8 (propylene glycol is used as an inert dispersion medium to reduce the viscosity of BDDE), and ultrasonically disperse for 15 min (frequency 40 kHz, power 200 W) to form a uniform predispersed solution; ② Slowly add a 1.5% NaOH aqueous solution pre-cooled to 4 °C to the homogeneous pre-dispersion solution at a dropping rate of 3 drops per second, and stir at a speed of 280 rpm while dropping. When the pH stabilizes at 8.2, add 0.1 wt% mercaptoethanol, stir for 8 min, and then raise the temperature to 50 °C to form a BDDE microemulsion; The mercapto group (-SH) of mercaptoethanol can undergo a ring-opening addition reaction with the epoxy group of BDDE, preferentially capturing free BDDE that has not participated in cross-linking to form a non-toxic thioether compound; S3. Room temperature pre-activation (promote the stretching of HA chains); ① Dissolve the purified HA powder (3 g) in 100 mL of deionized water (pre-heated to 25 °C in advance), and stir until a 3% HA solution is formed; ② Add the pre-dispersed BDDE microemulsion (cross-linking agent: HA mass ratio 1:200), and at the same time add 0.12% vitamin C (antioxidant, inhibit HA degradation); ③ Stir at a low speed (150 rpm) at room temperature for 40 min to allow the HA molecular chains to fully adsorb the cross-linking agent droplets; S4. Medium temperature directional cross-linking (improve the effective cross-linking rate); ① Dropwise add 3 M NaOH solution to adjust the pH to 9.0 ± 0.5; ② Slowly raise the temperature to 45 °C at a rate of 5 °C / min, maintain the stirring rate at 300 rpm (promote the rupture of the BDDE microemulsion, release BDDE to react with HA hydroxyl groups), and keep warm for 15 min; S5. Gradient temperature rise and curing (homogenize the cross-linking network); ① Raise the temperature to 52 °C at a rate of 8 °C / h; ② Raise the temperature to 57 °C at a rate of 2 °C / min; Through gradient temperature rise, the distal HA molecular chains gradually participate in cross-linking, eliminating local over-cross-linking and forming a gel network with a uniform pore size distribution; S6. Neutralization and preliminary impurity removal; ① Add 0.1 M hydrochloric acid to adjust the pH to 6.8 - 7.2 (neutralize the excess alkali solution and terminate the reaction); ② Add 1% pretreated activated carbon (soak the activated carbon in 5% nitric acid for 24 h, wash with water until neutral and dry at 120 °C to generate more carboxyl and hydroxyl groups on the surface of the activated carbon, improving the adsorption capacity for polar molecules containing epoxy groups), stir and adsorb for 45 min to remove residual cross-linking agents and degradation products; ③ Centrifuge (3000 rpm, 10 min) to remove the activated carbon particles and collect the supernatant; S7. Gradient dialysis purification (dual residual control); ① Primary dialysis: Load the supernatant into a dialysis bag with a molecular weight cut-off of 100,000, and dialyze it in deionized water containing 0.03 M β-cyclodextrin for 12 h (change the water every 4 h); ② Secondary dialysis: Replace the dialysis bag with a molecular weight cut-off of 50,000, and dialyze it in PBS buffer containing 3% bovine serum albumin for 24 h (through the binding of protein-drug interaction sites, the residual BDDE in covalent bonds detaches from the HA chain and enters the dialysis solution); ③ Tertiary dialysis: Dialyze with deionized water for 8 h to remove buffer salt ions. Finally, the free HA in the gel is <0.05 mg / mL, and the residual BDDE is <5 ppm; S8. Molding and sterilization to obtain sodium hyaluronate gel (denoted as HA Gel ①); ① Extrude the purified supernatant gel through a 200-mesh sieve to remove large particle impurities and form a uniform paste; ② Sterilize by γ-ray irradiation (dose 25 kGy) to avoid the destruction of the cross-linked network by high-temperature sterilization; ③ Sterile filling into a medical syringe, nitrogen filling and sealing, and storing at 4°C.

[0023] Example 2: This example provides a method for preparing sodium hyaluronate gel, including the following steps: S1. Purification of sodium hyaluronate (HA); ① Dissolve sodium hyaluronate (crude product) (molecular weight 1800 - 2200 kDa) in phosphate buffer at pH 7.4 with a concentration of 20 mg / mL, and stir until completely dissolved into a gel; ② Pass through a Protein A affinity chromatography column at a flow rate of 4 mL / min, and collect the breakthrough solution; ③ Ultrafilter the breakthrough solution (molecular weight cut-off 300,000) to remove small molecule impurities, and cycle ultrafiltration 2 times until the volume is concentrated to 1 / 4 of the original volume; ④ Lyophilize the concentrated solution to obtain purified HA powder; S2. Predispersion of cross-linking agent; ① Mix BDDE and propylene glycol at a volume ratio of 1:5, and ultrasonically disperse for 10 min (frequency 40 kHz, power 200 W) to form a uniform predispersed solution; ② Slowly dropwise add pre-cooled 1% NaOH aqueous solution at 4°C to the uniform predispersed solution at a dropping rate of 1 drop / second, and stir at a speed of 200 rpm while dropping. When the pH stabilizes at 9.6, add 0.05 wt% mercaptoethanol, stir for 5 min and then heat up to 45°C to form a BDDE microemulsion; S3. Room temperature pre-activation; ① Dissolve the purified HA powder (1 g) in 100 mL of deionized water (pre-heated to 25°C in advance), and stir to form a 1% HA solution; ② Add the pre-dispersed BDDE microemulsion (crosslinking agent: HA mass ratio 1:150), and at the same time add 0.08% vitamin C; ③ Stir at low speed (120 rpm) for 30 min at room temperature; S4. Medium-temperature directional crosslinking; ① Dropwise add 1M NaOH solution to adjust the pH to 9.0 ± 0.5; ② Slowly heat up to 42 °C at a rate of 3 °C / min, maintain the stirring rate at 260 rpm, and keep warm for 10 min; S5. Gradient heating and curing; ① Heat up to 50 °C at a rate of 5 °C / h; ② Heat up to 55 °C at a rate of 1 °C / min; S6. Neutralization and preliminary impurity removal; ① Add 0.1M hydrochloric acid to adjust the pH to 6.8 - 7.2; ② Add 0.5% pretreated activated carbon (the activated carbon is soaked in 5% nitric acid for 24 h, washed with water until neutral, and dried at 120 °C to generate more carboxyl and hydroxyl groups on the surface of the activated carbon, improving the adsorption capacity for polar molecules containing epoxy groups), stir and adsorb for 30 min to remove residual crosslinking agent and degradation products; ③ Centrifuge (3000 rpm, 10 min) to remove the activated carbon particles and collect the supernatant; S7. Gradient dialysis purification; ① First dialysis: Load the supernatant into a dialysis bag with a molecular weight cut-off of 100,000, and dialyze it in deionized water containing 0.01M β-cyclodextrin for 12 h (change water every 4 h); ② Second dialysis: Replace the dialysis bag with a molecular weight cut-off of 50,000, and dialyze it in PBS buffer containing 1% bovine serum albumin for 24 h; ③ Third dialysis: Dialyze with deionized water for 8 h to remove buffer salt ions; S8. Molding and sterilization to obtain sodium hyaluronate gel (denoted as HA Gel②); ① Extrude and form the purified supernatant gel through a 200-mesh sieve to remove large particle impurities and form a uniform paste; ② Sterilize by γ-ray irradiation (dose 25 kGy) to avoid damage to the crosslinking network by high-temperature sterilization; ③ Sterile filling into a medical syringe, filling with nitrogen and sealing, and storing at 4 °C.

[0024] Example 3: This example provides a method for preparing sodium hyaluronate gel, including the following steps: S1. Purification of sodium hyaluronate (HA); ① Dissolve sodium hyaluronate (crude product) (molecular weight 1800 - 2200 kDa) in phosphate buffer at pH 8.2 with a concentration of 28 mg / mL, and stir until completely dissolved into a gel; ② Pass through a Protein A affinity chromatography column at a flow rate of 5 mL / min, and collect the breakthrough solution; ③ Ultrafilter the breakthrough solution (molecular weight cut-off 300,000) to remove small molecule impurities, and cycle ultrafiltration 3 times until the volume is concentrated to 1 / 5 of the original volume; ④ Lyophilize the concentrated solution to obtain purified HA powder; S2. Predispersion of crosslinking agent; ① Mix BDDE and propylene glycol at a volume ratio of 1:6, and ultrasonically disperse for 12 min (frequency 40 kHz, power 200 W) to form a uniform predispersed solution; ② Slowly add 1.3% NaOH aqueous solution precooled to 4°C to the uniform predispersed solution at a dropping rate of 2 drops / second, and stir at a speed of 220 rpm while dropping. When the pH stabilizes at 8.8, add 0.08 wt% mercaptoethanol, stir for 6 min and then heat up to 48°C to form a BDDE microemulsion; S3. Room temperature pre-activation; ① Dissolve purified HA powder (1.8 g) in 100 mL of deionized water (preheated to 25°C in advance), and stir to form a 1.8% HA solution; ② Add the predispersed BDDE microemulsion (mass ratio of crosslinking agent to HA is 1:170), and at the same time add 0.1% vitamin C; ③ Stir at low speed (140 rpm) at room temperature for 38 min; S4. Medium temperature directional crosslinking; ① Add 2 M NaOH solution to adjust the pH to 9.0 ± 0.5; ② Slowly heat up to 43°C at a rate of 4°C / min, maintain the stirring rate at 280 rpm, and keep warm for 12 min; S5. Gradient temperature curing; ① Heat up to 51°C at a rate of 6°C / h; ② Heat up to 56°C at a rate of 1°C / min; S6. Neutralization and preliminary impurity removal; ① Add 0.1 M hydrochloric acid to adjust the pH to 6.8 - 7.2; ② Add 0.6% pretreated activated carbon (soak the activated carbon in 5% nitric acid for 24 h, wash with water until neutral and dry at 120°C to generate more carboxyl and hydroxyl groups on the surface of the activated carbon, enhancing the adsorption capacity for polar molecules containing epoxy groups), stir and adsorb for 42 min to remove residual crosslinking agent and degradation products; ③ Centrifuge (3000 rpm, 10 min) to remove activated carbon particles, and collect the supernatant; S7. Gradient dialysis purification; ① Primary dialysis: Load the supernatant into a dialysis bag with a molecular weight cut-off of 100,000 and dialyze it in deionized water containing 0.02 M β-cyclodextrin for 12 h (change the water every 4 h); ② Secondary dialysis: Replace the dialysis bag with a molecular weight cut-off of 50,000 and dialyze it in PBS buffer containing 2% bovine serum albumin for 24 h; ③ Tertiary dialysis: Dialyze with deionized water for 8 h to remove buffer salt ions; S8. Molding and sterilization to obtain sodium hyaluronate gel (denoted as HA Gel③); ① Extrude and mold the purified supernatant gel through a 200-mesh sieve to remove large particle impurities and form a uniform paste; ② Sterilize by γ-ray irradiation (dose 25 kGy) to avoid damage to the cross-linking network by high-temperature sterilization; ③ Sterile filling into a medical syringe, nitrogen filling and sealing, and storing at 4°C.

[0025] Comparative Example 1: The difference between this comparative example and Example 3 is that S6 and S7 were not carried out.

[0026] Comparative Example 2: The difference between this comparative example and Example 3 is that in S2, mercaptoethanol was added only when the pH was stable at 11; in S4, 2 M NaOH solution was added dropwise to adjust the pH to 11.

[0027] Comparative Example 3: The difference between this comparative example and Example 3 is that S6 and S7 were not carried out, and in S2, mercaptoethanol was added only when the pH was stable at 11; in S4, 2 M NaOH solution was added dropwise to adjust the pH to 11.

[0028] Experimental Example: 1. Detect the change in molecular weight; Detection method: Gel Permeation Chromatography (GPC).

[0029] Principle: Separate HA fragments with different molecular weights by GPC, and quantitatively analyze the molecular weight distribution in combination with a refractive index detector (RI) or a multi-angle laser light scattering detector (MALLS).

[0030] Sample preparation: Dissolve the sodium hyaluronate gel in 0.1 M NaCl solution, with a concentration of about 1 - 2 mg / mL, and ultrasonically treat until completely dissolved.

[0031] Chromatographic column: TSKgel G6000PWXL (300 mm × 7.8 mm).

[0032] Mobile phase: 0.1 M NaCl solution, flow rate 0.5 mL / min.

[0033] Column temperature: 30°C.

[0034] Standard curve: A standard curve was established using HA standards of known molecular weights.

[0035] Detection: Inject the sample, record the chromatogram, and calculate the number-average molecular weight (Mn) and weight-average molecular weight (Mw).

[0036] Among them, the number-average molecular weight (Mn) reflects the lower limit of the average chain length of HA molecular chains and is more sensitive to low-molecular-weight components (a small number of short-chain molecules can significantly lower Mn). If HA undergoes β-elimination degradation (such as main-chain cleavage under alkaline conditions), the number of short-chain fragments increases, and Mn will decrease significantly. The weight-average molecular weight (Mw) reflects the upper limit of the average chain length of HA molecular chains and is more sensitive to high-molecular-weight components (a small number of long-chain molecules can significantly increase Mw). In crosslinked gels, Mw is related to the crosslinking density and network structure of molecular chains. A high Mw usually corresponds to more complex entanglements or crosslinks.

[0037] Therefore, the molecular weight often refers to the weight-average molecular weight (Mw).

[0038] 2. Viscoelasticity test; Detection method: Rotational rheometer.

[0039] Principle: Evaluate the viscoelasticity of the gel by measuring the storage modulus (G') and loss modulus (G").

[0040] Sample preparation: Evenly apply the gel on a parallel plate (diameter 25 mm, gap 1 mm).

[0041] Frequency sweep: 0.1 - 10 Hz, strain 1%.

[0042] Temperature: 25 °C.

[0043] Data analysis: Start the frequency sweep program, record the calculated storage modulus (G', elastic modulus, unit Pa) and loss modulus (G", viscous modulus, unit Pa) at each frequency point. At least 3 parallel samples of each sample should be tested, and the average values of G' and G" should be taken.

[0044] 3. DDE residue detection; Detection method: High-performance liquid chromatography - tandem mass spectrometry (HPLC-MS / MS).

[0045] Principle: Separate BDDE using HPLC and perform high-sensitivity detection in combination with the multiple reaction monitoring (MRM) mode of mass spectrometry.

[0046] Sample pretreatment: Weigh 0.1 g of the gel sample, add 1 mL of acetonitrile, vortex for 10 minutes, centrifuge (10,000 rpm, 10 minutes), and take the supernatant.

[0047] Extract it repeatedly 2 times, combine the supernatant, blow it dry with nitrogen, and re-dissolve it to 1 mL with 0.1% formic acid aqueous solution.

[0048] Chromatographic column: C18 column (150 mm × 2.1 mm, 3.5 μm).

[0049] 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).

[0050] Flow rate: 0.3 mL / min.

[0051] Column temperature: 30 °C.

[0052] Ion source: Electrospray ionization (ESI+).

[0053] Monitoring ion pairs: m / z165.1 → 123.1 (quantitative ion), m / z165.1 → 87.1 (qualitative ion).

[0054] Fragmentation voltage: 35 V, collision energy: 15 eV.

[0055] Standard curve: Establish a standard curve using BDDE standard.

[0056] Calculation: Calculate the content of BDDE in the sample (ppm) according to the peak area.

[0057] The results are shown in the following table: Item 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 Storage modulus G’ (Pa) 360 87 120 117 45 37 Loss modulus G” (Pa) 152 74 89 85 21 18 BDDE content (ppm) 0.23 0.25 0.24 1.05 0.38 1.34 As can be seen from the above table, the present invention realizes the removal of residual crosslinking agent BDDE through pretreatment of activated carbon for impurity removal and gradient dialysis purification with β-cyclodextrin + PBS buffer + deionized water; by coordinating the above impurity removal steps with the stable control of pH at weakly alkaline during the preparation process of BDDE microemulsion and the directional crosslinking process, a synergistic effect of removing residual crosslinking agent BDDE can be achieved.

[0058] By stably controlling the pH at weakly alkaline during the preparation process of BDDE microemulsion and the directional crosslinking process, although it has no obvious effect on the commonly used molecular weight index, 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.

[0059] In addition, the present invention can also control the number of short-chain fragments by controlling the pH, and then adjust the elastic modulus and viscous modulus of sodium hyaluronate gel to adapt to different application scenarios; For example, for hyaluronic acid injection solution used in the treatment of osteoarthritis, the elastic modulus is mostly 50 - 150 Pa, and the viscous modulus is usually 30 - 100 Pa; For cross-linked hyaluronic acid products used in areas with high support requirements such as the nasal dorsum and supraorbital arch, the elastic modulus is usually 350 - 400 Pa, and the viscous modulus is usually 50 - 150 Pa; For products used for superficial skin moisturization or drug sustained release, the elastic modulus and viscous modulus are as low as 1 - 10 Pa.

[0060] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and 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 embodiments of the present invention.

Claims

1. A method for preparing sodium hyaluronate gel, characterized in that, It includes the following steps: S1. Sodium hyaluronate purification; Dissolve the crude HA in phosphate buffer solution and stir until it is completely dissolved into a gel; Pass through a Protein A affinity chromatography column and collect the flow-through; Ultrafilter the flow-through to remove small molecule impurities, and perform cyclic ultrafiltration and concentration; Lyophilize the concentrated solution to obtain purified HA powder; S2. Crosslinker pre-dispersion; Mix 1,4-butanediol diglycidyl ether with propylene glycol to form a uniform pre-dispersed solution; Dropwise add NaOH solution to the uniform pre-dispersed solution. When the pH stabilizes at 8.2 - 9.6, add mercaptoethanol and heat up to form a BDDE microemulsion; S3. Room temperature pre-activation; Dissolve the purified HA powder in deionized water to form an HA solution; Add the pre-dispersed BDDE microemulsion and simultaneously add vitamin C; Stir at low speed at room temperature; S4. Medium temperature directional crosslinking; Dropwise add NaOH solution to adjust the pH to 9.0 ± 0.5; Slowly heat up; S5. Gradient heating and curing; Slowly heat up and then accelerate the heating; S6. Neutralization and preliminary impurity removal; Add hydrochloric acid to adjust the pH to neutral; Add pretreated activated carbon and stir for adsorption; Centrifuge to remove the activated carbon particles and collect the supernatant; S7. Gradient dialysis purification; First dialysis: Load the supernatant into a dialysis bag and dialyze it in deionized water containing β-cyclodextrin; Second dialysis: Dialyze it in PBS buffer solution containing bovine serum albumin; Third dialysis: Dialyze it with deionized water; S8. Molding and sterilization to obtain sodium hyaluronate gel.

2. The preparation method of the sodium hyaluronate gel according to claim 1, wherein Specifically, S1 is as follows: Dissolve the crude HA with a molecular weight of 1800 - 2200 kDa in phosphate buffer solution with a pH of 7.4 - 8.5 and a concentration of 20 - 30 mg / mL, and stir until it is completely dissolved into a gel; Pass through a Protein A affinity chromatography column at a flow rate of 4 - 6 mL / min and collect the flow-through; Ultrafilter the flow-through to remove small molecule impurities, and perform cyclic ultrafiltration 2 - 3 times until the volume is concentrated to 1 / 5 - 1 / 4 of the original volume; Lyophilize the concentrated solution to obtain purified HA powder.

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

4. The preparation method of the sodium hyaluronate gel according to claim 1, wherein, Specifically, S3 is as follows: Dissolve the purified HA powder in deionized water and stir until a 1 - 3% HA solution is formed; Add the pre-dispersed BDDE microemulsion, and the mass ratio of the crosslinker to HA is 1:150 - 200. At the same time, add 0.08 - 0.12% vitamin C; Stir at low speed at 120 - 150 rpm at room temperature for 30 - 40 min to allow the HA molecular chains to fully adsorb the crosslinker microdroplets.

5. The preparation method of the sodium hyaluronate gel according to claim 1, characterized in that Specifically, S4 is as follows: Dropwise add 1 - 3 M NaOH solution to adjust the pH to 9.0 ± 0.5; Heat up slowly at 3 - 5 °C / min to 42 - 45 °C, maintain the stirring rate at 260 - 300 rpm, and keep warm for 10 - 15 min.

6. The preparation method of the sodium hyaluronate gel according to claim 1, wherein Specifically, S5 is: heat up at a rate of 5 - 8 °C / h to 50 - 52 °C; Heat up at a rate of 1 - 2 °C / min to 55 - 57 °C.

7. The preparation method of the sodium hyaluronate gel according to claim 1, characterized in that, Specifically, S6 is: add hydrochloric acid to adjust the pH to 6.8 - 7.2; Add 0.5 - 1% pretreated activated carbon, stir and adsorb for 30 - 45 min to remove residual cross-linking agents and degradation products; Centrifuge to remove activated carbon particles and collect the supernatant.

8. The preparation method of the sodium hyaluronate gel according to claim 2, wherein, Specifically, S7 is: primary dialysis: put the supernatant into a dialysis bag and dialyze it in deionized water containing 0.01 - 0.03 M β-cyclodextrin; Secondary dialysis: dialyze it in PBS buffer containing 1 - 3% bovine serum albumin; Tertiary dialysis: dialyze it with deionized water to remove buffer salt ions.

9. A sodium hyaluronate gel prepared by the preparation method of the sodium hyaluronate gel according to any one of claims 1 - 8.

10. Use of the sodium hyaluronate gel according to claim 9 in the preparation of an intra-articular injection, an ophthalmic viscoelastic agent, a medical aesthetic filler and / or a skin care product moisturizing gel.

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