Gel material, method of preparation and use thereof

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, in particular to a gel material, its preparation method and use. [Background technology]

[0002] Hyaluronic acid or sodium hyaluronate (abbreviated as hyaluronic acid, HA), also known as hyaluronan acid, is a disaccharide glycosaminoglycan composed of D-glucuronic acid through its β-1,4 glycosidic bond and N-acetylglucosamine through β-1,3 glycosidic bond. It is widely used in the field of cosmetics or ophthalmic surgery, and can also be used as a soft tissue filler to repair wrinkles and some soft tissue defects. Hyaluronic acid is a natural substance in the body, and has good biocompatibility and certain biological activity. However, exogenous hyaluronic acid is degraded by hyaluronidase in the body, which causes a shorter residence time in the body, shortening the therapeutic effect, and multiple injections are required to achieve the therapeutic effect. In order to prevent the decomposition of hyaluronic acid by hyaluronidase, it is necessary to crosslink hyaluronic acid molecules with a chemical crosslinking agent to form a spatial network structure. The dense and rigid network structure prevents the decomposition 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 sold on the market are mainly divided into two types: one is a diepoxy crosslinker, mainly 1,4-butanediol diglycidyl ether (BDDE), and the other is an unsaturated sulfone crosslinker, mainly divinyl sulfone (DVS). The mechanisms of these two types of crosslinkers are similar, and both use an alkaline catalyst as a precondition to catalyze the addition reaction between the hydroxyl group (-OH) of hyaluronic acid and the crosslinker to complete the crosslinking, where the -OH in hyaluronic acid undergoes a ring-opening addition reaction with BDDE to complete the crosslinking, while DVS undergoes a Michael addition reaction with the -OH in hyaluronic acid to complete the crosslinking. However, both of these crosslinkers have high biological toxicity, and the unreacted monomers or crosslinking reaction byproducts are potentially carcinogenic. Because hyaluronic acid gel is a long-term implantable medical device, there is a lack of long-term data tracking with a sufficient number of samples to prove the biological safety of this product in clinical aspects. Regarding the current market demand, extending the residence time of hyaluronic acid gel in the body and enhancing the viscoelasticity of hyaluronic acid gel are both the main 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 enhance viscoelasticity. However, when using traditional BDDE or DVS crosslinkers, the increased amount of crosslinker increases people's concerns about the safety of this kind of product.

[0004] Under this premise, choosing non-toxic crosslinking agents to prepare hyaluronic acid gel becomes more common among more hyaluronic acid gel manufacturers around the world. At present, non-toxic amino acid crosslinking agents are used in hyaluronic acid gel, and Chinese patent application CN10105713211 has disclosed a crosslinked sodium hyaluronate gel prepared with 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 CN106188609, CN106188584, and CN111732741 have disclosed a crosslinked sodium hyaluronate gel prepared with 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 a crosslinking agent. The above patent applications all disclose sodium hyaluronate gel prepared by using diaminoamino acids such as lysine or arginine as crosslinking agent. Take lysine or arginine crosslinking agent as an example, the performance difference of the crosslinked hyaluronate hydrogel during moist heat sterilization is large. Here, CN106188609, CN106188584 and CN111732741 do not specifically reflect that crosslinked hyaluronate can be moist heat sterilized at 121 ℃ for 15 minutes. CN10105713211 discloses a lysine crosslinked hyaluronate gel that can be moist heat sterilized, but according to the information disclosed in the patent application, the minimum decomposition rate of this kind of gel after moist heat sterilization is already more than 40%, indicating that this kind 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 according to the method of the present invention, the gel disclosed in the patent application only has two-site cross-linking, not multi-site cross-linking, 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. In addition, the elastic modulus loss rate of this type of gel after moist heat sterilization is as high as 23%, which indicates that this type of gel has poor resistance to moist heat sterilization.In addition, the paper published by Xiang Mei Yan et al. (Journal of Biomaterials Applications, 2011, 27(2):179-186) discloses hyaluronic acid hydrogel crosslinked with hexamethylenediamine, but the elastic modulus of the gel also decreases by more than 20% after sterilization. The main reasons are as follows: Binary amino molecules such as lysine, arginine, and hexamethylenediamine only have two amino crosslinking sites, and when crosslinking occurs, amide bonds are formed with the carboxyl groups of hyaluronic acid to complete the crosslinking. However, due to the small number of crosslinking sites in the molecule, the crosslinked network structure formed cannot provide sufficient thermal stability protection for the hyaluronic acid main chain, and in addition, the amide bond itself has poor thermal stability, so when embodied in the gel, it shows a high decomposition rate after moist heat sterilization and poor thermal stability.

[0005] Endogenous polyamines mainly refer to polyamines synthesized or produced during metabolism in the human body environment, and the main endogenous polyamines include spermine, spermidine and putrescine. In the literature (Madeo et al., Science 359, 410, 2018), it is reported that endogenous polyamines, namely spermidine, have special physiological effects, including but not limited to regulating circadian rhythm, improving hypertension, protecting the cardiovascular system, preventing Alzheimer's disease, enhancing immunity, anti-cancer, anti-aging, etc. The physiological activity 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, prevents 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 generates its physiological activities are as follows. Spermidine binds to polycationic (-NH 3+) is an aliphatic amine that exists in a polyprotonated form under physiological pH conditions and has strong biological activity. Nucleic acids containing acidic residues, phospholipids, acidic proteins, pectic polysaccharides containing carboxyl or sulfate, and neurotransmitters and hormones with similar structures (e.g., dopamine, epinephrine, serotonin, thyroid hormone, etc.) are possible spermidine binding targets. In terms of binding to nucleic acids, most polyamines exist in the form of polyamine-RNA complexes in cells, and the main role of spermidine is related to RNA structural changes and translation, for example, by affecting each step of protein synthesis by affecting the secondary structure of mRNA, tRNA, and rRNA. 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 a large number of negatively charged proteins, changing the spatial structure of the protein and thereby affecting its physiological function. These include, for example, protein kinases / phosphatases (important 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 crosslinking agents 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 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 site between endogenous polyamine and hyaluronic acid, and by controlling the crosslinking reaction site, affect each performance parameter of the obtained crosslinked hyaluronic acid gel, control the release rate of endogenous polyamine such as spermidine during the decomposition process of hyaluronic acid gel, and sustain the physiological activity effect of endogenous polyamine such as spermidine.

[0008] The technical solutions provided by the present invention are as follows: In one aspect, the present invention provides a gel material, the gel material is obtained mainly by crosslinking endogenous polyamines with hyaluronic acid, the endogenous polyamines include 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 realizes for the first time that endogenous polyamines and hyaluronic acid are crosslinked at three or four sites to form an active star-shaped network structure by multi-site crosslinking.

[0009] The present invention selects endogenous polyamine spermine or spermidine as a multi-site crosslinking agent, forming a dense network structure, and thus improving the thermal stability of the amide bond crosslinked 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 crosslinking sites are the same as those of amino acids such as lysine and arginine, so there is no possibility of achieving multi-site crosslinking.

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

[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, the crosslinking reaction efficiency is higher than 80%, and more preferably, the crosslinking reaction efficiency is 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, the method comprising: The method includes the steps of adjusting the pH of a mixed solution of hyaluronic acid and endogenous polyamine to 4.50 to 6.50, and adding an activator, thereby crosslinking the hyaluronic acid and the endogenous polyamine at two sites, three sites, or four sites to obtain the gel material; However, the endogenous polyamines include spermine and / or spermidine.

[0018] Spermidine and spermine have amino and imino groups, where spermidine contains one imino and two amino groups, and spermine contains two amino and two imino groups. The present invention has found that by adjusting the pH value of the mixed solution in which hyaluronic acid and endogenous polyamines are dissolved, the reaction sites of spermine or spermidine with hyaluronic acid can be controlled, and a two-site crosslinked hydrogel and a three-site or four-site crosslinked active star-shaped network gel can be obtained. During the reaction process, the amino site reaction of spermine or spermidine (i.e., two-site reaction) can be adjusted by the pH of the solution, or the amino group and the imino group can be reacted together (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 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 are reactive sites, and in particular, the proportions of residual amino groups and residual imino groups in the gel obtained by the three-site or four-site crosslinking are both less than 20%.

[0019] The present invention found that by controlling the pH value of the solution at 5.00-5.49, the reaction activity of imino groups can be improved, and three-site reaction or four-site reaction can be relatively stably realized, and by controlling the pH value of the solution at 4.50-4.99 or 5.50-6.50, the reaction activity of imino groups can be provided, and the cooperative reaction participation of multiple sites can be relatively stably realized, and an active star-shaped network gel can be prepared. Therefore, the present invention has obtained a hyaluronic acid hydrogel with imino groups as crosslinking sites for the first time, and found that hydrogels with different crosslinking sites can obtain different gel properties even with the same crosslinking degree.

[0020] In the present invention, when the pH of the mixed solution is controlled to 5.00 to 5.49, a relatively stable three-site reaction or four-site reaction can be achieved, and the pH includes, but is not limited to, 5.00, 5.10, 5.20, 5.30, 5.40, or 5.49. When the pH of the mixed solution is controlled to 4.50 to 4.99 or 5.50 to 6.50, a relatively stable two-site crosslinking reaction can be achieved, and the pH includes, but is 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, or 6.50. Outside the pH range of the present invention, the experimental results are not ideal, and the pH value range of the present invention is a pH condition suitable for the method of the present invention obtained through extensive experiments.

[0021] The method of the present invention is an endogenous polyamine (spermine and permidine) multisite active reaction technology (abbreviated as Spermidine / Spermine Multisite Active Reaction Technology, SMART). The method of the present invention can control the crosslinking site between endogenous polyamine and hyaluronic acid, and by controlling the crosslinking reaction site, it affects each performance parameter of the obtained crosslinked hyaluronic acid gel. In addition, it can control the release rate of endogenous polyamine such as spermidine during the decomposition process of hyaluronic acid gel, and can sustain the physiological activity effect of endogenous polyamine 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 by triphenylphosphine and bromide include phosphonium salts formed by triphenylphosphine and carbon tetrabromide, phosphonium salts formed by 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] In the activator, when using a water-soluble carbodiimide activator, it is necessary to use an auxiliary agent at the same time to improve the crosslinking reaction efficiency. Preferably, the auxiliary agent includes any one or more of N-hydroxysuccinimide (NHS), sulfonated N-hydroxysuccinimide (Sulfo-NHS), tert-butanol and 1-hydroxybenzotriazole (HOBt); more preferably, the amount of the auxiliary agent added is 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%. By using a water-soluble carbodiimide and an auxiliary agent in combination, the crosslinking reaction efficiency can reach about 70%.

[0027] Among the three activators, namely, phosphonium bromide salt formed by triphenylphosphine and bromide, carbonium salt, and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride, the carbonium salt has the highest crosslinking efficiency of 80-95%, among which the crosslinking efficiency when using HATU catalyst is as high as 95%, followed by the crosslinking efficiency of 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 (1): [ka]

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

[0030] In the crosslinking reaction, when performing two-site crosslinking (the main reactive site of spermine or spermidine is an amino group), the amount of the activator added is 200-280% of the substance amount of the endogenous polyamine, including but not limited to 210%, 220%, 230%, 240%, 250%, 260%, 270% and 280%; when performing three-site and / or four-site crosslinking (the imino site is also involved in the reaction), the amount of the activator added is 300-550% of the substance 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 sites of the crosslinking agent spermine or spermidine, and when the above activators are used, each molecule of the activator activates one carboxyl group and, in addition, can carry out an amide coupling reaction with one amino group (or imino group).

[0033] In one embodiment, in the crosslinking reaction, when the endogenous polyamine is spermine, the amount of spermine added is 0.3-35% of the mass of the hyaluronic acid, including 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 0.5-40% 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 from 100 KDa (kilodaltons) to 3000 KDa, and preferably, the hyaluronic acid is hyaluronic acid prepared by microbial fermentation.

[0037] In one embodiment, in the crosslinking reaction, when the activator comprises 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] In the reaction process, the two-site crosslinking gel and the three-site or four-site crosslinking active star-shaped network gel can be controlled by different reaction temperatures and the mixing ratio of activator and spermine or spermidine. The imino group is subject to the steric hindrance effect, so it has a lower reactivity than the primary amino group. Therefore, in the two-site reaction involving only the primary amino group, only a low temperature and a small amount of activator are required to complete the crosslinking reaction; however, when imino needs to be involved in the reaction, it is necessary to increase the reaction temperature and extend the reaction time to increase the crosslinking reaction efficiency of imino in order to increase the reaction activity of imino.

[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-shaped network), and (4) is the cross-linking reaction between hyaluronic acid and spermidine (three-site star-shaped 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, and obtain two-site crosslinked gels and three- or four-site crosslinked active star-shaped network gels by controlling one or more of the reaction conditions, such as pH, temperature, and the type and amount of active agent.

[0041] In one embodiment, the gel material is further eluted with an eluent, 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 the grinding is 1:1 to 5; Furthermore, the particle size of the gel after pulverization is 10 to 500 μm.

[0042] In one embodiment, the method further comprises the step of reconstituting and moist heat sterilizing the gel material after drying; Preferably, the reconstituted solution is a phosphate buffer, more preferably, the mass concentration of the phosphate buffer salt in the phosphate buffer is 5 to 40 mg / mL; Furthermore, the temperature of the moist heat sterilization is 120 to 130° C., and the time of the moist heat sterilization 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 repeating washing. The drying can be performed by vacuum drying, and the organic solvent can be removed by drying. The gel can be crushed into small particles to ensure the efficiency of elution of the residual active agent during several washing processes.

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

[0045] In one specific embodiment, the method further comprises: (a) dissolving hyaluronic acid and endogenous polyamines directly 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 break up the gel particles, washing several times to remove residual activator, and vacuum drying to remove the organic solvent; (d) Redissolving the vacuum-dried gel particles in a phosphate buffer solution, filling a prefilled syringe, and sterilizing 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 the minimum value can be controlled within 10%, the rheological performance of the gel before sterilization is effectively maintained, and the thermal stability of the hyaluronic acid gel is greatly improved.

[0047] The present invention not only designs polyamines such as spermidine as hyaluronic acid crosslinking agents, but also uses the entire hyaluronic acid gel as a sustained release gel of polyamines such as spermidine, that is, during the natural decomposition process in the body, the gel can continuously and normally release polyamine substances such as spermidine.Three-site or four-site active star-shaped network gel of the present invention can stably release spermidine monomers through the enzymatic induction of hyaluronidase, and continuously exert the bioactivity effect of spermidine.

[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 injections for soft tissue filling, soft tissue repair or for 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 greatly improves the thermal stability of the hyaluronic acid gel. (3) The preparation method of the present invention makes it possible to control the number of cross-linking reaction sites, 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 decomposition process of hyaluronic acid gel, and can sustain the physiological activity of endogenous polyamines such as spermidine. (5) The present invention also improves the ease of use of the gel in the fields of soft tissue filling, soft tissue repair, medical beauty, etc., making it suitable for popularization and application. [Brief description 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 briefly introduces drawings required for the description of 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 creative work. [Figure 1] 1 is a 1H NMR spectrum of spermidine cross-linked hyaluronic acid provided by the present invention. [Diagram 2] FIG. 1 is a schematic diagram of cell proliferation in spermidine-crosslinked hyaluronic acid provided by the present invention. [Diagram 3] 1 is a curve showing the amount of spermidine released together with crosslinked hyaluronic acid in a spermidine-crosslinked hyaluronic acid gel under pyrolysis conditions provided by the present invention. [Figure 4] 1 is a curve showing the amount of spermidine released together with cross-linked hyaluronic acid in a spermidine-cross-linked hyaluronic acid gel under enzyme conditions provided by the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] The technical solutions of the present invention will be described clearly and completely 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. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art 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 Weigh out 3.0g sodium hyaluronate (molecular weight 900KDa, contains 7.4mmol of hyaluronic acid repeating unit), add 98mL purified water, and the concentration of hyaluronic acid after complete dissolution is 30mg / mL. Add 0.03g spermine (molar number is 0.15mmol) to the hyaluronic acid solution, and in this case, spermine content accounts for 1% of the mass of hyaluronic acid (2% of the mole number of hyaluronic acid repeating unit). Use 6mol / L hydrochloric acid solution to adjust the pH value of the hyaluronic acid solution to about 6.20, then add and stir evenly, add 0.32mmol HATU, which is 213% of the mole number of spermine, continue to stir evenly, seal and put into a 25℃ incubator to react for 24 hours. After the reaction is completed, 40 mL of anhydrous ethanol is added, and the gel particles are crushed using an IKA T25 high shear disperser at a crushing speed of 10,000 rpm / min for 5 minutes. After the crushing is completed, 200 mL of anhydrous ethanol is added to completely dehydrate the gel. The precipitate is separated, and the precipitate is washed five times in succession with 200 mL of anhydrous ethanol, and then placed in a vacuum oven, and then vacuum dried at a vacuum degree of -0.09 MPa and 40°C for 24 hours. After complete drying, 1.0 g of dry gel is taken, and a total of 50 mL of phosphate buffer solution with a concentration of 10 mg / mL and pH 7.0 is added to obtain a hydrogel with a hyaluronic acid concentration of 20 mg / mL. After the gel is completely swollen, the gel is filled into a prefilled syringe and sterilized with moist heat at 121°C for 15 minutes to obtain the final product gel. The particle size D0.5 of the gel is 158 μm, and D0.9 is 196 μm.

[0054] Example 2 Example 2. Preparation of three-site crosslinked hydrogel of spermidine and hyaluronic acid by HATU catalysis Weigh out 3.0g sodium hyaluronate (molecular weight 900KDa, contains 7.4mmol of hyaluronic acid repeating unit), add 98mL purified water, and the concentration of hyaluronic acid after complete dissolution is 30mg / mL. Add 0.022g 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 unit). Add 0.48mmol HATU, which is 320% of the mole number of spermidine, and stir evenly, and use 6mol / L hydrochloric acid solution to adjust the pH value of the hyaluronic acid solution to about 5.40, then add and stir, seal and put into a 50℃ incubator to react for 14 hours. After the reaction is completed, 40 mL of anhydrous ethanol is added, and the gel particles are crushed using an IKA T25 high shear disperser at a crushing speed of 10,000 rpm / min for 5 minutes. After the crushing is completed, 200 mL of anhydrous ethanol is added to completely dehydrate the gel. The precipitate is separated, and the precipitate is washed five times in succession with 200 mL of anhydrous ethanol, and then placed in a vacuum oven, and then vacuum dried at a vacuum degree of -0.09 MPa and 40°C for 24 hours. After complete drying, 1.0 g of dry gel is taken, and a total of 50 mL of phosphate buffer solution with a concentration of 10 mg / mL and a pH of 7.0 is added to obtain a hydrogel with a hyaluronic acid concentration of 20 mg / mL. After the gel is completely swollen, the gel is filled into a prefilled syringe and sterilized with moist heat at 121°C for 15 minutes to obtain the final product gel. The particle size D0.5 of the gel is 143 μm, and D0.9 is 201 μm.

[0055] Example 3 Example 3. Preparation of four-site crosslinked hydrogel of spermine and hyaluronic acid by HATU catalysis 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 the concentration of hyaluronic acid after complete dissolution is 30mg / mL. Add 0.03g of spermine (molar number is 0.15mmol) to the hyaluronic acid solution, in which case the spermine content accounts for 1% of the mass of hyaluronic acid (2% of the mole number of hyaluronic acid repeating structural units). 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 5.30, continue to stir evenly, seal and place in a 60℃ incubator to react for 14 hours. After the reaction is completed, add 40mL of acetone, and use an IKA T25 high shear disperser to crush the gel particles at a grinding speed of 10000rpm / min for 5 minutes continuously. After the grinding is completed, 200 mL of acetone is added continuously to completely dehydrate the gel. The precipitate is separated, and the precipitate is washed five times in succession with 200 mL of acetone, then placed in a vacuum oven, and then vacuum dried at a vacuum degree of -0.09 MPa and 40°C for 24 hours. After complete drying, 1.0 g of dry gel is taken, and a total of 50 mL of phosphate buffer solution with a concentration of 10 mg / mL and pH 7.0 is added to obtain a hydrogel with a hyaluronic acid concentration of 20 mg / mL. After the gel is completely swollen, the gel is 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 D0.5 of the gel is 139 μm, and D0.9 is 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 completed, use a rotary evaporator to remove the dichloromethane to obtain triphenylphosphonium bromide succinimide salt, which is sealed and ready for use.

[0057] Weigh out 3.0g sodium hyaluronate (molecular weight 900KDa, contains 7.4mmol of hyaluronic acid repeating unit), add 98mL purified water, the concentration of hyaluronic acid after completely dissolving is 30mg / mL, add 0.022g spermidine (molar number is 0.15mmol) to hyaluronic acid solution, in this case spermidine content is 0.7% of the mass of hyaluronic acid (2% of the mole number of hyaluronic acid repeating unit). 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, use 6mol / L hydrochloric acid solution to adjust the pH value of hyaluronic acid solution to about 6.00, then add and stir, seal and put in a 25℃ incubator to react for 24 hours. After the reaction is completed, 40 mL of acetone is added, and the gel particles are crushed using an IKA T25 high shear disperser at a crushing speed of 10,000 rpm / min for 5 minutes. After the crushing is completed, 200 mL of acetone is added to completely dehydrate the gel. The precipitate is separated, washed with 200 mL of acetone 5 times in succession, and then placed in a vacuum oven, and then vacuum dried at a vacuum degree of -0.09 MPa and 40°C for 24 hours. After complete drying, 1.0 g of dry gel is taken, and a total of 50 mL of phosphate buffer solution with a concentration of 10 mg / mL and pH 7.0 is added to obtain a hydrogel with a hyaluronic acid concentration of 20 mg / mL. After the gel is completely swollen, the gel is filled into a prefilled syringe and sterilized with moist heat at 121°C for 15 minutes to obtain the final product gel. The particle size D0.5 of the gel 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 Weigh out 3.0g sodium hyaluronate (molecular weight 900KDa, contains 7.4mmol of hyaluronic acid repeating unit), add 98mL purified water, and the concentration of hyaluronic acid after completely dissolving is 30mg / mL. Add 0.022g spermidine (molar number is 0.15mmol) to hyaluronic acid solution, and in this case, spermidine content accounts for 0.7% of the mass of hyaluronic acid (2% of the mole number of hyaluronic acid repeating unit). Add 0.48mmol of triphenylphosphonium bromide succinimide salt obtained by the method in Example 4, which is 320% of the mole number of spermidine, and stir evenly, and use 6mol / L hydrochloric acid solution to adjust the pH value of hyaluronic acid solution to about 5.20, then add and stir evenly, seal and put into 40℃ incubator to react for 24 hours. After the reaction is completed, 40 mL of acetone is added, and the gel particles are crushed using an IKA T25 high shear disperser at a crushing speed of 10,000 rpm / min for 5 minutes. After the crushing is completed, 200 mL of acetone is added to completely dehydrate the gel. The precipitate is separated, washed with 200 mL of acetone 5 times in succession, and then placed in a vacuum oven, and then vacuum dried at a vacuum degree of -0.09 MPa and 40°C for 24 hours. After complete drying, 1.0 g of dry gel is taken, and a total of 50 mL of phosphate buffer solution with a concentration of 10 mg / mL and pH 7.0 is added to obtain a hydrogel with a hyaluronic acid concentration of 20 mg / mL. After the gel is completely swollen, the gel is filled into a prefilled syringe and sterilized with moist heat at 121°C for 15 minutes to obtain the final product gel. The particle size D0.5 of the gel 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 sodium hyaluronate (molecular weight 900KDa, containing 7.4mmol of hyaluronic acid repeating structural unit), add 98mL purified water, the concentration of hyaluronic acid after complete dissolution is 30mg / mL, add 0.022g spermidine (mol number is 0.15mmol) to the hyaluronic acid solution, in this case spermidine content is 0.7% of the mass of hyaluronic acid (2% of the mole number of hyaluronic acid repeating structural unit). Add 0.48mmol DMTMM, which is 320% of the mole number of spermidine, and stir evenly, use 6mol / L hydrochloric acid solution to adjust the pH value of the hyaluronic acid solution to about 5.10, then add and stir evenly, seal and put into a 40℃ incubator to react for 24 hours. After the reaction is completed, 40 mL of anhydrous ethanol is added, and the gel particles are crushed using an IKA T25 high shear disperser at a grinding speed of 10,000 rpm / min for 5 minutes. After the grinding is completed, 200 mL of anhydrous ethanol is added to completely dehydrate the gel. The precipitate is separated, and the precipitate is washed five times in succession with 200 mL of anhydrous ethanol, and then placed in a vacuum oven, and then vacuum dried at a vacuum degree of -0.09 MPa and 40°C for 24 hours. After complete drying, 1.0 g of dry gel is taken, and a total of 50 mL of phosphate buffer solution with a concentration of 10 mg / mL and pH 7.0 is added to obtain a hydrogel with a hyaluronic acid concentration of 20 mg / mL. After the gel is completely swollen, the gel is filled into a prefilled syringe and sterilized with moist heat at 121°C for 15 minutes to obtain the final product gel. The particle size D0.5 of the gel 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 sodium hyaluronate (molecular weight 900KDa, contains 7.4mmol of hyaluronic acid repeating unit), add 98mL purified water, the concentration of hyaluronic acid after complete dissolution is 30mg / mL, add 0.03g spermine (molar number is 0.15mmol) to the hyaluronic acid solution, in this case spermine content is 1% of the mass of hyaluronic acid (2% of the mole number of hyaluronic acid repeating unit). Add 0.65mmol EDC, which is 433% of the mole number of spermine, and add NHS, which is 20% of the mass of EDC, and stir evenly, use 6mol / L hydrochloric acid solution to adjust the pH value of the hyaluronic acid solution to about 5.45, continue to stir evenly, seal and put into a 60℃ incubator to react for 14 hours. After the reaction is completed, 40 mL of anhydrous ethanol is added, and the gel particles are crushed using an IKA T25 high shear disperser at a crushing speed of 10,000 rpm / min for 5 minutes. After the crushing is completed, 200 mL of anhydrous ethanol is added to completely dehydrate the gel. The precipitate is separated, and the precipitate is washed five times in succession with 200 mL of anhydrous ethanol, and then placed in a vacuum oven, and then vacuum dried at a vacuum degree of -0.09 MPa and 40°C for 24 hours. After complete drying, 1.0 g of dry gel is taken, and a total of 50 mL of phosphate buffer solution with a concentration of 10 mg / mL and pH 7.0 is added to obtain a hydrogel with a hyaluronic acid concentration of 20 mg / mL. After the gel is completely swollen, the gel is filled into a prefilled syringe and sterilized with moist heat at 121°C for 15 minutes to obtain the final product gel. The particle size D0.5 of the gel is 125 μm, and D0.9 is 193 μm.

[0061] Example 8 Example 8, HATU-catalyzed combination of spermidine and spermine involved in the preparation of multi-site crosslinked hydrogels of hyaluronic acid Weigh out 3.0g sodium hyaluronate (molecular weight 900KDa, contains 7.4mmol of hyaluronic acid repeating unit), add 98mL purified water, and the concentration of hyaluronic acid after completely dissolving is 30mg / mL. Then add 0.022g spermidine (molar number is 0.15mmol) and 0.03g spermine (molar number is 0.15mmol) to the hyaluronic acid solution, and in this case, the spermidine content or spermine content respectively accounts for 2% of the mole number of hyaluronic acid repeating unit. Add 0.48mmol HATU, which is 320% of the mole number of spermidine, then add 0.65mmol HATU, which is 433% of the mole number of spermine, and stir evenly, and use 6mol / L hydrochloric acid solution to adjust the pH value of hyaluronic acid solution to about 5.00, then add and stir evenly, seal and put into 50℃ incubator to react for 14 hours. After the reaction is completed, 40 mL of anhydrous ethanol is added, and the gel particles are subsequently ground using an IKA T25 high shear disperser at a grinding speed of 10,000 rpm / min for 5 minutes. After the grinding is completed, 200 mL of anhydrous ethanol is added continuously to completely dehydrate the gel. The precipitate is separated, and the precipitate is washed five times in succession with 200 mL of anhydrous ethanol, and then placed in a vacuum oven, and then vacuum dried at a vacuum degree of -0.09 MPa and 40°C for 24 hours. After complete drying, 1.0 g of dry gel is taken, and a total of 50 mL of phosphate buffer with a concentration of 10 mg / mL and pH 7.0 is added to obtain a hydrogel with a hyaluronic acid concentration of 20 mg / mL. After the gel is completely swollen, the gel is filled into a prefilled syringe and sterilized with moist heat at 121°C for 15 minutes to obtain the final product gel. The particle size D0.5 of the gel 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 sodium hyaluronate (molecular weight 120KDa), add 35mL purified water, and the concentration of hyaluronic acid after complete dissolution is 150mg / mL. Add 2.4g 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. On the premise of three-site crosslinking, use 6mol / L hydrochloric acid solution to adjust the pH value of the hyaluronic acid solution to about 5.2, then add and stir evenly, add 66.4mmol EDC, which is 400% of the mole number of spermidine, and at the same time add 2.4g Sulfo-NHS (20% of EDC mass), continue to stir evenly, seal, and put into a 60℃ blow dry oven to react for 24 hours. After the reaction is completed, 40 mL of anhydrous ethanol is added, and the gel particles are crushed using an IKA T25 high shear disperser at a grinding speed of 10,000 rpm / min for 5 minutes. After the grinding is completed, 200 mL of anhydrous ethanol is added to completely dehydrate the gel. The precipitate is separated, and the precipitate is washed five times in succession with 200 mL of anhydrous ethanol, and then placed in a vacuum oven, and then vacuum dried at a vacuum degree of -0.09 MPa and 40°C for 24 hours. After complete drying, 1.0 g of dry gel is collected, and a total of 20 mL of phosphate buffer solution with a concentration of 10 mg / mL and a pH of 7.0 is added. After the gel is completely swollen, the gel is filled into a prefilled syringe and sterilized with moist heat at 121°C for 15 minutes to obtain the final product gel. The particle size D0.5 of the gel is 145 μm, and D0.9 is 213 μm.

[0063] Example 10 Example 10. Preparation of four-site crosslinked hydrogel of spermine and hyaluronic acid by HATU catalysis Weigh out 0.5g of sodium hyaluronate (molecular weight 2800KDa), add 38.5mL of purified water, and the concentration of hyaluronic acid after complete dissolution is 13.3mg / mL. Add 1.5mg of spermine (molar number is 7.4μmol) to the hyaluronic acid solution, in which case the spermine content accounts for 0.3% of the mass of hyaluronic acid. On the premise of 4-site crosslinking, use 6mol / L hydrochloric acid solution to adjust the pH value of the hyaluronic acid solution to about 5.2, then add and stir, add 29.6μmol of HATU, which is 400% of the mole number of spermine, continue to stir evenly, seal, and place in a 30℃ air drying oven to react for 24 hours. After the reaction is completed, add 40mL of acetone, and use an IKA T25 high shear disperser to crush the gel particles at a grinding speed of 10000rpm / min for 5 minutes. After the grinding is completed, continue to add 200mL of acetone to completely dehydrate the gel. The precipitate is separated, washed five times with 200mL of acetone, and then placed in a vacuum oven and vacuum dried at a vacuum of -0.09MPa and 40℃ for 24 hours. After complete drying, 0.4g of dry gel is taken, and a total of 400mL of phosphate buffer solution with a concentration of 10mg / mL and a pH of 7.0 is added. After the gel is completely swollen, the gel is filled into a prefilled syringe and sterilized with moist heat at 121℃ for 15 minutes to obtain the final gel product. The particle size D0.5 of the gel is 156μm, and D0.9 is 231μm.

[0064] Example 11 Example 11. Preparation of three-site crosslinked hydrogel of spermidine and hyaluronic acid by HATU catalysis Weigh out 0.5g of sodium hyaluronate (molecular weight 2800KDa), add 38.5mL of purified water, and the concentration of hyaluronic acid after complete dissolution is 13.3mg / mL. Add 2.5mg of spermidine (molar number is 17.2μmol) to the hyaluronic acid solution, in which case the spermidine content accounts for 0.5% of the mass of hyaluronic acid. On the premise of three-site crosslinking, use 6mol / L hydrochloric acid solution to adjust the pH value of the hyaluronic acid solution to about 5.2, then add and stir, add 51.6μmol of HATU, which is 300% of the mole number of spermidine, continue to stir evenly, seal, and place in a 30℃ air drying oven to react for 24 hours. After the reaction is completed, add 40mL of acetone, and use an IKA T25 high shear disperser to crush the gel particles at a grinding speed of 10000rpm / min for 5 minutes continuously. After the grinding is completed, 200mL of acetone is added continuously to completely dehydrate the gel. The precipitate is separated, washed five times with 200mL of acetone, and then placed in a vacuum oven, and then vacuum dried at a vacuum degree of -0.09MPa and 40°C for 24 hours. After complete drying, 0.4g of dry gel is taken, and a total of 200mL of phosphate buffer solution with a concentration of 10mg / mL and a pH of 7.0 is added. After the gel is completely swollen, the gel is 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 D0.5 of the gel is 125μm, and D0.9 is 201μm.

[0065] Example 12 Example 12: EDC-catalyzed cross-linking of spermine and hyaluronic acid at four sites Weigh out 6g of sodium hyaluronate (molecular weight 120KDa), add 35mL of purified water, and the concentration of hyaluronic acid after complete dissolution is 150mg / mL. Add 2.1g of spermine (molar number is 10.4mmol) to the hyaluronic acid solution, in which the spermine content accounts for 35% of the mass of hyaluronic acid. On the premise of 4-site crosslinking, use 6mol / L hydrochloric acid solution to adjust the pH value of the hyaluronic acid solution to about 5.2, then add and stir, add 57.2mmol of EDC, which is 550% of the mole number of spermine, and at the same time add 2.0g of HOBt (20% of the mass of EDC), continue to stir evenly, seal, and place in a 60℃ air drying oven to react for 24 hours. After the reaction is completed, add 40mL of anhydrous ethanol, and use an IKA T25 high shear disperser to crush the gel particles at a grinding speed of 10000rpm / min for 5 minutes. After the grinding is completed, 200 mL of anhydrous ethanol is added continuously to completely dehydrate the gel. The precipitate is separated, and the precipitate is washed five times in succession with 200 mL of anhydrous ethanol, then placed in a vacuum oven, and then vacuum dried at a vacuum degree of -0.09 MPa and 40°C for 24 hours. After complete drying, 1.0 g of dry gel is taken, and a total of 20 mL of phosphate buffer solution with a concentration of 10 mg / mL and a pH of 7.0 is added. After the gel is completely swollen, the gel is 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 D0.5 of the gel is 148 μm, and D0.9 is 197 μm.

[0066] Example 13 Example 13. Preparation of two-site crosslinked hydrogel of spermidine and hyaluronic acid under slightly acidic conditions by HATU catalysis Weigh out 3.0g sodium hyaluronate (molecular weight 900KDa, containing 7.4mmol of hyaluronic acid repeating unit), add 98mL purified water, and the concentration of hyaluronic acid after complete dissolution is 30mg / mL. Add 0.022g 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 unit). Add 0.48mmol HATU, which is 320% of the mole number of spermidine, and stir evenly, and use 6mol / L hydrochloric acid solution to adjust the pH value of the hyaluronic acid solution to about 4.60, then add and stir evenly, seal and put into a 50℃ incubator to react for 14 hours. After the reaction is completed, 40 mL of anhydrous ethanol is added, and the gel particles are crushed using an IKA T25 high shear disperser at a crushing speed of 10,000 rpm / min for 5 minutes. After crushing is completed, 200 mL of anhydrous ethanol is added to completely dehydrate the gel. The precipitate is separated, and the precipitate is washed five times in succession with 200 mL of anhydrous ethanol, and then placed in a vacuum oven, and then vacuum dried at a vacuum degree of -0.09 MPa and 40°C for 24 hours. After complete drying, 1.0 g of dry gel is taken, and a total of 50 mL of phosphate buffer solution with a concentration of 10 mg / mL and pH 7.0 is added to obtain a hydrogel with a hyaluronic acid concentration of 20 mg / mL. After the gel is completely swollen, the gel is filled into a prefilled syringe and sterilized with moist heat at 121°C for 15 minutes to obtain the final product gel. The particle size D0.5 of the gel 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 strong acidic conditions Weigh out 3.0g sodium hyaluronate (molecular weight 900KDa, contains 7.4mmol of hyaluronic acid repeating unit), add 98mL purified water, the concentration of hyaluronic acid after complete dissolution is 30mg / mL, add 0.022g spermidine (molar number is 0.15mmol) to hyaluronic acid solution, in this case spermidine content is 0.7% of the mass of hyaluronic acid (2% of the mole number of hyaluronic acid repeating unit). Add 0.48mmol HATU, which is 320% of the mole number of spermidine, and stir evenly, use 6mol / L hydrochloric acid solution to adjust the pH value of hyaluronic acid solution to about 3.20, then add and stir evenly, seal and put in 50℃ incubator to react for 14 hours. After the reaction is completed, 40 mL of anhydrous ethanol is added, and the gel particles are subsequently ground using an IKA T25 high shear disperser at a grinding speed of 10,000 rpm / min for 5 minutes. After the grinding is completed, 200 mL of anhydrous ethanol is added continuously to completely dehydrate the gel. The precipitate is separated, and the precipitate is washed five times in succession with 200 mL of anhydrous ethanol, and then placed in a vacuum oven, and then vacuum dried at a vacuum degree of -0.09 MPa and 40°C for 24 hours. After complete drying, 1.0 g of dry gel is taken, and a total of 50 mL of phosphate buffer with a concentration of 10 mg / mL and pH 7.0 is added to obtain a hydrogel with a hyaluronic acid concentration of 20 mg / mL. After the gel is completely swollen, the gel is filled into a prefilled syringe and sterilized with moist heat at 121°C for 15 minutes to obtain the final product gel. The particle size D0.5 of the gel is 143 μm, and D0.9 is 201 μm.

[0068] Comparative Example 2 Comparative Example 2: Crosslinking reaction of spermine and hyaluronic acid using EDC catalyst In this comparative example, the gel is prepared according to the method disclosed in US9907739B2. Weigh out 3.0g sodium hyaluronate (molecular weight 900KDa, containing 7.4mmol of hyaluronic acid repeating structural unit), add 98mL purified water, and the concentration of hyaluronic acid after complete dissolution is 30mg / mL. Add 0.03g spermine (molar number is 0.15mmol) to the hyaluronic acid solution, and in this case, the spermine content accounts for 1% of the mass of hyaluronic acid (2% of the mole number of hyaluronic acid repeating structural unit). Use 6mol / L hydrochloric acid solution to adjust the pH value of the hyaluronic acid solution to about 6.2, stir evenly, add 7.4mmol of EDC, which is 100% of the mole number of hyaluronic acid structural unit and 49 times the mole number of spermine, and at the same time add HOBt, which is 20% of the mass of EDC, continue to stir evenly, seal and put into a 25℃ incubator to react for 24 hours. After the reaction is completed, 40 mL of anhydrous ethanol is added, and the gel particles are crushed using an IKA T25 high shear disperser at a crushing speed of 10,000 rpm / min for 5 minutes. After the crushing is completed, 200 mL of anhydrous ethanol is added to completely dehydrate the gel. The precipitate is separated, and the precipitate is washed five times in succession with 200 mL of anhydrous ethanol, and then placed in a vacuum oven, and then vacuum dried at a vacuum degree of -0.09 MPa and 40°C for 24 hours. After complete drying, 1.0 g of dry gel is taken, and a total of 50 mL of phosphate buffer solution with a concentration of 10 mg / mL and a pH of 7.0 is added to obtain a hydrogel with a hyaluronic acid concentration of 20 mg / mL. After the gel is completely swollen, the gel is filled into a prefilled syringe and sterilized with moist heat at 121°C for 15 minutes to obtain the final product gel. The particle size D0.5 of the gel is 155 μm, and D0.9 is 221 μm.

[0069] Example 14 Example 14: Detection of cross-linking efficiency of spermine or spermidine 2.0 ml of the hyaluronic acid gel obtained in Examples 1-8 and Comparative Examples 1-2 is taken out, 50 ml of anhydrous ethanol is added to each gel, and the gel is stirred for 30 minutes until the gel turns into a white precipitate. The precipitate is separated and vacuum dried at a vacuum degree of -0.09 MPa and 40°C for 24 hours to remove the ethanol. The gel is taken out, 10 mL of 0.5 mol / L sulfuric acid solution is added, and the gel is treated at 90°C for 2 hours, and then neutralized to pH=7 with 6 mol / L sodium hydroxide solution. The solution is placed in a vacuum drying oven and vacuum dried at a vacuum degree of -0.09 MPa and 60°C for 24 hours to remove excess water. The gel is dissolved in deuterium water 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 of H NMR spectrum, the actual molar ratio of spermidine and hyaluronic acid can be obtained by the integrated peak, 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, the selected characteristic H atom of spermidine is the α-H of amino group or imino group, the number of hydrogen atoms is 8 in total, the selected characteristic H atom of spermine is the α-H of amino group or imino group, the number of H atoms is 12 in total, the integration range of H atoms of spermine and spermidine is the same, 2.5-2.85 ppm, the selected characteristic H atom of hyaluronic acid is the α-H of carbonyl group in acetamide group, the number of H atoms is 3 in total, the integration range is 1.85-2.05 ppm, and when integrating, the integrated peak area of ​​the carbonyl group α-H of hyaluronic acid is 3, the integrated peak area of ​​the amino group and imino α-H of spermidine or spermine can be obtained. The calculation formula is as follows: Actual molar ratio of spermidine bridges = α-H integrated peak area of ​​amino and imino groups in spermidine / 8; Actual crosslinking molar ratio of spermine = α-H integrated peak area of ​​amino and imino groups of spermine / 12; Crosslinking efficiency = (actual crosslinking molar ratio / theoretical crosslinking molar ratio) x 100%.

[0071] The crosslinking efficiencies of Examples 1 to 8 are shown in Table 1, and the nuclear magnetic spectra are shown in FIG. [Table 1]

[0072] As can be seen from the analysis of the data in Table 1, when two-site crosslinking occurs, the crosslinking efficiency is lower than that of multi-site crosslinking. As can be seen from the comparison of Examples 1 and 4, which perform two-site crosslinking, with Examples 2, 3, and 5, the two-site crosslinking efficiency is generally lower than that of multi-site crosslinking, which may be due to the influence of three conditions, namely the amount of activator added, pH value, and temperature, which results in a lower crosslinking efficiency during two-site crosslinking; similarly, when performing multi-site crosslinking, the activation efficiency of each activator is in the order of HATU>triphenylphosphonium salt>DMTMM>EDC+auxiliary agent. Here, the activation efficiency of HATU is as high as nearly 95%, while the activation efficiency of EDC+NHS is only 75%. Under the same conditions, the crosslinking efficiency of spermine and spermidine is close, with spermine being slightly lower, which is believed to be due to the high imino content of spermine, and the reactivity of the imino group is not as good as that of primary amino groups due to the large steric hindrance. In addition, according to the analysis of the comparative examples, 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, and the crosslinking efficiency is reduced. When the test temperature is lower than the test temperature for multi-site crosslinking, even if the reaction is carried out according to the conditions for multi-site crosslinking, the final crosslinking efficiency is similar to that of two-site crosslinking, and the reaction efficiency is lower.

[0073] Example 15 Example 15: Effect of spermidine cross-linked hyaluronic acid gel on cell proliferation L-929 cells are seeded in cell culture medium, and 1% penicillin-streptomycin solution and 10% fetal bovine serum solution are added. The L-929 cells are 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 is transferred to a 96-well plate, cured with an ultraviolet lamp, and sterilized. Thereafter, L-929 cell culture medium is added to the wells containing the spermidine cross-linked hyaluronic acid gel, and 1 mL of trypsin solution containing 0.1% EDTA is added, and the number of L-929 cells in each well is adjusted to 1 x 10 5 Alternatively, place 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 h, respectively, 100 μL of MTT aqueous solution (concentration 5 mg mL) was added to each well. -1 ) and place in an incubator to continue culturing for 4 hours. Then, remove the MTT solution, add 150 μL of dimethyl sulfoxide to dissolve the formazan crystals, and use a microplate reader to detect the absorbance of the solution at a wavelength of 490 nm to measure the degree of cell proliferation in L-929 by 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, and as can be seen from this figure, the cell viability of spermidine cross-linked hyaluronic acid gel at 24, 48 and 72 hours is 112%, 123% and 138%, respectively. As can be seen from the cell viability of these two types of gels, spermidine cross-linked hyaluronic acid hydrogel has no cytotoxicity, and the phenomenon that the cell viability increases with time indicates that the hydrogel has a certain 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 are divided into two types, one before sterilization and one after sterilization, and 2.0 mL of each is taken, and the elastic modulus (G') of the gel is detected using a TA DHR-2 type plate rheometer, and the elastic modulus loss rate is calculated. Here, the G' loss rate is calculated by the following formula. G' loss rate = (G' before sterilization - G' after sterilization) / G' before sterilization, The parameters of the rheometer are 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] G' loss rate can be considered as a characterization index of gel thermal stability, 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, G' loss rate has a certain relationship with crosslinking efficiency in Example 14, where, under similar crosslinking degree and crosslinking efficiency (Examples 2 and 3), the thermal stability of spermidine crosslinked hyaluronic acid and spermine crosslinked hyaluronic acid is similar, but spermine has a higher elastic modulus and higher thermal stability, which may be due to the fact that the crosslinked network structure formed by the four-site crosslinking of spermine is more dense, and the same theory can be used for three-site crosslinked spermidine gel and two-site crosslinked spermidine gel, i.e., 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. In addition, the concentration of hyaluronic acid in the crosslinked hyaluronic acid hydrogel also has a great influence on the thermal stability of the gel, and the same crosslinking sites as those in Examples 2 and 3 are used in Examples 10 and 11, but the concentrations of hyaluronic acid in the crosslinked hyaluronic acid hydrogels in Examples 10 and 11 are 1 mg / mL and 2 mg / mL, respectively, while the corresponding concentrations in Examples 2 and 3 are 20 mg / mL, which leads to the G' being significantly different from each other and the decrease in G' being large. In addition, the amount of activator added also has a great influence on the performance of the hydrogel, and when other conditions are the same in Example 1 and Example 13, the amount of HATU activator added in Example 13 is reduced by 28.6% (i.e., 164.8 μmol) compared with Example 1, which leads to the G' being obviously reduced in Example 13 compared with Example 1, and the decrease rate of G' being obviously increased compared with Example 1.

[0078] (Example 17) Example 17: Detection of spermidine release in gels during hyaluronic acid decomposition under pyrolysis conditions In Example 2, 2mL of the final gel product is taken for each sample, sealed in a vial, and then placed in a 125°C blow dry oven to carry out accelerated hydrolysis. Every 15 minutes, three samples are taken in parallel, cooled to about 25°C, and then the pH is adjusted to 12 with sodium hydroxide. 20mL of chromatography grade acetone is added, and filtered through a 0.2μm filter membrane. The acetone filtrate is then injected into an Agilent 7890B gas chromatograph, using an SGE H2 capillary column (30m×0.53mm×1.0μm), and the chromatography conditions are as follows: Sample inlet temperature 220°C, pressure 40, separation flow rate 30, split ratio 10:1, separation time 0.8, 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 350mL / min, H2 flow rate 35mL / min, make-up N2 flow rate 30mL / min.

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

[0080] The purpose of this experiment is to simulate the release process of spermidine monomers due to hydrolysis of cross-linked sites during the thermal decomposition process of hyaluronic acid. However, in normal physiological environments, the decomposition process of cross-linked hyaluronic acid takes a long time, from several months in the shortest case to more than a year in the longest case. Therefore, in this experiment, an experiment was adopted to detect the amount of spermidine released during accelerated hydrolysis under high-temperature conditions above 100°C using spermidine cross-linked hyaluronic acid gel, thereby allowing the decomposition process of hyaluronic acid and the release process of spermidine to be observed in a short time. As shown in Figure 3, spermidine is released slowly with the decomposition process of hyaluronic acid, but the initial release rate is slow, and in the later stage, with the collapse of the cross-linked hyaluronic acid network, the release rate of spermidine is significantly accelerated and the cumulative release amount is also significantly improved, which indicates that the release of spermidine in the gel is initially slow and then fast. This experiment also proves that the cross-linking technology in the present invention is an active reaction technology, which can continuously release prototype spermidine monomer during the continuous decomposition process of cross-linked hyaluronic acid, thereby proving that the gel can continuously release spermidine in the body and exert the unique bioactive effects of spermidine.

[0081] (Example 18) Example 18: Detection of spermidine release in gels during hyaluronic acid degradation under enzymatic conditions In Example 2, 2mL of the final gel product was taken for each sample and sealed in a vial. Then, 1000u hyaluronidase was added to each sample to carry out accelerated hydrolysis. Three samples were taken in parallel every 30 minutes. After cooling to about 25℃, 0.5mL of the supernatant was taken out, and the pH was adjusted to 12 with sodium hydroxide. 20mL of chromatographic grade acetone was added, and filtered through a 0.2μm filter membrane. The acetone filtrate was then injected into an Agilent 7890B gas chromatograph. An SGE H2 capillary column (30m×0.53mm×1.0μm) was used. The chromatographic conditions were as follows: Sample inlet temperature 220°C, pressure 40, separation flow rate 30, split ratio 10:1, separation time 0.8, 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 350mL / min, H2 flow rate 35mL / min, make-up N2 flow rate 30mL / min.

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

[0083] The purpose of this experiment is to simulate the release process of spermidine monomer by gel decomposition during the enzymatic decomposition process of hyaluronic acid.As shown in Figure 4, spermidine is slowly released with the decomposition process of hyaluronic acid, and the release rate of spermidine hardly changes during the whole decomposition process of hyaluronic acid gel, which shows that the hyaluronic acid gel obtained by crosslinking with spermidine can release spermidine uniformly and stably during enzymatic decomposition.This experiment can also prove that the crosslinking technology in the present invention is an active reaction technology, and can continuously release prototype spermidine monomer during the continuous decomposition process of crosslinked hyaluronic acid, thereby proving that the gel can sustainably release spermidine in the body and exert the unique bioactive effect of spermidine.

[0084] (Example 19) Example 19. Detection of remaining active sites in two-site and multi-site cross-linked gels Take about 2g of ninhydrin, add 100ml of purified water to dissolve, mix uniformly, and store in a dark place at 2-8℃ for use. Take 54.6g of sodium acetate, add 20ml of 1mol / L acetic acid solution to dissolve, add water until diluted to 500ml for use. Take spermidine standard solution and spermine standard solution, place them in a 10ml measuring flask, dilute to a specified volume with purified water, shake uniformly, prepare spermidine standard working solution of 500μg / mL and spermine standard working solution of 500μg / mL, and when used, dilute to 10, 50, 100, 200μg / mL, and use the original 500μg / ml standard working solution as the calibration curve.

[0085] 1 mL of each of the hydrogels before moist heat sterilization in Examples 1 to 8 and Comparative Examples 1 and 2, and the above spermine and spermidine series standard solutions, was taken, and 2.0 mL of the above acetic acid-sodium acetate buffer solution and 2.0 mL of ninhydrin solution were added in that order, the solution was 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 solution was diluted to 25 mL, mixed uniformly, and the mixed solution was 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 in the same manner.

[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 are two-site crosslinking, the main reactive group is the amino group, and the reaction efficiency is as high as 89.66% (100%-amino group residual amount, the same below). The lowest reaction efficiency of the amino group is also more than 80%, and the reaction efficiency of the imino group does not exceed 15%, so that under these conditions, two-site crosslinking reaction mainly involving the amino group occurs. In Examples 2, 3, and 8, the reaction efficiency of the amino group and the imino group in spermidine or spermine both reach 90% or more, and in Examples 5 to 7, the reaction efficiency of the imino group also exceeds 85%. This indicates that under these conditions, most spermidine undergoes three-site crosslinking and spermine undergoes four-site crosslinking. The reliability of the three-site and four-site crosslinking in the present invention is further proven. From the detection of the comparative examples, it is found that outside the conditions specified in the present invention, the reaction efficiency of the imino group of spermidine or spermine decreases to 10% or less, and the reaction efficiency of the amino group also decreases to a certain extent. Here, 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, those skilled in the art should understand that the technical solutions described in the above embodiments can be modified or equivalently substituted for some or all of the technical features thereof, and such modifications or equivalent substitutions do 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 crosslinking endogenous polyamines with hyaluronic acid, the endogenous polyamines comprising spermine and / or spermidine, and the crosslinking between the endogenous polyamines and the hyaluronic acid comprising two-site crosslinking, three-site crosslinking or four-site crosslinking; 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. 2. The gel material according to claim 1, wherein 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 15%, and further wherein 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%.

3. 3. The gel material according to claim 1, wherein 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 further wherein 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%.

4. the crosslinking efficiency of the crosslinking reaction between the endogenous polyamine and hyaluronic acid is greater than 75%; Furthermore, the crosslinking reaction efficiency between the endogenous polyamine and hyaluronic acid is higher than 80%, The gel material according to claim 1 or 2, further characterized in that the crosslinking reaction efficiency between the endogenous polyamine and hyaluronic acid is higher than 85%.

5. 1. A method for preparing a gel material, comprising: adjusting the pH of the mixture 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; However, when the activator contains a carbonium salt and / or a phosphonium bromide salt formed by triphenylphosphine and bromide, the temperature of the crosslinking reaction is 10° C. to 60° C., and the time of the crosslinking reaction is 14 hours to 24 hours; A method for preparing a gel material, characterized in that, 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° C. to 60° C., and the time of the crosslinking reaction is 14 hours to 24 hours.

6. The method for preparing a gel material according to claim 5, 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.

7. 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, 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 to obtain the gel material, Alternatively, the method for preparing a gel material according to claim 5, further comprising the steps of: adjusting the pH of a mixed solution of hyaluronic acid and endogenous polyamine to 5.00-5.49; adding an activator, water-soluble carbodiimide and / or 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride; setting the crosslinking reaction temperature at 40°C-60°C; and setting the crosslinking reaction time at 14 hours-24 hours, so that the hyaluronic acid and the endogenous polyamine undergo a three-site crosslinking reaction or a four-site crosslinking reaction to obtain the gel material.

8. 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)-biphenylphosphine; 8. The method according to any one of claims 5 to 7, characterized in that the compound comprises one or more of the following: 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.

9. The preparation method according to any one of claims 5 to 7, characterized in that when a water-soluble carbodiimide activator is used, it further comprises using it in combination with an auxiliary agent, preferably the auxiliary agent comprises any one or more of N-hydroxysuccinimide, sulfonated N-hydroxysuccinimide, tert-butanol and 1-hydroxybenzotriazole.

10. In the crosslinking reaction, when two-site crosslinking is performed, the amount of the activator added is 200% to 280% of the substance amount of the endogenous polyamine; The preparation method according to any one of claims 5 to 7, characterized in that, when three-site crosslinking and / or four-site crosslinking are performed, the amount of the activator added is 300% to 550% of the substance amount of the endogenous polyamine, 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.

11. In the crosslinking reaction, when the endogenous polyamine is spermine, the amount of spermine added is 0.3% to 35% of the mass of the hyaluronic acid, and when the endogenous polyamine is spermidine, the amount of spermidine added is 0.5% to 40% of the mass of the hyaluronic acid; Furthermore, the mass concentration of hyaluronic acid in the reaction aqueous solution is 10 mg / mL to 150 mg / mL, The preparation method according to any one of claims 5 to 7, further characterized in that the mass concentration of hyaluronic acid in the finally obtained crosslinked hyaluronic acid hydrogel is 1 mg / mL to 50 mg / mL.

12. The gel material is further eluted with an eluent, crushed and 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 the grinding is 1:1-5; The preparation method according to any one of claims 5 to 7, further characterized in that the particle size of the gel after grinding is between 10 μm and 500 μm.

13. The method further comprises the steps of redissolving and moist heat sterilizing the gel material after drying; Preferably, the reconstitution solution is a phosphate buffer, more preferably, the mass concentration of phosphate buffer salts in the phosphate buffer is 5 mg / mL to 40 mg / mL; The preparation method according to claim 12, further characterized in that the temperature of the moist heat sterilization is 120°C to 130°C, and the time of the moist heat sterilization is preferably 15 minutes to 45 minutes.

14. Use of a gel material according to any one of claims 1 to 4 or obtained by the preparation method according to any one of claims 5 to 13 in the preparation of a tissue filling and repair material or a drug carrier.

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