Injectable interlocking cross-linked hydrogel of hyaluronan or salt thereof, preparation method therefor and use thereof

Abstract

An injectable interlocking cross-linked hydrogel of hyaluronan or a salt thereof, a preparation method therefor and the use thereof. The preparation process of the hydrogel comprises a two-step cross-linking step of the hyaluronan or the salt thereof with a crosslinking agent, such as an endogenous polyamine, and a dialysis step. In the two-step cross-linking, the first cross-linking step forms a network structure, and the second cross-linking step further forms an interlocking structure. The prepared hydrogel is subjected to moist heat sterilization. The prepared hydrogel has the advantages of a strong anti-degradation ability, excellent viscosity, good support, easy injection, high safety, and easy reconstitution, and thus meets the clinical requirements for injectable products. Moreover, the whole process is simple, convenient and rapid to operate, and is also easy to industrialize.

Description

Injectable interlocking cross-linked hyaluronic acid or its saline gel, preparation method and application Technical Field

[0001] The present invention relates to the field of biomedical materials, and in particular to an injectable interlocking cross-linked hyaluronic acid or saline gel thereof, a preparation method and application thereof. Background Art

[0002] Hyaluronic acid (HA) and its salts are highly biocompatible, macromolecular polysaccharide compounds that are widely distributed in the extracellular matrix of connective tissue in animals and humans and have been commercialized for use in medicine and cosmetics. A large amount of HA is distributed in human skin tissue, where it plays a role in water retention, maintaining extracellular space, promoting cell repair, bonding the skin tissue interface, and providing lubrication. Therefore, it is often used as a filler matrix for facial rejuvenation and contour repair treatments. However, unmodified HA has drawbacks when injected into skin tissue, such as poor mechanical properties, low viscosity, and a short in vivo retention time. Optimizing its properties through chemical methods is a common strategy for the application of sodium hyaluronate.

[0003] Most commercially available hyaluronic acid fillers use BDDE as a cross-linking agent, and it is well known that even very low concentrations of BDDE residues may be genotoxic. Due to the safety risks associated with BDDE, the annual dosage of commercially available dermal filler products is limited to no more than 20 mL per year (e.g. ), the maximum dose per use is limited to 6 mL (e.g. ). Therefore, safer and more effective cross-linking agents are urgently needed for the future development of hyaluronic acid fillers.

[0004] Patent CN113499480 reports a physical and chemical double-network hydrogel for subdermal fillers, its preparation method, and application. In this patent, HA is first cross-linked and modified using chemical methods, and then the cross-linked gel is secondary cross-linked using physical freeze-thaw cycles. Although the molecular lattice of the polymer material changes during the freeze-drying process, physical cross-linking is still not as stable as chemical bonds. Patent CN113896915 reports the use of endogenous polyamines including spermine and / or spermidine as new cross-linking agents to prepare cross-linked sodium hyaluronate. However, the gel prepared by this process has relatively low viscosity and relatively large thrust. At the same time, under this cross-linking system, more ester bond structures will be formed. When the sodium hyaluronate content is low (such as less than 15 mg / mL), "hydrogel separation" is likely to occur, thus limiting its clinical use as an injectable filler product. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the present invention aims to provide an injectable interlocking cross-linked hyaluronic acid or its saline gel, a preparation method and application.

[0006] The injectable, cross-linked hyaluronic acid or saline hydrogel of the present invention utilizes double cross-linked interlocking technology (DCIT) to form an interlocking network structure in a two-step chemical cross-linking reaction. First, hyaluronic acid or its salt and an endogenous polyamine cross-linker form a network-like gel in the first step, followed by a second cross-linking step to form an interlocking hydrogel.

[0007] The present invention is achieved through the following technical solutions.

[0008] In a first aspect, the present invention provides an injectable interlocked cross-linked hyaluronic acid or its saline hydrogel, which is obtained by a two-step cross-linking reaction. First, hyaluronic acid or its salt and a cross-linking agent are subjected to a first cross-linking reaction to form a network structure. Subsequently, hyaluronic acid or its salt and a cross-linking agent are added and interspersed in the network structure, and a second cross-linking reaction is carried out to obtain an injectable interlocked cross-linked hyaluronic acid or its saline hydrogel.

[0009] The hyaluronic acid or its salt is selected from one or more of sodium hyaluronate, zinc hyaluronate, potassium hyaluronate and calcium hyaluronate, preferably sodium hyaluronate.

[0010] The molecular weight of the hyaluronic acid or its salt is 500-2600 KDa.

[0011] The cross-linking agent is an endogenous polyamine, including spermidine, spermine and its derivatives.

[0012] The particle size D50 of the hyaluronic acid or its saline gel is 50 to 650 μm.

[0013] The concentration of hyaluronic acid or its salt in the injectable interlocked cross-linked hyaluronic acid or its salt gel is 10-25 mg / mL.

[0014] In the first cross-linking reaction, the molar ratio of the cross-linking agent to the hyaluronic acid or its salt is 0.01 to 0.1, and in the second cross-linking reaction, the molar ratio of the cross-linking agent to the hyaluronic acid or its salt is 0.01 to 0.1.

[0015] The first cross-linking step in the two-step cross-linking forms a network structure of hyaluronic acid or its salt, and the second cross-linking step further forms an interlocking structure, thereby achieving an upgrade from an interpenetrating structure to an interlocking structure.

[0016] Wherein, the molar amount of hyaluronic acid or its salt = the mass of hyaluronic acid or its salt used / the molecular weight of the disaccharide repeating unit of hyaluronic acid or its salt 403;

[0017] Preferably, the cross-linking process requires the addition of a catalyst, which is selected from one or more of carbodiimide, phosphonium bromide salt formed by triphenylphosphine and bromide, carbonium salt and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM).

[0018] In the first cross-linking reaction, the molar ratio of the catalyst to the hyaluronic acid or its salt is 0.6-3. In the second cross-linking reaction, the molar ratio of the catalyst to the hyaluronic acid or its salt is 0.03-0.3.

[0019] Furthermore, after the two-step cross-linking reaction, a dialysis step is further included to obtain the injectable interlocked cross-linked hyaluronic acid or its saline gel.

[0020] Preferably, the dialysis is performed first with an alkaline dialysis fluid and then with a neutral dialysis fluid.

[0021] The interlocked cross-linked hyaluronic acid or its saline gel needs to be dialyzed under appropriate alkaline concentration conditions to remove unstable ester bonds, ultimately obtaining the injectable interlocked cross-linked hyaluronic acid or its saline gel. This has the advantages of strong degradation resistance, excellent viscosity, good support, easy push injection, and high safety, and can be used for subcutaneous injection filling.

[0022] In a second aspect, the present invention provides a method for preparing the injectable interlocked cross-linked hyaluronic acid or saline gel thereof according to the first aspect, comprising the following steps:

[0023] (1) mixing a cross-linking agent with hyaluronic acid or a salt thereof to prepare a mixed solution A of the cross-linking agent and hyaluronic acid or a salt thereof;

[0024] (2) adding a catalyst to the mixed solution A of the cross-linking agent and hyaluronic acid or its salt to perform a first cross-linking reaction to obtain a cross-linked gel with a network structure;

[0025] (3) mixing a cross-linking agent with hyaluronic acid or a salt thereof to prepare a mixed solution B of the cross-linking agent and hyaluronic acid or a salt thereof;

[0026] (4) adding the mixed solution B obtained in step (3) to the network structure cross-linked gel obtained in step (2), performing a second cross-linking reaction under the action of a catalyst to obtain a cross-linked gel with an interlocking structure, and dialyzing the gel.

[0027] The interlocking cross-linked hyaluronic acid or its saline gel of the present invention adopts double cross-linked interlock technology (DCIT). In the first cross-linking process, the carboxyl groups in the hyaluronic acid or its salt (hereinafter referred to as HA) molecules and the multiple amino sites of spermine or spermidine can form amide bonds under the action of a catalyst to form a network structure; at the same time, the carboxyl groups in the HA molecules and the hydroxyl groups activated by the catalyst can form ester bonds, which have a placeholder effect; then the HA solution, cross-linking agent and catalyst are added to carry out the second cross-linking process; during this process, the HA molecules and the cross-linking agent can be evenly inserted into the network structure formed in the first step under the stirring force, and then amide bonds are formed under the action of the catalyst to form an interlocking structure.

[0028] Preferably, the cross-linking agent in steps (1) and (3) is an endogenous polyamine, and more preferably includes spermidine, spermine and its derivatives.

[0029] Preferably, in steps (1) and (3), the cross-linking agent is first dissolved in water and the pH is adjusted to 6.0 to 6.3.

[0030] The hyaluronic acid or its salt in steps (1) and (3) is selected from one or more of sodium hyaluronate, zinc hyaluronate, potassium hyaluronate and calcium hyaluronate, preferably sodium hyaluronate.

[0031] Furthermore, the molecular weight of hyaluronic acid or its salt is 500 to 2600 KDa.

[0032] Preferably, the mixing and dissolving time in steps (1) and (3) is 3 to 5 hours, for example: 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours.

[0033] In the mixed solution A and the mixed solution B, the mass concentration of the hyaluronic acid or its salt is 1% to 5%.

[0034] Preferably, in the mixed solution A and the mixed solution B, the molar ratio of the crosslinker to hyaluronic acid or its salt is 0.01-0.1, and the amount of the crosslinker in the two solutions can be the same or different, for example: 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1.

[0035] More preferably, the molar ratio of the cross-linking agent to the hyaluronic acid or its salt in step (1) is lower than the molar ratio of the cross-linking agent to the hyaluronic acid or its salt in step (3), that is, the cross-linking degree of the first step reaction is lower than the cross-linking degree of the second step reaction. This is because the first step cross-linking reaction prepares a relatively loose first network structure, which makes it easy for the hyaluronic acid or its salt (HA) molecules added in the second step to penetrate. After the second step cross-linking reaction, spatial structures of different levels are constructed, which is more conducive to the formation of an interlocking structure.

[0036] Preferably, the cross-linking process requires a catalyst, and the catalysts in steps (2) and (4) are selected from one or more of carbodiimide, phosphonium bromide salt formed by triphenylphosphine and bromide, carbonium salt and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM), and the two may be the same or different.

[0037] Preferably, the catalyst in steps (2) and (4) is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and an auxiliary agent is added to improve the efficiency of the cross-linking reaction.

[0038] Preferably, the auxiliary agent is selected from any one or more of N-hydroxysuccinimide (NHS), sulfonated N-hydroxysuccinimide (Sulfo-NHS), tert-butyl alcohol, and 1-hydroxybenzotriazole (HOBt), more preferably NHS. The molar ratio of the auxiliary agent to the catalyst is 0.2 to 0.4.

[0039] Preferably, the molar ratio of the catalyst in step (2) to the hyaluronic acid or its salt in the mixed solution A is 0.6-3, for example, 0.6, 1, 1.5, 2, 2.5, 3, more preferably 1-2.

[0040] The cross-linking reaction time in step (2) is 1 to 3 hours, for example: 1 hour, 2 hours, 3 hours.

[0041] In the first cross-linking reaction of step (2), a larger amount of catalyst is added to form more ester bonds, which plays a sufficient "placeholder" role, which is beneficial to improving the cross-linking efficiency of the second cross-linking reaction. At the same time, the amount of catalyst used in the second cross-linking reaction is reduced, so that the cross-linking reaction can construct more interlocking structures under the reaction conditions of low-dose catalyst and safe cross-linking agent to improve the anti-degradation performance of the final product.

[0042] Preferably, in step (4), the molar ratio of the catalyst to the hyaluronic acid or its salt in the mixed solution B is 0.03 to 0.3, for example: 0.03, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3.

[0043] The cross-linking reaction time in step (4) is 1 to 24 hours, for example: 1 hour, 2 hours, 3 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours.

[0044] Preferably, the dialysis in step (4) is a two-step dialysis, the first step being alkaline dialysis and the second step being neutral dialysis.

[0045] In order to fully form an interlocking structure gel, it is necessary to form an ester bond structure substance (including EDC active ester, NHS active ester, ester formed by HA hydroxyl and carboxyl groups, etc.) in advance in the first cross-linking reaction. However, the ester bond structure substance is unstable and will affect the stability of the gel product. The present invention uses a later alkaline dialysis treatment to open the ester bond formed in advance during the alkaline dialysis process, releasing a linear HA chain. This part of the HA chain not only plays a lubricating role and improves the injectability of the product, but also the HA chain can be further entangled or interlaced with the formed interlocking structure, thereby enhancing the intermolecular interaction force and improving the viscosity of the product.

[0046] Preferably, the molar ratio of the hydroxide in the alkaline dialysate to the total amount of hyaluronic acid or its salt used in steps (1) and (3) (the total amount in mixed solution A and mixed solution B) is 1:1 to 10:1, for example: 1:1, 5:1, 10:1.

[0047] The solute of the alkaline dialysate is selected from sodium hydroxide or potassium hydroxide, and the solvent of the alkaline dialysate is any one or more combinations of purified water, physiological saline or phosphate buffer.

[0048] In the two-step cross-linking reaction process, the unstable ester bond structure substances produced can be destroyed after the alkaline dialysis process, which can improve the resistance to moist heat sterilization and storage stability of the final gel product; at the same time, alkaline conditions will also destroy the glycosidic bond of HA to a certain extent. Therefore, it is necessary to control the molar ratio of hydroxide to hyaluronic acid or its salt in the alkaline dialysis fluid. If the molar amount of hydroxide is low, the unstable ester bond structure substances cannot be effectively removed, and the stability of the hydrogel product cannot be guaranteed; if the molar amount of hydroxide is high, the glycosidic bond of HA will be destroyed, the network structure of the cross-linked HA will not be stable enough and easily degraded, or the HA concentration in the product will decrease too quickly, which cannot meet clinical injection requirements.

[0049] Further preferably, in the alkaline dialysis, the volume ratio of the dialysate to the cross-linked gel of the interlocking structure is 15:1 to 25:1, the dialysis temperature is 10 to 40°C, for example: 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C; the dialysis time is 1 to 10h, for example: 1h, 2h, 4h, 6h, 8h, 10h.

[0050] Preferably, in the neutral dialysis, the dialysate includes any one or more combinations of purified water, physiological saline or phosphate buffer, and the pH of the neutral dialysate may be 6.0 to 7.6.

[0051] Further preferably, in the neutral dialysis, the volume ratio of the dialysate to the cross-linked gel of the interlocking structure is 15:1 to 30:1, for example: 15:1, 17:1, 20:1, 23:1, 25:1, 27:1, 30:1; the dialysis temperature is 10 to 40°C, and the dialysis time is 1 to 20 hours.

[0052] Before alkaline dialysis treatment, the cross-linked gel with an interlocking structure obtained by the second cross-linking reaction is cut into small pieces with an equivalent diameter of 0.02 to 1 cm, for example: 0.02 cm, 0.05 cm, 0.1 cm, 0.15 cm, 0.2 cm, 0.25 cm, 0.3 cm, 0.35 cm, 0.4 cm, 0.45 cm, 0.5 cm, 0.55 cm, 0.6 cm, 0.65 cm, 0.7 cm, 0.75 cm, 0.8 cm, 0.85 cm, 0.9 cm, 0.95 cm, and 1 cm.

[0053] Preferably, the molecular weight cut-off (MWCO) of the dialysis bag is 1 to 100 KDa, for example: 1 KDa, 5 KDa, 10 KDa, 20 KDa, 30 KDa, 40 KDa, 50 KDa, 60 KDa, 70 KDa, 80 KDa, 90 KDa, 100 KDa.

[0054] Optionally, the preparation method includes step (5), wherein the dialysis is followed by dilution, crushing, filling, and moist heat sterilization to obtain the injectable interlocking cross-linked hyaluronic acid or its saline gel.

[0055] Preferably, the dilution solution for the interlocking cross-linked gel in step (5) is selected from one or more of purified water, physiological saline, a phosphate-balanced solution, or a phosphate buffer solution. When purified water is used, sodium dihydrogen phosphate or sodium hydrogen phosphate is used to adjust the osmotic pressure of the system to within the range of 200 mOsmol / kg to 400 mOsmol / kg and the pH value to within the range of 6.0 to 7.6.

[0056] Preferably, in step (5), lidocaine hydrochloride is added to the diluted cross-linked gel with an interlocking structure, and then the gel is crushed. The addition of lidocaine hydrochloride can reduce the pain during injection of hyaluronic acid or its saline gel.

[0057] Preferably, the concentration of lidocaine hydrochloride is 1-5 mg / mL, for example: 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL.

[0058] Preferably, the particle size D50 of the gel after crushing in step (5) is 50 to 450 μm, and the particle size D90 is 100 to 650 μm.

[0059] Preferably, the moist heat sterilization parameters in step (5) are 116-121° C. and 15-30 min.

[0060] Preferably, in step (5), the concentration of hyaluronic acid or its salt in the injectable interlocking cross-linked hyaluronic acid or its saline gel is 10-25 mg / mL, for example: 10 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 23 mg / mL, 25 mg / mL.

[0061] In a third aspect, the present invention provides the use of the injectable interlocked cross-linked hyaluronic acid or its saline gel described in the first aspect, or the injectable interlocked cross-linked hyaluronic acid or its saline gel obtained according to the preparation method described in the second aspect, in the preparation of tissue filling and repair materials or drug carriers for non-therapeutic purposes.

[0062] Specifically, the tissue filling can be used to eliminate wrinkles (such as wrinkles around the eyes, forehead wrinkles, frown lines, perioral wrinkles, nasolabial folds, tear grooves, nasolabial folds, neck wrinkles, hand wrinkles, stretch marks, etc.), anti-aging, eliminate scars, wound repair, intraoperative and postoperative venous hemostasis, etc.

[0063] The tissue repair material can be a bone tissue repair material, a cartilage tissue repair material, a corneal tissue repair material, a cardiovascular tissue repair material, a liver tissue repair material, etc.

[0064] The drug uses interlocked cross-linked hyaluronic acid or its saline gel as a carrier to achieve the purposes of sustained release, controlled release, targeted drug delivery, etc.

[0065] Beneficial effects of the present invention:

[0066] 1. The interlocking cross-linked hydrogel of the present invention uses endogenous polyamines as cross-linking agents, and compared with existing gels, the gel has higher safety.

[0067] 2. The interlocking structure cross-linked hydrogel of the present invention adopts double cross-linked interlock technology (DCIT), and undergoes two cross-linking reactions in succession. By controlling the amount of cross-linking agent and catalyst, the ester bond placeholder role is fully utilized in the first cross-linking reaction, thereby improving the efficiency of the second cross-linking reaction and realizing the upgrade from interpenetrating network to interlocking structure; further, through the alkaline dialysis process, the ester bond originally used for placeholder in the cross-linking reaction process is destroyed, and the released HA chain not only enhances the injectability of the product, but also further entangles with the interlocking structure, enhancing the interaction force between molecules. The prepared gel exhibits high support performance and viscosity as well as excellent resistance to enzymatic hydrolysis, meeting the clinical demand for injectable products.

[0068] Third, the interlocking cross-linked hydrogel of the present invention utilizes a highly efficient alkaline dialysis process, enabling rapid removal of processing aids and ensuring that the hyaluronic acid or salt concentration in the post-dialysis gel is higher than that in the intended product. This eliminates the need for subsequent concentration steps, improving production efficiency while also preventing damage to the polymer lattice and the resulting difficulty in redissolution during the concentration process. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] FIG1 is a schematic diagram showing the gel cross-linking reaction process of the present invention;

[0070] FIG2 shows the appearance of the gel product of the present invention, wherein A represents the sample of Example 2 and B represents the sample of Comparative Example 1. DETAILED DESCRIPTION

[0071] The technical solutions of the present invention are described clearly and completely below in conjunction with the embodiments and accompanying drawings. The described embodiments are only a portion of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0072] It should be noted that, unless otherwise specified, the experimental methods and reagents used in the embodiments of the present invention are conventional experimental methods and reagents in the art.

[0073] Example 1 Preparation of Injectable Interlocking Cross-linked Sodium Hyaluronate Hydrogel

[0074] (1) Prepare an 8% aqueous solution of spermidine at pH 6.0 and refrigerate until ready for use. Weigh 0.9 g of this solution and disperse it in 250 mL of purified water. Add 5 g of sodium hyaluronate powder (molar ratio of spermidine to sodium hyaluronate: 0.04, HA concentration: 20 mg / mL) and stir until completely dissolved. Dissolve for 3 h.

[0075] (2) Then, 2.37 g of EDC (the molar ratio of EDC to sodium hyaluronate was 1.00) and 0.48 g of NHS were weighed and added to the above mixed solution. After stirring at room temperature for 40 min, the system was placed in a 40°C environment for cross-linking reaction for 3 h to obtain a network cross-linked gel.

[0076] (3) Weigh 0.9 g of spermidine solution and disperse it in 250 mL of purified water. Add 5 g of sodium hyaluronate powder (the molar ratio of spermidine to sodium hyaluronate is 0.04, and the concentration of HA is 20 mg / mL) and stir until it is completely dissolved. The dissolution time is 3 h.

[0077] (4) The mixed solution from step (3) was added to the network cross-linked gel from step (2). After stirring for 10 minutes, the mixture was pulverized using a homogenizer at 10,000 rpm for 3 minutes, followed by a 5-minute pause, and repeated five times. 0.284 g of EDC (the molar ratio of EDC to sodium hyaluronate was 0.12) and 0.057 g of NHS were then weighed and added to the system. After further stirring at room temperature for 40 minutes, the system was placed at 40°C for a cross-linking reaction for 16 hours to obtain an interlocking cross-linked gel.

[0078] Use scissors to cut the interlocking cross-linked gel into small pieces with an equivalent diameter of 0.5 cm, place them in a dialysis bag, seal the ends of the dialysis bag with clips, and place them in 9L of phosphate solution containing 2g of sodium hydroxide (the molar ratio of hydroxide to sodium hyaluronate is 2) for dialysis. The dialysis temperature is 40°C. After 2 hours, replace it with 9L of neutral phosphate solution for dialysis. The dialysis temperature is 25°C, and the solution is changed every 3 hours. The dialysis is carried out overnight for a total of 20 hours. Before dialysis, accurately weigh the mass of the blank dialysis bag (M0) and the dialysis bag after sample loading (M1). After dialysis, accurately weigh the mass of the dialysis bag and gel (M2). The initial HA concentration (N) is 20 mg / mL. Calculate the sodium hyaluronate content (C) of the gel after dialysis:

[0079] (5) Dilute the mixture to a concentration of 15 mg / mL of sodium hyaluronate at concentration C, add lidocaine hydrochloride, and use the same diluent as the neutral dialysate. The content of lidocaine hydrochloride in the gel is 3 mg / mL. The reconstituted gel is crushed and filled into a pre-filled syringe. Sterilize the mixture by moist heat at 121°C for 15 minutes to obtain an injectable interlocking cross-linked sodium hyaluronate hydrogel.

[0080] Example 2 Preparation of Injectable Interlocking Cross-linked Sodium Hyaluronate Hydrogel

[0081] (1) Prepare an 8% aqueous solution of spermidine at pH 6.0 and refrigerate until ready for use. Weigh 0.68 g of this solution and disperse it in 250 mL of purified water. Add 5 g of sodium hyaluronate powder (molar ratio of spermidine to sodium hyaluronate: 0.03, HA concentration: 20 mg / mL) and stir until completely dissolved. Dissolve for 3 h.

[0082] (2) 2.37 g of EDC (the molar ratio of EDC to sodium hyaluronate is 1.00) and 0.48 g of NHS were weighed and added to the above mixed solution. After stirring at room temperature for 40 min, the system was placed in a 40°C environment for cross-linking reaction for 3 h to obtain a network cross-linked gel.

[0083] (3) Weigh 1.13 g of spermidine solution and disperse it in 250 mL of purified water. Add 5 g of sodium hyaluronate powder (the molar ratio of spermidine to sodium hyaluronate is 0.05, and the concentration of HA is 20 mg / mL) and stir until it is completely dissolved. The dissolution time is 3 h.

[0084] (4) The mixed solution from step (3) was added to the network cross-linked gel from step (2). After stirring for 10 minutes, the mixture was pulverized using a homogenizer at 10,000 rpm for 3 minutes, followed by a 5-minute pause. This was repeated five times. 0.355 g of EDC (the molar ratio of EDC to sodium hyaluronate was 0.15) and 0.071 g of NHS were then added to the system. Stirring was continued at room temperature for 40 minutes, and the system was placed at 40°C for a cross-linking reaction for 16 hours to obtain an interlocking cross-linked gel.

[0085] Use scissors to cut the interlocking cross-linked gel into small pieces with an equivalent diameter of 0.5 cm, place them in a dialysis bag, seal the ends of the dialysis bag with clips, and place them in 9L of phosphate solution containing 2g of sodium hydroxide (the molar ratio of hydroxide to sodium hyaluronate is 2) for dialysis. The dialysis temperature is 40°C. After 2 hours, replace it with 9L of neutral phosphate solution for dialysis. The dialysis temperature is 25°C, and the solution is changed every 3 hours. The dialysis is carried out overnight for a total of 20 hours. Before dialysis, accurately weigh the mass of the blank dialysis bag (M0) and the dialysis bag after sample loading (M1). After dialysis, accurately weigh the mass of the dialysis bag and gel (M2). The initial HA concentration (N) is 20 mg / mL. Calculate the sodium hyaluronate content (C) of the gel after dialysis:

[0086] (5) Dilute the mixture to a concentration of 15 mg / mL of sodium hyaluronate at concentration C, add lidocaine hydrochloride, and use the same diluent as the neutral dialysate. The content of lidocaine hydrochloride in the gel is 3 mg / mL. The reconstituted gel is crushed and filled into a pre-filled syringe. Sterilize the mixture by moist heat at 121°C for 15 minutes to obtain an injectable interlocking cross-linked sodium hyaluronate hydrogel.

[0087] Example 3 Preparation of Injectable Interlocking Cross-linked Sodium Hyaluronate Hydrogel

[0088] (1) Prepare an 8% aqueous solution of spermidine at pH 6.0 and refrigerate until ready for use. Weigh 1.13 g of this solution and disperse it in 250 mL of purified water. Add 5 g of sodium hyaluronate powder (molar ratio of spermidine to sodium hyaluronate: 0.05, HA concentration: 20 mg / mL) and stir until completely dissolved. Dissolve for 3 h.

[0089] (2) Then, 3.56 g of EDC (the molar ratio of EDC to sodium hyaluronate was 1.5) and 0.713 g of NHS were weighed and added to the above mixed solution. After stirring at room temperature for 40 min, the system was placed in a 40°C environment for cross-linking reaction for 3 h to obtain a network cross-linked gel.

[0090] (3) Weigh 0.68 g of spermidine solution and disperse it into 250 mL of purified water. Add 5 g of sodium hyaluronate powder (the molar ratio of spermidine to sodium hyaluronate is 0.03, and the concentration of HA is 20 mg / mL) and stir until it is completely dissolved. The dissolution time is 3 h.

[0091] (4) The mixed solution from step (3) was added to the network cross-linked gel from step (2). After stirring for 10 minutes, the mixture was pulverized using a homogenizer at 10,000 rpm for 3 minutes, followed by a 5-minute pause. This was repeated five times. 0.213 g of EDC (the molar ratio of EDC to sodium hyaluronate was 0.09) and 0.043 g of NHS were then weighed and added to the system. After further stirring at room temperature for 40 minutes, the system was placed at 40°C for a cross-linking reaction for 16 hours to obtain an interlocking cross-linked gel.

[0092] Use scissors to cut the interlocking cross-linked gel into small pieces with an equivalent diameter of 0.5 cm, place them in a dialysis bag, seal the ends of the dialysis bag with clips, and place them in 9L of phosphate solution containing 2g of sodium hydroxide (the molar ratio of hydroxide to sodium hyaluronate is 2) for dialysis. The dialysis temperature is 40°C. After 2 hours, replace it with 9L of neutral phosphate solution for dialysis. The dialysis temperature is 25°C, and the solution is changed every 3 hours. The dialysis is carried out overnight for a total of 20 hours. Before dialysis, accurately weigh the mass of the blank dialysis bag (M0) and the dialysis bag after sample loading (M1). After dialysis, accurately weigh the mass of the dialysis bag and gel (M2). The initial HA concentration (N) is 20 mg / mL. Calculate the sodium hyaluronate content (C) of the gel after dialysis:

[0093] (5) Dilute the mixture to a concentration of 15 mg / mL of sodium hyaluronate at concentration C, add lidocaine hydrochloride, and use the same diluent as the neutral dialysate. The content of lidocaine hydrochloride in the gel is 3 mg / mL. The reconstituted gel is crushed and filled into a pre-filled syringe. Sterilize the mixture by moist heat at 121°C for 15 minutes to obtain an injectable interlocking cross-linked sodium hyaluronate hydrogel.

[0094] Example 4 Preparation of Injectable Interlocking Cross-linked Sodium Hyaluronate Hydrogel

[0095] (1) Prepare an 8% aqueous solution of spermidine at pH 6.0 and refrigerate until ready for use. Weigh 0.68 g of this solution and disperse it in 250 mL of purified water. Add 5 g of sodium hyaluronate powder (molar ratio of spermidine to sodium hyaluronate: 0.03, HA concentration: 20 mg / mL) and stir until completely dissolved. Dissolve for 3 h.

[0096] (2) 1.422 g of EDC (the molar ratio of EDC to sodium hyaluronate is 0.6) and 0.285 g of NHS were weighed and added to the above mixed solution. After stirring at room temperature for 40 min, the system was placed in a 40°C environment for cross-linking reaction for 3 h to obtain a network cross-linked gel.

[0097] (3) Weigh 1.13 g of spermidine solution and disperse it in 250 mL of purified water. Add 5 g of sodium hyaluronate powder (the molar ratio of spermidine to sodium hyaluronate is 0.05, and the concentration of HA is 20 mg / mL) and stir until it is completely dissolved. The dissolution time is 3 h.

[0098] (4) The mixed solution from step (3) was added to the network cross-linked gel from step (2). After stirring for 10 minutes, the mixture was pulverized using a homogenizer at 10,000 rpm for 3 minutes, followed by a 5-minute pause. This was repeated five times. 0.355 g of EDC (the molar ratio of EDC to sodium hyaluronate was 0.15) and 0.071 g of NHS were then weighed and added to the system. After further stirring at room temperature for 40 minutes, the system was placed at 40°C for a cross-linking reaction for 16 hours to obtain a cross-linked gel with an interlocking structure.

[0099] Use scissors to cut the cross-linked gel with an interlocking structure into small pieces with an equivalent diameter of 0.5 cm, place them in a dialysis bag, seal the ends of the dialysis bag with clips, and place them in 9L of phosphate solution containing 2g of sodium hydroxide (the molar ratio of hydroxide to sodium hyaluronate is 2) for dialysis. The dialysis temperature is 40°C. After 2 hours, it is replaced with 9L of neutral phosphate solution for dialysis. The dialysis temperature is 25°C, and the solution is changed every 3 hours. The dialysis is carried out overnight for a total of 20 hours. Before dialysis, the mass of the blank dialysis bag (M0) and the dialysis bag after sample loading (M1) are accurately weighed. After dialysis, the mass of the dialysis bag and gel (M2) is accurately weighed. The initial HA concentration (N) is 20mg / mL. The sodium hyaluronate content (C) of the gel after dialysis is calculated as follows:

[0100] (5) Dilute the mixture to a concentration of 15 mg / mL of sodium hyaluronate at concentration C, add lidocaine hydrochloride, and use the same diluent as the neutral dialysate. The content of lidocaine hydrochloride in the gel is 3 mg / mL. The reconstituted gel is crushed and filled into a pre-filled syringe. Sterilize the mixture by moist heat at 121°C for 15 minutes to obtain an injectable interlocking cross-linked sodium hyaluronate hydrogel.

[0101] Example 5 Preparation of Injectable Interlocking Cross-linked Sodium Hyaluronate Hydrogel

[0102] (1) Prepare an 8% aqueous solution of spermine at pH 6.0 and refrigerate until ready for use. Weigh 0.94 g of this solution and disperse it in 250 mL of purified water. Add 5 g of sodium hyaluronate powder (molar ratio of spermine to sodium hyaluronate: 0.03, HA concentration: 20 mg / mL) and stir until completely dissolved. Dissolve for 3 hours.

[0103] (2) 4.74 g of EDC (the molar ratio of EDC to sodium hyaluronate is 2.00) and 0.95 g of NHS were weighed and added to the above mixed solution. After stirring at room temperature for 40 min, the system was placed in a 40°C environment for cross-linking reaction for 3 h to obtain a network cross-linked gel.

[0104] (3) Weigh 1.88 g of spermine solution and disperse it in 250 mL of purified water. Add 5 g of sodium hyaluronate powder (the molar ratio of spermine to sodium hyaluronate is 0.06, and the concentration of HA is 20 mg / mL) and stir until it is completely dissolved. The dissolution time is 3 h.

[0105] (4) The mixed solution from step (3) was added to the network cross-linked gel from step (2). After stirring for 10 minutes, the mixture was pulverized using a homogenizer at 10,000 rpm for 3 minutes, followed by a 5-minute pause, and repeated 5 times. 0.427 g of EDC (the molar ratio of EDC to sodium hyaluronate was 0.18) and 0.086 g of NHS were then weighed and added to the above system. After further stirring at room temperature for 40 minutes, the system was placed in a 40°C environment for cross-linking reaction for 16 hours to obtain a cross-linked gel with an interlocking structure.

[0106] Use scissors to cut the interlocking cross-linked gel into small pieces with an equivalent diameter of 0.5 cm, place them in a dialysis bag, seal the ends of the dialysis bag with clips, and place them in 9L of phosphate solution containing 4g of sodium hydroxide (the molar ratio of hydroxide to sodium hyaluronate is 8) for dialysis. The dialysis temperature is 40°C. After 2 hours, it is replaced with 9L of neutral phosphate solution for dialysis. The dialysis temperature is 25°C, and the solution is changed every 3 hours. The dialysis is carried out overnight for a total of 20 hours. Before dialysis, the mass of the blank dialysis bag (M0) and the dialysis bag after sample loading (M1) are accurately weighed. After dialysis, the mass of the dialysis bag and gel (M2) is accurately weighed. The initial HA concentration (N) is 20 mg / mL. The sodium hyaluronate content (C) of the gel after dialysis is calculated as follows:

[0107] (5) Dilute the mixture to a concentration of 15 mg / mL of sodium hyaluronate at concentration C, add lidocaine hydrochloride, and use the same diluent as the neutral dialysate. The content of lidocaine hydrochloride in the gel is 3 mg / mL. The reconstituted gel is crushed and filled into a pre-filled syringe. Sterilize the mixture by moist heat at 121°C for 15 minutes to obtain an injectable interlocking cross-linked sodium hyaluronate hydrogel.

[0108] Comparative Example 1 Preparation of Cross-linked Sodium Hyaluronate Hydrogel

[0109] (1) Prepare an 8% concentration of spermidine aqueous solution with a pH of 6.0 and refrigerate until ready for use. Weigh 1.13 g of this solution and disperse it in 250 mL of purified water. Add 5 g of sodium hyaluronate powder (the molar ratio of spermidine to sodium hyaluronate is 0.05, and the concentration of HA is 20 mg / mL) and stir until completely dissolved. The dissolution time is 3 h. Then weigh 0.356 g of EDC (the molar ratio of EDC to sodium hyaluronate is 0.15) and 0.071 g of NHS and add them to the above mixed solution. After stirring at room temperature for 40 min, the system is placed in a 40°C environment for cross-linking reaction for 3 h to prepare a single cross-linking degree gel (component one).

[0110] Separately, 0.68g of spermidine solution was dispersed in 250mL of purified water. 5g of sodium hyaluronate powder was added (molar ratio of spermidine to sodium hyaluronate: 0.03, HA concentration: 20mg / mL) and stirred for complete dissolution for 3 hours. 0.213g of EDC (molar ratio of EDC to sodium hyaluronate: 0.09) and 0.043g of NHS were then added to the sodium hyaluronate gel system. After stirring at room temperature for 40 minutes, the system was placed at 40°C for a cross-linking reaction for 3 hours to produce a single-crosslinking gel (Component 2).

[0111] (2) Mix the above-mentioned component 1 and component 2 gels in a mass ratio of 1:1 in the same beaker, cut the gel into small pieces with an equivalent diameter of 0.5 cm with scissors, put them into a dialysis bag, seal the two ends of the dialysis bag with clips, and place them in 9L of phosphate solution containing 2g of sodium hydroxide (the molar ratio of hydroxide to sodium hyaluronate is 2) for dialysis. The dialysis temperature is 40℃. After 2h, change to 9L of neutral phosphate solution for dialysis. The dialysis temperature is 25℃. The solution is changed every 3h and the dialysis is carried out overnight for a total of 20h. Accurately weigh the mass of the blank dialysis bag (M0) and the mass of the dialysis bag after sample loading (M1) before dialysis. Accurately weigh the mass of the dialysis bag and gel after dialysis (M2). The initial HA concentration (N) is 20mg / mL. Calculate the sodium hyaluronate content (C) of the gel after dialysis:

[0112] (3) Dilute the sodium hyaluronate to a concentration of 15 mg / mL at concentration C, add lidocaine hydrochloride, and use the same diluent as the neutral dialysate. The content of lidocaine hydrochloride in the gel is 3 mg / mL. The reconstituted cross-linked gel is crushed and filled into a pre-filled syringe. Sterilize by moist heat at 121°C for 15 minutes to obtain a cross-linked sodium hyaluronate hydrogel.

[0113] Comparative Example 2 Preparation of Cross-linked Sodium Hyaluronate Hydrogel

[0114] An interlocking cross-linked gel was prepared using the process parameters described in Example 2, except that alkaline dialysis was omitted. During the dialysis step, dialysis was performed using only 9 L of neutral phosphate solution at 25°C, with the solution changed every 3 hours. Dialysis was continued overnight for a total of 20 hours. The reconstituted gel was crushed, filled into prefilled syringes, and sterilized by moist heat at 121°C for 15 minutes to obtain a cross-linked sodium hyaluronate hydrogel.

[0115] Comparative Example 3 Preparation of Cross-linked Sodium Hyaluronate Hydrogel

[0116] (1) Prepare an 8% concentration of spermidine aqueous solution with a pH of 6.0 and refrigerate until ready for use. Weigh 2.25 g of this solution and disperse it in 500 mL of purified water. Add 10 g of sodium hyaluronate powder (the molar ratio of spermidine to sodium hyaluronate is 0.05, and the concentration of HA is 20 mg / mL) and stir until completely dissolved. The dissolution time is 3 h. Then weigh 0.711 g of EDC (the molar ratio of EDC to sodium hyaluronate is 0.15) and 0.143 g of NHS and add them to the above mixed solution. After stirring at room temperature for 40 min, the system is placed in a 40°C environment for cross-linking reaction for 16 h to prepare a single cross-linking degree gel.

[0117] (2) Use scissors to cut the gel into small pieces with an equivalent diameter of 0.5 cm, put them into a dialysis bag, seal the two ends of the dialysis bag with clips, and place it in 9L of phosphate solution containing 2g of sodium hydroxide (the molar ratio of hydroxide to sodium hyaluronate is 2) for dialysis. The dialysis temperature is 40℃, and the solution is changed every 2 hours. Then it is replaced with 9L of neutral phosphate solution for dialysis. The dialysis temperature is 25℃, and the solution is changed every 3 hours. The dialysis is carried out overnight for a total of 20 hours. Before dialysis, the mass of the blank dialysis bag (M0) and the mass of the dialysis bag after sample loading (M1) are accurately weighed. After dialysis, the mass of the dialysis bag and gel (M2) is accurately weighed. The initial HA concentration (N) is 20mg / mL. The sodium hyaluronate content (C) of the gel after dialysis is calculated as follows:

[0118] (3) Dilute the sodium hyaluronate to a concentration of 15 mg / mL at concentration C, add lidocaine hydrochloride, and use the same diluent as the neutral dialysate. The content of lidocaine hydrochloride in the gel is 3 mg / mL. The reconstituted cross-linked gel is crushed and filled into a pre-filled syringe. Sterilize by moist heat at 121°C for 15 minutes to obtain a cross-linked sodium hyaluronate hydrogel.

[0119] Comparative Example 4 Preparation of Cross-linked Sodium Hyaluronate Gel

[0120] (1) Prepare an 8% aqueous solution of spermidine at pH 6.0 and refrigerate until ready for use. Weigh 0.68 g of this solution and disperse it in 250 mL of purified water. Add 5 g of sodium hyaluronate powder (molar ratio of spermidine to sodium hyaluronate: 0.03, HA concentration: 20 mg / mL) and stir until completely dissolved. Dissolve for 3 h.

[0121] (2) 0.711 g of EDC (the molar ratio of EDC to sodium hyaluronate is 0.3) and 0.143 g of NHS were added to the above mixed solution. After stirring at room temperature for 40 min, the system was placed in a 40°C environment for cross-linking reaction for 3 h to obtain a network cross-linked gel.

[0122] (3) Weigh 1.13 g of spermidine solution and disperse it in 250 mL of purified water. Add 5 g of sodium hyaluronate powder (the molar ratio of spermidine to sodium hyaluronate is 0.05, and the concentration of HA is 20 mg / mL) and stir until it is completely dissolved. The dissolution time is 3 h.

[0123] (4) The mixed solution from step (3) was added to the network cross-linked gel from step (2). After stirring for 10 minutes, the mixture was pulverized using a homogenizer at 10,000 rpm for 3 minutes, followed by a 5-minute pause. This was repeated five times. 0.355 g of EDC (the molar ratio of EDC to sodium hyaluronate was 0.15) and 0.071 g of NHS were then weighed and added to the system. Stirring was continued at room temperature for 40 minutes, and the system was placed at 40°C for a cross-linking reaction for 16 hours to produce a cross-linked gel.

[0124] Use scissors to cut the cross-linked gel into small pieces with an equivalent diameter of 0.5 cm, place them in a dialysis bag, seal the ends of the dialysis bag with clips, and place them in 9L of phosphate solution containing 2g of sodium hydroxide (the molar ratio of hydroxide to sodium hyaluronate is 2) for dialysis. The dialysis temperature is 40°C. After 2 hours, replace it with 9L of neutral phosphate solution for dialysis. The dialysis temperature is 25°C, and the solution is changed every 3 hours. The dialysis is carried out overnight for a total of 20 hours. Before dialysis, accurately weigh the mass of the blank dialysis bag (M0) and the dialysis bag after sample loading (M1). After dialysis, accurately weigh the mass of the dialysis bag and gel (M2). The initial HA concentration (N) is 20mg / mL. Calculate the sodium hyaluronate content (C) of the gel after dialysis:

[0125] (5) Dilute the mixture to a concentration of 15 mg / mL of sodium hyaluronate at concentration C, add lidocaine hydrochloride, and use the same diluent as the neutral dialysate. The content of lidocaine hydrochloride in the gel is 3 mg / mL. The reconstituted gel is crushed and filled into a pre-filled syringe. Sterilize the mixture by moist heat at 121°C for 15 minutes to obtain a cross-linked sodium hyaluronate hydrogel.

[0126] Performance test 1 Elastic modulus test

[0127] The elastic modulus of the hydrogel samples of Examples 1 to 5 and Comparative Examples 1 to 4 before and after sterilization was measured using a rheometer.

[0128] Table 1 Elastic modulus of samples before and after sterilization

[0129] Note: Before sterilization, the gel is "water-gel separation" and cannot be tested.

[0130] As can be seen from Table 1, the elastic modulus of the hydrogel samples of the embodiment is relatively high before and after sterilization, and the elastic modulus loss rate is almost within 12%. They have good resistance to moist heat sterilization and meet the requirements for clinical use.

[0131] Among them, the elastic modulus of the hydrogel sample of Example 1 before and after sterilization is relatively high, and the loss rate is only 7.5%, with high support performance and stability. This is because while the first step of cross-linking forms a network structure, the carboxyl group in the HA molecule forms an ester bond with the activated hydroxyl group, which plays a placeholder effect; then the HA and cross-linking agent added can be evenly inserted into the network structure formed in the first step, and then a secondary cross-linking is carried out under the action of a catalyst to obtain a cross-linked gel with an interlocking structure. Furthermore, during the alkaline dialysis process, the released HA can further enhance the intermolecular force and bonding force of the cross-linked network, so that the gel product has a high elastic modulus, good support performance, and strong resistance to moist heat sterilization.

[0132] The elastic modulus of the hydrogel sample in Example 2 was higher than that in Example 1, and the loss rate was even lower, at only 6%. This is because the crosslinking degree in the first crosslinking reaction was low, resulting in a looser crosslinked network structure that was more conducive to the subsequent interpenetration and entanglement of HA molecules. This allowed for a tight interlocking structure between networks at different levels, resulting in better support performance and greater resistance to moist heat sterilization.

[0133] The elastic modulus of the hydrogel sample of Example 3 is slightly lower than that of Example 1. This is because although an interlocking structure can be formed after a two-step reaction, under the condition of similar overall cross-linking degree, a larger amount of cross-linking agent is used in the first cross-linking reaction, the cross-linking degree is higher, and the cross-linked network structure formed is denser, and the interpenetration performance of HA molecules is weaker. Therefore, the intermolecular force is slightly reduced, and the sample shows a lower elastic modulus.

[0134] The elastic modulus of the hydrogel sample in Example 2 is higher than that in Example 4. This is because in the first crosslinking reaction, the amount of catalyst added in Example 2 is higher, which can form complete HA occupancy. As a result, the gel product has more interlocking structures, a denser network structure, and better gel support performance. In contrast, the amount of catalyst added in Example 4 is slightly lower, so the carboxyl sites of HA are not fully occupied, which in turn reduces the proportion of interlocking structures in the crosslinked gel and slightly reduces the gel support performance. This also shows that in the first crosslinking step, the optimal method is "complete occupancy."

[0135] In addition, it can be seen from the data of Example 5 that the use of spermine as a cross-linking agent can also achieve a good cross-linking effect, forming an interlocking cross-linked hyaluronic acid or its saline gel, and the supporting performance and resistance to moist heat sterilization meet the requirements of clinical use.

[0136] It can be seen from Example 3 and Comparative Example 1 that the elastic modulus of the hydrogel sample of Comparative Example 1 before and after sterilization is relatively low. At the same time, the elastic modulus loss rate of the sample of Comparative Example 1 is significantly different from that of the sample of Example 3, which is as high as 24%. The main reason is that Comparative Example 1 is a physical mixing rather than an interlocking cross-linked gel prepared by two-step chemical cross-linking. The force of the physical mixing of the two gel structures is relatively small, so the elastic modulus of the gel product is relatively low, and its resistance to moist heat sterilization is also relatively weak.

[0137] At the same time, comparing the data of Example 2 and Comparative Example 2, the sample of Comparative Example 2 was in a "water-gel separation" state before sterilization, making it difficult to detect the elastic modulus data. This is because in the EDC / NHS catalytic system, ester bonds are inevitably generated, which easily causes the cross-linked gel to be supersaturated. During the dialysis process, alkaline dialysis was not used, but only neutral dialysis was used. Therefore, the ester bonds were not effectively destroyed, resulting in the sample showing a "water-gel separation" state when the HA concentration was low (15 mg / mL), which could not meet the actual clinical use requirements. The sample of Example 2 was treated with appropriate alkaline dialysis to destroy most of the ester bonds while retaining the glycosidic bonds of HA, which not only improved the gel's resistance to moist heat sterilization, but also solved the "water-gel separation" problem at the current HA concentration.

[0138] Meanwhile, the post-sterilization elastic modulus data show that the two gel products have the same degree of crosslinking, but the elastic modulus of Example 2 (329 Pa) is slightly lower than that of Comparative Example 2 (401 Pa). This is because Comparative Example 2 did not undergo alkaline dialysis, and even after moist heat sterilization, some ester bonds remained in the gel, resulting in a higher elastic modulus. However, ester bonds are extremely unstable, which can adversely affect the storage stability of the product and also restrict the viscosity of the gel product (see Performance Test 3).

[0139] It can be seen from the data of Example 2 and Comparative Example 3 that the interlocking structure design of Example 2 provides higher support performance to the product compared with the ordinary single-crosslinked gel, which is more conducive to tissue repair in areas that require shaping; while the single-crosslinked hydrogel sample of Comparative Example 3, simply increasing the crosslinking degree will result in excessive pushing force of the hydrogel sample, and therefore its clinical use is limited (see Performance Test 5).

[0140] It can be seen from the data of Example 2 and Comparative Example 4 that when the amount of EDC / NHS added during the first cross-linking reaction is too low, the HA carboxyl sites that do not participate in the cross-linking reaction are not occupied by EDC / NHS, so that the free HA will consume the cross-linking agent added in the second step, reducing the cross-linking reaction efficiency of HA interspersed in the first network and reducing the formation of interlocking structures, thereby resulting in a significant decrease in the support of the hydrogel sample.

[0141] Performance test 2 Esterase degradation experiment

[0142] Prepare a 100 U / mL esterase solution (carboxylesterase, purchased from Sigma) in purified water and set aside. Weigh 0.618 g of boric acid and 0.01 g of sodium hydroxide and dissolve them in 1 L of purified water to obtain a buffer solution with a pH of 8.65, set aside. Add 300 μL of the esterase stock solution and 700 μL of the boric acid buffer solution to 2 mL of each hydrogel sample from Example 2 and Comparative Example 2, mix thoroughly, and analyze the enzymatic hydrolysis curve.

[0143] The elastic modulus of the samples was measured over degradation time using a rheometer with the following settings: 7200 seconds in the Oscillation Time mode at 37°C, strain: 1%, and frequency: 0.9 Hz. Degradation rates were measured over degradation periods of 10, 30, 60, 90, and 120 minutes. The results are shown in Tables 2 and 3.

[0144] Table 2 Degradation rate of samples before sterilization under the action of esterase

[0145] Table 3. Degradation rate of sterilized samples under esterase

[0146] As can be seen from the pre-sterilization data in Table 2, compared with Comparative Example 2, the degradation rate of the hydrogel sample of Example 2 before sterilization was significantly lower than that of the hydrogel sample of Comparative Example 2. After 120 minutes of enzymatic hydrolysis, the degradation rate was only 6.2%. This is because Example 2 used alkaline dialysis to eliminate most of the ester bonds generated in the cross-linking process, so there was less substrate for esterase. In contrast, the hydrogel sample of Comparative Example 2, which did not undergo alkaline dialysis treatment, retained a large number of ester bonds. Under the action of esterase, the degradation rate of the hydrogel was high, even reaching 60%.

[0147] As can be seen from the post-sterilization data in Table 3, the degradation rates of the hydrogel samples in Example 2 and Comparative Example 2 were both reduced due to the destruction of ester bonds during the moist heat sterilization process. However, the degradation rate of the hydrogel sample in Comparative Example 2 was still significantly higher than that in Example 2. This is because Comparative Example 2 originally contained a relatively large number of unstable ester bonds and a large number of ester bond bases. Although moist heat sterilization destroys ester bonds, relatively more ester bonds remain in the final product. After 120 minutes of esterase treatment, the degradation rate was 15%, significantly higher than the 1.1% degradation rate of the sample in Example 2. Furthermore, after 60 minutes of esterase treatment, the degradation rate of the hydrogel sample in Example 2 remained almost unchanged. However, the degradation rate of the hydrogel sample in Comparative Example 2 continued to increase with increasing esterase time, indicating that the presence of ester bonds can affect the storage stability of the product. At the same time, due to the large number of unstable ester bond substances, the modulus of the product will be significantly reduced during storage, which can also affect the clinical use of the product. Therefore, the present invention can eliminate the unstable ester bonds generated during the cross-linking process through appropriate alkaline dialysis, which is beneficial to improving the storage stability of the product.

[0148] Performance Test 3 Viscosity

[0149] (1) Drop weight method

[0150] The hydrogel samples of Examples 1-4 and Comparative Examples 1-4 were filled into 1 mL prefilled syringes (bubbles removed). A chlorobutyl rubber piston, push rod, and booster were installed, followed by a 27G needle. The syringes were then clamped with appropriate fixtures. A motor was activated to move the upper and lower fixtures into position. The push rod was then pushed at a constant speed of 30 mm / min. When a constant force was achieved, the gel was extruded from the needle until it broke and fell, forming one sample. Ten samples were collected and weighed, and the average weight was calculated. A higher weight indicates better viscosity. The results are shown in Table 4.

[0151] Table 4 Viscosity Characterization

[0152] The results in Table 4 show that the interlocking cross-linked hydrogels prepared by two-step chemical cross-linking (Examples 1-4) have higher viscosity than the hydrogels using only physical mixing of gels with different cross-linking degrees (Comparative Example 1). This is because after the two-step cross-linking reaction, the molecular chains formed by the cross-linking reaction between sodium hyaluronate and the cross-linker molecules are interlocked, and the three-dimensional network structure is interwoven, which greatly improves the intermolecular binding force and molecular tension of HA, resulting in a denser cross-linked HA network structure with an interlocking structure, thus exhibiting excellent viscosity.

[0153] Compared with Example 2, the hydrogel sample of Comparative Example 4 did not form an effective ester bond in the first cross-linking process, which reduced the HA cross-linking efficiency and made it difficult to release the linear HA chain after dialysis. Therefore, the interaction force between the cross-linked HA network structure was weak, and the viscosity of the hydrogel sample of Comparative Example 4 was lower.

[0154] (2) Appearance

[0155] The appearance of the samples of Example 2 and Comparative Example 1 in (1) was observed, and the results are shown in Figure 2. As can be seen from Figure 2, the product appearance further shows that the viscosity of the interlocking cross-linked hydrogel sample prepared in Example 2 (A in Figure 2) is better than that of the hydrogel sample of Comparative Example 1 (B in Figure 2). Higher viscosity is crucial for the clinical use of the product, which is manifested in good ductility after injection, easy plasticity, strong ability to bind to tissues, and no displacement, making the injection filling effect more natural and lasting longer.

[0156] Performance test 4: in vitro degradation

[0157] 1.0 ml of 30 U / mL hyaluronidase solution (hyaluronidase purchased from Sigma) was added to 2.0 ml of the hydrogel samples of Examples 1-4 and Comparative Examples 1-4. After manual stirring to ensure uniform mixing of the hydrogel sample and the enzyme solution, the elastic modulus of the hydrogel sample was measured using a rheometer over a 2-hour period (the Peltier plate temperature of the rheometer must be equilibrated to 4°C before sample loading). The change in elastic modulus was used to characterize the enzymatic hydrolysis rate. The results are shown in Table 5.

[0158] Table 5 Degradation rate (%)

[0159] From the above simulated in vivo degradation experimental data, it can be seen that after 120 minutes, the hydrogel samples of Examples 1-4 have significantly higher resistance to enzymatic degradation than those of Comparative Examples 1, 3, and 4, indicating that the interlocking structure can significantly increase the retention time of the product in the body after injection, fully exerting its long-lasting filling effect. At the same time, comparing the degradation rates of Example 2 and Comparative Example 4, it can be seen that during the preparation of the sample of Comparative Example 4, the first cross-linking step was not occupied by EDC / NHS, resulting in low efficiency of the second cross-linking step, almost unable to form an interlocking structure, and at the same time, unable to utilize the HA chains formed after alkaline dialysis to further enhance the intermolecular forces of the interlocking network structure, resulting in weak resistance to enzymatic degradation.

[0160] Comparative Example 2 was not subjected to alkaline dialysis treatment, which had little effect on the anti-enzymatic effect of the hydrogel sample, so the degradation rate was slightly lower than that of Example 2, but had a greater impact on the viscosity and stability of the product (performance tests 2 and 3).

[0161] Performance test 5 Pushing force

[0162] The hydrogel samples of Examples 1-5 and Comparative Examples 1-4 were taken respectively, and the pushing force of each sample was tested. The results are shown in Table 6.

[0163] Table 6 Pushing force

[0164] Note: (1) The pushing force test method refers to the method in YY / T 0962-2021; (2) The injection needle used in the test is 27G×1 / 2.

[0165] As can be seen from Table 6, the hydrogel samples of Examples 1 to 5 have a relatively low extrusion force, which facilitates clinical push injection. Specifically, the extrusion force of the hydrogel sample of Example 2 is lower than that of the hydrogel sample of Comparative Example 2. This is because alkaline dialysis releases linear sodium hyaluronate structures when breaking ester bonds, which acts like adding free HA, providing a lubricating effect and making the product easier to push.

[0166] At the same time, the pushing force of the hydrogel sample in Example 2 was lower than that of the hydrogel sample in Comparative Example 3. This is because, compared to the hydrogel prepared through a single crosslinking reaction, the hydrogel prepared through a two-step chemical crosslinking reaction forms a spatial structure with different levels of interpenetration. This allows the released free HA molecules to interpenetrate more fully, resulting in a tighter spatial structure. This improves the product's support and makes it easier to push, meeting clinical injection requirements. Similarly, in Comparative Example 4, since the initial crosslinking did not form an HA space-occupying effect, the free HA had already participated in the crosslinking reaction, and the HA interspersed in the network was difficult to release. Therefore, the pushing force was also higher.

[0167] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention specification, or any direct or indirect application in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. An injectable interlocked cross-linked hyaluronic acid or its saline gel, characterized in that: The hydrogel is obtained through a two-step crosslinking reaction. First, hyaluronic acid or its salt and a crosslinking agent are subjected to the first crosslinking reaction to form a network structure. Subsequently, the added hyaluronic acid or its salt and the crosslinking agent are interspersed in the network structure, and the second crosslinking reaction is carried out to obtain an injectable interlocked crosslinked hyaluronic acid or its salt hydrogel.

2. The injectable interlocking crosslinked hyaluronic acid or its saline gel according to claim 1, characterized in that: The crosslinking agent is an endogenous polyamine, including spermidine, spermine and their derivatives; The hyaluronic acid or its salt is selected from one or more of sodium hyaluronate, zinc hyaluronate, potassium hyaluronate and calcium hyaluronate; The molecular weight of the hyaluronic acid or its salt is 500 - 2600KDa.

3. The injectable interlocked crosslinked hyaluronic acid or its saline gel according to claim 2, characterized in that: The hyaluronic acid or its salt is sodium hyaluronate.

4. The injectable interlocking cross-linked hyaluronic acid or its saline gel according to any one of claims 1-3, characterized in that: The particle size of the injectable interlocked crosslinked hyaluronic acid or its salt hydrogel is 50 - 650μm; In the injectable interlocked crosslinked hyaluronic acid or its salt hydrogel, the concentration of hyaluronic acid or its salt is 10 - 25mg / mL.

5. A method for preparing an injectable interlocked crosslinked hyaluronic acid or its saline gel, characterized in that: The preparation method includes the following steps: (1) Mix the crosslinking agent with hyaluronic acid or its salt to obtain a mixed solution A of the crosslinking agent and hyaluronic acid or its salt; (2) Add a catalyst to the mixed solution A of the crosslinking agent and hyaluronic acid or its salt, and carry out the first crosslinking reaction to obtain a network structure crosslinked gel; (3) Mix the crosslinking agent with hyaluronic acid or its salt to obtain a mixed solution B of the crosslinking agent and hyaluronic acid or its salt; (4) Add the mixed solution B prepared in step (3) to the network structure crosslinked gel prepared in step (2), and under the action of a catalyst, carry out the second crosslinking reaction to obtain an interlocked structure crosslinked gel, and perform dialysis treatment.

6. The preparation method according to claim 5, characterized in that: In steps (1) and (3), the crosslinking agent is an endogenous polyamine, including spermidine, spermine and their derivatives; In steps (1) and (3), the hyaluronic acid or its salt is selected from one or more of sodium hyaluronate, zinc hyaluronate, potassium hyaluronate and calcium hyaluronate.

7. The preparation method according to claim 5, characterized in that: The mass concentration of the hyaluronic acid or its salt in the mixed solution A and the mixed solution B is 1% - 5%.

8. The preparation method according to claim 5, characterized in that: The molar ratio of the crosslinking agent to the hyaluronic acid or its salt in the mixed solution A and the mixed solution B is 0.01 - 0.

1.

9. The preparation method according to claim 8, characterized in that: The molar ratio of the crosslinking agent to the hyaluronic acid or its salt in step (1) is lower than that in step (3).

10. The preparation method according to claim 5, characterized in that: In steps (2) and (4), the catalyst is selected from one or more of carbodiimide, phosphonium bromide formed by triphenylphosphine and bromide, carbonium salt and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM).

11. The preparation method according to claim 10, characterized in that: In step (2), the molar ratio of the catalyst to the hyaluronic acid or its salt in the mixed solution A is 0.6 - 3, and the crosslinking time is 1 - 3h; and / or In step (4), the molar ratio of the catalyst to the hyaluronic acid or its salt in the mixed solution B is 0.03 - 0.3, and the crosslinking reaction time is 1 - 24h.

12. The preparation method according to claim 5, characterized in that: In the dialysis in step (4), first dialyze with an alkaline dialysis solution, and then dialyze with a neutral dialysis solution; In the alkaline dialysate, the molar ratio of hydroxide ion to the total hyaluronic acid or its salt used in steps (1) and (3) is 1:1 to 10:1, the alkaline dialysis temperature is 10 to 40 °C, and the dialysis duration is 1 to 10 h; and / or The neutral dialysis temperature is 10 to 40 °C, and the dialysis duration is 1 to 20 h.

13. Use of the injectable interlocked cross-linked hyaluronic acid or its saline hydrogel according to any one of claims 1-4 or the injectable interlocked cross-linked hyaluronic acid or its saline hydrogel obtained by the preparation method according to any one of claims 5-12 in the preparation of a tissue filling and repairing material or a drug carrier for non-therapeutic purposes.

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