Injectable interlocking cross-linked hyaluronic acid or hydrogel thereof, preparation method and application

Abstract

The invention relates to injectable interlocking cross-linked hyaluronic acid or hydrogel thereof, a preparation method and application. The preparation process of the hydrogel comprises a two-step cross-linking step of hyaluronic acid or salt thereof and a cross-linking agent endogenous polyamine and a dialysis step. In the two steps of crosslinking, the first step of crosslinking forms a net-shaped structure, and the second step of crosslinking further forms an interlocking structure. The hydrogel prepared through the technology is subjected to moist heat sterilization, high in degradation resistance, excellent in viscosity, good in supporting performance, easy to push and inject, high in safety and easy to redissolve, the clinical requirements for injection products are met, the whole technology is easy, convenient and rapid to operate, and industrialization is easy to achieve.

Description

Technical Field

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

[0002] Hyaluronic acid (abbreviated as HA) and its salts are macromolecular polysaccharide compounds with high biocompatibility, widely distributed in the extracellular matrix of connective tissue cells of animals and humans, and have been commercially applied in the fields of medicine and beauty. A large amount of hyaluronic acid is distributed in the skin tissue of the human body, which plays functions such as water retention, maintaining the extracellular space, promoting cell repair, bonding the internal interface in the skin tissue, and lubrication. Therefore, it is often used as a filling matrix for facial rejuvenation and contour repair treatment. However, unmodified hyaluronic acid injected into the skin tissue has disadvantages such as poor mechanical properties, low viscosity, and short retention time in the body. Optimizing its properties by 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. As is well known, even extremely low concentrations of BDDE residues may be genotoxic. Considering the safety risks associated with BDDE, the annual dose of commercially available skin filler products is limited to no more than 20 mL per year (e.g. ), and the maximum dose per use is limited to 6 mL (e.g. ). Therefore, a safer and more effective cross-linking agent is an urgent need for the future development of hyaluronic acid-based fillers.

[0004] Patent CN113499480 reported a physical and chemical double-network hydrogel for subcutaneous fillers, its preparation method and applications. In this patent, HA was first cross-linked and modified by chemical methods, and then physical freeze-thaw was used to achieve secondary cross-linking of the cross-linked gel. Although the molecular lattice of the polymer material will change during the freeze-drying process, the physical cross-linking is still not as stable as chemical bonds. Patent CN113896915 reported the use of endogenous polyamines including spermine and / or spermidine as novel cross-linking agents to prepare cross-linked sodium hyaluronate, but the gel prepared by this process has relatively low viscosity and relatively large thrust. At the same time, in this cross-linking system, more ester bond structures will be formed. When the content of sodium hyaluronate is low (such as less than 15 mg / mL), "water-gel separation" is likely to occur, so it is relatively limited when used clinically as an injectable filling product. Summary of the Invention

[0005] Aiming at the deficiencies of the existing processes, the purpose of the present invention is to provide an injectable interlocked cross-linked hyaluronic acid or its saline gel, a preparation method and applications thereof.

[0006] The injectable interlocked cross-linked hyaluronic acid or its saline gel of the present invention adopts the double cross-linked interlock technology (DCIT) to form an interlocked network structure during the two-step chemical cross-linking reaction. First, hyaluronic acid or its salt and the cross-linking agent endogenous polyamine form a reticular structure gel through the first cross-linking, and then an interlocked structure hydrogel is formed through the second cross-linking.

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

[0008] In the first aspect, the present invention provides an injectable interlocked cross-linked hyaluronic acid or its saline gel, which is obtained through a two-step cross-linking reaction. First, hyaluronic acid or its salt and the cross-linking agent are subjected to the first cross-linking reaction to form a reticular structure, and then the subsequently added hyaluronic acid or its salt and the cross-linking agent are interspersed in the reticular structure to carry out the second cross-linking reaction, thereby obtaining the injectable interlocked cross-linked hyaluronic acid or its saline gel.

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

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

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

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

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

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

[0015] In the two-step cross-linking, the first cross-linking forms a reticular structure, and the second cross-linking further forms an interlocked structure, realizing the upgrade from an interpenetrating structure to an interlocked structure.

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

[0017] Preferably, a catalyst is added during the crosslinking process, and 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).

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

[0019] Furthermore, after the two-step crosslinking reaction, a dialysis step is further included to obtain the injectable interlocked crosslinked hyaluronic acid or its saline hydrogel.

[0020] Preferably, the dialysis is first carried out with an alkaline dialysis solution and then with a neutral dialysis solution.

[0021] The interlocked crosslinked hyaluronic acid or its saline hydrogel needs to and is dialyzed under appropriate alkali concentration conditions to remove unstable ester bonds to obtain the injectable interlocked crosslinked hyaluronic acid or its saline hydrogel. It has the advantages of strong anti-degradation ability, excellent viscosity, good supportability, easy injection, high safety, etc., and can be used for subcutaneous injection filling.

[0022] In the second aspect, the present invention provides a preparation method of the injectable interlocked crosslinked hyaluronic acid or its saline hydrogel described in the first aspect, and the specific steps are as follows:

[0023] (1) Mix a crosslinking agent with hyaluronic acid or its salt to obtain a mixed solution A of the crosslinking agent and hyaluronic acid or its salt;

[0024] (2) Add a catalyst to the mixed solution A of the crosslinking agent and hyaluronic acid or its salt to carry out the first crosslinking reaction to obtain a reticular structure crosslinked gel;

[0025] (3) Mix a crosslinking agent with hyaluronic acid or its salt to obtain a mixed solution B of the crosslinking agent and hyaluronic acid or its salt;

[0026] (4) Add the mixed solution B prepared in step (3) to the reticular 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.

[0027] The interlocked cross-linked hyaluronic acid or its saline gel of the present invention adopts a double cross-linked interlock technology (Double cross-linked interlock technology, abbreviated as DCIT). In the first cross-linking process, carboxyl groups in HA molecules and multiple amino sites of spermidine or norspermidine can form amide bonds under the action of a catalyst to form a network structure; at the same time, carboxyl groups in HA molecules and hydroxyl groups activated by the catalyst can form ester bonds, playing an occupancy effect; then HA solution, cross-linking agent and catalyst are added for the second cross-linking; during this process, HA molecules and cross-linking agents can be uniformly inserted into the network structure formed in the first step along with the stirring force, and then amide bonds are formed under the action of the catalyst to form an interlocked structure.

[0028] Preferably, the cross-linking agents in steps (1) and (3) are endogenous polyamines, and further preferably include spermidine, spermine and their 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 - 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, and preferably sodium hyaluronate.

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

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

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

[0034] Preferably, in the mixed solution A and the mixed solution B, the molar ratio of the cross-linking agent to the hyaluronic acid or its salt is 0.01 - 0.1, and the two 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 that in step (3), that is, the cross-linking degree of the first reaction is lower than that of the second reaction. This is because a relatively loose first network structure is prepared in the first cross-linking reaction, making it easy for the hyaluronic acid or its salt (HA) molecules added in the second step to interpenetrate. After the second cross-linking reaction, different levels of spatial structures are constructed, which is more conducive to the formation of an interlocked structure.

[0036] Preferably, the crosslinking 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-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM), and the two can 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 simultaneously to improve the crosslinking reaction efficiency.

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

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

[0040] The crosslinking reaction time in step (2) is 1 - 3 h, such as 1 h, 2 h, 3 h.

[0041] In the first-step crosslinking reaction of step (2), a larger amount of catalyst added can form more ester bonds, playing a full "occupation" role, which is beneficial to improving the crosslinking efficiency of the second-step crosslinking reaction. At the same time, the amount of catalyst used in the second-step crosslinking reaction is reduced, enabling the crosslinking reaction to construct more interlocked structures under the reaction conditions of low-dose catalyst and safe crosslinking agent to improve the anti-degradation performance of the final product.

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

[0043] The crosslinking reaction time in step (4) is 1 - 24 h, such as 1 h, 2 h, 3 h, 8 h, 12 h, 16 h, 20 h, 24 h.

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

[0045] In order to fully form an interlocked structure gel, it is necessary to form an ester bond structure substance that occupies a position in advance (including EDC active ester, NHS active ester, ester formed by HA hydroxyl group and carboxyl group, etc.) in the first cross-linking reaction. However, the ester bond structure substance is unstable and will also affect the stability of the gel product. In the present invention, through subsequent alkaline dialysis treatment, the ester bond formed by occupying a position in advance is opened during alkaline dialysis, releasing linear HA chains. This part of the HA chains not only plays a lubricating role and improves the injectability of the product, but also can further entangle or interpenetrate with the formed interlocked structure, enhancing the intermolecular interaction force and improving the viscosity of the product.

[0046] Preferably, the molar ratio of hydroxide ions in the alkaline dialysis solution to the total 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 dialysis solution is selected from sodium hydroxide or potassium hydroxide, and the solvent of the alkaline dialysis solution is any one or a combination of purified water, physiological saline or phosphate buffer solution.

[0048] During the two-step cross-linking reaction process, the unstable ester bond structure substance generated can, after the alkaline dialysis process, destroy the above-mentioned unstable ester bond structure, and can improve the ability of the final gel product to withstand heat and humidity sterilization and storage stability; at the same time, the glycosidic bond of HA will also be damaged to a certain extent under alkaline conditions. Therefore, it is necessary to control the molar ratio of hydroxide ions to hyaluronic acid or its salt in the alkaline dialysis solution. If the molar amount of hydroxide ions is low, the unstable ester bond structure substance cannot be effectively removed, and the stability of the hydrogel product cannot be guaranteed; if the molar amount of hydroxide ions is high, the glycosidic bond of HA will be damaged, the network structure of cross-linked HA is not stable enough and is prone to degradation, or the concentration of HA in the product decreases too fast to meet the clinical injection requirements.

[0049] More preferably, in the alkaline dialysis, the volume ratio of the dialysis solution to the cross-linked gel of the interlocked 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 10 h, for example: 1 h, 2 h, 4 h, 6 h, 8 h, 10 h.

[0050] Preferably, in the neutral dialysis, the dialysis solution includes any one or a combination of purified water, physiological saline or phosphate buffer solution, and the pH of the neutral dialysis solution can be 6.0 to 7.6.

[0051] Further preferably, in the neutral dialysis, the volume ratio of the dialysis fluid to the crosslinked gel of the interlocking structure is 15:1 to 30:1, such as: 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 h.

[0052] Before the alkaline dialysis treatment, the crosslinked gel of the interlocking structure obtained from the second crosslinking reaction is cut into small pieces with an equivalent diameter of 0.02 to 1 cm, such as: 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, 1 cm.

[0053] Preferably, the dialysis bag cut-off molecular weight (MWCO) is 1 to 100 KDa, such as: 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), and after dialysis, it is diluted, crushed, filled, and sterilized by moist heat to obtain the injectable interlocked crosslinked hyaluronic acid or its saline gel.

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

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

[0057] Preferably, the concentration of lidocaine hydrochloride is 1 to 5 mg / mL, such as: 1 mg / mL, 2 mg / mL, 3 mg / ml, 4 mg / mL, 5 mg / mL.

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

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

[0060] Preferably, in step (5), in the injectable interlocked cross-linked hyaluronic acid or its saline gel, the concentration of hyaluronic acid or its salt 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 for removing wrinkles (such as periorbital wrinkles, forehead wrinkles, glabellar wrinkles, perioral wrinkles, nasolabial folds, tear troughs, nasolabial folds, neck wrinkles, hand wrinkles, stretch marks, etc.), anti-aging, scar removal, 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 purposes such as sustained release, controlled release, and targeted drug delivery.

[0065] Advantages of the present invention:

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

[0067] Second, the interlocked structure cross-linked hydrogel of the present invention adopts a double cross-linked interlock technology (Double cross-linked interlock technology, abbreviated as DCIT), which undergoes two cross-linking reactions successively. By controlling the amounts of the cross-linking agent and the catalyst, the ester bond occupancy effect is fully utilized in the first cross-linking reaction, improving the efficiency of the second cross-linking reaction and realizing the upgrade from an interpenetrating network to an interlocked structure; further through an alkaline dialysis process, the ester bonds originally used for occupancy in the cross-linking reaction process are destroyed, and the released HA chains not only enhance the injectability of the product, but also further entangle with the interlocked structure, enhancing the intermolecular interaction force. The prepared gel exhibits high support performance, viscosity, and excellent anti-enzymatic hydrolysis ability, meeting the clinical requirements for injection products.

[0068] 3. The interlocked structure crosslinked hydrogel of the present invention uses an efficient alkaline dialysis process, and can also remove processing aids within a short time, ensuring that the concentration of hyaluronic acid or its salt in the gel after dialysis is higher than that of hyaluronic acid or its salt in the expected product. This not only eliminates the subsequent concentration process, improves production efficiency, but also avoids the destruction of the polymer lattice and the phenomenon of difficult redissolution during the concentration process. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 The figure shows a schematic diagram of the gel crosslinking reaction process of the present invention;

[0070] Figure 2 The figure shows the appearance of the gel product of the present invention, where A represents the sample of Example 2 and B represents the sample of Comparative Example 1. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0071] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments and the drawings. The described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, other embodiments obtained by those of ordinary skill in the art without creative efforts belong to 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] Preparation of Injectable Interlocked Crosslinked Sodium Hyaluronate Hydrogel in Example 1

[0074] (1) Prepare a spermidine aqueous solution with a concentration of 8% and a pH of 6.0, and store it refrigerated for later use. Weigh 0.9 g of this solution and disperse it in 250 mL of purified water. Add 5 g of sodium hyaluronate powder thereto (the molar ratio of spermidine to sodium hyaluronate is 0.04, and the concentration of HA is 20 mg / mL), and stir to completely dissolve it. The dissolution time is 3 h.

[0075] (2) Then weigh 2.37 g of EDC (the molar ratio of EDC to sodium hyaluronate is 1.00) and 0.48 g of NHS and add them to the above mixed solution. After stirring at room temperature for 40 min, place the system in a crosslinking reaction at 40 °C for 3 h to obtain a network crosslinked 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 thereto (the molar ratio of spermidine to sodium hyaluronate is 0.04, and the concentration of HA is 20 mg / mL), and stir to completely dissolve it. The dissolution time is 3 h.

[0077] (4) Add the mixed solution from step (3) to the reticular crosslinked gel from step (2), stir for 10 min, then crush it with a homogenizer at 10,000 rpm for 3 min, with an interval of 5 min, and repeat 5 times. Then weigh 0.284 g of EDC (the molar ratio of EDC to sodium hyaluronate is 0.12) and 0.057 g of NHS and add them to the aforementioned system. Continue to stir at room temperature for 40 min, and then place the system in an environment at 40 °C for crosslinking reaction for 16 h to obtain an interlocked structure crosslinked gel.

[0078] Use scissors to cut the interlocked structure crosslinked gel into small pieces with an equivalent diameter of 0.5 cm, put them into a dialysis bag, seal both ends of the dialysis bag with clips, and place it in a 9 L phosphate solution containing 2 g of sodium hydroxide (the molar ratio of hydroxide to sodium hyaluronate is 2) for dialysis. The dialysis temperature is 40 °C. After 2 h, replace it with a 9 L neutral phosphate solution for dialysis, and the dialysis temperature is 25 °C. Change the solution once every 1 - 3 h, and carry out overnight dialysis for one night, with a total dialysis time of 20 h. Accurately weigh the blank dialysis bag (M0) and the mass of the dialysis bag after loading the sample (M1) before dialysis, and accurately weigh the mass of the dialysis bag and the gel (M2) after dialysis. The initial HA concentration (N) is 20 mg / mL, and calculate the sodium hyaluronate content (C) of the gel after dialysis:

[0079]

[0080] (5) Dilute and compound to a sodium hyaluronate concentration of 15 mg / mL with the concentration C, add lidocaine hydrochloride, and the diluent is the same as the neutral dialysis solution. The content of lidocaine hydrochloride in the gel is 3 mg / mL. Crush the compounded gel and fill it into a pre-filled syringe, and sterilize it by moist heat at 121 °C for 15 min to obtain an injectable interlocked crosslinked sodium hyaluronate hydrogel.

[0081] Preparation of Injectable Interlocked Crosslinked Sodium Hyaluronate Hydrogel in Example 2

[0082] (1) Prepare a spermidine aqueous solution with a concentration of 8% and a pH of 6.0, and store it refrigerated for later use. Weigh 0.68 g of this solution and disperse it in 250 mL of purified water, then add 5 g of sodium hyaluronate powder to it (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.

[0083] (2) Weigh 2.37 g of EDC (the molar ratio of EDC to sodium hyaluronate is 1.00) and 0.48 g of NHS and add them to the above mixed solution. Stir at room temperature for 40 min, and then place the system in an environment at 40 °C for crosslinking reaction for 3 h to obtain a reticular crosslinked gel.

[0084] (3) Weigh 1.13 g of spermidine solution and disperse it in 250 mL of purified water. Add 5 g of sodium hyaluronate powder thereto (the molar ratio of spermidine to sodium hyaluronate is 0.05, and the concentration of HA is 20 mg / mL), and stir to completely dissolve it. The dissolution time is 3 h.

[0085] (4) Add the mixed solution in step (3) to the reticular cross-linked gel in step (2), stir for 10 min, then crush it with a homogenizer at 10,000 rpm for 3 min, with an intermittent time of 5 min, and repeat 5 times. Then weigh 0.355 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-mentioned system. Continue to stir at room temperature for 40 min, and then place the system in a 40 °C environment for cross-linking reaction for 16 h to obtain an interlocked structure cross-linked gel.

[0086] Use scissors to cut the interlocked structure cross-linked gel into small pieces with an equivalent diameter of 0.5 cm, put them into a dialysis bag, seal both ends of the dialysis bag with clips, and place it in a 9 L phosphate solution containing 2 g of sodium hydroxide for dialysis. The dialysis temperature is 40 °C. After 2 h, replace it with a 9 L neutral phosphate solution for dialysis. The dialysis temperature is 25 °C, and the solution is changed once every 1 - 3 h, and overnight dialysis is carried out for one night, with a total dialysis time of 20 h. Accurately weigh the blank dialysis bag (M0) and the mass of the dialysis bag after loading the sample (M1) before dialysis, and accurately weigh the mass of the dialysis bag and the gel (M2) after dialysis. The initial HA concentration (N) is 20 mg / mL, and calculate the sodium hyaluronate content (C) of the gel after dialysis:

[0087]

[0088] (5) Dilute and compound to a sodium hyaluronate concentration of 15 mg / mL with the concentration C, add lidocaine hydrochloride, and the dilution solution is the same as the neutral dialysis solution. The content of lidocaine hydrochloride in the gel is 3 mg / mL. Crush the compounded gel and fill it into a pre-filled syringe, and sterilize it by moist heat at 121 °C for 15 min to obtain an injectable interlocked cross-linked sodium hyaluronate hydrogel.

[0089] Preparation of Injectable Interlocked Cross-Linked Sodium Hyaluronate Hydrogel in Example 3

[0090] (1) Prepare an 8% concentration aqueous solution of spermidine with pH = 6.0 and store it in the refrigerator for later use. Weigh 1.13 g of this solution and disperse it in 250 mL of purified water. Add 5 g of sodium hyaluronate powder thereto (the molar ratio of spermidine to sodium hyaluronate is 0.05, and the concentration of HA is 20 mg / mL), and stir to completely dissolve it. The dissolution time is 3 h.

[0091] (2) Then weigh 3.56 g of EDC (the molar ratio of EDC to sodium hyaluronate is 1.5) and 0.713 g of NHS and add them to the above mixed solution. After stirring at room temperature for 40 min, place the system in an environment of 40 °C for cross-linking reaction for 3 h to obtain a reticular cross-linked gel.

[0092] (3) Weigh 0.68 g of spermidine solution and disperse it in 250 mL of purified water. Add 5 g of sodium hyaluronate powder thereto (the molar ratio of spermidine to sodium hyaluronate is 0.03, and the concentration of HA is 20 mg / mL), and stir to completely dissolve it. The dissolution time is 3 h.

[0093] (4) Add the mixed solution in step (3) to the reticular cross-linked gel in step (2). After stirring for 10 min, crush it with a homogenizer at 10000 rpm for 3 min, with an intermittent time of 5 min, and repeat 5 times. Then weigh 0.213 g of EDC (the molar ratio of EDC to sodium hyaluronate is 0.09) and 0.043 g of NHS and add them to the aforementioned system. Continue to stir at room temperature for 40 min, and then place the system in an environment of 40 °C for cross-linking reaction for 16 h to obtain an interlocked structure cross-linked gel.

[0094] Use scissors to cut the interlocked structure cross-linked gel into small pieces with an equivalent diameter of 0.5 cm, put them into a dialysis bag, seal both ends of the dialysis bag with clips, and place it in a phosphate solution containing 2 g of sodium hydroxide in 9 L for dialysis. The dialysis temperature is 40 °C, and the solution is changed every 2 h. Then change it to a neutral phosphate solution in 9 L for dialysis. The dialysis temperature is 25 °C, and the solution is changed every 1 - 3 h, and overnight dialysis is carried out for a total of 20 h. Accurately weigh the blank dialysis bag (M0) and the mass of the dialysis bag after loading the sample (M1) before dialysis. After dialysis, accurately weigh the mass of the dialysis bag and the gel (M2). The initial HA concentration (N) is 20 mg / mL, and calculate the sodium hyaluronate content (C) of the gel after dialysis:

[0095]

[0096] (5) Dilute and compound to a sodium hyaluronate concentration of 15 mg / mL with the concentration C, add lidocaine hydrochloride, and the diluent is the same as the neutral dialysis solution. The content of lidocaine hydrochloride in the gel is 3 mg / mL. Crush the compounded gel and fill it into a pre-filled syringe, and sterilize it by moist heat at 121 °C for 15 min to obtain an injectable interlocked cross-linked sodium hyaluronate hydrogel.

[0097] Preparation of Injectable Interlocked Cross-Linked Sodium Hyaluronate Hydrogel in Example 4

[0098] (1) Prepare an aqueous solution of spermidine with a concentration of 8% and a pH of 6.0, and store it refrigerated for later use. Weigh 0.68 g of this solution and disperse it in 250 mL of purified water. Add 5 g of sodium hyaluronate powder to it (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.

[0099] (2) Weigh 1.422 g of EDC (the molar ratio of EDC to sodium hyaluronate is 0.6) and 0.285 g of NHS, and add them to the above mixed solution. After stirring at room temperature for 40 min, place this system in an environment at 40 °C for cross-linking reaction for 3 h to obtain a reticular cross-linked gel.

[0100] (3) Weigh 1.13 g of spermidine solution and disperse it in 250 mL of purified water. Add 5 g of sodium hyaluronate powder to it (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.

[0101] (4) Add the mixed solution in step (3) to the reticular cross-linked gel in step (2). After stirring for 10 min, crush it with a homogenizer at 10000 rpm for 3 min, with an interval of 5 min, and repeat 5 times. Then weigh 0.355 g of EDC (the molar ratio of EDC to sodium hyaluronate is 0.15) and 0.071 g of NHS, add them to the aforementioned system, continue to stir at room temperature for 40 min, and then place this system in an environment at 40 °C for cross-linking reaction for 16 h to obtain a cross-linked gel with an interlocking structure.

[0102] Use scissors to cut the cross-linked gel with an interlocking structure into small pieces with an equivalent diameter of 0.5 cm, put them into a dialysis bag, seal both ends of the dialysis bag with clips, and place it in a 9 L phosphate solution containing 2 g of sodium hydroxide for dialysis. The dialysis temperature is 40 °C. After 2 h, replace it with a 9 L neutral phosphate solution for dialysis. The dialysis temperature is 25 °C, and the solution is changed once every 1 - 3 h, and overnight dialysis is carried out for a total of 20 h. Accurately weigh the blank dialysis bag (M0) and the mass of the dialysis bag after loading the sample (M1) before dialysis. After dialysis, accurately weigh the mass of the dialysis bag and the gel (M2). The initial HA concentration (N) is 20 mg / mL, and calculate the sodium hyaluronate content (C) of the gel after dialysis:

[0103]

[0104] (5) Dilute and compound it to a sodium hyaluronate concentration of 15 mg / mL with a concentration of C, add lidocaine hydrochloride, and the dilution solution is the same as the neutral dialysis solution. The content of lidocaine hydrochloride in the gel is 3 mg / mL. Crush the compounded gel and fill it into a pre-filled syringe, and sterilize it by moist heat at 121 °C for 15 min to obtain an injectable interlocking cross-linked sodium hyaluronate hydrogel.

[0105] Preparation of Injectable Interlocked Crosslinked Sodium Hyaluronate Hydrogel in Example 5

[0106] (1) Prepare an aqueous spermine solution with a concentration of 8% and a pH of 6.0, and store it refrigerated. Weigh 0.94 g of this solution and disperse it in 250 mL of purified water. Add 5 g of sodium hyaluronate powder thereto (the molar ratio of spermine 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.

[0107] (2) Weigh 4.74 g of EDC (the molar ratio of EDC to sodium hyaluronate is 2.00) and 0.95 g of NHS, add them to the above mixed solution, stir at room temperature for 40 min, and then place the system in a 40 °C environment for crosslinking reaction for 3 h to obtain a reticular crosslinked gel.

[0108] (3) Weigh 1.88 g of spermine solution and disperse it in 250 mL of purified water. Add 5 g of sodium hyaluronate powder thereto (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.

[0109] (4) Add the mixed solution in step (3) to the reticular crosslinked gel in step (2), stir for 10 min, then crush it with a homogenizer at 10000 rpm for 3 min, with an intermittent time of 5 min, and repeat 5 times. Then weigh 0.427 g of EDC (the molar ratio of EDC to sodium hyaluronate is 0.18) and 0.086 g of NHS, add them to the above-mentioned system, continue to stir at room temperature for 40 min, and then place the system in a 40 °C environment for crosslinking reaction for 16 h to obtain a crosslinked gel with an interlocked structure.

[0110] Use scissors to cut the crosslinked gel with an interlocked structure into small pieces with an equivalent diameter of 0.5 cm, put them into a dialysis bag, seal both ends of the dialysis bag with clips, and place it in a 9 L phosphate solution containing 4 g of sodium hydroxide for dialysis. The dialysis temperature is 40 °C. After 2 h, replace it with a 9 L neutral phosphate solution for dialysis. The dialysis temperature is 25 °C, and the solution is changed once every 1 - 3 h, and overnight dialysis is carried out for one night, with a total dialysis time of 20 h. Accurately weigh the blank dialysis bag (M0) and the mass of the dialysis bag after loading the sample (M1) before dialysis, and accurately weigh the mass of the dialysis bag and the gel (M2) after dialysis. The initial HA concentration (N) is 20 mg / mL, and calculate the sodium hyaluronate content (C) of the gel after dialysis:

[0111]

[0112] (5) Dilute and compound to a sodium hyaluronate concentration of 15 mg / mL at a concentration of C, add lidocaine hydrochloride, and the diluent is the same as the neutral dialysis solution. The content of lidocaine hydrochloride in the gel is 3 mg / mL. Crush the compounded gel and fill it into a prefilled syringe, and sterilize it by moist heat at 121 °C for 15 min to obtain an injectable interlocked cross-linked sodium hyaluronate hydrogel.

[0113] Preparation of cross-linked sodium hyaluronate hydrogel in Comparative Example 1

[0114] (1) Prepare an aqueous solution of spermidine with a concentration of 8% and a pH of 6.0, and store it refrigerated for later use. Weigh 1.13 g of this solution and disperse it in 250 mL of purified water. Add 5 g of sodium hyaluronate powder thereto (the molar ratio of spermidine to sodium hyaluronate is 0.05, and the concentration of HA is 20 mg / mL), and stir to completely dissolve it. 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, place the system in a 40 °C environment for cross-linking reaction for 3 h to obtain a gel with a single cross-linking degree (Component 1).

[0115] In addition, weigh 0.68 g of spermidine solution and disperse it in 250 mL of purified water. Add 5 g of sodium hyaluronate powder thereto (the molar ratio of spermidine to sodium hyaluronate is 0.03, and the concentration of HA is 20 mg / mL), and stir to completely dissolve it. The dissolution time is 3 h. Then weigh 0.213 g of EDC (the molar ratio of EDC to sodium hyaluronate is 0.09) and 0.043 g of NHS and add them to the above sodium hyaluronate gel system. After stirring at room temperature for 40 min, place the system in a 40 °C environment for cross-linking reaction for 3 h to obtain a gel with a single cross-linking degree (Component 2).

[0116] (2) Mix the above 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 both ends of the dialysis bag with a clip, and place it in a phosphate solution containing 2 g of sodium hydroxide in 9 L for dialysis. The dialysis temperature is 40 °C, and the solution is changed once every 2 h. Then change it to a neutral phosphate solution in 9 L for dialysis. The dialysis temperature is 25 °C, and the solution is changed once every 1 - 3 h, and overnight dialysis is carried out for one night, with a total dialysis time of 20 h. Accurately weigh the blank dialysis bag (M0) and the mass of the dialysis bag after loading the sample (M1) before dialysis, and accurately weigh the mass of the dialysis bag and the gel (M2) after dialysis. The initial HA concentration (N) is 20 mg / mL, and calculate the sodium hyaluronate content (C) of the gel after dialysis:

[0117]

[0118] (3) Dilute and compound to a concentration of 15 mg / mL of sodium hyaluronate with concentration C, add lidocaine hydrochloride, and the diluent is the same as the neutral dialysis solution. The content of lidocaine hydrochloride in the gel is 3 mg / mL. Crush the compounded crosslinked gel, fill it into a pre-filled syringe, and sterilize it by moist heat at 121 °C for 15 minutes to obtain the crosslinked sodium hyaluronate hydrogel.

[0119] Preparation of crosslinked sodium hyaluronate hydrogel in Comparative Example 2

[0120] Prepare the interlocked crosslinked gel according to the process parameters in Example 2, with the only difference being that alkaline dialysis is not used. In the dialysis process, only dialyze with 9 L of neutral phosphate solution, the dialysis temperature is 25 °C, change the solution once every 1 - 3 h, and experience overnight dialysis for one night, with a total dialysis time of 20 h. Crush the compounded gel, fill it into a pre-filled syringe, and sterilize it by moist heat at 121 °C for 15 minutes to finally obtain the crosslinked sodium hyaluronate hydrogel.

[0121] Preparation of crosslinked sodium hyaluronate hydrogel in Comparative Example 3

[0122] (1) Prepare an aqueous solution of spermidine with a concentration of 8% and a pH of 6.0, and store it refrigerated for later use. Weigh 2.25 g of this solution and disperse it in 500 mL of purified water. Add 10 g of sodium hyaluronate powder to it (the molar ratio of spermidine to sodium hyaluronate is 0.05, and the concentration of HA is 20 mg / mL), and stir to completely dissolve it. 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 minutes, place the system in a 40 °C environment for crosslinking reaction for 16 h to obtain a gel with a single crosslinking degree.

[0123] (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 both ends of the dialysis bag with clips, and place it in 9 L of phosphate solution containing 2 g of sodium hydroxide for dialysis. The dialysis temperature is 40 °C, change the solution once every 2 h, and then change to 9 L of neutral phosphate solution for dialysis. The dialysis temperature is 25 °C, change the solution once every 1 - 3 h, and experience overnight dialysis for one night, with a total dialysis time of 20 h. Accurately weigh the blank dialysis bag (M0) and the mass of the dialysis bag after loading the sample (M1) before dialysis, and accurately weigh the mass of the dialysis bag and the gel (M2) after dialysis. The initial HA concentration (N) is 20 mg / mL, and calculate the sodium hyaluronate content (C) of the gel after dialysis:

[0124]

[0125] (3) Dilute and compound to a sodium hyaluronate concentration of 15 mg / mL at a concentration of C, add lidocaine hydrochloride, and the diluent is the same as the neutral dialysis solution. The content of lidocaine hydrochloride in the gel is 3 mg / mL. Crush the compounded crosslinked gel, fill it into a prefilled syringe, and sterilize it by moist heat at 121 °C for 15 min to obtain the crosslinked sodium hyaluronate hydrogel.

[0126] Preparation of crosslinked sodium hyaluronate gel in Comparative Example 4

[0127] (1) Prepare an aqueous solution of spermidine with a concentration of 8% and a pH of 6.0, and store it refrigerated for later use. Weigh 0.68 g of this solution and disperse it in 250 mL of purified water. Add 5 g of sodium hyaluronate powder thereto (the molar ratio of spermidine to sodium hyaluronate is 0.03, and the concentration of HA is 20 mg / mL), and stir to completely dissolve it. The dissolution time is 3 h.

[0128] (2) Weigh 0.711 g of EDC (the molar ratio of EDC to sodium hyaluronate is 0.3) and 0.143 g of NHS, add them to the above mixed solution, stir at room temperature for 40 min, and then place the system in a 40 °C environment for crosslinking reaction for 3 h to obtain a reticular crosslinked gel.

[0129] (3) Weigh 1.13 g of spermidine solution and disperse it in 250 mL of purified water. Add 5 g of sodium hyaluronate powder thereto (the molar ratio of spermidine to sodium hyaluronate is 0.05, and the concentration of HA is 20 mg / mL), and stir to completely dissolve it. The dissolution time is 3 h.

[0130] (4) Add the mixed solution in step (3) to the reticular crosslinked gel in step (2), stir for 10 min, then crush it with a homogenizer at 10000 rpm for 3 min, with an interval of 5 min, and repeat 5 times. Then weigh 0.355 g of EDC (the molar ratio of EDC to sodium hyaluronate is 0.15) and 0.071 g of NHS, add them to the above system, continue to stir at room temperature for 40 min, and then place the system in a 40 °C environment for crosslinking reaction for 16 h to obtain a crosslinked gel.

[0131] Use scissors to cut the crosslinked gel into small pieces with an equivalent diameter of 0.5 cm, put them into a dialysis bag, seal both ends of the dialysis bag with clips, and place it in a 9 L phosphate solution containing 2 g of sodium hydroxide for dialysis. The dialysis temperature is 40 °C. After 2 h, replace it with a 9 L neutral phosphate solution for dialysis. The dialysis temperature is 25 °C, and the solution is changed once every 1 - 3 h, and overnight dialysis is carried out for one night, with a total dialysis time of 20 h. Accurately weigh the blank dialysis bag (M0) and the mass of the dialysis bag after loading the sample (M1) before dialysis. After dialysis, accurately weigh the mass of the dialysis bag and the gel (M2). The initial HA concentration (N) is 20 mg / mL, and calculate the sodium hyaluronate content (C) of the gel after dialysis:

[0132]

[0133] (5) Dilute and compound to a concentration of 15 mg / mL of sodium hyaluronate at concentration C, add lidocaine hydrochloride, and the diluent is the same as the neutral dialysis solution. The content of lidocaine hydrochloride in the gel is 3 mg / mL. Crush the compounded gel and fill it into a pre-filled syringe, and sterilize it by moist heat at 121 °C for 15 min to obtain cross-linked sodium hyaluronate hydrogel.

[0134] Performance testing 1 Elastic modulus test

[0135] Use a rheometer to detect the elastic modulus of the hydrogel samples before and after sterilization in Examples 1-5 and Comparative Examples 1-4.

[0136] Table 1 Elastic modulus of samples before and after sterilization

[0137]

[0138] Note: Before sterilization, the gel showed "water-gel separation" and could not be tested.

[0139] It can be seen from Table 1 that the elastic modulus of the hydrogel samples in the examples is relatively high before and after sterilization, and the elastic modulus loss rate is almost within 12%, with good moist heat sterilization resistance, all meeting the clinical use requirements.

[0140] Among them, the elastic modulus of the hydrogel sample in Example 1 is relatively high before and after sterilization, and the loss rate is only 7.5%, with high support performance and stability. This is because while the first-step cross-linking forms a network structure, the carboxyl group in the HA molecule forms an ester bond with the activated hydroxyl group, playing an occupancy effect; then the added HA and cross-linking agent can be uniformly inserted into the network structure formed in the first step, and then secondary cross-linking occurs under the action of a catalyst to obtain a cross-linked gel with an interlocking structure. Further, during the alkali dialysis process, the released HA can further enhance the intermolecular force and binding force of the cross-linked network, making the gel product have a high elastic modulus, good support performance, and strong moist heat sterilization resistance.

[0141] The elastic modulus of the hydrogel sample in Example 2 is higher than that in Example 1, and the loss rate is lower, only 6%. This is because the cross-linking degree of the first-step cross-linking reaction is low, and the formed cross-linked network structure is relatively loose, which is more conducive to the interpenetration and entanglement of subsequent HA molecules, making it easier to form a tight interlocking structure between different levels of networks, so the support performance is better and the moist heat sterilization resistance is stronger.

[0142] The elastic modulus of the hydrogel sample of Example 3 is slightly lower than that of Example 1. This is because although an interlocked structure can be formed through two-step reactions, when the overall crosslinking degree is similar, the amount of crosslinking agent used in the first crosslinking reaction is relatively large, resulting in a relatively high crosslinking degree. The formed crosslinked network structure is relatively dense, and the interpenetrating performance of HA molecules is weak. Therefore, the intermolecular force decreases slightly, and the sample shows a slightly lower elastic modulus.

[0143] The elastic modulus of the hydrogel sample of Example 1 is higher than that of Example 4. This is because in the first crosslinking reaction, the amount of catalyst added in Example 1 is relatively high, which can form a complete occupancy of HA. Therefore, the hydrogel product has more interlocked structures, the network structure is denser, and the support performance of the gel is better. In Example 4, the amount of catalyst added is slightly lower, so the carboxyl sites of HA are not completely occupied, which reduces the proportion of interlocked structures in the crosslinked gel and slightly reduces the support performance of the gel. This also shows that the best way in the first crosslinking is "complete occupancy".

[0144] In addition, from the data of Example 5, it can be seen that using spermine as a crosslinking agent can also achieve good crosslinking effects, forming an interlocked crosslinked hyaluronic acid or its salt hydrogel, and both the support performance and the resistance to moist heat sterilization meet the clinical use requirements.

[0145] It can be seen from Example 3 and Comparative Example 1 that the elastic modulus of the hydrogel sample of Comparative Example 1 is relatively low before and after sterilization. At the same time, there is a significant difference in the elastic modulus loss rate between the sample of Comparative Example 1 and the sample of Example 3, up to 24%. The main reason is that Comparative Example 1 is a physical mixture rather than an interlocked crosslinked gel prepared by two-step chemical crosslinking. The force between the two gel structures in the physical mixture is small, so the elastic modulus of the gel product is relatively low, and the ability to resist moist heat sterilization is also weak.

[0146] At the same time, comparing the data of Example 2 and Comparative Example 2, before sterilization, the sample of Comparative Example 2 was in a "water-gel separation" state, and it was difficult to detect the elastic modulus data. This is because in the EDC / NHS catalytic system, ester bonds will inevitably be generated, which easily makes the crosslinked gel in a supersaturated state. During the dialysis process, only neutral dialysis was used instead of alkaline dialysis, so the ester bonds could not be effectively broken, resulting in the sample showing a "water-gel separation" state at a relatively low HA concentration (15 mg / mL), which could not meet the actual clinical use requirements. The sample of Example 2 can retain the glycosidic bond of HA while breaking most of the ester bonds through appropriate alkaline dialysis treatment, which can not only improve the ability of the gel to resist moist heat sterilization but also solve the "water-gel separation" problem at the current HA concentration.

[0147] Meanwhile, from the elastic modulus data after sterilization, it can be seen that the crosslinking degrees of the two gel products are the same, 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 was not subjected to alkaline dialysis, and even after autoclaving, some ester bonds remained in the gel, resulting in a higher elastic modulus. However, ester bonds are extremely unstable, which will have an adverse impact on the storage stability of the product and also restrict the viscosity of the gel product (see Performance Test 3).

[0148] From the data of Example 2 and Comparative Example 3, it can be seen that compared with the ordinary single-crosslinked gel, the interlocking structure design of Example 2 brings higher support performance to the product, which is more conducive to the tissue repair of the parts that need shaping; while for the single-crosslinked hydrogel sample of Comparative Example 3, simply increasing the crosslinking degree will lead to excessive pushing force of the hydrogel sample, so its clinical use is limited (see Performance Test 5).

[0149] From the data of Example 2 and Comparative Example 4, it can be seen that when the amount of EDC / NHS added in the first crosslinking reaction process is too low, the carboxyl sites of HA that did not participate in the crosslinking reaction are not occupied by EDC / NHS, so that the free HA will consume the crosslinking agent added in the second step, reducing the crosslinking reaction efficiency of HA interspersed in the first network and reducing the formation of the interlocking structure, thereby significantly reducing the support of the hydrogel sample.

[0150] Performance Test 2 Esterase Degradation Experiment

[0151] Prepare a 100 U / mL esterase solution (carboxylesterase, purchased from Sigma) with purified water for later use. 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 pH = 8.65 for later use. Take 2 mL of the hydrogel sample of Example 2 and the hydrogel sample of Comparative Example 2 respectively, add 300 μL of the esterase stock solution and 700 μL of the boric acid buffer solution to them, mix well, and then detect the enzymatic hydrolysis curve.

[0152] Use a rheometer to set the following program to test the change of the elastic modulus of the sample with the degradation time: Under the condition of 37 °C, use the Oscillation Time mode of the rheometer to test for 7200 s; deformation (Strain): 1%; frequency (Frequency): 0.9 Hz. Investigate the degradation rates at 10 min, 30 min, 60 min, 90 min, and 120 min of degradation. The results are shown in Table 2 and Table 3.

[0153] Table 2 Degradation Rates of Samples Before Sterilization under the Action of Esterase

[0154]

[0155] Table 3 Degradation Rates of Samples After Sterilization under the Action of Esterase

[0156]

[0157] As can be seen from the data before sterilization in Table 2, compared with Comparative Example 2, the degradation rate of the hydrogel sample before sterilization in Example 2 is significantly lower than that of the hydrogel sample in Comparative Example 2. After 120 min of enzymatic hydrolysis treatment, the degradation rate is only 6.2%. This is because in Example 2, alkaline dialysis was used to eliminate most of the ester bonds generated in the cross-linking process. Therefore, there are fewer substrates for esterase; while a large number of ester bonds are retained in the hydrogel sample of Comparative Example 2 that was not treated with alkaline dialysis. Under the action of esterase, the degradation rate of the hydrogel is high, even reaching 60%.

[0158] From the data after sterilization in Table 3, it can be known that since the ester bonds will be destroyed during the moist heat sterilization process, the degradation rates of the hydrogel samples in Example 2 and Comparative Example 2 both decrease. However, the degradation rate of the hydrogel sample in Comparative Example 2 is still significantly higher than that in Example 2. The reason is that there are originally more unstable ester bonds in Comparative Example 2, and the number of ester bond bases is large. Although the ester bonds will be destroyed during the moist heat sterilization, the ester bonds retained in the final product are relatively more. After 120 min of esterase treatment, the degradation rate is 15%, which is significantly higher than the degradation rate of 1.1% of the sample in Example 2. Further, after 60 min of esterase treatment, the degradation rate of the hydrogel sample in Example 2 hardly changes. However, for the hydrogel sample in Comparative Example 2, as the esterase treatment time increases, the degradation rate still gradually increases, which also indicates that the presence of ester bonds will affect the storage stability of the product. At the same time, due to the large amount of unstable ester bond substances, the modulus of the product will be greatly reduced during storage, which will also affect the clinical use of the product. Therefore, through appropriate alkaline dialysis, the present invention can eliminate the unstable ester bonds generated in the cross-linking process, which is beneficial to improving the storage stability of the product.

[0159] Performance Test 3 Viscosity

[0160] (1) Drop Weight Method

[0161] The hydrogel samples of Examples 1-4 and Comparative Examples 1-4 were filled into 1 mL prefilled syringes (removing air bubbles), and after installing butyl chloride rubber pistons, push rods, and boosters, and then installing a 27G needle, they were clamped with an appropriate fixture. The motor was started to move the upper and lower fixtures to an appropriate position, and the push rod was pushed at a constant speed of 30 mm / min. When a constant force was obtained, the gel was extruded from the needle until it dropped and disconnected, which was one sample. A total of 10 samples were collected and weighed, and the average weight of the samples was calculated. The greater the weight, the better the viscosity. The results are shown in Table 4.

[0162] Table 4 Viscosity Characterization

[0163]

[0164] Table 4 results show that the interlocked cross-linked hydrogels prepared by two-step chemical cross-linking (Examples 1-4) have higher viscosity compared to the hydrogels prepared by physically mixing gels with different degrees of cross-linking only (Comparative Example 1). This is because, after undergoing two-step cross-linking reactions, the molecular chains formed by the cross-linking reaction between sodium hyaluronate and cross-linking agent molecules are interlocked, and the three-dimensional network structures interpenetrate each other, resulting in a significant increase in both the intermolecular binding force and molecular tension of HA molecules. The cross-linked HA network structure with an interlocked structure is denser, thus exhibiting excellent viscosity.

[0165] In Comparative Example 2, alkaline dialysis was not carried out, so there are more residual ester bonds, the thermal stability of the hydrogel sample is poor, the network structure is unstable, which is not conducive to the establishment of the product shelf life.

[0166] Compared with Example 2, in the hydrogel sample of Comparative Example 4, since no effective occupancy ester bonds were formed during the first-step cross-linking process, the cross-linking efficiency of HA was reduced, and it was difficult to release linear HA chains after dialysis. Therefore, the interaction force between the cross-linked HA network structures is weak, so the viscosity of the hydrogel sample in Comparative Example 2 is low.

[0167] (2) Appearance

[0168] Observe the appearance of the samples of Example 2 and Comparative Example 1 in (1), and the results are shown in Figure 2 . From Figure 2 it can be seen that from the product appearance, it further shows that the viscosity of the interlocked cross-linked hydrogel sample prepared in Example 2 ( Figure 2 A therein) is better than that of the hydrogel sample in Comparative Example 1 ( Figure 2 B therein). Higher viscosity is crucial for the clinical use of the product, which is manifested as good ductility after injection, easy to spread and plasticize, strong binding ability with tissues, no displacement, making the injection filling effect more natural and the maintenance time longer.

[0169] Performance Test 4 In vitro Degradation

[0170] Take 1.0 mL of 30 U / mL hyaluronidase solution (hyaluronidase was purchased from sigma company) and add it to 2.0 mL of the hydrogel samples of Examples 1-4 and Comparative Examples 1-4. After manual stirring and mixing (ensure uniform mixing of the hydrogel sample and the enzyme solution), use a rheometer to measure the elastic modulus of the hydrogel sample within 2 h (the Peltier plate temperature of the rheometer needs to be balanced to 4 °C before loading), and the enzymatic hydrolysis rate is characterized by the change value of the elastic modulus. The results are shown in Table 5.

[0171] Table 5 Degradation Rate (%)

[0172]

[0173] From the above in vitro degradation experiment data, it can be seen that after 120 min, the anti-enzymatic degradation ability of the hydrogel samples of Examples 1-4 is significantly higher than that of Comparative Examples 1, 3, and 4, indicating that the interlocking structure can significantly improve the retention time of the product in vivo after injection and give full play to its long-acting filling effect. At the same time, by comparing the degradation rates of Example 2 and Comparative Example 4, it can be seen that in the preparation process of the sample of Comparative Example 4, no EDC / NHS occupation occurred in the first-step crosslinking, resulting in a low efficiency of the second-step crosslinking. Almost no interlocking structure could be formed, and at the same time, the intermolecular force of the interlocking network structure could not be further enhanced by using the HA chains formed after alkali dialysis. Therefore, the anti-enzymatic degradation ability is weak.

[0174] Comparative Example 2 was not treated with alkali dialysis, which had little effect on the anti-enzymatic degradation of the hydrogel sample. Therefore, the degradation rate was slightly lower than that of Example 2, but it had a greater impact on the viscosity, stability, etc. of the product (Performance Tests 2 and 3).

[0175] Performance Test 5 Pushing Force

[0176] 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.

[0177] Table 6 Pushing Force

[0178]

[0179]

[0180] Note: (1) The pushing force test method refers to the method in YY / T 0962-2021; (2) The injection needle used for testing was 27G×1 / 2.

[0181] As can be seen from Table 6, the pushing forces of the hydrogel samples of Examples 1-5 are small, which is beneficial for clinical injection. Among them, the pushing force of the hydrogel sample of Example 2 is less than that of the hydrogel sample of Comparative Example 2. The reason is that alkali dialysis releases the linear structure of sodium hyaluronate when breaking the ester bond, which is equivalent to adding free HA and plays a lubricating role, making the product easier to inject.

[0182] At the same time, the pushing force of the hydrogel sample of Example 2 is less than that of the hydrogel sample of Comparative Example 3. The reason is that compared with the hydrogel prepared by only one crosslinking, the hydrogel prepared by two-step chemical crosslinking forms different levels of spatial structures, making the released free HA molecules interpenetrate more fully. Furthermore, the spatial structure is more compact, improving the product's supportability and making it easier to inject, meeting the requirements of clinical injection use. Similarly, in Comparative Example 4, due to the lack of HA occupation effect in the previous crosslinking, the free HA has participated in the crosslinking reaction and the HA interspersed in the network is difficult to release. Therefore, the pushing force is also high.

[0183] The above are only embodiments of the present invention, and do not thereby limit the patent scope of the present invention. Any equivalent structural transformation made by using the content of the specification of the present invention, or directly or indirectly applied in other related technical fields, shall similarly be included within 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, preferably sodium hyaluronate; The molecular weight of the hyaluronic acid or its salt is 500 - 2600 KDa.

3. The injectable interlocking cross-linked hyaluronic acid or its saline gel according to claim 1 or 2, characterized in that: The particle size of the injectable interlocked crosslinked hyaluronic acid or its salt hydrogel is 50 - 650 μm; In the injectable interlocked crosslinked hyaluronic acid or its salt hydrogel, the concentration of hyaluronic acid or its salt is 10 - 25 mg / mL.

4. A method for preparing an injectable interlocked cross-linked 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.

5. The preparation method according to claim 4, characterized in that: In steps (1) and (3), the crosslinking agent is an endogenous polyamine, including spermidine, spermine and their derivatives; 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.

6. The preparation method according to claim 4, characterized in that: The mass concentration of hyaluronic acid or its salt in the mixed solution A and the mixed solution B is 1% - 5%.

7. The preparation method according to claim 4, characterized in that: The molar ratio of the crosslinking agent to hyaluronic acid or its salt in the mixed solution A and the mixed solution B is 0.01 - 0.

1.

8. The preparation method according to claim 7, characterized in that: The molar ratio of the crosslinking agent to hyaluronic acid or its salt in step (1) is lower than that in step (3).

9. The preparation method according to claim 4, 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).

10. The preparation method according to claim 9, characterized in that: In step (2), the molar ratio of the catalyst to hyaluronic acid or its salt in the mixed solution A is 0.6 - 3, and the crosslinking time is 1 - 3 h; and / or In step (4), the molar ratio of the catalyst to hyaluronic acid or its salt in the mixed solution B is 0.03 - 0.3, and the crosslinking reaction time is 1 - 24 h.

11. According to the preparation method described in claim 4, 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 dialysis solution, the molar ratio of hydroxide to the total hyaluronic acid or its salt used in steps (1) and (3) is 1:1 - 10:1, the alkaline dialysis temperature is 10 - 40 °C, and the dialysis duration is 1 - 10 h; and / or The neutral dialysis temperature is 10 to 40 °C, and the dialysis duration is 1 to 20 h.

12. Use of the injectable interlocked cross-linked hyaluronic acid or its saline gel according to any one of claims 1-3, or the injectable interlocked cross-linked hyaluronic acid or its saline gel obtained by the preparation method according to any one of claims 4-11, in the preparation of a tissue filling and repair material or a drug carrier for non-therapeutic purposes.

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