Gel materials, methods for preparing same and uses

Abstract

The present disclosure provides a gel material, its preparation method and use. The gel material is obtained by crosslinking endogenous polyamines with hyaluronic acid, the endogenous polyamines include spermine and / or spermidine, and the crosslinking between endogenous polyamines and hyaluronic acid includes two-site crosslinking, three-site crosslinking or four-site crosslinking. The reactive crosslinking sites can be controlled to affect various performances of the gel, as well as the degradation and release rate of polyamines. The gel has a low elastic modulus loss rate after moist heat sterilization, and can effectively maintain the rheological performance of the gel before sterilization, greatly improving the thermal stability of the hyaluronic acid gel, and also improving the usability of the gel in fields such as soft tissue filling, soft tissue repair and medical beauty.

Description

[Technical Field]

[0001] The present invention relates to the technical field of biomedical materials, and in particular to gel materials, their preparation methods and uses. [Background technology]

[0002] Hyaluronic acid or sodium hyaluronate (HA), also known as hyaluronan acid, is a glycosaminoglycan consisting of a disaccharide unit composed of D-glucuronic acid linked via a β-1,4 glycosidic bond to N-acetylglucosamine via a β-1,3 glycosidic bond. It is widely used in cosmetics and ophthalmic surgery, and can also be used as a soft tissue filler to repair wrinkles and certain soft tissue defects. Hyaluronic acid is a natural substance found in the body, has good biocompatibility, and possesses certain biological activity. However, exogenous hyaluronic acid is degraded by hyaluronidase in the body, resulting in a shorter retention time in the body and a shorter therapeutic effect, requiring multiple injections to achieve therapeutic results. In order to prevent the degradation of hyaluronic acid by hyaluronidase, it is necessary to crosslink hyaluronic acid molecules with a chemical crosslinking agent to form a spatial network structure. This dense and rigid network structure prevents the degradation of hyaluronic acid by hyaluronidase, extends the retention time of exogenous hyaluronic acid in the body, ensures biocompatibility, and brings about good therapeutic effects.

[0003] Currently, crosslinkers for crosslinked hyaluronic acid on the market are primarily divided into two types: diepoxy crosslinkers, primarily 1,4-butanediol diglycidyl ether (BDDE), and unsaturated sulfone crosslinkers, primarily divinyl sulfone (DVS). These two crosslinkers have similar mechanisms. Both use an alkaline catalyst as a precondition to catalyze the addition reaction between the hydroxyl groups (-OH) of hyaluronic acid and the crosslinker to complete the crosslinking. The -OH groups in hyaluronic acid undergo a ring-opening addition reaction with BDDE to complete the crosslinking, while DVS undergoes a Michael addition reaction with the -OH groups in hyaluronic acid to complete the crosslinking. However, both crosslinkers have high biological toxicity, and unreacted monomers or crosslinking by-products are potentially carcinogenic. Because hyaluronic acid gel is a long-term implantable medical device, there is a lack of long-term data from a sufficient number of samples to demonstrate the biological safety of this product. In terms of current market demand, extending the residence time of hyaluronic acid gel in the body and increasing the viscoelasticity of hyaluronic acid gel are both major trends in the industry. The current method that can effectively extend the residence time of hyaluronic acid gel in the body is to increase the degree of crosslinking, but at the same time, increasing the degree of crosslinking can also improve viscoelasticity. However, when using traditional BDDE or DVS crosslinkers, the increased amount of crosslinker added raises people's concerns about the safety of this type of product.

[0004] Under this premise, the selection of non-toxic crosslinkers for preparing hyaluronic acid gels has become more common among hyaluronic acid gel manufacturers worldwide. Currently, non-toxic amino acid crosslinkers are used in hyaluronic acid gels. Chinese Patent Application No. CN10105713211 discloses crosslinked sodium hyaluronate gels prepared using 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride as a condensing agent and lysine and its derivatives or arginine and its derivatives as a crosslinking agent. Chinese Patent Applications Nos. CN106188609, CN106188584, and CN111732741 disclose crosslinked sodium hyaluronate gels prepared using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride as a condensing agent, N-hydroxysuccinimide as a catalyst, and arginine and its derivatives, lysine and its derivatives (including polylysine) as crosslinking agents. The above patent applications all disclose sodium hyaluronate gels prepared using diaminoamino acids such as lysine or arginine as crosslinkers. Taking lysine or arginine as an example, the crosslinked hyaluronate hydrogels exhibit significant differences in performance during moist heat sterilization. CN106188609, CN106188584, and CN111732741 do not specifically reflect that crosslinked hyaluronate can be moist heat sterilized at 121°C for 15 minutes. CN10105713211 discloses a lysine-crosslinked hyaluronate gel that can be moist heat sterilized. However, according to the information disclosed in the patent application, the minimum degradation rate of this type of gel after moist heat sterilization is already over 40%, indicating that this type of gel has poor resistance to moist heat sterilization. US9907739B2 discloses the cross-linking reaction between spermine or spermidine and hyaluronic acid. However, when detected based on the method of the present invention, the gel disclosed in this patent application is only two-site cross-linked, not multi-site cross-linked, that is, the cross-linking site is only the amino group of spermine or spermidine, and the imino group is not involved in the reaction. Furthermore, the elastic modulus loss rate of this type of gel after moist heat sterilization is as high as 23%, which also indicates that this type of gel has poor resistance to moist heat sterilization.In addition, a paper published by Xiang Mei Yan et al. (Journal of Biomaterials Applications, 2011, 27(2):179-186) discloses a hyaluronic acid hydrogel crosslinked with hexamethylenediamine, and the elastic modulus of the gel also decreased by more than 20% after sterilization. The main reasons for this are as follows: binary amino molecules such as lysine, arginine, and hexamethylenediamine only have two amino crosslinking sites. When crosslinking occurs, an amide bond is formed with the carboxyl group of hyaluronic acid to complete the crosslinking. However, due to the limited number of crosslinking sites in the molecule, the formed crosslinked network structure cannot provide sufficient thermal stability protection for the hyaluronic acid main chain. In addition, the amide bond itself has poor thermal stability. Therefore, when embodied in a gel, it has a high decomposition rate after moist heat sterilization, indicating poor thermal stability.

[0005] Endogenous polyamines primarily refer to polyamines synthesized or produced during metabolism in the human body. The main endogenous polyamines include spermine, spermidine, and putrescine. (Madeo et al., Science 359, 410, 2018) reported that endogenous polyamines, namely spermidine, have specific physiological functions, including but not limited to regulating circadian rhythm, improving hypertension, protecting the cardiovascular system, preventing Alzheimer's disease, strengthening the immune system, anti-cancer, and anti-aging. The physiological effects of spermidine are as follows: 1) Kidneys: Reduces tension and prevents senility; 2) Heart: Lowers blood pressure and prevents arteriosclerosis; 3) Brain: Prevents memory decline, Alzheimer's disease, and protects the nerves; 4) Bones: Prevents bone loss due to ovariectomy; 5) Skeletal muscles: Increases the temperature of aged muscles and prevents muscle diseases; 6) Whole organism: Prolongs the lifespan of the organism; 7) Immune system: Improves immune activity after vaccination, improves directed immunity against cancer, and prevents fatal sepsis; 8) Liver: Prevents liver fibrosis and carcinogenesis. The main mechanisms by which spermidine produces its physiological activity are as follows: Spermidine binds to polycationic (-NH 3+Spermidine is an aliphatic amine that exists in a polyprotonated form under physiological pH conditions and has strong biological activity. Potential targets for spermidine binding include nucleic acids containing acidic residues, phospholipids, acidic proteins, pectic polysaccharides containing carboxyl or sulfate groups, and neurotransmitters and hormones with similar structures (e.g., dopamine, epinephrine, serotonin, thyroid hormone, etc.). In terms of binding to nucleic acids, most polyamines exist in the form of polyamine-RNA complexes within cells. Spermidine's primary role is related to RNA structural changes and translation, for example, by affecting the secondary structure of mRNA, tRNA, and rRNA, thereby influencing each stage of protein synthesis. Spermidine can also form stable bridges between double-helical DNA strands, reducing the access of ROS and other DNA-damaging factors and protecting DNA from heat denaturation and X-ray irradiation. In terms of binding to proteins, spermidine can bind to many negatively charged proteins, altering their spatial structure and thereby affecting their physiological functions. These include, for example, protein kinases / phosphatases (key links in many signal transduction pathways), enzymes involved in histone methylation and acetylation (which affect gene expression by altering epigenetics), and ion channel receptors (e.g., AMPA and AMDA receptors).

[0006] Currently, there are few reports on the use of endogenous polyamines such as spermidine as crosslinkers for hyaluronic acid gels, and there are also few reports on the crosslinking reaction conditions between endogenous polyamines and hyaluronic acid or the performance of the crosslinked hydrogels.In light of this, the present invention is provided. Summary of the Invention

[0007] The object of the present invention is to provide a gel material, its preparation method and use. The preparation method of the present invention can control the crosslinking sites between endogenous polyamines and hyaluronic acid, and by controlling the crosslinking reaction sites, it can affect the performance parameters of the resulting crosslinked hyaluronic acid gel, and control the release rate of endogenous polyamines such as spermidine during the degradation process of the hyaluronic acid gel, so that the physiological activity effects of endogenous polyamines such as spermidine can be sustained.

[0008] The technical solutions provided by the present invention are as follows: In one aspect, the present invention provides a gel material, which is obtained by crosslinking endogenous polyamines with hyaluronic acid, the endogenous polyamines including spermine (a tetraamino compound) and / or spermidine (a triamino compound), and the crosslinking between the endogenous polyamines and the hyaluronic acid includes two-site crosslinking, three-site crosslinking, or four-site crosslinking. In particular, the present invention is the first to achieve three-site or four-site crosslinking between endogenous polyamines and hyaluronic acid to form an active star-shaped network structure through multi-site crosslinking.

[0009] The present invention selects the endogenous polyamine spermine or spermidine as a multi-site cross-linking agent, which forms a dense network structure and improves the thermal stability of the amide-bond cross-linked hyaluronic acid gel thus formed. The reason why the present invention does not use endogenous diamines such as putrescine is as follows: diamines such as putrescine are highly toxic, and their cross-linking sites are the same as those of amino acids such as lysine and arginine, making it impossible to achieve multi-site cross-linking.

[0010] In one embodiment, the present invention provides a gel material, the gel material being obtained by cross-linking endogenous polyamines with hyaluronic acid, the endogenous polyamines comprising spermine and / or spermidine, and the cross-linking between the endogenous polyamines and the hyaluronic acid comprising two-site cross-linking, three-site cross-linking, or four-site cross-linking.

[0011] Furthermore, the proportion of residual amino groups in the gel obtained by the two-site crosslinking is less than 20%, preferably, in the two-site crosslinking, the proportion of residual amino groups in the gel obtained by crosslinking is less than 15%, and more preferably, the proportion of residual amino groups in the gel obtained by the two-site crosslinking is less than 10%.

[0012] Furthermore, the proportion of residual amino groups and residual imino groups in the gel obtained by three-site crosslinking or four-site crosslinking is both less than 20%.

[0013] Furthermore, the proportion of residual amino groups in the gel obtained by crosslinking through the three-site crosslinking or four-site crosslinking is less than 15%, and preferably, the proportion of residual amino groups in the gel obtained by crosslinking through the three-site crosslinking or four-site crosslinking is less than 10%.

[0014] Furthermore, the proportion of residual imino groups in the gel obtained by crosslinking through the three-site crosslinking or four-site crosslinking is less than 15%, and preferably, the proportion of residual imino groups in the gel obtained by crosslinking through the three-site crosslinking or four-site crosslinking is less than 10%.

[0015] Furthermore, in the crosslinking reaction between the endogenous polyamines and hyaluronic acid, the crosslinking reaction efficiency of two-site crosslinking, three-site crosslinking, or four-site crosslinking is higher than 75%, preferably higher than 80%, and more preferably higher than 85%.

[0016] Furthermore, the gel obtained by the crosslinking has a modulus loss (G' loss) of less than 22%, preferably a modulus loss (G' loss) of less than 15%, and more preferably a modulus loss (G' loss) of less than 10%.

[0017] In another aspect, the present invention provides a method for preparing a gel material, said method comprising: adjusting the pH of a mixed solution of hyaluronic acid and endogenous polyamine to 4.50-6.50, and adding an activator, thereby causing a two-site, three-site, or four-site cross-linking reaction between the hyaluronic acid and the endogenous polyamine to obtain the gel material; However, the endogenous polyamines include spermine and / or spermidine.

[0018] Spermidine and spermine contain an amino group and an imino group, with spermidine containing one imino group and two amino groups, and spermine containing two amino groups and two imino groups. The present inventors discovered that by adjusting the pH of a mixed solution containing hyaluronic acid and endogenous polyamines, the reaction sites between spermine or spermidine and hyaluronic acid can be controlled, resulting in two-site crosslinked hydrogels and three- or four-site crosslinked activated star-shaped network gels. During the reaction process, the pH of the solution can be adjusted to control the reaction at the amino site of spermine or spermidine (i.e., two-site reaction), or to allow both the amino and imino groups to react (i.e., multi-site reaction). The two-site crosslinking described in the present invention means that when hyaluronic acid is crosslinked with spermine or spermidine, the amino groups in spermine or spermidine are the main reactive sites, and in particular, the proportion of residual amino groups in the gel obtained by the two-site crosslinking is less than 20%. The three-site crosslinking or four-site crosslinking described in the present invention means that when hyaluronic acid is crosslinked with spermine or spermidine, both the amino groups and imino groups in spermine or spermidine become reactive sites, and in particular, the proportions of residual amino groups and residual imino groups in the gel obtained by the three-site crosslinking or four-site crosslinking are both less than 20%.

[0019] In the present invention, the pH value of the solution is controlled to 5.00 to 5.49, thereby improving the reactivity of the imino group and realizing a three-site reaction or a four-site reaction relatively stably. Active star-shaped network gels can be prepared; By controlling the pH value of the solution to 4.50 to 4.99 or 5.50 to 6.50, a Amino reaction activity Improved, 2 Relatively stable joint reaction participation of the sites do Therefore, the present invention has achieved for the first time a hyaluronic acid hydrogel with imino groups as crosslinking sites, and has found that hydrogels with different crosslinking sites can have different gel properties even with the same degree of crosslinking.

[0020] In the present invention, a relatively stable three-site or four-site reaction can be achieved by controlling the pH of the mixed solution between 5.00 and 5.49, including, but not limited to, 5.00, 5.10, 5.20, 5.30, 5.40, and 5.49. A relatively stable two-site crosslinking reaction can be achieved by controlling the pH of the mixed solution between 4.50 and 4.99 or between 5.50 and 6.50, including, but not limited to, 4.50, 4.60, 4.70, 4.80, 4.90, 4.99, 5.50, 5.60, 5.70, 5.80, 5.90, 6.00, 6.10, 6.20, 6.30, 6.40, and 6.50. Experimental results are not ideal outside the pH range of the present invention. The pH range of the present invention is a pH condition suitable for the method of the present invention, which was determined through extensive experimentation.

[0021] The method of the present invention is an endogenous polyamine (spermine and permidine) multisite active reaction technology (Spermidine / Spermine Multisite Active Reaction Technology, abbreviated as SMART). The method of the present invention can control the crosslinking sites between endogenous polyamines and hyaluronic acid, and by controlling the crosslinking reaction sites, it can affect the performance parameters of the resulting crosslinked hyaluronic acid gel. Furthermore, it can control the release rate of endogenous polyamines such as spermidine during the degradation process of the hyaluronic acid gel, thereby sustaining the physiological activity of endogenous polyamines such as spermidine.

[0022] In one embodiment, an activator is added to the two-site crosslinking reaction, the three-site crosslinking reaction, or the four-site crosslinking reaction, Preferably, the activating agent comprises one or more of a water-soluble carbodiimide, a phosphonium bromide salt formed by triphenylphosphine and bromide, a carbonium salt, and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM).

[0023] In one embodiment, the water-soluble carbodiimide activator comprises 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide, 1,3-bis[di(methoxymethyl)methyl]carbodiimide, and the like, or salts thereof, and mixtures of one or more thereof.

[0024] In one embodiment, the phosphonium bromide salts formed with triphenylphosphine and bromide include phosphonium salts formed with triphenylphosphine and carbon tetrabromide, phosphonium salts formed with triphenylphosphine and N-bromosuccinimide, etc. For the phosphonium bromide salts, the required phosphonium salt is obtained from triphenylphosphine and bromide in dichloromethane by conventional methods.

[0025] In one embodiment, the carbonium salt comprises a mixture of one or more of O-(7-azabenzotriazol-1-yl)-bis(dimethylamino)carbonium hexafluorophosphate (HATU), O-(benzotriazol-1-yl)-bis(dimethylamino)carbonium hexafluorophosphate (HBTU), O-(5-chlorobenzotriazol-1-yl)-bis(dimethylamino)carbonium hexafluorophosphate (HCTU), O-(benzotriazol-1-yl)-bis(dimethylamino)carbonium tetrafluoroborate (TBTU), O-(N-succinimino)-bis(dimethylamino)carbonium tetrafluoroborate (TSTU), and 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethylurea tetrafluoroborate (TNTU).

[0026] When using a water-soluble carbodiimide activator, it is necessary to use a co-agent in combination to improve the crosslinking reaction efficiency. Preferably, the co-agent includes any one or more of N-hydroxysuccinimide (NHS), sulfonated N-hydroxysuccinimide (Sulfo-NHS), tert-butanol, and 1-hydroxybenzotriazole (HOBt). More preferably, the co-agent is added in an amount of 10-30% of the mass of the carbodiimide, including, but not limited to, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, and 30%. The use of a water-soluble carbodiimide and a co-agent in combination can increase the crosslinking reaction efficiency to approximately 70%.

[0027] Among the three activators, phosphonium bromide salt formed by triphenylphosphine and bromide, carbonium salt, and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride, carbonium salt had the highest crosslinking efficiency at 80-95%, with the crosslinking efficiency when using the HATU catalyst reaching 95%, followed by phosphonium bromide salt and DMTMM, with a crosslinking efficiency of 70-85%.

[0028] In a preferred embodiment, the carbonium salt HATU is used as the reaction activator. The molecular formula structure of HATU is as follows: [ka]

[0029] The mechanism by which HATU participates in the amide condensation reaction is as follows. [ka]

[0030] In the crosslinking reaction, when two-site crosslinking (the main reactive site of spermine or spermidine is an amino group) is performed, the amount of the activator added is 200 to 280% of the amount of the endogenous polyamine, including, but not limited to, 210%, 220%, 230%, 240%, 250%, 260%, 270%, and 280%; when three-site and / or four-site crosslinking (the imino site also participates in the reaction) is performed, the amount of the activator added is 300 to 550% of the amount of the endogenous polyamine, including, but not limited to, 320%, 350%, 370%, 390%, 400%, 420%, 450%, 470%, 500%, 520%, and 550%.

[0031] Preferably, when the endogenous polyamine is spermine, the amount of the activator added is 400 to 550% of the substance amount of the spermine, and when the endogenous polyamine is spermidine, the amount of the activator added is 300 to 400% of the substance amount of the spermidine.

[0032] The amount of activator added is related to the amino crosslinking site of the crosslinker spermine or spermidine. When using the above activator, each molecule of the activator activates one carboxyl group, and in addition: The activated carboxyl group is An amide coupling reaction can be carried out with one amino group (or imino group).

[0033] In one embodiment, in the cross-linking reaction, when the endogenous polyamine is spermine, the amount of spermine added accounts for 0.3 to 35% of the mass of the hyaluronic acid, including, but not limited to, 0.4%, 0.5%, 0.8%, 1%, 3%, 5%, 8%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, and 35%. When the endogenous polyamine is spermidine, the amount of spermidine added is 0.5 to 40% of the mass of hyaluronic acid, including, but not limited to, 0.5%, 0.8%, 1%, 3%, 5%, 8%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 34%, 38%, and 40%.

[0034] Furthermore, the mass concentration of hyaluronic acid in the aqueous solution of the reaction system is 10 to 150 mg / mL, including, but not limited to, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, and 150 mg / mL.

[0035] Furthermore, the mass concentration of hyaluronic acid in the finally obtained crosslinked hyaluronic acid hydrogel is 1 to 50 mg / mL, including, but not limited to, 1, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 mg / mL.

[0036] In one embodiment, the molecular weight of the hyaluronic acid is 100 KDa (kilodaltons) to 3000 KDa, and preferably, the hyaluronic acid is prepared by microbial fermentation.

[0037] In one embodiment, in the crosslinking reaction, when the activator includes a carbonium salt and / or a phosphonium bromide salt formed by triphenylphosphine and bromide, the temperature of the crosslinking reaction is 10 to 60°C, and the time of the crosslinking reaction is 14 to 24 hours; When the activator contains a water-soluble carbodiimide and / or 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride, the temperature of the crosslinking reaction is 40 to 60° C., and the time of the crosslinking reaction is 14 to 24 hours.

[0038] During the reaction process, the formation of two-site crosslinked gels and three- or four-site crosslinked activated star-shaped network gels can be controlled by varying the reaction temperature and the ratio of activator to spermine or spermidine. Due to steric hindrance, imino groups are less reactive than primary amino groups. Therefore, in two-site reactions involving only primary amino groups, lower temperatures and less activator are required to complete the crosslinking reaction. However, when imino groups are required to participate in the reaction, the reaction temperature must be increased and the reaction time must be extended to increase the reactivity of the imino groups and improve the efficiency of the imino crosslinking reaction.

[0039] The cross-linking reactions between spermidine and hyaluronic acid and between spermine and hyaluronic acid are as follows, where (3) is the cross-linking reaction between hyaluronic acid and spermine (four-site star network), and (4) is the cross-linking reaction between hyaluronic acid and spermidine (three-site star network). [ka] [ka]

[0040] The present invention uses SMART multi-site active reaction technology to prepare hyaluronic acid gels, which take full advantage of the different activities of the amino and imino groups in spermine or spermidine molecules. By controlling one or more of the reaction conditions, such as pH, temperature, and the type and amount of active agent, two-site crosslinked gels and three-site or four-site crosslinked active star-shaped network gels can be obtained, respectively.

[0041] In one embodiment, the gel material is further eluted with an eluant, crushed, dried, Preferably, the eluent is an organic solvent, more preferably, the organic solvent is a soluble alcohol or a soluble ketone, more preferably, the organic solvent is ethanol or acetone; Furthermore, the volume ratio of the gel to the eluent in the reaction system during pulverization is 1:1 to 1:5; Furthermore, the particle size of the gel after pulverization is 10 to 500 μm.

[0042] In one embodiment, the method further comprises reconstituting and moist heat sterilizing the dried gel material; Preferably, the reconstituted solution is a phosphate buffer solution, and more preferably, the mass concentration of the phosphate buffer salt in the phosphate buffer solution is 5 to 40 mg / mL; Furthermore, the temperature for the moist heat sterilization is 120 to 130°C, and the moist heat sterilization time is preferably 15 to 45 minutes.

[0043] In one specific embodiment, the residual active agent can be removed by eluting the gel with an organic solvent, crushing the gel particles, and repeatedly washing. The drying can be performed by vacuum drying, and the organic solvent can be removed by drying. Crushing the gel into small particle sizes can ensure the efficiency of eluting the residual active agent during multiple washing processes.

[0044] In one specific embodiment, the vacuum-dried gel particles are redissolved in a phosphate buffer solution, filled into a pre-filled syringe, and then sterilized by moist heat to obtain the final gel product. The pH value of the phosphate buffer solution is in the range of 6.8 to 7.6. The concentration of the hyaluronic acid gel in the final product is 1 to 35 mg / mL.

[0045] In one specific embodiment, the method comprises: (a) directly dissolving hyaluronic acid and endogenous polyamines in water and adjusting the pH of the solution; (b) adding an activator to complete the cross-linking reaction between spermidine and / or spermine and hyaluronic acid; (c) adding an organic eluent to pulverize the gel particles, washing them several times to remove residual activator, and then vacuum drying to remove the organic solvent; (d) Redissolving the vacuum-dried gel particles in a phosphate buffer solution, filling them into a pre-filled syringe, and sterilizing them with moist heat to obtain the final gel product.

[0046] After the hyaluronic acid gel obtained by the method of the present invention is subjected to moist heat sterilization, the elastic modulus loss rate is low and can be controlled to a minimum value within 10%, the rheological properties of the gel before sterilization are effectively maintained, and the thermal stability of the hyaluronic acid gel is greatly improved.

[0047] The present invention relates to a method for producing a compound such as spermidine. endogenous Designing polyamines as crosslinkers for hyaluronic acid Along with this, During the body's natural degradation process, the gel is degraded by the release of hydroxybenzoates such as spermidine. endogenous Polyamines Slowly sustainably release The three- or four-site activated star network gels of the present invention can be synthesized by the enzymatic induction of hyaluronidase to form spermidine. of It releases spermidine stably, allowing the biological activity of spermidine to be sustained.

[0048] The present invention further provides a gel material obtained by the above preparation method.

[0049] In another aspect, the present invention further provides the use of said gel material or a gel material obtained by said preparation method in the field of tissue filling and repair materials or drug carriers, for example for the preparation of pharmaceutical, medical aesthetic and cosmetic products, such as cosmetic injectables for soft tissue filling, soft tissue repair or removing facial skin wrinkles.

[0050] The beneficial effects are as follows: (1) The gel material provided by the present invention realizes multi-site cross-linking between endogenous polyamines and hyaluronic acid, forming a denser network structure. (2) The hyaluronic acid gel obtained by the present invention has a low elastic modulus loss rate after moist heat sterilization (the minimum can be controlled within 10%), effectively maintains the rheological properties of the gel before sterilization, and significantly improves the thermal stability of the hyaluronic acid gel. (3) The preparation method of the present invention allows the number of cross-linking reaction sites to be controlled, thereby affecting the performance of the prepared cross-linked hyaluronic acid gel. (4) The present invention can control the release rate of endogenous polyamines such as spermidine during the degradation process of hyaluronic acid gel, thereby enabling the physiological activity of endogenous polyamines such as spermidine to be sustained. (5) The present invention also improves the ease of use of the gel in the fields of soft tissue filling, soft tissue repair, medical cosmetics, etc., making it suitable for widespread use and application. [Brief explanation of the drawings]

[0051] In order to more clearly describe the specific embodiments of the present invention or the technical solutions of the prior art, the following will briefly introduce the drawings necessary for describing the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative work. [Figure 1] 1 is a 1H NMR spectrum of spermidine-crosslinked hyaluronic acid provided by the present invention. [Figure 2] FIG. 1 is a schematic diagram of cell proliferation in spermidine-crosslinked hyaluronic acid provided by the present invention. [Figure 3] 1 is a curve showing the release amount of spermidine together with cross-linked hyaluronic acid in a spermidine-cross-linked hyaluronic acid gel under thermal decomposition conditions provided by the present invention. [Figure 4] 1 is a curve showing the release amount of spermidine together with cross-linked hyaluronic acid in a spermidine-cross-linked hyaluronic acid gel under the enzymatic conditions provided by the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0052] The technical solutions of the present invention will be described clearly and completely below with reference to the following embodiments, and it is obvious that the described embodiments are only some of the embodiments of the present invention, and are not all of the embodiments, and all other embodiments that can be obtained by those skilled in the art based on the embodiments of the present invention without creative work fall within the scope of protection of the present invention.

[0053] Example 1 Example 1: Preparation of two-site crosslinked hydrogel of spermine and hyaluronic acid by HATU catalyst 3.0 g of sodium hyaluronate (molecular weight 900 kDa, containing 7.4 mmol of hyaluronic acid repeating structural units) was weighed out, and 98 mL of purified water was added. After complete dissolution, the concentration of hyaluronic acid was 30 mg / mL. 0.03 g of spermine (molar number 0.15 mmol) was added to the hyaluronic acid solution. In this case, the spermine content accounted for 1% of the mass of hyaluronic acid (2% of the mole number of hyaluronic acid repeating structural units). 6 mol / L hydrochloric acid solution was used to adjust the pH value of the hyaluronic acid solution to approximately 6.20, and then uniformly Stir, add 0.32 mmol of HATU (213% of the moles of spermine), continue stirring until uniform, seal, and place in an incubator at 25°C for 24 hours. After the reaction is complete, add 40 mL of absolute ethanol and crush the gel particles using an IKA T25 high-shear disperser at a grinding speed of 10,000 rpm / min for 5 minutes. After crushing, add 200 mL of absolute ethanol to completely dehydrate the gel. Separate the precipitate and wash it five times with 200 mL of absolute ethanol. Then place in a vacuum oven and dry it at a vacuum of -0.09 MPa at 40°C for 24 hours. After complete drying, 1.0 g of the dried gel was collected and a total of 50 mL of 10 mg / mL phosphate buffer solution at pH 7.0 was added to obtain a hydrogel with a hyaluronic acid concentration of 20 mg / mL. After the gel was fully swollen, it was loaded into a prefilled syringe and sterilized with moist heat at 121°C for 15 minutes to obtain the final gel. The particle size of the gel, D0.5, was 158 μm and D0.9, was 196 μm.

[0054] Example 2 Example 2: Preparation of three-site crosslinked hydrogel of spermidine and hyaluronic acid by HATU catalyst Weigh out 3.0g of sodium hyaluronate (molecular weight 900KDa, containing 7.4mmol of hyaluronic acid repeating structural units), add 98mL of purified water, and after complete dissolution, the concentration of hyaluronic acid becomes 30mg / mL. Add 0.022g of spermidine (molar number is 0.15mmol) to the hyaluronic acid solution, and in this case, the spermidine content accounts for 0.7% of the mass of hyaluronic acid (2% of the mole number of hyaluronic acid repeating structural units). Add 0.48mmol of HATU, which is 320% of the mole number of spermidine, and stir evenly. Adjust the pH value of the hyaluronic acid solution to about 5.40 using 6mol / L hydrochloric acid solution, and then Stir The mixture was then sealed and placed in a 50°C incubator for 14 hours. After the reaction was complete, 40 mL of absolute ethanol was added, and the gel particles were milled using an IKA T25 high-shear disperser at a milling speed of 10,000 rpm / min for 5 minutes. After milling was complete, 200 mL of absolute ethanol was added to completely dehydrate the gel. The precipitate was separated and washed five times with 200 mL of absolute ethanol. It was then placed in a vacuum oven and vacuum-dried at -0.09 MPa and 40°C for 24 hours. After complete drying, 1.0 g of the dried gel was collected and added to a total of 50 mL of 10 mg / mL pH 7.0 phosphate buffer to obtain a hydrogel with a hyaluronic acid concentration of 20 mg / mL. After the gel was completely swollen, it was loaded into a prefilled syringe and sterilized with moist heat at 121°C for 15 minutes to obtain the final gel product. The particle size of the gel D0.5 is 143 μm and D0.9 is 201 μm.

[0055] Example 3 Example 3. Preparation of HATU-catalyzed four-site crosslinked hydrogel of spermine and hyaluronic acid 3.0 g of sodium hyaluronate (molecular weight 900 kDa, containing 7.4 mmol of hyaluronic acid repeating units) was weighed out and added to 98 mL of purified water until the hyaluronic acid concentration reached 30 mg / mL. 0.03 g of spermine (0.15 mmol) was added to the hyaluronic acid solution, which accounted for 1% of the mass of hyaluronic acid (2% of the moles of hyaluronic acid repeating units). 0.65 mmol of HATU (433% of the moles of spermine) was added and stirred thoroughly. The pH of the hyaluronic acid solution was adjusted to approximately 5.30 using 6 mol / L hydrochloric acid solution, and the mixture was then stirred thoroughly. The mixture was then sealed and placed in an incubator at 60°C for 14 hours. After the reaction was complete, 40 mL of acetone was added and the gel particles were milled using an IKA T25 high-shear disperser at a milling speed of 10,000 rpm / min for 5 minutes. After grinding, 200 mL of acetone was added continuously to completely dehydrate the gel. The precipitate was separated and washed five times with 200 mL of acetone. It was then placed in a vacuum oven and dried at a vacuum of -0.09 MPa and 40°C for 24 hours. After complete drying, 1.0 g of the dried gel was collected and a total of 50 mL of 10 mg / mL pH 7.0 phosphate buffer solution was added to obtain a hydrogel with a hyaluronic acid concentration of 20 mg / mL. After the gel was completely swollen, it was loaded into a prefilled syringe and sterilized with moist heat at 121°C for 15 minutes to obtain the final gel. The particle size of the gel, D0.5, was 139 μm and D0.9, was 186 μm.

[0056] Example 4 Example 4: Preparation of two-site crosslinked hydrogel of spermidine and hyaluronic acid catalyzed by triphenylphosphonium bromide succinimide salt Dissolve 0.2 mol of triphenylphosphine and 0.2 mol of N-bromosuccinimide in 1000 mL of dichloromethane and stir at 20-25°C for 24 hours. After the reaction is complete, remove the dichloromethane using a rotary evaporator to obtain triphenylphosphonium bromide succinimide salt, which is then sealed and ready for use.

[0057] Weigh out 3.0g of sodium hyaluronate (molecular weight 900KDa, containing 7.4mmol of hyaluronic acid repeating structural units), add 98mL of purified water, and after complete dissolution, the concentration of hyaluronic acid becomes 30mg / mL. Add 0.022g of spermidine (molar number is 0.15mmol) to the hyaluronic acid solution, and in this case, the spermidine content accounts for 0.7% of the mass of hyaluronic acid (2% of the mole number of hyaluronic acid repeating structural units). Add 0.32mmol of triphenylphosphonium bromide succinimide salt obtained in the above step, which is 213% of the mole number of spermidine, and stir evenly. Adjust the pH value of the hyaluronic acid solution to about 6.00 using 6mol / L hydrochloric acid solution, and then Stir The mixture was then sealed and placed in an incubator at 25°C for 24 hours. After the reaction was complete, 40 mL of acetone was added, and the gel particles were milled using an IKA T25 high-shear disperser at a milling speed of 10,000 rpm / min for 5 minutes. After milling was complete, 200 mL of acetone was added to completely dehydrate the gel. The precipitate was separated and washed five times with 200 mL of acetone. It was then placed in a vacuum oven and vacuum-dried at -0.09 MPa and 40°C for 24 hours. After complete drying, 1.0 g of the dried gel was collected and added to a total of 50 mL of 10 mg / mL phosphate buffer solution at pH 7.0 to obtain a hydrogel with a hyaluronic acid concentration of 20 mg / mL. After the gel was completely swollen, it was loaded into a prefilled syringe and sterilized with moist heat at 121°C for 15 minutes to obtain the final gel product. The particle size of the gel D0.5 is 141 μm and D0.9 is 191 μm.

[0058] Example 5 Example 5. Preparation of three-site crosslinked hydrogel of spermidine and hyaluronic acid catalyzed by triphenylphosphonium bromide succinimide salt 3.0g of sodium hyaluronate (molecular weight 900KDa, containing 7.4mmol of hyaluronic acid repeating structural units) was weighed out, and 98mL of purified water was added, and the concentration of hyaluronic acid after complete dissolution was 30mg / mL. 0.022g of spermidine (molar number 0.15mmol) was added to the hyaluronic acid solution, and in this case, the spermidine content accounted for 0.7% of the mass of hyaluronic acid (2% of the mole number of hyaluronic acid repeating structural units). 0.48mmol of triphenylphosphonium bromide succinimide salt obtained by the method in Example 4, which is 320% of the mole number of spermidine, was added, and the mixture was stirred uniformly. The pH value of the hyaluronic acid solution was adjusted to about 5.20 using 6mol / L hydrochloric acid solution, and then uniformly The mixture was stirred, sealed, and placed in a 40°C incubator for 24 hours. After the reaction was complete, 40 mL of acetone was added, and the gel particles were milled using an IKA T25 high-shear disperser at a milling speed of 10,000 rpm / min for 5 minutes. After milling was complete, 200 mL of acetone was added to completely dehydrate the gel. The precipitate was separated and washed five times with 200 mL of acetone. It was then placed in a vacuum oven and vacuum-dried at -0.09 MPa and 40°C for 24 hours. After complete drying, 1.0 g of the dried gel was collected and added to a total of 50 mL of 10 mg / mL phosphate buffer solution at pH 7.0 to obtain a hydrogel with a hyaluronic acid concentration of 20 mg / mL. After the gel was completely swollen, it was loaded into a prefilled syringe and sterilized with moist heat at 121°C for 15 minutes to obtain the final gel product. The particle size of the gel D0.5 is 133 μm and D0.9 is 178 μm.

[0059] Example 6 Example 6. Preparation of three-site crosslinked hydrogel of spermidine and hyaluronic acid catalyzed by DMTMM Weigh out 3.0g of sodium hyaluronate (molecular weight 900kDa, containing 7.4mmol of hyaluronic acid repeating structural units), add 98mL of purified water, and after complete dissolution, the concentration of hyaluronic acid becomes 30mg / mL. Add 0.022g of spermidine (molar number is 0.15mmol) to the hyaluronic acid solution, in this case, the spermidine content is 0.7% of the mass of hyaluronic acid (2% of the mole number of hyaluronic acid repeating structural units). Add 0.48mmol of DMTMM, which is 320% of the mole number of spermidine, and stir uniformly. Adjust the pH value of the hyaluronic acid solution to about 5.10 using 6mol / L hydrochloric acid solution, then uniformly The mixture was stirred, sealed, and placed in a 40°C incubator for 24 hours. After the reaction was complete, 40 mL of absolute ethanol was added, and the gel particles were milled using an IKA T25 high-shear disperser at a milling speed of 10,000 rpm / min for 5 minutes. After milling was complete, 200 mL of absolute ethanol was added to completely dehydrate the gel. The precipitate was separated and washed five times with 200 mL of absolute ethanol. It was then placed in a vacuum oven and vacuum-dried at -0.09 MPa and 40°C for 24 hours. After complete drying, 1.0 g of the dried gel was collected and added to a total of 50 mL of 10 mg / mL phosphate buffer solution at pH 7.0 to obtain a hydrogel with a hyaluronic acid concentration of 20 mg / mL. After the gel was completely swollen, it was loaded into a prefilled syringe and sterilized with moist heat at 121°C for 15 minutes to obtain the final gel product. The particle size of the gel D0.5 is 144 μm and D0.9 is 206 μm.

[0060] Example 7 Example 7. Preparation of EDC-catalyzed four-site crosslinked hydrogel of spermine and hyaluronic acid Weigh out 3.0g of sodium hyaluronate (molecular weight 900kDa, containing 7.4mmol of hyaluronic acid repeating units), add 98mL of purified water, and after complete dissolution, the concentration of hyaluronic acid becomes 30mg / mL. Add 0.03g of spermine (0.15mmol) to the hyaluronic acid solution, in which the spermine content is 1% of the mass of hyaluronic acid (2% of the moles of hyaluronic acid repeating units). Add 0.65mmol of EDC, which is 433% of the moles of spermine, and then add NHS, which is 20% of the mass of EDC. Stir thoroughly, and adjust the pH of the hyaluronic acid solution to about 5.45 using 6mol / L hydrochloric acid solution. Continue stirring thoroughly, seal, and place in an incubator at 60℃ for 14 hours to react. After the reaction was complete, 40 mL of absolute ethanol was added, and the gel particles were milled using an IKA T25 high-shear disperser at a milling speed of 10,000 rpm for 5 minutes. After milling, 200 mL of absolute ethanol was added to completely dehydrate the gel. The precipitate was separated and washed five times with 200 mL of absolute ethanol. It was then placed in a vacuum oven and dried at a vacuum of -0.09 MPa and 40°C for 24 hours. After complete drying, 1.0 g of the dried gel was collected and a total of 50 mL of 10 mg / mL pH 7.0 phosphate buffer solution was added to obtain a hydrogel with a hyaluronic acid concentration of 20 mg / mL. After the gel was fully swollen, it was loaded into a prefilled syringe and sterilized with moist heat at 121°C for 15 minutes to obtain the final gel. The particle size of the gel, D0.5, was 125 μm, and D0.9 was 193 μm.

[0061] Example 8 Example 8: HATU-catalyzed combination of spermidine and spermine contributes to the preparation of multi-site cross-linked hydrogels of hyaluronic acid Weigh out 3.0g of sodium hyaluronate (molecular weight 900KDa, containing 7.4mmol of hyaluronic acid repeating structural units), add 98mL of purified water, and after complete dissolution, the concentration of hyaluronic acid becomes 30mg / mL. Then, add 0.022g of spermidine (molar number is 0.15mmol) and 0.03g of spermine (molar number is 0.15mmol) to the hyaluronic acid solution, and in this case, the spermidine content and spermine content respectively account for 2% of the mole number of hyaluronic acid repeating structural units. Add 0.48mmol of HATU, which is 320% of the mole number of spermidine, and then add 0.65mmol of HATU, which is 433% of the mole number of spermine, and stir evenly. Use 6mol / L hydrochloric acid solution to adjust the pH value of the hyaluronic acid solution to about 5.00, and then uniformly The mixture was stirred, sealed, and placed in an incubator at 50°C for 14 hours. After the reaction was complete, 40 mL of absolute ethanol was added, and the gel particles were milled using an IKA T25 high-shear disperser at a milling speed of 10,000 rpm / min for 5 minutes. After milling was complete, 200 mL of absolute ethanol was added continuously to completely dehydrate the gel. The precipitate was separated and washed five times with 200 mL of absolute ethanol. It was then placed in a vacuum oven and vacuum-dried at -0.09 MPa and 40°C for 24 hours. After complete drying, 1.0 g of the dried gel was collected and a total of 50 mL of 10 mg / mL pH 7.0 phosphate buffer solution was added to obtain a hydrogel with a hyaluronic acid concentration of 20 mg / mL. After the gel was completely swollen, it was loaded into a prefilled syringe and sterilized with moist heat at 121°C for 15 minutes to obtain the final gel product. The particle size of the gel D0.5 is 134 μm and D0.9 is 236 μm.

[0062] Example 9 Example 9: EDC-catalyzed three-site crosslinking of spermidine and hyaluronic acid Weigh out 6g of sodium hyaluronate (molecular weight 120KDa), add 35mL of purified water, and after complete dissolution, the concentration of hyaluronic acid becomes 150mg / mL. Add 2.4g of spermidine (molar number is 16.6mmol) to the hyaluronic acid solution, and in this case, the spermidine content accounts for 40% of the mass of hyaluronic acid. Assuming three-site crosslinking, adjust the pH value of the hyaluronic acid solution to about 5.2 using 6mol / L hydrochloric acid solution, and then uniformly Stir, add 66.4 mmol of EDC (400% moles of spermidine), and simultaneously add 2.4 g of Sulfo-NHS (20% of the EDC mass). Continue stirring until uniform, seal, and place in a 60°C fan oven for 24 hours. After the reaction is complete, add 40 mL of absolute ethanol and grind the gel particles using an IKA T25 high-shear disperser at a grinding speed of 10,000 rpm / min for 5 minutes. After grinding, add 200 mL of absolute ethanol to completely dehydrate the gel. Separate the precipitate and wash it five times with 200 mL of absolute ethanol. Then, place in a vacuum oven and dry it at a vacuum of -0.09 MPa at 40°C for 24 hours. After complete drying, 1.0 g of the dried gel was collected and 20 mL of 10 mg / mL phosphate buffer solution at pH 7.0 was added. After the gel had completely swollen, it was filled into a prefilled syringe and sterilized with moist heat at 121°C for 15 minutes to obtain the final gel. The particle size of the gel, D0.5, was 145 μm and D0.9, 213 μm.

[0063] Example 10 Example 10: Preparation of four-site crosslinked hydrogel of spermine and hyaluronic acid by HATU catalyst Weigh out 0.5g of sodium hyaluronate (molecular weight 2800KDa), add 38.5mL of purified water, and after complete dissolution, the concentration of hyaluronic acid becomes 13.3mg / mL. Add 1.5mg of spermine (molar number is 7.4μmol) to the hyaluronic acid solution, and in this case, the spermine content accounts for 0.3% of the mass of hyaluronic acid. Assuming four-site crosslinking, use 6mol / L hydrochloric acid solution to adjust the pH value of the hyaluronic acid solution to about 5.2, and then Stir29.6 μmol of HATU (400% spermine moles) was added, and the mixture was stirred uniformly. The container was sealed and placed in a 30°C air-dried oven for 24 hours. After the reaction was complete, 40 mL of acetone was added, and the gel particles were pulverized using an IKA T25 high-shear disperser at 10,000 rpm / min for 5 minutes. After pulverization was complete, 200 mL of acetone was added to completely dehydrate the gel. The precipitate was separated and washed five times with 200 mL of acetone. The mixture was then placed in a vacuum oven and vacuum-dried at -0.09 MPa and 40°C for 24 hours. After complete drying, 0.4 g of dry gel was collected and a total of 400 mL of 10 mg / mL phosphate buffer solution at pH 7.0 was added. After the gel was completely swollen, the gel was filled into a prefilled syringe and sterilized with moist heat at 121°C for 15 minutes to obtain the final gel product. The particle size of the gel D0.5 is 156 μm and D0.9 is 231 μm.

[0064] Example 11 Example 11. Preparation of three-site crosslinked hydrogel of spermidine and hyaluronic acid by HATU catalyst Weigh out 0.5g of sodium hyaluronate (molecular weight 2800KDa), add 38.5mL of purified water, and after complete dissolution, the concentration of hyaluronic acid becomes 13.3mg / mL. Add 2.5mg of spermidine (molar number is 17.2μmol) to the hyaluronic acid solution, and in this case, the spermidine content accounts for 0.5% of the mass of hyaluronic acid. Assuming three-site crosslinking, use 6mol / L hydrochloric acid solution to adjust the pH value of the hyaluronic acid solution to about 5.2, and then Stir51.6 μmol of HATU (300% spermidine moles) was added, and the mixture was stirred uniformly. The container was sealed and placed in a 30°C air-dried oven for 24 hours. After the reaction was complete, 40 mL of acetone was added, and the gel particles were pulverized using an IKA T25 high-shear disperser at a pulverization speed of 10,000 rpm / min for 5 minutes. After pulverization was complete, 200 mL of acetone was added to completely dehydrate the gel. The precipitate was separated and washed five times with 200 mL of acetone. It was then placed in a vacuum oven and vacuum-dried at -0.09 MPa and 40°C for 24 hours. After complete drying, 0.4 g of dry gel was collected and a total of 200 mL of 10 mg / mL phosphate buffer solution at pH 7.0 was added. After the gel was completely swollen, it was filled into a prefilled syringe and sterilized with moist heat at 121°C for 15 minutes to obtain the final gel product. The particle size of the gel D0.5 is 125 μm and D0.9 is 201 μm.

[0065] Example 12 Example 12: EDC-catalyzed four-site crosslinking of spermine and hyaluronic acid Weigh out 6g of sodium hyaluronate (molecular weight 120KDa), add 35mL of purified water, and after complete dissolution, the concentration of hyaluronic acid becomes 150mg / mL. Add 2.1g of spermine (molar number is 10.4mmol) to the hyaluronic acid solution, and in this case, the spermine content accounts for 35% of the mass of hyaluronic acid. Assuming four-site crosslinking, adjust the pH value of the hyaluronic acid solution to about 5.2 using 6mol / L hydrochloric acid solution, and then StirAdd 57.2 mmol of EDC (550% of the spermine moles) and 2.0 g of HOBt (20% of the EDC mass) simultaneously. Stir evenly, seal, and place in a 60°C fan oven for 24 hours. After the reaction is complete, add 40 mL of absolute ethanol and crush the gel particles using an IKA T25 high-shear disperser at 10,000 rpm / min for 5 minutes. After crushing, add 200 mL of absolute ethanol to completely dehydrate the gel. Separate the precipitate and wash it five times with 200 mL of absolute ethanol. Then, place in a vacuum oven and dry it at -0.09 MPa and 40°C for 24 hours. After complete drying, 1.0 g of the dried gel was collected and 20 mL of 10 mg / mL phosphate buffer solution at pH 7.0 was added. After the gel had completely swollen, it was filled into a prefilled syringe and sterilized with moist heat at 121°C for 15 minutes to obtain the final gel. The particle size of the gel, D0.5, was 148 μm and D0.9, 197 μm.

[0066] Example 13 Example 13. Preparation of HATU-catalyzed two-site crosslinked hydrogel of spermidine and hyaluronic acid under slightly acidic conditions Weigh out 3.0g of sodium hyaluronate (molecular weight 900kDa, containing 7.4mmol of hyaluronic acid repeating structural units), add 98mL of purified water, and after complete dissolution, the concentration of hyaluronic acid becomes 30mg / mL. Add 0.022g of spermidine (molar number is 0.15mmol) to the hyaluronic acid solution, and in this case, the spermidine content accounts for 0.7% of the mass of hyaluronic acid (2% of the mole number of hyaluronic acid repeating structural units). Add 0.48mmol of HATU, which is 320% of the mole number of spermidine, and stir evenly. Adjust the pH value of the hyaluronic acid solution to about 4.60 using 6mol / L hydrochloric acid solution, and then uniformlyThe mixture was stirred, sealed, and placed in an incubator at 50°C for 14 hours. After the reaction was complete, 40 mL of absolute ethanol was added, and the gel particles were milled using an IKA T25 high-shear disperser at a milling speed of 10,000 rpm / min for 5 minutes. After milling was complete, 200 mL of absolute ethanol was added to completely dehydrate the gel. The precipitate was separated and washed five times with 200 mL of absolute ethanol. It was then placed in a vacuum oven and vacuum-dried at -0.09 MPa and 40°C for 24 hours. After complete drying, 1.0 g of the dried gel was collected and a total of 50 mL of 10 mg / mL phosphate buffer solution at pH 7.0 was added to obtain a hydrogel with a hyaluronic acid concentration of 20 mg / mL. After the gel was completely swollen, it was loaded into a prefilled syringe and sterilized with moist heat at 121°C for 15 minutes to obtain the final gel product. The particle size of the gel D0.5 is 138 μm and D0.9 is 210 μm.

[0067] (Comparative Example 1) Comparative Example 1, Preparation of crosslinked hydrogel of spermidine and hyaluronic acid by HATU catalysis under strongly acidic conditions Weigh out 3.0g of sodium hyaluronate (molecular weight 900kDa, containing 7.4mmol of hyaluronic acid repeating structural units), add 98mL of purified water, and after complete dissolution, the concentration of hyaluronic acid becomes 30mg / mL. Add 0.022g of spermidine (molar number is 0.15mmol) to the hyaluronic acid solution, and in this case, the spermidine content accounts for 0.7% of the mass of hyaluronic acid (2% of the mole number of hyaluronic acid repeating structural units). Add 0.48mmol of HATU, which is 320% of the mole number of spermidine, and stir evenly. Adjust the pH value of the hyaluronic acid solution to about 3.20 using 6mol / L hydrochloric acid solution, and then uniformlyThe mixture was stirred, sealed, and placed in an incubator at 50°C for 14 hours. After the reaction was complete, 40 mL of absolute ethanol was added, and the gel particles were milled using an IKA T25 high-shear disperser at a milling speed of 10,000 rpm / min for 5 minutes. After milling was complete, 200 mL of absolute ethanol was added continuously to completely dehydrate the gel. The precipitate was separated and washed five times with 200 mL of absolute ethanol. It was then placed in a vacuum oven and vacuum-dried at -0.09 MPa and 40°C for 24 hours. After complete drying, 1.0 g of the dried gel was collected and a total of 50 mL of 10 mg / mL pH 7.0 phosphate buffer solution was added to obtain a hydrogel with a hyaluronic acid concentration of 20 mg / mL. After the gel was completely swollen, it was loaded into a prefilled syringe and sterilized with moist heat at 121°C for 15 minutes to obtain the final gel product. The particle size of the gel D0.5 is 143 μm and D0.9 is 201 μm.

[0068] (Comparative Example 2) Comparative Example 2: EDC-catalyzed crosslinking reaction of spermine and hyaluronic acid In this comparative example, the gel is prepared according to the method disclosed in US9907739B2. 3.0g of sodium hyaluronate (molecular weight 900kDa, containing 7.4mmol of hyaluronic acid repeating units) was weighed out and 98mL of purified water was added. After complete dissolution, the concentration of hyaluronic acid was 30mg / mL. 0.03g of spermine (0.15mmol) was added to the hyaluronic acid solution. In this case, the spermine content was 1% of the mass of hyaluronic acid (2% of the moles of hyaluronic acid repeating units). The pH of the hyaluronic acid solution was adjusted to approximately 6.2 using 6mol / L hydrochloric acid solution, and after uniform stirring, 7.4mmol of EDC was added, which is 100% of the moles of hyaluronic acid repeating units and 49 times the moles of spermine. At the same time, HOBt, equivalent to 20% of the mass of EDC, was added. The mixture was then stirred uniformly, sealed, and placed in an incubator at 25℃ for 24 hours. After the reaction was complete, 40 mL of absolute ethanol was added, and the gel particles were milled using an IKA T25 high-shear disperser at a milling speed of 10,000 rpm for 5 minutes. After milling, 200 mL of absolute ethanol was added to completely dehydrate the gel. The precipitate was separated and washed five times with 200 mL of absolute ethanol. It was then placed in a vacuum oven and dried at a vacuum of -0.09 MPa and 40°C for 24 hours. After complete drying, 1.0 g of the dried gel was collected and a total of 50 mL of 10 mg / mL pH 7.0 phosphate buffer solution was added to obtain a hydrogel with a hyaluronic acid concentration of 20 mg / mL. After the gel was fully swollen, it was loaded into a prefilled syringe and sterilized with moist heat at 121°C for 15 minutes to obtain the final gel. The particle size of the gel, D0.5, was 155 μm, and D0.9, was 221 μm.

[0069] Example 14 Example 14: Detection of cross-linking efficiency of spermine or spermidine 2.0 ml of each hyaluronic acid gel obtained in Examples 1-8 and Comparative Examples 1-2 was taken, and 50 ml of absolute ethanol was added to each gel. Stirring was performed for 30 minutes until the gel turned into a white precipitate. The precipitate was separated and vacuum-dried at -0.09 MPa and 40°C for 24 hours to remove the ethanol. The gel was taken out, and 10 mL of 0.5 mol / L sulfuric acid solution was added to each gel. After heating at 90°C for 2 hours, the gel was neutralized to pH 7 with 6 mol / L sodium hydroxide solution. The solution was placed in a vacuum drying oven and vacuum-dried at -0.09 MPa and 60°C for 24 hours to remove excess water. The gel was dissolved in deuterium oxide at a concentration of 10 mg / mL and analyzed using a 400M nuclear magnetic resonance spectrometer. 1 Scan the H spectrum.

[0070] 1 By integrating the key characteristic peaks in the H NMR spectrum, the actual molar ratio of spermidine to hyaluronic acid at the integrated peaks can be obtained, and the crosslinking efficiency can be obtained by comparing the actual molar ratio with the theoretical molar ratio (calculated based on the actual supply). Here, selected characteristic H atoms of spermidine are linked to the amino group. and The α-H of the imino group, and the total number of hydrogen atoms is eight, and the selected characteristic H atom of spermine is the amino group and The α-H of imino group is the total number of H atoms is 12, and the integral range of the H atoms of spermine and spermidine is the same, 2.5~2.85ppm. The selected characteristic H atom of hyaluronic acid is the α-H of the carbonyl group in acetamide group, the total number of H atoms is 3, and the integral range is 1.85~2.05ppm. When integrating, the integral peak area of ​​the carbonyl group α-H of hyaluronic acid is taken as 3, and the integral peak area of ​​the amino group and imino α-H of spermidine or spermine can be calculated.Its calculation formula is as follows: Actual molar ratio of spermidine crosslinks = α-H integral peak area of ​​amino and imino groups in spermidine / 8. Actual crosslinking molar ratio of spermine = α-H integral peak area of ​​amino and imino groups of spermine / 12. Crosslinking efficiency = (actual crosslinking molar ratio / theoretical crosslinking molar ratio) × 100%.

[0071] Examples 1 to 8 and Comparative Examples 1-2 The cross-linking efficiency is shown in Table 1, and the nuclear magnetic spectrum is shown in Figure 1. [Table 1]

[0072] As can be seen from an analysis of the data in Table 1, when two-site crosslinking occurs, the crosslinking efficiency is lower than that of multi-site crosslinking. Comparing Examples 1 and 4, which involve two-site crosslinking, with Examples 2, 3, and 5 shows that the two-site crosslinking efficiency is generally lower than that of multi-site crosslinking. This may be due to the influence of three conditions: the amount of activator added, pH value, and temperature, which result in lower crosslinking efficiency during two-site crosslinking. Similarly, when multi-site crosslinking is performed, the activation efficiency of each activator is HATU > triphenylphosphonium salt > DMTMM > EDC + auxiliary. Here, the activation efficiency of HATU is as high as 95%, while that of EDC + NHS is only 75%. Under the same conditions, the crosslinking efficiencies of spermine and spermidine are similar, with spermine being slightly lower. This is likely due to spermine's higher imino content, which is less reactive than primary amino groups due to greater steric hindrance. Furthermore, analysis of comparative examples has shown that when the pH value of the multi-site is out of range, the reaction activity of the amino group and the imino group is inhibited, resulting in a low crosslinking efficiency; and when the test temperature is lower than that of the multi-site crosslinking, even if the reaction is carried out according to the conditions for the multi-site crosslinking, the final crosslinking efficiency will be the same as that of the two-site crosslinking, and the reaction efficiency will be lower.

[0073] Example 15 Example 15: Effect of spermidine cross-linked hyaluronic acid gel on cell proliferation L-929 cells were seeded in cell culture medium, and 1% penicillin-streptomycin solution and 10% fetal bovine serum solution were added. The L-929 cells were incubated at 37°C for 3 days in a humidified cell culture incubator containing 5% carbon dioxide. Next, the spermidine three-site cross-linked hyaluronic acid gel obtained in Example 2 was transferred to a 96-well plate, cured under an ultraviolet lamp, and sterilized. Then, L-929 cell culture medium was added to the wells containing the spermidine cross-linked hyaluronic acid gel, and 1 mL of trypsin solution containing 0.1% EDTA was added until the number of L-929 cells in each well reached 1 x 10. 5 Alternatively, place the cells in a cell culture incubator to promote cell growth.

[0074] Cell proliferation in spermidine-crosslinked hyaluronic acid gels was measured by the MTT assay. After culturing for 24, 48, and 72 hours, 100 μL of MTT solution (5 mg mL) was added to each well. -1 ) and place in an incubator for 4 hours. Next, remove the MTT solution and add 150 μL of dimethyl sulfoxide to dissolve the formazan crystals. The absorbance of the solution is measured at 490 nm using a microplate reader to measure the degree of cell growth in the L-929 gel. The cell viability is calculated according to the following formula: Cell viability (%)=(As / Ac)×100%, Here, As is the absorbance of the sample solution at 570 nm, and Ac is the absorbance of the blank control at 570 nm.

[0075] The results of cell proliferation are shown in Figure 2. As can be seen from this figure, the cell viability of the spermidine-crosslinked hyaluronic acid gel at 24, 48, and 72 hours was 112%, 123%, and 138%, respectively. As can be seen from the cell viability of these two gels, the spermidine-crosslinked hyaluronic acid hydrogel was not cytotoxic, and the phenomenon of cell viability increasing over time indicates that the hydrogel exerts some effect of promoting cell proliferation.

[0076] Example 16 Example 16: Testing the rheological properties of spermine or spermidine cross-linked hyaluronic acid gels The hyaluronic acid gels obtained in Examples 1 to 13 and Comparative Example 2 were divided into two groups, one before sterilization and one after sterilization, and 2.0 mL of each was sampled. The elastic modulus (G') of the gel was measured using a TA DHR-2 plate rheometer, and the elastic modulus loss factor was calculated. Here, the G' loss factor was calculated using the following formula: G' loss rate = (G' before sterilization - G' after sterilization) / G' before sterilization, The rheometer parameters were as follows: operating gap: 1000 mm, load gap: 45000 m, operating temperature: 37°C, deformation: 1%, frequency: 0.9 Hz, operating time: 60 s. The rheological data of each gel are shown in Table 2. [Table 2]

[0077] The G' loss rate can be considered as a characterization index of the thermal stability of a gel; the lower the G' loss rate, the higher the thermal stability of the gel. As can be seen from the data in Table 2, taking Examples 1-7 as an example, the G' loss rate has a certain relationship with the crosslinking efficiency in Example 14. Here, under similar crosslinking degrees and crosslinking efficiencies (Examples 2 and 3), the thermal stabilities of spermidine-crosslinked hyaluronic acid and spermine-crosslinked hyaluronic acid are similar, but spermine has a higher modulus and thermal stability. This may be due to the denser crosslinked network structure formed by the four-site crosslinking of spermine. The same theory can be applied to the three-site crosslinked spermidine gel and the two-site crosslinked spermidine gel; that is, the thermal stability of the gel with two-site crosslinking is lower than that of the gel with three-site crosslinking. Comparing the four activators, the order of gel thermal stability is HATU > triphenylphosphonium salt > DMTMM > EDC + auxiliary agent, which is the same as in Example 14. The concentration of hyaluronic acid in the crosslinked hyaluronic acid hydrogel also significantly affected the thermal stability of the gel. Examples 10 and 11 used the same crosslinking sites as Examples 2 and 3. However, the concentrations of hyaluronic acid in the crosslinked hyaluronic acid hydrogels of Examples 10 and 11 were 1 mg / mL and 2 mg / mL, respectively, while the corresponding concentrations in Examples 2 and 3 were 20 mg / mL. This resulted in significant differences in G' and a larger decrease in G'. The amount of activator added also significantly affected the performance of the hydrogel. Under the same conditions as in Example 1 and Example 13, the amount of HATU activator added in Example 13 was reduced by 28.6% (i.e., 164.8 μmol) compared to Example 1. As a result, G' was significantly reduced in Example 13 compared to Example 1, and the rate of decrease in G' was significantly increased compared to Example 1.

[0078] Example 17 Example 17: Detection of spermidine release in gels during hyaluronic acid degradation under pyrolysis conditions 2 mL of the final gel product in Example 2 was taken per sample, sealed in a vial, and then placed in a 125°C air-dried oven for accelerated hydrolysis. Three samples were taken in parallel every 15 minutes. After cooling to about 25°C, the pH was adjusted to 12 with sodium hydroxide, 20 mL of chromatography-grade acetone was added, and the mixture was filtered through a 0.2 μm filter membrane. The acetone filtrate was injected into an Agilent 7890B gas chromatograph and analyzed by SGE H. - Two capillary columns (30 m × 0.53 mm × 1.0 μm) were used, and the chromatographic conditions were as follows: sample inlet temperature 220°C, pressure 40 psi , separation flow rate 30 mL / min , split ratio 10:1, separation time 0.8 minutes Column temperature: held at 100°C for 0.5 minutes, increased to 180°C at 20°C / min and held for 1 minute; Detector (FID): temperature 250°C, air flow rate 350 mL / min, H2 flow rate 35 mL / min, makeup N2 flow rate 30 mL / min.

[0079] The total decomposition time was 90 min, and a total of six detection points were deployed. The detection results are shown in Figure 3.

[0080] The purpose of this experiment was to simulate the release of spermidine monomers due to hydrolysis of crosslinked sites during the thermal decomposition of hyaluronic acid. However, under normal physiological conditions, the decomposition of crosslinked hyaluronic acid takes a long time, from several months to over a year. Therefore, in this experiment, we used spermidine-crosslinked hyaluronic acid gel to measure the amount of spermidine released during accelerated hydrolysis at high temperatures above 100°C, allowing us to quickly observe the decomposition and release of spermidine. As shown in Figure 3, spermidine is released slowly during the decomposition of hyaluronic acid, but the initial release rate slows. In the later stages, as the crosslinked hyaluronic acid network collapses, the spermidine release rate significantly accelerates, and the cumulative release amount also increases significantly. This indicates that spermidine release within the gel is initially slow and then rapidly accelerated. This experiment also proves that the cross-linking technology of the present invention is an active reaction technology, and that the prototype spermidine monomer can be continuously released during the continuous degradation process of cross-linked hyaluronic acid, thereby proving that the gel can continuously release spermidine in the body and exert the unique physiological activity effects of spermidine.

[0081] Example 18 Example 18: Detection of spermidine release in gels during hyaluronic acid degradation under enzymatic conditions 2 mL of the final gel product in Example 2 was taken for each sample. Each sample Add 1000u of hyaluronidase to , sealed in a vial, and then Accelerated hydrolysis was performed, and three samples were withdrawn in parallel every 30 minutes. After cooling to approximately 25°C, 0.5 mL of the supernatant was withdrawn, the pH was adjusted to 12 with sodium hydroxide, 20 mL of chromatography-grade acetone was added, and the acetone filtrate was filtered through a 0.2 μm filter membrane. The acetone filtrate was injected into an Agilent 7890B gas chromatograph using an SGE H2 capillary column (30 m × 0.53 mm × 1.0 μm). The chromatographic conditions were as follows: sample inlet temperature 220°C, pressure 40 psi , separation flow rate 30 mL / min , split ratio 10:1, separation time 0.8 minutes Column temperature: held at 100°C for 0.5 minutes, increased to 180°C at 20°C / min and held for 1 minute; Detector (FID): temperature 250°C, air flow rate 350 mL / min, H2 flow rate 35 mL / min, makeup N2 flow rate 30 mL / min.

[0082] The total decomposition time was 180 min, and a total of six detection points were deployed. The detection results are shown in Figure 4.

[0083] The purpose of this experiment was to simulate the release process of spermidine monomers due to gel degradation during the enzymatic degradation of hyaluronic acid. As shown in Figure 4, spermidine was slowly released as hyaluronic acid degraded, and the spermidine release rate remained almost unchanged throughout the entire degradation process of the hyaluronic acid gel. This indicates that the hyaluronic acid gel obtained by cross-linking with spermidine can release spermidine uniformly and stably during enzymatic degradation. This experiment also proves that the cross-linking technology used in this invention is an active reaction technology, and that the prototype spermidine monomer can be continuously released during the continuous degradation process of cross-linked hyaluronic acid, thereby demonstrating that the gel can sustainably release spermidine in the body and exert the unique physiological effects of spermidine.

[0084] Example 19 Example 19: Detection of remaining active sites in two-site and multi-site cross-linked gels Take approximately 2 g of ninhydrin and dissolve it in 100 ml of purified water. Mix thoroughly and store in the dark at 2-8°C for use. Take 54.6 g of sodium acetate and dissolve it in 20 ml of 1 mol / L acetic acid solution. Add water to dilute to 500 ml for use. Take spermidine standard solution and spermine standard solution, place them in 10 ml volumetric flasks, dilute to the specified volume with purified water, and shake thoroughly to prepare 500 μg / mL spermidine standard working solutions and 500 μg / mL spermine standard working solutions. When using, dilute them to 10, 50, 100, or 200 μg / mL. Use the original 500 μg / mL standard working solution as the calibration curve.

[0085] 1 mL of each of the hydrogels of Examples 1 to 8 and Comparative Examples 1 and 2 before moist heat sterilization, and the spermine and spermidine series standard solutions, was taken, and 2.0 mL of the acetic acid-sodium acetate buffer solution and 2.0 mL of ninhydrin solution were added in that order. The containers were then stoppered, mixed thoroughly and uniformly, heated in a water bath at 70°C for 30 minutes, removed and rapidly cooled to room temperature. Purified water was added until the total volume was 25 mL, and the mixture was mixed thoroughly. The mixed solution was then taken, and the absorbance of the amino group derivative at a wavelength of 565 nm was measured, while the absorbance of the imino group derivative at a wavelength of 400 nm was detected. Purified water was also used for blank calibration.

[0086] Calculate the percentage of residual amino or imino groups according to the following formula: Residual amount ratio = (A0 - A i ) / A0×100% where A0 is the absorbance value measured by the control solution and Ai is the absorbance value measured by the sample. Here, one spermidine molecule contains one imino group and two amino groups, and one spermine molecule contains two imino groups and two amino groups.

[0087] The actual detection results are shown in Table 3. [Table 3]

[0088] As can be seen from the results in Table 3, Examples 1 and 4 show two-site crosslinking, with the main reactive group being the amino group. The reaction efficiency is high at 89.66% (100% minus the amount of remaining amino groups, hereinafter the same). The lowest reaction efficiency of the amino group exceeded 80%, while the reaction efficiency of the imino group did not exceed 15%. Therefore, under these conditions, two-site crosslinking reactions mainly involve amino groups. In Examples 2, 3, and 8, the reaction efficiencies of the amino and imino groups in spermidine or spermine both reached 90% or higher, and in Examples 5 to 7, the reaction efficiency of the imino group exceeded 85%. This indicates that under these conditions, most spermidine undergoes three-site crosslinking, and spermine undergoes four-site crosslinking. This further demonstrates the reliability of the three-site and four-site crosslinking in the present invention. The comparative experiments revealed that, outside the conditions specified in the present invention, the reaction efficiency of the imino group in spermidine or spermine falls below 10%, and the reaction efficiency of the amino group also declines to some extent. In Comparative Example 2, the total amount of activator added was 49 times that of the crosslinker, but due to inappropriate reaction conditions such as pH and reaction temperature, none of the imino groups of spermine could participate in the reaction, and the reaction efficiency of the amino groups did not exceed 60%.

[0089] Finally, for illustrative purposes, the above embodiments are only used to describe the technical solutions of the present invention, and are not intended to limit the same. The present invention will be described in more detail with reference to the above embodiments. However, it should be understood that those skilled in the art can modify the technical solutions described in the above embodiments or make equivalent substitutions for some or all of the technical features thereof, and these modifications or equivalent substitutions will not cause the essence of the corresponding technical solutions to depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A gel material, the gel material being obtained by cross-linking endogenous polyamines with hyaluronic acid, the endogenous polyamines comprising spermine and / or spermidine, and the cross-linking between the endogenous polyamines and the hyaluronic acid comprising two-site cross-linking, three-site cross-linking, or four-site cross-linking; the proportion of residual amino groups in the gel obtained by the two-site crosslinking is less than 20%; A gel material characterized in that the proportion of residual amino groups and residual imino groups in the gel obtained by the three-site crosslinking or four-site crosslinking is both less than 20%.

2. The proportion of residual amino groups in the gel obtained by crosslinking through the two-site crosslinking, three-site crosslinking, or four-site crosslinking is less than 10%, 2. The gel material according to claim 1, wherein the proportion of residual imino groups in the gel obtained by the three-site crosslinking or four-site crosslinking is less than 10%.

3. 3. The gel material according to claim 1, wherein the cross-linking efficiency of the cross-linking reaction between the endogenous polyamines and hyaluronic acid is higher than 75%.

4. A method for preparing the gel material of claim 1, comprising: adjusting the pH of the mixed solution of hyaluronic acid and endogenous polyamine to 4.5-6.5, and adding an activator, thereby causing a two-site, three-site or four-site cross-linking reaction between the hyaluronic acid and the endogenous polyamine to obtain the gel material; the endogenous polyamines include spermine and / or spermidine; provided that when the activator contains a carbonium salt and / or a phosphonium bromide salt formed by triphenylphosphine and bromide, the crosslinking reaction temperature is 10°C to 60°C, and the crosslinking reaction time is 14 hours to 24 hours; 2. The method for preparing a gel material according to claim 1, wherein the activator comprises a water-soluble carbodiimide and / or 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride, and the crosslinking reaction temperature is 40°C to 60°C, and the crosslinking reaction time is 14 hours to 24 hours.

5. 5. The method for preparing a gel material according to claim 4, characterized in that the pH of the mixed solution of hyaluronic acid and endogenous polyamine is adjusted to 4.50-4.99 or 5.50-6.50, an activator carbonium salt and / or a phosphonium bromide salt formed by triphenylphosphine and bromide is added, the crosslinking reaction temperature is 10°C-60°C, and the crosslinking reaction time is 14 hours-24 hours, so that the hyaluronic acid and the endogenous polyamine undergo a two-site crosslinking reaction to obtain the gel material.

6. The pH of the mixed solution of hyaluronic acid and endogenous polyamine is adjusted to 5.00-5.49, and an activator carbonium salt and / or a phosphonium bromide salt formed by triphenylphosphine and bromide is added, and the crosslinking reaction temperature is 10°C-60°C, and the crosslinking reaction time is 14 hours-24 hours, so that the hyaluronic acid and the endogenous polyamine undergo a three-site or four-site crosslinking reaction, and the gel material is obtained. Alternatively, the method for preparing a gel material according to claim 4, further comprising adjusting the pH of a mixed solution of hyaluronic acid and endogenous polyamine to 5.00-5.49, adding an activator such as water-soluble carbodiimide and / or 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride, carrying out a crosslinking reaction at a temperature of 40°C-60°C for a time of 14-24 hours, thereby allowing a three-site crosslinking reaction or a four-site crosslinking reaction to occur between the hyaluronic acid and the endogenous polyamine, thereby obtaining the gel material.

7. The water-soluble carbodiimide includes one or more of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide, 1,3-bis[bis(methoxymethyl)methyl]carbodiimide, or salts thereof; the phosphonium bromide salt formed by triphenylphosphine and bromide includes one or more of phosphonium salt formed by triphenylphosphine and carbon tetrabromide, and phosphonium salt formed by triphenylphosphine and N-bromosuccinimide; the carbonium salt is O-(7-azabenzotriazol-1-yl)-bis ​​...

7. The method of any one of claims 4 to 6, characterized in that the compound comprises one or more of the following: O-(benzotriazol-1-yl)-bis(dimethylamino)carbonium hexafluorophosphate, O-(benzotriazol-1-yl)-bis(dimethylamino)carbonium hexafluorophosphate, O-(5-chlorobenzotriazol-1-yl)-bis(dimethylamino)carbonium hexafluorophosphate, O-(benzotriazol-1-yl)-bis(dimethylamino)carbonium tetrafluoroborate, O-(N-succinimino)-bis(dimethylamino)carbonium tetrafluoroborate, and 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethylurea tetrafluoroborate.

8. The preparation method according to any one of claims 4 to 6, characterized in that when a water-soluble carbodiimide activator is used, it further comprises using an auxiliary agent in combination, wherein the auxiliary agent comprises any one or more of N-hydroxysuccinimide, sulfonated N-hydroxysuccinimide, tert-butanol, and 1-hydroxybenzotriazole.

9. In the crosslinking reaction, when two-site crosslinking is performed, the amount of the activator added is 200% to 280% of the amount of the endogenous polyamine; The method according to any one of claims 4 to 6, wherein when three-site crosslinking and / or four-site crosslinking is performed, the amount of the activator added is 300% to 550% of the amount of substance of the endogenous polyamine, when the endogenous polyamine is spermine, the amount of the activator added is 400% to 550% of the amount of substance of the spermine, and when the endogenous polyamine is spermidine, the amount of the activator added is 300% to 400% of the amount of substance of the spermidine.

10. The preparation method according to any one of claims 4 to 6, characterized in that, in the crosslinking reaction, if the endogenous polyamine is spermine, the amount of spermine added accounts for 0.3% to 35% of the mass of the hyaluronic acid, and if the endogenous polyamine is spermidine, the amount of spermidine added accounts for 0.5% to 40% of the mass of the hyaluronic acid.

11. The mass concentration of hyaluronic acid in the mixed solution is 10 mg / mL to 150 mg / mL, The method according to any one of claims 4 to 6, wherein the mass concentration of hyaluronic acid in the final crosslinked hyaluronic acid hydrogel obtained is 1 mg / mL to 50 mg / mL.

12. The preparation method according to any one of claims 4 to 6, characterized in that the gel material is further eluted with an eluent, crushed, dried, redissolved and moist heat sterilized.

13. 10. Use of the gel material of claim 1 in the preparation of a tissue filling and repair material or a drug carrier.

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