A method for removing residual crosslinking agent bdde of crosslinked hyaluronic acid hydrogel

Abstract

This invention relates to a method for removing residual BDDE, a crosslinking agent, from crosslinked hyaluronic acid hydrogels, belonging to the field of gel formulation technology. The invention employs a two-step strategy of "composite solution treatment combined with stabilizer sterilization," with its core feature being the phased, multi-mechanism synergistic removal of BDDE residues. First, the composite solution treatment, through the synergistic action of inorganic salts and / or quaternary ammonium salts with organic solvents, degrades the free epoxy groups of ineffective crosslinked BDDE; simultaneously, the crosslinked hyaluronic acid gel is precipitated, separating the free-state crosslinking agent BDDE. Second, the obtained crosslinked hyaluronic acid gel precipitate is co-sterilized with a stabilizer. The active groups in the stabilizer, such as amino and hydroxyl groups, react with the free epoxy groups of the residual BDDE, achieving in-situ and simultaneous residue removal in the final sterilization step, simplifying the process.

Description

Technical Field

[0001] This invention relates to a method for removing residual BDDE, a crosslinking agent in crosslinked hyaluronic acid hydrogels, and belongs to the field of gel formulation technology. Background Technology

[0002] Hyaluronic acid (HA) is a natural linear anionic acidic polysaccharide widely found in the vitreous humor, joint spaces, and skin of humans and animals. Its disaccharide units are cross-linked by D-glucuronic acid and N-acetylglucosamine via β-1,3-glycosidic bonds, and the disaccharide units are linked by β-1,4-glycosidic bonds. Due to its excellent biocompatibility, biodegradability, and hydrophilicity, hyaluronic acid and its hydrogels have been extensively studied in the biomedical field. Common biomedical applications of hyaluronic acid hydrogels include joint viscoelasticity supplements, ocular surface lubricants, tissue engineering scaffolds, surgical implant materials, and drug delivery carriers. Although hyaluronic acid itself can form highly viscoelastic solutions through molecular chain entanglement, its linear non-crosslinked form has poor mechanical properties. Without the introduction of crosslinking agents, it cannot form stable hydrogels and is easily degraded by hyaluronidase in vivo, limiting its support effect and effective survival time in vivo. By linking hyaluronic acid molecules in and out of each other using cross-linking technology, it is possible to improve the support capacity of hyaluronic acid fillers, slow down the degradation rate, and enhance their durability.

[0003] The common method for cross-linking hyaluronic acid is to use 1,4-butanediol diglycidyl ether (BDDE) as a cross-linking agent to form a hydrogel. In cross-linked hyaluronic acid hydrogels using BDDE as the cross-linking agent, BDDE exists in three states: First, both ends of the BDDE cross-linking agent are connected to hyaluronic acid molecules, representing effective cross-linking modification; second, one end of the BDDE cross-linking agent is connected to a hyaluronic acid molecule, while the other end has a free epoxy group, representing ineffective modification; third, neither end of the BDDE cross-linking agent is cross-linked with hyaluronic acid, remaining in a small, free molecule state. In the first type of effectively cross-linked BDDE, both epoxy groups react with the hydroxyl groups on hyaluronic acid, resulting in no toxic side effects, and it is subsequently excreted through metabolism. However, the second and third types of ineffective cross-linking modification and the free BDDE state are referred to as BDDE residues, as one or both ends of these residues contain reactive epoxy groups that can react with human biomolecules, producing irritation, allergies, or even more serious side effects. Ineffective BDDE modification, due to its covalent cross-linking with hyaluronic acid hydrogel at one end, persists in the cross-linked hyaluronic acid filler material, making BDDE residue more difficult to remove. However, to improve the support strength of hyaluronic acid hydrogel and prolong its duration in vivo, its cross-linking degree must be increased, requiring the use of a certain amount of BDDE cross-linking agent. But increasing the amount of BDDE cross-linking agent inevitably leads to its free state, especially the ineffective modified state, remaining in the cross-linked hyaluronic acid hydrogel filler material. According to the national standard YY / T0962-2021 Cross-linked Sodium Hyaluronate Gel for Plastic Surgery, the residual amount of BDDE in injectable sodium hyaluronate gel should be less than 2 µg / g. However, for safety reasons, BDDE residue should be minimized as much as possible while ensuring product performance.

[0004] To address this challenge, research on BDDE crosslinking methods and effective BDDE removal processes for hyaluronic acid hydrogel fillers is a key research direction in this field. Chinese patent document CN 107880282 A (application number 201711246123.9) describes high-temperature washing with a carbonate buffer system at pH 8-9 to hydrolyze epoxy groups and remove BDDE residues. This method uses weakly alkaline conditions, which easily leads to the breakage of hyaluronic acid sugar chains in the hydrogel. Chinese patent document CN115710362 A (application number 202310026975.0) describes elution for 48 hours in a high osmotic pressure (500 m0smol / L) weakly alkaline PBS buffer (NaH2PO4 and Na2HPO4) to remove BDDE residues. This method not only still uses weakly alkaline conditions but also does not completely remove BDDE, leaving 0.36 µg / g of BDDE residue. Moreover, the buffer washing time in both patents is relatively long (36-48 hours), which is not conducive to efficient large-scale industrial production. Chinese patent document CN 117304561 A (application number 202311286123.1) describes a method for removing residual crosslinking agent BDDE from crosslinked hyaluronic acid aerogels using steam distillation. This method requires steam distillation at high temperatures, which can easily cause the breakage of hyaluronic acid sugar chains in the hydrogel. Therefore, this patent is only used for removing BDDE from hyaluronic acid aerogels, not from hyaluronic acid hydrogels. Furthermore, the method is not thorough in removing BDDE, leaving 0.46 µg / g of BDDE residue.

[0005] Therefore, there is an urgent need in the field to develop an efficient method for removing BDDE, especially BDDE residues in an ineffective modified state, from hyaluronic acid hydrogels, and to simplify the process and improve production efficiency in order to prepare anti-inflammatory (i.e., low toxicity and high biocompatibility) cross-linked hyaluronic acid hydrogels. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for removing residual BDDE, the crosslinking agent, from crosslinked hyaluronic acid hydrogels. This invention employs a two-step strategy of "composite solution treatment combined with stabilizer sterilization," with its core feature being the phased, multi-mechanism synergistic removal of BDDE residues. First, the composite solution treatment, through the synergistic action of inorganic salts and / or quaternary ammonium salts with organic solvents, degrades the free epoxy groups of ineffectively crosslinked BDDE; simultaneously, the crosslinked hyaluronic acid gel is precipitated, separating the free-state crosslinking agent BDDE. Second, the obtained crosslinked hyaluronic acid gel precipitate is co-sterilized with a stabilizer. The active groups in the stabilizer, such as amino and hydroxyl groups, react with the free epoxy groups of the residual BDDE, achieving in-situ and simultaneous residue removal during the final sterilization step, simplifying the process.

[0007] The technical solution adopted in this invention is as follows: A method for removing residual BDDE crosslinking agent from crosslinked hyaluronic acid hydrogels includes the following steps: (1) Composite solution treatment: After adjusting the pH of the cross-linked hyaluronic acid hydrogel to near neutral, it is contacted with a composite solution containing salt and organic solvent for 2 to 12 hours for synergistic precipitation treatment, and the resulting cross-linked hyaluronic acid hydrogel precipitate is collected. (2) Sterilization with stabilizer: The cross-linked hyaluronic acid hydrogel precipitate collected in step (1) is mixed with the stabilizer aqueous solution and sterilized by moist heat to obtain a cross-linked hyaluronic acid hydrogel product with BDDE residue removed.

[0008] According to a preferred embodiment of the present invention, the pH is adjusted to 6.5-7.5 in step (1).

[0009] According to a preferred embodiment of the present invention, hydrochloric acid solution is used to adjust the pH to near neutral.

[0010] More preferably, the mass concentration of the hydrochloric acid solution is 0.2-2%.

[0011] According to a preferred embodiment of the present invention, the organic solvent in step (1) is selected from at least one of polyethylene glycol, acetone, ethanol, and isoamyl alcohol, and the volume percentage concentration of the selected organic solvent is 50% to 100%, more preferably 70% to 95%.

[0012] According to a preferred embodiment of the present invention, the volume ratio of the cross-linked hyaluronic acid hydrogel to the organic solvent in the composite solution in step (1) is 1:(1~10), more preferably 1:(2~7).

[0013] According to a preferred embodiment of the present invention, the salt in step (1) is an inorganic salt and / or a quaternary ammonium salt, wherein the inorganic salt is selected from at least one of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium carbonate, and ammonium sulfate; and the quaternary ammonium salt is selected from at least one of hexadecylpyridine chloride, hexadecylpyridine bromide, and hexadecyltrimethylammonium bromide.

[0014] Further preferably, when the salt is an inorganic salt, the final concentration of the inorganic salt in the cross-linked hyaluronic acid hydrogel co-precipitation treatment system is 0.1~50% (w / v, g / mL), more preferably 0.5~15% (w / v, g / mL); when the salt is a quaternary ammonium salt, the final concentration of the quaternary ammonium salt in the cross-linked hyaluronic acid hydrogel co-precipitation treatment system is 0.01~5% (w / v, g / mL), more preferably 0.1~1.5% (w / v, g / mL); when the salt is both an inorganic salt and a quaternary ammonium salt, the final concentration of the inorganic salt is 0.5~10% (w / v, g / mL), and the final concentration of the quaternary ammonium salt is 0.1~1.0% (w / v, g / mL).

[0015] According to a preferred embodiment of the present invention, the conditions for the synergistic precipitation treatment in step (1) are: pressure 1~5 atm, temperature -20℃~30℃.

[0016] According to a preferred embodiment of the present invention, the cross-linked hyaluronic acid hydrogel in step (1) is prepared by the following steps: dissolving hyaluronic acid or its salt with a molecular weight of 10 kDa to 290 kDa in an alkaline solution, the final concentration of hyaluronic acid or its salt being 6 to 18%, and reacting it with the cross-linking agent BDDE at 30 to 60°C for 30 minutes to 2 hours to obtain the cross-linked hyaluronic acid hydrogel; wherein the mass ratio of BDDE to hyaluronic acid or its salt is 1 to 8:100.

[0017] More preferably, the alkaline solution is a 1% NaOH solution.

[0018] In step (1), in order to ensure sufficient contact between the cross-linked hyaluronic acid hydrogel and the composite solution, the cross-linked hyaluronic acid hydrogel can be pre-treated by crushing it into pieces of approximately 1 cm. 3 The resulting cross-linked hyaluronic acid hydrogel precipitate is in the size of a block; in addition, to fully separate the free cross-linking agent BDDE, the precipitate can be washed with 50%~100% ethanol.

[0019] According to a preferred embodiment of the present invention, the stabilizer in step (2) is a bioactive molecule containing amino and hydroxyl groups, selected from at least one of amino acids, monosaccharides, disaccharides, oligosaccharides, polyols, and vitamins.

[0020] More preferably, the stabilizer includes glycine, mannitol, glycerol, and trehalose.

[0021] According to a preferred embodiment of the present invention, the concentration of the stabilizer in the stabilizer aqueous solution in step (2) is 1-15%.

[0022] According to a preferred embodiment of the present invention, the solvent of the stabilizer aqueous solution in step (2) is water or PBS buffer at pH 7.2 and 0.01M.

[0023] According to a preferred embodiment of the present invention, the final concentration of hyaluronic acid in the mixed system of cross-linked hyaluronic acid hydrogel precipitate and stabilizer aqueous solution in step (2) is 2-5%.

[0024] According to a preferred embodiment of the present invention, before mixing the collected cross-linked hyaluronic acid precipitate with the stabilizer aqueous solution in step (2), it is further dried to reduce its free water content to less than 5%.

[0025] More preferably, the drying process is freeze-drying or oven drying.

[0026] More preferably, the freeze-drying temperature is below -20°C; and the drying temperature is 40°C to 70°C.

[0027] According to a preferred embodiment of the present invention, the moist heat sterilization in step (2) is sterilization at 121°C for 10 to 30 minutes.

[0028] The cross-linked hyaluronic acid hydrogel product prepared according to the above method has a BDDE residue of less than 0.12 µg / g.

[0029] The above-mentioned cross-linked hyaluronic acid hydrogel is used in the preparation of products for tissue filling, tissue repair or medical aesthetics.

[0030] According to a preferred embodiment of the present invention, the product is an injectable product.

[0031] The beneficial effects of this invention are: The core innovation of this invention lies in proposing an integrated process with multi-mechanism synergy and phased processing, which aims to efficiently, gently and thoroughly remove BDDE residues, especially ineffective modified parts, from cross-linked hyaluronic acid hydrogels, thereby preparing cross-linked hyaluronic acid hydrogel products with high safety and low toxicity.

[0032] 1. Synergistic and Efficient Deep Removal of BDDE Residues via Multiple Mechanisms: Targeting the most difficult-to-remove residual form of ineffective modified BDDE, this invention employs a two-step method combining "salt-organic solvent synergistic precipitation" and "stabilizer-sterilization synergistic reaction," integrating multiple chemical, physical, and thermochemical mechanisms to achieve deep and thorough BDDE removal. The first step utilizes an innovative composite solution treatment method, employing chemical degradation combined with physical separation to remove BDDE. Under mild conditions (neutral pH, low temperature, short time), free and single-end modified BDDE are efficiently transferred to the liquid phase for separation. The second step further removes BDDE by adding a stabilizer during standard moist heat sterilization. The final product contains BDDE residues reduced to <0.12 µg / g, far exceeding the <2 µg / g limit specified in the national standard YY / T0962-2021, and significantly lower than the residue levels reported in comparative patent literature (0.36~0.46 µg / g), greatly improving product safety.

[0033] 2. Overcoming the limitations of existing technologies and avoiding damage to the hyaluronic acid structure: Unlike existing technologies that commonly use weakly alkaline buffer solutions for prolonged washing, which easily leads to the breakage of hyaluronic acid glycan chains and low efficiency, this invention mainly operates under neutral conditions (pretreatment to adjust the pH to neutral), avoiding the weakly alkaline treatment environment throughout the process. This fundamentally prevents the hydrolysis and breakage of hyaluronic acid glycan chains due to alkaline catalysis, ensuring the structural integrity and mechanical stability of the hydrogel, and ensuring the clinical effectiveness of the product.

[0034] 3. Simple and efficient process, suitable for industrialization: The present invention adopts an integrated process of staged processing, which has a short processing time for composite solutions, does not require complex equipment, has mild operating conditions, simplifies the process, greatly improves production efficiency, reduces energy consumption and costs, and meets the needs of large-scale production.

[0035] The present invention provides a method for removing BDDE residues from crosslinking agents, which combines chemical, physical, and thermochemical mechanisms. It can not only remove free BDDE, but also effectively remove ineffective BDDE modifications attached to the long chains of hyaluronic acid, bringing the BDDE residue content to a level far below the national standard. Under mild, efficient, and alkaline-avoiding conditions, it achieves deep removal of BDDE residues from crosslinked hyaluronic acid hydrogel products, providing a reliable technical guarantee for the preparation of highly safe injectable crosslinked hyaluronic acid hydrogel products. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of the formation and injection of hyaluronic acid hydrogel.

[0037] Figure 2 This is a bar chart showing cell viability under the action of hyaluronic acid hydrogel extract.

[0038] Figure 3 A bar chart showing the solids retention rate of hyaluronic acid hydrogel after in vitro enzymatic hydrolysis.

[0039] Figure 4 This is a line graph showing the volume change of hyaluronic acid hydrogel during its in vivo existence.

[0040] Figure 5 HE staining image of the injection site tissue and inflammatory cell density count results after hyaluronic acid hydrogel injection. Detailed Implementation

[0041] The technical solution of the present invention will be further described below with reference to embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available materials for biological and chemical experiments. Unless otherwise specified, all percentages in the embodiments are mass percentages.

[0042] The method for determining the residual amount of BDDE in the cross-linked hyaluronic acid hydrogel in the examples was carried out according to the national standard YY / T 0962-2021.

[0043] Example 1 A method for preparing hyaluronic acid hydrogel includes the following steps: Prepare a 1% NaOH solution, add hyaluronic acid (molecular weight 150 kDa) to reach a final concentration of 10%, stir until dissolved, then add BDDE to reach a final concentration of 0.4%, stir thoroughly until completely dissolved, and carry out the cross-linking reaction in a 50°C water bath for 30 minutes, then let it stand at room temperature for 2 hours. After the cross-linking reaction is completed, break the cross-linked hyaluronic acid hydrogel into pieces of approximately 1 cm. 3 The hyaluronic acid hydrogel was neutralized by adding an equal volume of 1% HCl solution to 1% NaOH solution, adjusting the pH to neutral. Then, a composite solution containing sodium chloride, hexadecylpyridine chloride, and 100% (v / v) acetone was added. The volume of 100% acetone was twice the volume of the hyaluronic acid hydrogel. The final concentration of sodium chloride in the hyaluronic acid hydrogel treatment system was 10% (w / v, g / mL), and the final concentration of hexadecylpyridine chloride in the hyaluronic acid hydrogel treatment system was 0.1% (w / v, g / mL). The mixture was then subjected to treatment at 1 atm and 25°C. Slowly stir to ensure sufficient contact for 8 hours until precipitation is complete. Collect the precipitate, wash with 80% (v / v) ethanol, and sieve to obtain cross-linked hyaluronic acid hydrogel precipitate. Pre-cool the cross-linked hyaluronic acid hydrogel precipitate at -80℃ for 2 hours, then freeze-dry it at -50℃ until its free water content is less than 5%. Reconstitute the dried gel with PBS buffer (pH 7.2, 0.01M) containing 10% glycine to a final hyaluronic acid concentration of 2%. Fill syringes with the solution and sterilize by moist heat at 121℃ for 15 min to obtain the hyaluronic acid hydrogel product. Injection using a 26G needle is shown in the figure. Figure 1 .

[0044] Example 2 A method for preparing hyaluronic acid hydrogel includes the following steps: Prepare a 1% NaOH solution, add hyaluronic acid (molecular weight 80 kDa) to reach a final concentration of 15%, stir until dissolved, then add BDDE to reach a final concentration of 0.6%, stir thoroughly until completely dissolved, and carry out the cross-linking reaction in a 60℃ water bath for 30 minutes, then let it stand at room temperature for 5 hours; after the cross-linking reaction is completed, break the cross-linked hyaluronic acid hydrogel into pieces of approximately 1 cm. 3The hyaluronic acid hydrogel was neutralized by adding an equal volume of 1% HCl solution to 1% NaOH solution, adjusting the pH of the hydrogel to neutral. Then, a composite solution containing calcium chloride, hexadecyltrimethylammonium bromide, and 70% (v / v) isoamyl alcohol was added, with the volume of isoamyl alcohol equal to the volume of the hyaluronic acid hydrogel. The final concentration of calcium chloride in the hyaluronic acid hydrogel treatment system was 6% (w / v, g / mL), and the final concentration of hexadecyltrimethylammonium bromide in the hyaluronic acid hydrogel treatment system was 0.5% (w / v, g / mL). The mixture was then subjected to pressure... At 1 atm and 4°C, the mixture was slowly stirred to ensure sufficient contact for 12 hours until precipitation was complete. The precipitate was collected, washed with 80% (v / v) ethanol, and sieved to obtain cross-linked hyaluronic acid hydrogel precipitate. The cross-linked hyaluronic acid hydrogel precipitate was dried in a 60°C oven until its free water content was less than 5%. The dried gel was reconstituted with PBS buffer (pH 7.2, 0.01M) containing 10% mannitol to make the final concentration of hyaluronic acid 2%. The gel was then filled into syringes and sterilized by moist heat at 121°C for 15 min to obtain the hyaluronic acid hydrogel product.

[0045] Comparative Example 1 A method for preparing hyaluronic acid hydrogel includes the following steps: Prepare a 1% NaOH solution, add hyaluronic acid (molecular weight 150 kDa) to reach a final concentration of 10%, stir until dissolved, then add BDDE to reach a final concentration of 0.4%, stir thoroughly until completely dissolved, and carry out the cross-linking reaction in a 50°C water bath for 30 minutes, then let it stand at room temperature for 2 hours. After the cross-linking reaction is completed, break the cross-linked hyaluronic acid hydrogel into pieces of approximately 1 cm. 3 The hyaluronic acid hydrogel was neutralized by adding an equal volume of 1% HCl solution to 1% NaOH solution, adjusting the pH to neutral. Then, 3 volumes of PBS buffer (pH 7.2, 0.01M) were added for elution, changing the buffer every 8 hours for a total of 6 times. After elution, the cross-linked hyaluronic acid hydrogel precipitate was filtered out, pre-cooled at -80℃ for 2 hours, and then freeze-dried at -50℃ until the free water content was below 5%. The dried gel was then reconstituted with PBS buffer (pH 7.2, 0.01M) to a final hyaluronic acid concentration of 2%, filled into syringes, and sterilized by moist heat at 121℃ for 15 minutes to obtain the hyaluronic acid hydrogel product of Comparative Example 1.

[0046] Comparative Example 2: A method for preparing hyaluronic acid hydrogel includes the following steps: Prepare a 1% NaOH solution, add hyaluronic acid (molecular weight 150 kDa) to reach a final concentration of 10%, stir until dissolved, then add BDDE to reach a final concentration of 0.4%, stir thoroughly until completely dissolved, and carry out the cross-linking reaction in a 50°C water bath for 30 minutes, then let it stand at room temperature for 2 hours. After the cross-linking reaction is complete, break the cross-linked sodium hyaluronate gel into pieces of approximately 1 cm. 3 The hyaluronic acid hydrogel was neutralized by adding an equal volume of 1% HCl solution to 1% NaOH solution to adjust its pH to neutral. Then, 95% (v / v) ethanol was slowly added until a white precipitate was completely formed. The precipitate was washed with ethanol of the same concentration to obtain a cross-linked hyaluronic acid hydrogel. The cross-linked hyaluronic acid hydrogel was pre-cooled at -80℃ for 2 hours and then freeze-dried at -50℃ until its free water content was less than 5%. PBS buffer (pH 7.2, 0.01M) was added to the dried gel to reconstitute it, bringing the final concentration of hyaluronic acid to 2%. The gel was then filled into syringes and sterilized by moist heat at 121℃ for 15 min to obtain the hyaluronic acid hydrogel product of Comparative Example 2.

[0047] Example 3 For the hyaluronic acid hydrogels prepared in Examples 1 and 2 and Comparative Examples 1 and 2, the residual BDDE content in the cross-linked hyaluronic acid hydrogel precipitate obtained after the first step of residue removal and the hyaluronic acid hydrogel product obtained after the second step of residue removal were detected. The residual BDDE content was determined by the reaction of epoxides with nicotinamide according to the national standard YY / T 0962-2021, where fluorescence is generated under excitation light at 370 nm. The fluorescence intensity is directly proportional to the amount of epoxide, and the fluorescence intensity can be detected by a microplate reader at an emission wavelength of 430 nm. The detection results are shown in Table 1.

[0048] Table 1. Detection results of BDDE residue in hyaluronic acid hydrogel, unit: µg / g

[0049] The results in Table 1 show that the residual BDDE content in the hyaluronic acid hydrogel products obtained in Examples 1-2 was significantly lower than that in Comparative Examples 1-2, only 5.7% and 1.3% of the national standard of 2 µg / g, respectively. Specifically, in the cross-linked hyaluronic acid hydrogel precipitate obtained by the "salt-organic solvent co-precipitation" method in Example 1, the residual BDDE content was 0.27 µg / g, which was lower than that in the cross-linked hyaluronic acid hydrogel precipitates obtained by the "PBS buffer elution method" in Comparative Example 1 and the "ethanol precipitation method" in Comparative Example 2. This indicates that the "salt-organic solvent co-precipitation" method of the present invention is more effective in removing BDDE residue. Secondly, Example 1 also employed a "stabilizer-sterilization synergistic reaction" to remove BDDE residues in situ, especially the more difficult-to-remove BDDE, further reducing the BDDE residue level. The final product contained only 0.113 µg / g of BDDE residue. Meanwhile, Comparative Examples 1 and 2 used a "high-temperature sterilization reaction" to further remove BDDE residue. The removal amount in Comparative Example 1 was 0.8 µg / g, and in Comparative Example 2 it was 0.13 µg / g. This demonstrates that the "high-temperature sterilization reaction" can effectively remove BDDE residue. From the BDDE residue levels in the final products of Comparative Examples 1 and 2, it can be seen that the 0.8 µg / g BDDE removal amount in Comparative Example 1 represents the upper limit of what the "high-temperature sterilization reaction" can remove. The removal amount in Comparative Example 2 is much lower than that in Comparative Example 1, indicating that the amount of BDDE that can be removed by the "high-temperature sterilization reaction" in Comparative Example 2 is limited. Due to the limitations of the removal method, the more difficult-to-remove BDDE still remains, resulting in a final product containing only 0.36 µg / g of BDDE residue.

[0050] Example 4 The composite shear modulus (G*) of the hyaluronic acid hydrogel products obtained in Examples 1 and 2 and Comparative Examples 1 and 2 was measured, characterizing the material's resistance to shear strain. The samples were placed in a sample pan with a 20 mm parallel plate spacing of 1,500 μm on a modular dynamic rheometer. The rheometer was used to measure angular frequencies of 0.1–100 rad / s, strain frequencies of 1 Hz, and temperatures of 25 °C. At an angular frequency ω = 10 rad·s... -1 Under the given conditions, the results are shown in Table 2. A higher composite shear modulus reflects stronger product rigidity and better support within the product body.

[0051] Table 2. Composite Shear Modulus of Hyaluronic Acid Hydrogel Products

[0052] The results in Table 2 show that the composite shear modulus (G*) of the hyaluronic acid hydrogel products of Examples 1-2 is significantly higher than that of Comparative Examples 1-2. At a certain angular frequency, Examples 1 and 2 both have high composite shear modulus (G*), which are 110.72% and 33.70% higher than those of Comparative Examples 1 and 2, respectively, and 96.27% and 24.54% higher, respectively, indicating that the hyaluronic acid hydrogel products of these examples exhibit excellent mechanical properties.

[0053] Example 5 The cell compatibility of hyaluronic acid hydrogel products was evaluated using the CCK-8 assay. The hydrogel extract was prepared according to the People's Republic of China National Standard GB / T 14233.2—2025 Medical Infusion, Transfusion and Injection Equipment Test Methods Part 2 "Biological Test Methods" Extract Preparation Method, and was placed in a pressure steam sterilizer and extracted at (121±2)℃ for (1±0.1) hours. L929 cells (purchased from ATCC) were seeded in 96-well plates at a density of 1×10⁻⁶ cells per well. 4 Cells / wells were cultured in a cell culture incubator containing 5% CO2 at 37°C for 24 h. The experimental group was then replaced with DMEM medium containing 100 mg / mL hydrogel extract, while the control group was replaced with fresh DMEM medium. Cultured for another day. Subsequently, the liquid was removed, and 100 μL of DMEM medium and 10 μL of CCK-8 solution were added, followed by incubation for another 1 h. Absorbance was measured at 450 nm using a microplate reader. Five replicates were set up for each sample, and cell viability was calculated as follows: Cell viability (%) = (Absorbance of experimental group / Absorbance of control group) × 100% The experimental results are shown in Figure 2 The hyaluronic acid hydrogel products prepared in the examples and comparative examples did not have a significant inhibitory effect on human epithelial cells L929, indicating that the hyaluronic acid hydrogel products have high biocompatibility.

[0054] Example 6 The hyaluronic acid hydrogel products prepared in Examples 1 and 2 and Comparative Examples 1 and 2 were tested for their resistance to hyaluronidase degradation. After enzymatic hydrolysis, the free sodium hyaluronate content was determined using the carbazole sulfate method according to the national standard YY / T 0962-2021. 0.5 g of the hyaluronic acid hydrogel product was weighed and added to 2 mL of pH 7.0, 0.01 M PBS buffer and 2 mL of hyaluronidase solution (1200 U, 600 U / mL). ）After mixing thoroughly, place in a 37℃ water bath for 12 hours, then boil to remove enzymes. Centrifuge at 10000 rpm for 15 minutes, collect the supernatant, and measure the absorbance at 530 nm. Prepare a standard hyaluronic acid sample solution according to the carbazole colorimetric method in the national standard YY / T 0962-2021, measure the absorbance at 530 nm, plot a standard curve, and calculate the hyaluronic acid content based on the detected absorbance of the sample. This content is the content of hyaluronic acid degraded by hyaluronidase.

[0055] Solids retention rate after enzymatic hydrolysis (%) = (Initial content of cross-linked hyaluronic acid) (Content of degraded hyaluronic acid) / Initial content of cross-linked hyaluronic acid × 100% The experimental results are shown in Figure 3 The hyaluronic acid hydrogel products prepared in Examples 1 and 2 have a higher resistance to hyaluronidase degradation than those prepared in Comparative Examples 1 and 2. After enzymatic hydrolysis with high concentration of hyaluronidase solution, the solid content retained was 39.47% and 41.08%, respectively, while that retained was only 9.66% and 20.69%, respectively, in Comparative Examples 1 and 2.

[0056] Example 7 SD rats were divided into four groups of three rats each. After ether anesthesia, the backs of the rats were shaved, and the hyaluronic acid hydrogel products of Examples 1 and 2 and Comparative Examples 1 and 2 were injected subcutaneously at a dose of 200 µL / site. Each type of hydrogel was injected at three sites. The first day of injection was recorded as Day 1. The volume of hydrogel at the injection site was measured and recorded weekly using calipers. The appearance of the animal's skin and the animal's health status were also assessed and recorded. Changes in hydrogel volume are shown below. Figure 4 As shown, Examples 1 and 2 exhibited less in vivo swelling and more stable in vivo survival compared to Comparative Examples 1 and 2.

[0057] On day 11 post-injection, tissue samples were taken from the injection site. HE staining was performed on the injection site and surrounding skin tissue to assess the reaction. Results are shown below. Figure 5 The hyaluronic acid hydrogels prepared in Examples 1 and 2 showed less inflammatory cell infiltration than those in Comparative Examples 1 and 2, indicating that the hyaluronic acid hydrogel products prepared in Examples 1 and 2 have anti-inflammatory activity.

[0058] In summary, the preparation method and application of the hyaluronic acid hydrogel of the present invention have high cross-linking efficiency, can efficiently and deeply remove the cross-linking agent BDDE, and the obtained hyaluronic acid hydrogel product has a high composite shear modulus and a lower BDDE residue, which can effectively reduce the inflammatory response during tissue injection and improve the biosafety of the product.

Claims

1. A method for removing residual BDDE crosslinking agent from crosslinked hyaluronic acid hydrogels, characterized in that, Includes the following steps: (1) Composite solution treatment: After adjusting the pH of the cross-linked hyaluronic acid hydrogel to near neutral, it is contacted with a composite solution containing salt and organic solvent for 2 to 12 hours for synergistic precipitation treatment, and the resulting cross-linked hyaluronic acid hydrogel precipitate is collected. (2) Sterilization with stabilizer: The cross-linked hyaluronic acid hydrogel precipitate collected in step (1) is mixed with the stabilizer aqueous solution and sterilized by moist heat to obtain a cross-linked hyaluronic acid hydrogel product with BDDE residue removed.

2. The method as described in claim 1, characterized in that, Step (1) satisfies one or more of the following conditions: i. Adjust the pH to 6.5-7.5; ii. The pH is adjusted to near neutral using a hydrochloric acid solution; more preferably, the mass concentration of the hydrochloric acid solution is 0.2-2%; iii. The organic solvent is selected from at least one of polyethylene glycol, acetone, ethanol, and isoamyl alcohol, and the volume percentage concentration of the selected organic solvent is 50% to 100%, more preferably 70% to 95%; iv. The volume ratio of the cross-linked hyaluronic acid hydrogel to the organic solvent in the composite solution is 1:(1~10), more preferably 1:(2~7); v. The salt is an inorganic salt and / or a quaternary ammonium salt, wherein the inorganic salt is selected from at least one of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium carbonate, and ammonium sulfate; the quaternary ammonium salt is selected from at least one of hexadecylpyridine chloride, hexadecylpyridine bromide, and hexadecyltrimethylammonium bromide; more preferably, when the salt is an inorganic salt, the final concentration of the inorganic salt in the crosslinked hyaluronic acid hydrogel co-precipitation treatment system is 0.1~50% (w / v, g / mL), more preferably... 0.5~15% (w / v, g / mL); when the salt is a quaternary ammonium salt, the final concentration of the quaternary ammonium salt in the cross-linked hyaluronic acid hydrogel co-precipitation treatment system is 0.01~5% (w / v, g / mL), more preferably 0.1~1.5% (w / v, g / mL); when the salt is an inorganic salt and a quaternary ammonium salt, the final concentration of the inorganic salt is 0.5~10% (w / v, g / mL), and the final concentration of the quaternary ammonium salt is 0.1~1.0% (w / v, g / mL). vi. The conditions for the synergistic precipitation treatment are: pressure 1~5 atm, temperature -20℃~30℃; vii. The cross-linked hyaluronic acid hydrogel is prepared by the following steps: hyaluronic acid or its salt with a molecular weight of 10 kDa to 290 kDa is dissolved in an alkaline solution, the final concentration of hyaluronic acid or its salt is 6% to 18%, and reacted with the cross-linking agent BDDE at 30 to 60°C for 30 minutes to 2 hours to obtain the cross-linked hyaluronic acid hydrogel; wherein, the mass ratio of BDDE to hyaluronic acid or its salt is 1 to 8:100; more preferably, the alkaline solution is a 1% NaOH solution.

3. The method as described in claim 1, characterized in that, The stabilizer mentioned in step (2) is a bioactive molecule containing amino and hydroxyl groups, selected from at least one of amino acids, monosaccharides, disaccharides, oligosaccharides, polyols, and vitamins; More preferably, the stabilizer includes glycine, mannitol, glycerol, and trehalose.

4. The method as described in claim 1, characterized in that, The concentration of the stabilizer in the stabilizer aqueous solution mentioned in step (2) is 1~15%; Preferably, the solvent for the stabilizer aqueous solution in step (2) is water or a 0.01M PBS buffer at pH 7.

2.

5. The method as described in claim 1, characterized in that, In step (2), the final concentration of hyaluronic acid in the mixture of cross-linked hyaluronic acid hydrogel precipitate and stabilizer aqueous solution is 2-5%.

6. The method as described in claim 1, characterized in that, Before mixing the collected cross-linked hyaluronic acid precipitate with the stabilizer aqueous solution in step (2), it is also dried to reduce its free water content to less than 5%; More preferably, the drying process is freeze-drying or oven drying; More preferably, the freeze-drying temperature is below -20°C; and the drying temperature is 40°C to 70°C.

7. The method as described in claim 1, characterized in that, The moist heat sterilization described in step (2) is sterilization at 121℃ for 10~30 minutes.

8. The cross-linked hyaluronic acid hydrogel product prepared according to the method of claim 1 has a BDDE residue of less than 0.12 µg / g.

9. Use of the cross-linked hyaluronic acid hydrogel of claim 8 in the preparation of products for tissue filling, tissue repair or medical aesthetics.

10. The application as described in claim 9, characterized in that, The product in question is an injectable product.

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