Polyamino acid gel material, method for making same and use thereof

Abstract

The present invention provides a polyamino acid gel material, which is obtained by subjecting endogenous polyamines and polyamino acids to a preliminary cross-linking treatment under the action of an activator, followed by freeze-crosslinking, and then thawing at room temperature, and repeating the freeze-thaw cycle 0 to 3 times, where the polyamino acid is polyglutamic acid or polyaspartic acid, and the endogenous polyamine is spermine or spermidine.

Description

[Technical Field]

[0001] The present invention relates to the technical field of biomedical materials, and in particular to polyamino acid gel materials and methods for making and using the same. [Background technology]

[0002] Cryogels are a type of hydrogel that undergoes temperature-controllable polymerization below the freezing point of the solvent. By freezing solvent crystals as an interconnected pore-forming agent, a large amount of ice crystal templates are formed within the system as the temperature drops. After the templates melt, an interconnected macroporous gel is obtained. Its most distinctive feature is its interconnected macroporous structure, which can accelerate the supply of nutrients and the removal of metabolic products. Furthermore, the interconnected macroporosity can better simulate the in vivo environment and favor inward vascular growth. Common methods for introducing interpenetrating macroporous structures into hydrogel networks include the use of pore-forming agents that can disrupt the network structure, such as crystalline particles, immiscible solvents, or air bubbles. However, these pore-forming agents must be removed from the network. While pore-forming agents can be used to synthesize hydrogels with macroporous structures and interpenetrating network structures of hundreds of microns, residual pore-forming agents in the hydrogel can be potentially toxic and can form physical barriers that hinder cell penetration when used as biomaterials. Therefore, it is essential to fabricate cryogels that are biocompatible, free of toxic crosslinkers, and have good mechanical performance.

[0003] Polyamino acid-based hydrogels have attracted particular attention because they possess excellent water absorption and retention properties similar to synthetic polymers, as well as biocompatibility and biodegradability similar to natural polymers. Polyamino acid-based hydrogels primarily contain polyglutamic acid (PGA) and polyaspartic acid (PASP). Polyglutamic acid, also known as natto gum or polyglutamic acid, is a water-soluble, biodegradable, and non-toxic biopolymer. Polyglutamic acid is a novel green polymer material with excellent properties, including high molecular weight, easy dispersibility, non-toxicity, harmlessness, and edibility. Polyglutamic acid is non-immunogenic, completely degradable both in nature and in the human body, and safe and non-toxic for use in food, pharmaceuticals, and cosmetics. Therefore, it is a novel polymer material with great potential for development in fields such as tissue engineering. The main chain of polyglutamic acid contains many activated free side chain carboxyl groups (-COOH), has a unique polypeptide chain secondary structure and chiral properties, is easily modified, and has stronger water absorption than hyaluronic acid. Furthermore, it has very good biocompatibility and degradability; the decomposed small molecular amino acids do not accumulate in the body or cause toxicity or side effects, and have very good biocompatibility.

[0004] Polyaspartic acid (PASP), also known as PASP, is an environmentally friendly, biodegradable polymeric material that has attracted considerable attention. It can be obtained by dehydration condensation using biological or chemical methods. Its chemical structure is somewhat similar to that of proteins, with the two molecules connected by peptide bonds. The peptide bonds are easily degraded and broken by microbial activity, breaking the polymer into smaller molecular chains, ultimately degrading into water, carbon dioxide, and nitrogen-containing compounds. This makes it environmentally friendly and non-polluting. Polyaspartic acid hydrogel (PASP hydrogel) is a spatially networked material fabricated by crosslinking linear PASP molecular chains with a crosslinking agent. Its main chain contains numerous hydrophilic carboxyl groups and peptide bonds, and its side groups contain ether bonds and hydroxyl groups, both of which can bind water molecules. PASP molecular chains are flexible and extensible, with high water storage and retention properties, and are elastically deformable under external forces, exhibiting good flexibility and plasticity. Furthermore, as an amino acid polymer, its polypeptide backbone is similar to a protein structure, offering advantages such as good biocompatibility and easy bioabsorption. Research has shown that PASP can be digested and absorbed by proteases, is non-antigenic, and its metabolites are non-toxic. Therefore, PASP hydrogels have become a research hotspot due to their wide range of applications and broad market prospects in fields such as agriculture, horticulture, and biopharmaceuticals.

[0005] Looking at the current state of research on polyglutamic acid and polyaspartic acid hydrogels both at home and abroad, although both have achieved results in many fields, there are still certain deficiencies in performance such as strength, elasticity, toughness, and antibacterial properties. As a result, not only is the development of polyamino acid hydrogels restricted, but their biological effects are also significantly limited. Therefore, it is extremely important to prepare polyamino acid cryogels with high mechanical properties.

[0006] Endogenous polyamines refer to polyamines that can be synthesized in the human body or produced through metabolic processes. Major endogenous polyamines include spermine, spermidine, and putrescine. Spermidine, a naturally occurring small molecule widely present in the body, can combat rejection, suppress inflammation, promote cellular autophagy, and extend lifespan. Applying biomimetic design concepts to the bioactive molecule spermidine in fields such as biomedicine and tissue engineering can provide a novel solution to the immune rejection encountered during artificial tissue or organ transplantation, thereby achieving better physiological function and therapeutic effects. (Madeo et al., Science 359, 410, 2018) reported that the endogenous polyamine spermidine has special physiological functions, including, but not limited to, regulating circadian rhythm, improving hypertension, protecting the cardiovascular system, preventing Alzheimer's disease, enhancing immunity, anti-cancer, and even anti-aging. The physiological activity of spermidine is manifested in several areas: 1) Kidney: Reduces tension and prevents aging. 2) Heart: Lowers blood pressure and prevents arteriosclerosis. 3) Brain: Prevents memory decline, resists Alzheimer's disease, and has neuroprotective effects. 4) Bones: Prevents bone loss due to oophorectomy. 5) Skeletal muscles: Increases the temperature of aging muscles and prevents muscle diseases. 6) Whole organism: Prolongs the lifespan of the organism. 7) Immune system: Improves immune activity after vaccination, enhances cancer-directed immunity, and prevents fatal sepsis. 8) Liver: Prevents liver fibrosis and carcinogenesis. The main mechanism by which spermidine exerts its physiological activity is as follows: spermidine binds to polycations (-NH3 + ) fatty amines that exist in multiple protonated forms under physiological pH conditions and have very strong biological activity. Nucleic acids containing acidic residues, phospholipids, acidic proteins, pectin polysaccharides containing carboxyl groups or sulfates, and neurotransmitters and hormones with similar structures (e.g., dopamine, epinephrine, serotonin, thyroid hormone, etc.) are all potential targets for spermidine binding.

[0007] Crosslinked polyglutamic acid or polyaspartic acid cryogels are hydrophilic polymer network structures with high water absorption, relatively high mechanical strength, controllable degradation, and biocompatibility, making them ideal materials for fabricating wound dressings and tissue engineering scaffolds. Currently, there are few reports on the use of endogenous polyamines, such as spermidine, as crosslinkers for polyamino acid-based cryogels, and there are also few reports on the freeze-crosslinking reaction conditions between endogenous polyamines and polyglutamic acid or polyaspartic acid groups, and the performance of the resulting cryogels. In light of this, the present invention is proposed. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention aims to provide a polyamino acid gel material, in which spermine or spermidine, among endogenous polyamines, is selected as a crosslinking agent, and a polyamino acid gel is obtained through pre-crosslinking and freezing-thawing. The physical and chemical properties of the produced gel material can be adjusted by controlling the pre-crosslinking temperature and time, crosslinking degree, concentration, reaction temperature, reaction time, thawing temperature, thawing time, pH, and type of active agent. [Means for solving the problem]

[0009] The technical solutions provided by the present invention are as follows: In one aspect, the present invention provides a polyamino acid gel material, which is mainly obtained by pre-crosslinking endogenous polyamines and polyamino acids under the action of an active agent, followed by a freeze-thaw treatment to obtain a polyamino acid gel, and the crosslinking between the endogenous polyamines and the polyamino acids includes three-site crosslinking or four-site crosslinking.

[0010] Furthermore, the endogenous polyamines include spermidine (a triamino compound) and / or spermine (a tetraamino compound).

[0011] In the present invention, the endogenous polyamine spermine or spermidine is selected as the crosslinking agent, and a pre-crosslinking treatment allows the imino groups of spermidine or spermine to participate in the crosslinking reaction, thereby forming three- or four-site crosslinks and forming a dense interpenetrating network structure, improving the thermal stability of the polyamino acid gel crosslinked by amide bonds and reducing the safety risks caused by residual chemical crosslinkers. The present invention avoids the use of endogenous diamines such as putrescine because they are highly toxic, while endogenous diamines such as putrescine can only form two-site crosslinks and are unable to achieve multi-site crosslinking.

[0012] Furthermore, the polyamino acid is polyglutamic acid or / and polyaspartic acid.

[0013] Furthermore, the pore size of the gel material is 80 to 760 μm, including, but not limited to, 80 μm, 90 μm, 100 μm, 120 μm, 150 μm, 160 μm, 170 μm, 180 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 720 μm, 750 μm, or 760 μm.

[0014] and / or the porosity of the gel material is 95% or more, preferably 98% or more.

[0015] The present invention uses endogenous polyamines as crosslinking agents to prepare polyglutamic acid or lyspartic acid-based gels through pre-crosslinking and freeze-thaw processes, which have large pore sizes and higher porosity, making the cryogel more suitable for a wider range of applications.

[0016] Additionally, the activator includes one or more of a water-soluble carbodiimide, a carbonium salt, and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM).

[0017] The second aspect of the present invention further provides a method for making a polyamino acid gel material, the method comprising the steps of: The polyamino acid and the endogenous polyamine are mixed to obtain a mixed solution, the pH of the mixed solution is adjusted to 4.00 to 7.40, an activator is added, and after stirring, the solution is pre-crosslinked at 25 to 60°C for 5 to 60 minutes, and then freeze-crosslinked. The product after freeze-crosslinking is thawed, and the freeze-thaw process is repeated 0 to 3 times to obtain the gel material.

[0018] In order to complete the single-site crosslinking of spermine or spermidine with polyamino acids before freezing, pre-crosslinking is required before freezing. This means that one amino group of spermine or spermidine is involved in the reaction. The single-site crosslinking allows one end of the crosslinked spermine or spermidine molecule to be fixed on the molecular chain of the polyamino acid substance, which is fixed by ice crystals after freezing. This shortens the distance between them and the molecular chain of the polyamino acid substance, and also reduces the steric hindrance between them and the imino group. This increases the probability of collision between the imino group and the amino group at the other end of the spermine or spermidine and the carboxyl group of the adjacent molecular chain of the polyamino acid substance, improving the reaction efficiency and the probability of multi-site crosslinking (i.e., three-site crosslinking or four-site crosslinking). This results in the formation of a macroporous interpenetrating network structure cryogel with high porosity, while also enabling effective monitoring of the residual amount of spermidine or spermine.

[0019] If preliminary cross-linking treatment is not performed before freezing, the distance between the spermine or spermidine molecules and the polyamino acid molecular chain segments fixed by the ice crystals will be large after freezing, and the probability of collision between the imino groups, which have low cross-linking reaction activity, and the carboxyl groups of the molecular chains of the polyamino acid-based material will be extremely low, resulting in a decrease in the efficiency of the cross-linking process, and it will be impossible to form a structurally stable three- or four-site multi-site cross-linked gel and a stable interpenetrating network structure, and it will be impossible to achieve a good pore-forming effect.

[0020] Furthermore, if the preliminary cross-linking reaction treatment is carried out outside the above range, the preliminary cross-linking efficiency will be too high or too low, and the cross-linking efficiency of the subsequent freezing process will be too low, making it difficult to form multi-site cross-links, and therefore the network structure of the obtained gel will not be sufficiently dense and will have low stability.

[0021] Preferably, the freeze crosslinking is carried out at a temperature of -80 to -10°C for 2 to 48 hours, and the thawing is carried out at a temperature of 10 to 60°C for 0.5 to 8 hours.

[0022] Preferably, the endogenous polyamines include spermine (a tetraamino compound) and / or spermidine (a triamino compound); Preferably, the activator comprises one or more of a water-soluble carbodiimide, a carbonium salt, and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride; Preferably, the polyamino acid comprises polyglutamic acid or / and polyaspartic acid; And / or, the solvent of the mixed solution is one or a mixture of a plurality of solvents selected from water, a soluble alcohol, a soluble ketone, DMF, DMA, and DMSO, and preferably the solvent is water.

[0023] Preferably, the soluble alcohol comprises one or a mixture of more of methanol, ethanol, propanol or isopropanol; Preferably, the soluble ketone is acetone; Furthermore, the molecular weight of the polyamino acid may be within a normal range.

[0024] In some embodiments of the present invention, the solvent of the mixed solution is water.

[0025] In some embodiments of the present invention, the pH of the mixed solution is 4.00 to 7.40, for example, the pH is 4.00, 4.10, 4.15, 4.20, 4.25, 4.30, 4.35, 4.40, 4.45, 4.50, 4.60, 4.70, 4.80, 4.90, 5.00, 5.10, 5.20, 5.30, 5.40, 5.50, 5.60, 5.70, 5.80, 5.90 , 6.00, 6.10, 6.20, 6.30, 6.32, 6.35, 6.38, 6.40, 6.42, 6.45, 6.50, 6.55, 6.58, 6.60, 6.62, 6.65, 6.68, 6.70, 6.73, 6.75, 6.80, 6.85, 6.90, 6.95, 7.00, 7.05, 7.10, 7.20, 7.30 and 7.40.

[0026] Preferably, the pH of the mixed solution is adjusted to 4.00 to 7.40. By controlling the pH of the mixed solution within this range, the pore size and porosity of the resulting gel can be relatively stably controlled. If the pH of the solution is higher than 7.4, the solution system tends to be alkaline, which is likely to cause hydrolysis of the activated esters generated by the catalyst with polyglutamic acid and polyaspartic acid, significantly reducing the reaction efficiency between the polyamino acids and endogenous polyamines and further affecting the crosslinking efficiency. If the pH of the solution is lower than 4, the solution system tends to be acidic, which is likely to cause hydrolysis of the active agent, making it difficult to effectively complete the crosslinking reaction.

[0027] Preferably, when the pH of the mixed solution is controlled to 6.3 to 7.4, the resulting gel has a pore size of 80 to 154 μm and a porosity of about 95%, and the pH includes, but is not limited to, 6.30, 6.35, 6.40, 6.45, 6.50, 6.55, 6.60, 6.65, 6.70, 6.75, 6.80, 6.85, 6.88, 6.90, 7.00, 7.10, 7.15, 7.20, 7.25, 7.30, 7.35, or 7.40.

[0028] When the pH of the mixed solution is controlled to 5.40 to 6.20, the resulting gel has a pore size of 155 to 419 μm and a porosity of 95 to 96%, and the pH includes, but is not limited to, 5.40, 5.45, 5.50, 5.55, 5.58, 5.60, 5.65, 5.70, 5.75, 5.80, 5.85, 5.90, 5.95, 6.00, 6.05, 6.10, 6.15, or 6.20.

[0029] More preferably, when the pH of the mixed solution is controlled to 4.00 to 5.30, the resulting gel has a pore size of 420 to 760 μm and a porosity of 97 to 98%, and the pH includes, but is not limited to, 4.00, 4.05, 4.10, 4.15, 4.20, 4.25, 4.30, 4.35, 4.40, 4.45, 4.50, 4.55, 4.60, 4.65, 4.70, 4.75, 4.80, 4.85, 4.90, 4.95, 5.00, 5.10, 5.15, 5.20, 5.25, 5.30, or 5.30.

[0030] In some embodiments of the present invention, the freezing temperature is between -80°C and -10°C, and includes -80°C, -79°C, -78°C, -77°C, -76°C, -75°C, -74°C, -73°C, -72°C, -71°C, -70°C, -69°C, -68°C, -67°C, -66°C, -65°C, -64°C, -63°C, -62°C, -61°C, -60°C, -59°C, -58°C, -57°C, -56°C, -55°C, -54°C, -53°C, -52°C, -51°C, -50°C, -49°C, -48°C, -47°C, °C, -46°C, -45°C, -44°C, -43°C, -42°C, -41°C, -40°C, -39°C, -38°C, -37°C, -36°C, -35°C, -34°C, -33°C, -32°C, -31°C, -30°C, -29°C, -28°C, -27°C, -26°C, -25°C, -24°C, -23°C, -22°C, -21°C, -20°C, -19°C, -18°C, -17°C, -16°C, -15°C, -14°C, -13°C, -12°C, -11°C or -10°C.

[0031] Preferably, the freezing time is 2 to 48 hours, including but not limited to 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, or 48 hours.

[0032] Preferably, the thawing temperature is 10 to 60°C, including, but not limited to, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, or 60°C.

[0033] Preferably, the thawing time is 0.5 to 8 hours, including but not limited to 0.5 hours, 1 hour, 2 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, or 6 hours.

[0034] Furthermore, the water-soluble carbodiimide activator includes 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide, 1,3-bis[bis(methoxymethyl)methyl]carbodiimide, or the like, or a salt thereof, or a mixture of one or more thereof;

[0035] The carbonium salts include one or a mixture of more than one 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-succinimidyl)-bis(dimethylamino)carbonium tetrafluoroborate (TSTU), 2-(5-norbornene-2,3-dicarboximide)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU).

[0036] In some embodiments of the present invention, when the activator is a water-soluble carbodiimide activator, the method further comprises adding a co-agent to the mixed solution to improve the efficiency of the crosslinking reaction by using the activator and the co-agent in combination.

[0037] The coagent comprises one or more of N-hydroxysuccinimide (NHS), sulfonated N-hydroxysuccinimide (S-NHS), tert-butanol, and 1-hydroxybenzotriazole (HOBt);

[0038] More preferably, the amount of the auxiliary agent added is 10 to 50% 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%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, and 50%, and preferably, the amount of the auxiliary agent added is 15 to 50% of the mass of the carbodiimide.

[0039] Furthermore, when the endogenous polyamine is spermine, the amount of spermine added accounts for 1.0 to 62% of the molar amount of the polyamino acid structural unit, and is 1.0%, 1.5%, 2.2%, 2.5%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 13%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, Examples of the range include, but are not limited to, 28%, 29%, 30%, 31%, 32%, 32%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, and 62%, and preferably 9 to 18%.

[0040] When the endogenous polyamine is spermidine, the amount of spermidine added accounts for 0.5 to 52% of the molar amount of the polyamino acid structural unit, including, but not limited to, 0.5%, 0.8%, 1.0%, 1.5%, 2%, 2.5%, 3%, 5%, 6%, 7%, 8%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 34%, 38%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, and 52%, and is preferably 9 to 17%.

[0041] The mass concentration of the polyamino acid in the mixed solution is 10 to 160 mg / mL, including, but not limited to, 10, 20, 30, 40, 50, 55, 60, 70, 75, 80, 90, 100, 110, 120, 130, 140, 150, and 160 mg / mL.

[0042] The amount of the activator added is 0.5 to 550%, preferably 50 to 500%, and more preferably 50 to 300% of the molar amount of the polyamino acid structural unit.

[0043] Furthermore, the method further comprises the step of adding the thawed polyamino acid cryogel to a phosphate buffer solution having a concentration of 10 to 25 mg / mL, and performing moist heat sterilization to obtain a gel material; Preferably, the pH of the phosphate buffer is 7.0; Preferably, the moist heat sterilization temperature is 100 to 130°C, preferably 121°C, and the moist heat sterilization time is 10 to 30 minutes, preferably 15 minutes.

[0044] The present invention further provides a gel material produced by the above method.

[0045] A third aspect of the invention further provides the use of a gel material according to the first aspect or made by the method according to the second aspect in the manufacture of a medical, cosmetic or healthcare product.

[0046] The medical and cosmetic products include soft tissue filler materials, cartilage repair materials, tissue implant materials, coating layers for biomaterial implants; scaffold materials for tissue engineering, drug sustained release vehicles, drug targeting carriers, wound dressings, etc.

[0047] Preferably, the coating layer for biomaterial implants includes coating layers for breast fillers, catheters, cannulae, bone repair and replacement, cartilage substitutes, micropumps and other drug delivery devices, artificial organs and blood vessels, meshes for tissue reinforcement; Preferably, the soft tissue filler material comprises a medical filler material or joint lubricant for the face, neck, head, ear, breast, joints; More preferably, the filler material is an intradermal or subcutaneous implantable material used to improve skin texture, fill wrinkles, or restore volume to the face or body.

[0048] Preferably, the wound healing agent is a wound dressing; Preferably, said medical or cosmetic product also comprises a therapeutically active agent; Preferably, the therapeutically active agent comprises a chemotherapeutic agent or a biologically active factor; More preferably, said active agents include anti-inflammatory agents, antibiotics, analgesics, anesthetics, wound healing promoters, cell growth inhibitors, immunostimulants, immunosuppressants and antivirals; The health care products include capsules, tablets, health care drinks, etc. [Effects of the Invention]

[0049] The beneficial effects of the present invention are as follows:

[0050] (1) In order to control the gel reaction process, the gel obtained in the present invention is pre-crosslinked before freezing. This completes single-site crosslinking between spermine or spermidine and the polyamino acid substance before freezing, i.e., one amino group of spermine or spermidine is involved in the reaction. After freezing, one end of the crosslinked spermine or spermidine molecule is also fixed on the polymer chain of the polyamino acid substance fixed by ice crystals. This shortens the distance between them and the polymer chain of the polyamino acid substance, reducing the steric hindrance between them and the imino group. This increases the probability of collision between the imino group and the amino group at the other end of the spermine or spermidine and the carboxyl group of the adjacent molecular chain of the polyamino acid substance, improving the reaction efficiency and the probability of multi-site crosslinking. This results in the formation of a continuously interpenetrating macroporous gel with a porosity of 95% or more. Furthermore, by controlling the pre-crosslinking reaction process and reaction conditions, the overall reaction efficiency of the cryogel can be increased to 90% or more, and the amount of residual spermine or spermidine can be significantly reduced, which is of great significance for the safety of the clinical use of the product.

[0051] (2) The present invention produces porous gels with different pore size gradients by adjusting the pH value of the reaction system while controlling the pre-crosslinking reaction process. When the pH is 6.3 to 7.4, the pore size of the resulting gel is 80 to 154 μm; when the pH is 5.40 to 6.20, the pore size of the resulting gel is 155 to 419 μm; and when the pH is 4.00 to 5.30, the pore size of the resulting gel is 420 to 760 μm.

[0052] (3) The present invention uniquely selects endogenous polyamines and polyamino acids, and crosslinks them under pre-crosslinking and freeze-thaw conditions to produce a gel with a continuously interpenetrating porous structure. This unique structure is favorable for material exchange, promotes cell bioavailability and adhesion, and has high water absorption capacity. The entire process is gentle and controllable, with a high yield. This has potential research value and broad application prospects in the fields of biomaterials and tissue engineering. [Brief explanation of the drawings]

[0053] 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 necessary for describing the specific embodiments or the prior art. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without any creative efforts.

[0054] 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 necessary for describing the specific embodiments or the prior art. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without any creative efforts.

[0055] [Figure 1(a)] FIG. 1 is a SEM test image of gel C6 provided by the present invention. [Figure 1(b)] FIG. 1(b) is a partially enlarged view of FIG. [Figure 2(a)] FIG. 1 is a SEM test image of gel B1 provided by the present invention. [Figure 2(b)] FIG. 2(b) is a partially enlarged view of FIG. [Figure 3(a)] FIG. 1 is a SEM test image of gel B2 provided by the present invention. [Figure 3(b)] FIG. 3(b) is a partially enlarged view of FIG. [Figure 4(a)] FIG. 1 is a SEM test image of gel B3 provided by the present invention. [Figure 4(b)] FIG. 4(b) is a partially enlarged view of FIG. [Figure 5(a)] FIG. 1 is a SEM test image of gel B4 provided by the present invention. [Figure 5(b)] FIG. 5(b) is a partially enlarged view of FIG. [Figure 6] FIG. 1 is a porosity test diagram of gels C1 to C7 and gels B1 to B4 provided by the present invention. [Figure 7] 1 shows the swelling ratio test results of gels C1 to C7 and gels B1 to B4 provided by the present invention. [Figure 8] 1 shows the decomposition curves of gels C1 to C7 provided by the present invention. [Figure 9] 1 shows the stress-strain curves of gel C6 and gel B1 provided by the present invention. [Figure 10] 1 shows test results of the effects of gels C1 to C7 provided by the present invention on cell proliferation. DETAILED DESCRIPTION OF THE INVENTION

[0056] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0057] In the present invention, the term "crosslinking" refers to the process of changing a chemically reactive linear polymer into a polymer with a three-dimensional network structure through chemical reaction, which is often used in polymer modification.

[0058] In the present invention, the term "crosslinking agent" refers to a reagent that can generate chemical bonds between linear molecules and link the linear molecules to each other to form a network structure, and is used to improve the strength of polymeric materials.

[0059] In the present invention, the term "multi-site cross-linking" refers to three-site or four-site cross-linking rather than two-site cross-linking, where three-site cross-linking specifically refers to spermidine as the cross-linking agent, and four-site cross-linking specifically refers to spermine as the cross-linking agent.

[0060] In the present invention, the term "mixed solution" refers to a solution in which a non-crosslinked polyamino acid substance dissolved in a solvent is mixed with dissolved endogenous polyamines.

[0061] In the present invention, the term "room temperature" refers to 25°C ± 5°C.

[0062] The technical solutions of the present invention will be described clearly and completely below in conjunction with the embodiments, but obviously, the described embodiments are only a part of the embodiments of the present invention, and all other embodiments that can be obtained by those skilled in the art based on the embodiments of the present invention without any creative efforts fall within the protection scope of the present invention.

[0063] Example 1 Preparation of Gel C1 by DMTMM-catalyzed four-site cross-linking of spermine and polyglutamic acid 0.34 g of spermine (1.6 mmol) was weighed into a beaker, followed by 75 mL of purified water. After complete dissolution, 4.0 g of polyglutamic acid (1500 kDa, 27 mmol of polyglutamic acid repeating units) was weighed. The spermine content was 8.5% of the polyglutamic acid mass (i.e., 5.9% of the polyglutamic acid repeating units). The pH of the polyglutamic acid solution was adjusted to approximately 4.0 using 2 mol / L hydrochloric acid and 2 mol / L sodium hydroxide solutions. DMTMM solution (1.81 g of DMTMM, 5 mL of deionized water) was added, stirred uniformly, and sealed. The mixture was pre-crosslinked at 25°C for 60 minutes, then transferred to -80°C for 16 hours. The cryogel was then thawed at 25°C for 8 hours to obtain the initial cryogel product. Purified water was then added, and the gel was pulverized using a homogenizer, filtered under suction, washed six times, and the resulting filter cake was freeze-dried. 1 g of the freeze-dried gel was weighed, and a total of 50 mL of 20 mg / mL phosphate buffer solution at pH 7.0 was added. After the gel completely swelled, it was filled into a 1 mL or 2 mL pre-filled syringe and sterilized under moist heat at 121 °C for 15 minutes to obtain the final product, Gel C1.

[0064] Example 2 Preparation of Gel C2 by EDC-catalyzed Three-site Crosslinking of Spermidine and Polyglutamic Acid 0.32 g of spermidine (2.2 mmol) was weighed into a beaker, and then 52.14 mL of purified water was added to completely dissolve it. Then, 4.0 g of polyglutamic acid (having a molecular weight of 1500 kDa and containing 27 mmol of polyglutamic acid repeating structural units) was weighed out. At this time, the spermidine content accounted for 8% of the mass of polyglutamic acid (i.e., 8.1% of the number of moles of polyglutamic acid repeating structural units). The pH of the polyglutamic acid solution was adjusted to approximately 5.3 using 2 mol / L hydrochloric acid and 2 mol / L sodium hydroxide solutions. An aqueous solution of EDC and NHS (1.25 g EDC, 0.25 g NHS, 5 ml deionized water) was added, stirred evenly, and sealed. The mixture was pre-crosslinked at 40°C for 20 minutes, then transferred to -60°C for 8 hours, thawed at 25°C for 4 hours, frozen at -20°C for 16 hours, and finally thawed at 40°C for 3 hours to obtain the initial cryogel product. Purified water was then added, and the gel was pulverized using a homogenizer. It was then suction filtered and washed six times, and the resulting filter cake was freeze-dried. One gram of the freeze-dried gel was weighed, and a total of 50 mL of phosphate buffer solution with a concentration of 20 mg / mL and a pH of 7.0 was added. After the gel had completely swollen, it was filled into a 1 mL or 2 mL pre-filled syringe and subjected to moist heat sterilization at 121°C for 15 minutes to obtain the final product, Gel C2.

[0065] Example 3 Preparation of Gel C3 by HATU-catalyzed four-site cross-linking of spermine and polyglutamic acid 0.50 g of spermine (2.4 mmol) was weighed into a beaker, followed by 30 mL of purified water. After complete dissolution, 4.0 g of polyglutamic acid (1500 kDa, 27 mmol of polyglutamic acid repeating units) was weighed. The spermine content was 12.5% ​​of the polyglutamic acid mass (i.e., 8.9% of the polyglutamic acid repeating units). The pH of the polyglutamic acid solution was adjusted to approximately 6.0 using 2 mol / L hydrochloric acid and 2 mol / L sodium hydroxide solutions. An aqueous solution of HATU (46.56 g of HATU, 10 mL of deionized water) was added, stirred uniformly, and sealed. The solution was pre-crosslinked at 30°C for 40 minutes, then transferred to -40°C for 24 hours, and finally thawed at 30°C for 6 hours to obtain the cryogel initial product. Purified water was then added, and the gel was pulverized using a homogenizer, filtered under suction, washed six times, and the resulting filter cake was freeze-dried. 1 g of the freeze-dried gel was weighed, and a total of 50 mL of 20 mg / mL phosphate buffer solution at pH 7.0 was added. After the gel completely swelled, it was filled into a 1 mL or 2 mL pre-filled syringe and sterilized under moist heat at 121 °C for 15 minutes to obtain the final product, Gel C3.

[0066] Example 4 Preparation of Gel C4 by HATU-catalyzed Three-site Crosslinking of Spermidine and Polyglutamic Acid 0.39 g of spermidine (2.7 mmol) was weighed into a beaker, followed by 29.78 mL of purified water. After complete dissolution, 4.0 g of polyglutamic acid (1500 kDa molecular weight, 27 mmol of polyglutamic acid repeating units) was weighed. The spermidine content was 9.8% of the polyglutamic acid mass (i.e., 10.0% of the polyglutamic acid repeating units). The pH of the polyglutamic acid solution was adjusted to approximately 6.2 using 2 mol / L hydrochloric acid and 2 mol / L sodium hydroxide solutions. An aqueous solution of HATU (4.14 g of HATU, 5 mL of deionized water) was added, stirred uniformly, and sealed. The mixture was pre-crosslinked at 35°C for 30 minutes, then transferred to -30°C for 48 hours, and finally thawed at 35°C for 4 hours to obtain the cryogel initial product. Purified water was then added, and the gel was pulverized using a homogenizer, filtered under suction, washed six times, and the resulting filter cake was freeze-dried. 1 g of the freeze-dried gel was weighed, and a total of 50 mL of 20 mg / mL phosphate buffer solution at pH 7.0 was added. After the gel completely swelled, it was filled into a 1 mL or 2 mL pre-filled syringe and sterilized under moist heat at 121 °C for 15 minutes to obtain the final product, Gel C4.

[0067] Example 5 Preparation of Gel C5 by EDC-catalyzed Four-site Cross-linking of Spermine and Polyglutamic Acid 0.62 g of spermine (3.0 mmol) was weighed into a beaker, and then 23.57 mL of purified water was added to completely dissolve it. Then, 4.0 g of polyglutamic acid (having a molecular weight of 1500 kDa and containing 27 mmol of polyglutamic acid repeating structural units) was weighed out. At this time, the spermine content accounted for 15.5% of the mass of polyglutamic acid (i.e., 11.1% of the number of moles of polyglutamic acid repeating structural units). The pH of the polyglutamic acid solution was adjusted to approximately 5.0 using 2 mol / L hydrochloric acid and 2 mol / L sodium hydroxide solutions. An aqueous solution of EDC and NHS (2.30 g EDC, 0.46 g NHS, 5 ml deionized water) was added, stirred evenly, and sealed. The mixture was pre-crosslinked at 45°C for 15 minutes, then transferred to -60°C for 8 hours, thawed at 25°C for 4 hours, frozen at -20°C for 16 hours, and finally thawed at 45°C for 2 hours to obtain the cryogel. Purified water was then added, and the gel was pulverized using a homogenizer. It was then suction filtered and washed six times, and the resulting filter cake was freeze-dried. One gram of the freeze-dried gel was weighed, and a total of 50 mL of a 20 mg / mL phosphate buffer solution at pH 7.0 was added. After the gel had completely swelled, it was filled into a 1 mL or 2 mL pre-filled syringe and subjected to moist heat sterilization at 121°C for 15 minutes to obtain the final product, Gel C5.

[0068] Example 6 Preparation of Gel C6 by DMTMM-catalyzed four-site cross-linking of spermine and polyglutamic acid 0.84 g of spermine (4.1 mmol) was weighed into a beaker, and then 23.77 mL of purified water was added to completely dissolve it. Then, 4.0 g of polyglutamic acid (having a molecular weight of 1500 kDa and containing 27 mmol of polyglutamic acid repeating structural units) was weighed out. At this time, the spermine content accounted for 21.00% of the mass of polyglutamic acid (i.e., 15.2% of the number of moles of polyglutamic acid repeating structural units). The pH of the polyglutamic acid solution was adjusted to approximately 5.5 using 2 mol / L hydrochloric acid and 2 mol / L sodium hydroxide solutions. A DMTMM aqueous solution (11.29 g DMTMM, 7 ml deionized water) was added, stirred uniformly, and sealed. The mixture was pre-crosslinked at 54°C for 10 minutes, then transferred to -50°C for 4 hours, thawed at 25°C for 4 hours, then transferred to -10°C for 24 hours, and finally thawed at 54°C for 1 hour to obtain the initial cryogel product. Purified water was then added, and the gel was pulverized using a homogenizer. After suction filtration and washing six times, the resulting filter cake was lyophilized. One gram of lyophilized gel was weighed, and a total of 50 ml of 20 mg / ml phosphate buffer solution at pH 7.0 was added. After the gel had fully swelled, it was loaded into a 1 ml or 2 ml prefilled syringe and sterilized at 121°C for 15 minutes using moist heat to obtain the final product, Gel C6.

[0069] Example 7 Preparation of Gel C7 by EDC-catalyzed Three-site Crosslinking of Spermidine and Polyaspartic Acid 0.87 g of spermidine (6.0 mmol) was weighed into a beaker, and then 28.33 mL of purified water was added to completely dissolve it. Then, 4.0 g of polyaspartic acid (molecular weight 4000 kDa, containing 30 mmol of polyaspartic acid repeating structural units) was weighed out. At this time, the spermidine content accounted for 21.75% of the mass of polyaspartic acid (i.e., 20.0% of the moles of polyglutamic acid repeating structural units). The pH of the polyaspartic acid solution was adjusted to approximately 7.4 using 2 mol / L hydrochloric acid and 2 mol / L sodium hydroxide solutions. An EDC aqueous solution (11.52 g EDC, 2.3 g NHS, 5 ml deionized water) was added, stirred evenly, and sealed. The mixture was pre-crosslinked at 60°C for 8 minutes, then transferred to -65°C for 2 hours, thawed at 25°C for 4 hours, frozen at -30°C for 48 hours, and finally thawed at 60°C for 0.5 hours to obtain the cryogel initial product. Purified water was then added, and the gel was pulverized using a homogenizer. It was then suction filtered and washed six times, and the resulting filter cake was freeze-dried. One gram of the freeze-dried gel was weighed, and a total of 50 mL of a 20 mg / mL phosphate buffer solution at pH 7.0 was added. After the gel had completely swollen, it was filled into a 1 mL or 2 mL pre-filled syringe and subjected to moist heat sterilization at 121°C for 15 minutes to obtain the final product, Gel C7.

[0070] Comparative Example 1 Preparation of Gel B1 by Two-site Crosslinking of Spermine and Polyglutamic Acid with DMTMM Catalyst The low-temperature freeze-thaw crosslinking process was the same as in Example 6, except that the preliminary crosslinking process of "preliminary crosslinking at 54°C for 10 minutes" was omitted, to obtain the final product Cryogel B1.

[0071] Comparative Example 2 Preparation of Gel B2 by Two-Site Crosslinking of Spermine and Polyaspartic Acid Using EDC Catalyst The low-temperature freeze-thaw crosslinking process was the same as in Example 7, except that the preliminary crosslinking process of "preliminary crosslinking at 60°C for 8 minutes" was omitted, to obtain the final product Cryogel B2.

[0072] Comparative Example 3 Preparation of Gel B3 by two-site cross-linking of spermine and polyglutamic acid using DMTMM catalyst The final product, Gel B3, was obtained in the same manner as in Example 6, except that the low-temperature freeze-thaw crosslinking step was "pre-crosslinked at 70°C for 5 minutes" after uniform stirring and sealing.

[0073] Comparative Example 4 Preparation of Gel B4 by two-site cross-linking of spermine and polyglutamic acid using DMTMM catalyst The final product, Gel B4, was obtained in the same manner as in Example 6, except that the low-temperature freeze-thaw crosslinking step was "pre-crosslinked at 15°C for 65 minutes" after uniform stirring and sealing.

[0074] Test Example 1: Measurement of rheological properties of gel The rheological properties of Gels C1-C7 and Gels B1-B4 prepared in Examples 1-7 and Comparative Examples 1-4 were tested as follows: 2 ml of each gel was taken before and after sterilization and tested using a rotational rheometer. The rheometer parameters were: operating gap 1000 mm, loading gap 45000 mm, operating temperature 37°C, deformation 1%, frequency 0.9 Hz, and operating time 60 s. The rheological data for each gel are shown in Table 2, where the G' (elastic modulus) loss factor was calculated as follows: G' loss rate = (G' before sterilization - G' after sterilization) / G' before sterilization,

[0075] Table 1 shows the elastic modulus test results for the gels obtained in Examples 1 to 7 and Comparative Examples 1 to 4. Gels C1 to C7 obtained through the pre-crosslinking and freeze-thaw crosslinking processes all had a G' value of 3500 Pa or higher after sterilization, and the G' loss rate was less than 3%. This is thought to be because three-site or four-site crosslinking of the polyamino acid gel occurs during the crosslinking reaction process, forming an interpenetrating network structure that is very dense and therefore very stable, can withstand moist heat sterilization, and has a small G' loss rate. Comparing the data of Examples 6-7 with those of Comparative Examples 1-2, it was found that the G' value after sterilization of C6 was 11.9 times that of B1, and the G' value after sterilization of C7 was 11.4 times that of B2. The G' loss rates of C6 and C7 were both less than 2%, much lower than those of B1 (56.22%) and B2 (56.67%). This may be because gels B1 and B2 were not pre-crosslinked, and the imino groups of spermine were hardly involved in the crosslinking reaction, resulting in low crosslinking efficiency. As a result, the crosslinking was only two-site crosslinking, resulting in a loose network structure and the inability to form a stable and dense interpenetrating network structure. This may be due to the significantly reduced gel stability and high G' loss rate. This further indicates that pre-crosslinking treatment can form multi-site crosslinks (3 or 4 sites), thereby improving the gel stability. The elastic modulus G' and its loss rate after sterilization in Comparative Examples 3 and 4 are also obviously higher than those in Example 6. This is thought to be because the preliminary cross-linking treatment in Comparative Examples 3 and 4 was not performed under appropriate conditions, so the cross-linking efficiency in the subsequent freezing process was too low, and the resulting gels were unlikely to form multi-site cross-links (3 or 4 sites), and therefore did not form a dense interpenetrating network structure, resulting in poor stability and therefore a relatively high loss rate of the elastic modulus G'.

[0076] [Table 1]

[0077] Test Example 2: SEM of gel and pore size measurement The pore structure of the gel was observed using a scanning electron microscope. Freeze-dried cryogel sponges were cut into small cubes with a blade, fixed to the microscope table with conductive adhesive, and sprayed with gold for 60 seconds. The results of the scanning electron microscope observations of cryogel sponges C6 and B1 to B4 are shown in Figures 1 to 5, respectively. Figure b is a partial enlargement of Figure a.

[0078] The pore sizes of the gels were statistically analyzed using ImageJ software and SEM images. Table 2 shows the pore size distributions for gels C1–C7 and gels B1–B4. As can be seen from Table 2, the pore sizes of gels C1–C7 all ranged from 80 to 760 μm. Comparing the data for gels B1 and C6, and those for gels B2 and C7, we found that the pore sizes of gels B1–B2, which were not pre-crosslinked, were very small, much smaller than those of gels C6 and C7. This indicates that the pre-crosslinking process has a certain effect on the formation of the macroporous structure of the gels. This is thought to be because the pre-crosslinking process can adjust the number of crosslinking reaction sites in the cryogels, thereby affecting their pore size. Furthermore, analysis of the results for C1–C7 reveals that cryogels fabricated with a pH between 4 and 5.3 yielded pore sizes between 420 and 760 μm; cryogels fabricated with a pH between 5.4 and 6.2 yielded pore sizes between 155 and 419 μm; and cryogels fabricated with a pH between 6.3 and 7.4 yielded pore sizes between 80 and 154 μm. Figures 1–5 further reveal that cryogels B1–B4 have small pore sizes and low porosity, while cryogels C6 possess an irregular, interconnected macroporous structure with high porosity, suggesting their potential use as cell scaffolds in tissue engineering. Pre-crosslinking before freezing also contributes to the formation of a porous structure. Failure to perform pre-crosslinking, or pre-crosslinking under appropriate conditions, significantly impacts subsequent crosslinking efficiency, making it difficult to form multi-site crosslinks and affecting the formation of the gel's pore structure.

[0079] [Table 2]

[0080] Test Example 3: Measurement of gel porosity Gels C1 to C7 and Gels B1 to B4 prepared in Examples 1 to 7 and Comparative Examples 1 to 4 were each weighed out in 2 g and placed on a 500-mesh dry sieve. The mass of the dry sieve was weighed and recorded as M0. NaCl solution (0.9%) was added to the sieve, and the gel was thoroughly immersed in the NaCl solution and allowed to swell completely overnight. The sieve was placed on lens cleaning paper, and excess water was wiped off. The combined mass of the swollen gel and sieve was weighed and recorded as M1. The sieve containing the gel was then oven-dried until completely dry, and the weight of the dried gel and sieve was weighed and recorded as M2. Finally, the porosity of the gel was calculated using the following formula: Gel porosity (%) = (M1-M2) / (M1-M0)*100%.

[0081] The measurement results of the porosity of the gels are shown in Figure 6. The porosity of gels C1 to C7 of the present invention is all 95% or more, while the porosity of gels B1 to B4 obtained in comparative examples 1 to 4 is 15% to 35%.

[0082] Test Example 4: Measurement of swelling ratio of gel To evaluate the water absorption performance of the gels, the mass of gels C1 to C7 and gels B1 to B4 after they reached a constant weight was recorded as W1. They were then immersed in a phosphate buffer solution with a pH of 7.0 to allow them to swell sufficiently. Samples were collected every 0.2 h, and the unabsorbed water was gently removed with filter paper and then weighed. The water absorption rate was determined by the saturation point (W). hyd The swelling ratio of the gel was calculated using the following formula: Swelling ratio = (W hyd -W1) / W1×100%

[0083] The swelling ratio test results for Gels C1-C7 and Gels B1-B4 are shown in Figure 7. The swelling ratios of Gels C1-C7 of the present invention ranged from 15.00% to 41.4%, while those of Gels B1-B4 ranged from only 1.8% to 5.0%. Gel C1 had the highest swelling ratio and absorbed water the fastest, reaching saturation the soonest. This is because Gel C1 has a larger pore size and higher porosity, allowing it to absorb more water faster and reach saturation sooner. The swelling ratios of Gels B1-B4 obtained in the comparative example were all low. This is because their small pore size and porosity resulted in a slower water absorption rate and a longer time to reach saturation. Due to their high water absorption, cryogels may be used as hemostatic materials in tissue engineering.

[0084] Test Example 5: Measurement of in vitro degradation performance of gel A 10 ml glass vial was taken, 1.0 ml of 75 U / ml enzyme solution was added, and 2.0 ml of gel sample was inserted without a needle. The mixture was then mixed in a vortex mixer for 60 seconds to ensure uniform mixing of the gel sample and enzyme solution. The dynamic viscosity (η, Pa·s) was measured at 37°C using a rheometer with a loading gap of 1000 μm, flow peak hold mode, and a test time of 14,400 seconds.

[0085] Figure 8 shows the degradation curves of gels C1 to C7. As can be seen from the curves, the gels are very stable, with degradation times of 90 minutes or more for all gels. However, among polyglutamic acid gels C1 to C6, C6 has the highest modulus of elasticity after sterilization and the longest degradation time, reaching 190 minutes. This shows that the degradation time of a gel is basically directly proportional to its modulus of elasticity. That is, the higher the modulus of elasticity of a gel, the more difficult it is to degrade and the longer the degradation time.

[0086] Test Example 6: Measurement of the mechanical properties of gel A suitable amount of gel (a cylindrical object 10 mm in diameter and approximately 10 mm in height) was taken and subjected to uniaxial tension testing using a universal electronic testing machine. A 1000 N tensile sensor was selected, the tension speed was 120 mm / min, and a zero reset was required before each tension test. After the experiment was completed, the force-displacement curve of the gel sample was obtained, and the stress and strain were then calculated according to the following equation to obtain the stress-strain curve: Stress: Stress=force / area, Strain: Strain=Displacement / 10*100%.

[0087] The mechanical properties of the gels were measured using the above test method. Figure 9 shows the stress-strain curves of Gel C6 and Gel B1. Gel C6 maintained its shape completely after 10 cycles of compression, and the stress at 42% strain was approximately 5N. In contrast, Gel B1 exhibited a stress of approximately 1.99N at 26% strain, at which point the gel failed to maintain its original shape and burst. The reason for this is believed to be as follows: the gel obtained through the pre-crosslinking and freeze-thaw crosslinking processes has a four-site crosslinked structure, which makes its structure denser and more stable, and therefore its mechanical performance during compression resistance is stronger. Gel B1, on the other hand, is two-site crosslinked, and its skeleton is not dense enough, resulting in a less stable structure and poor mechanical performance during compression resistance.

[0088] Test Example 7: Effect of gel on cell proliferation L-929 cells were cultured in MEM medium containing antibiotics (100 U / mL penicillin, 100 μg / mL streptomycin) and 10% serum at 37°C and 5% CO2 saturated humidity. Aseptic techniques were used for cell culture. When the cells grew to near confluence, they were digested with trypsin, collected, and the cell concentration was adjusted to 1 × 10 for the following tests. 5 Adjust to cells / mL.

[0089] Add 100 μL of the above cell suspension per well, for a total of 1 × 10 "The cells / well were added to a 96-well plate and cultured at 37°C and 5% CO2 for 24 hours. After the culture was completed, the medium in the culture plate was discarded, and the extracts from gels C1 to C7 and the blank control were added to each group. Each group had 6 wells, and the wells were cultured in an incubator at 37°C, 5% CO2, and saturated humidity. 1 mL of trypsin solution containing 0.1% EDTA was added to the L-929 cell culture medium, and 1 x 10 L929 cells were cultured. 4 Cells were seeded into a 96-well plate at 100 μL per well, and the plate was placed in a cell culture incubator to promote cell growth. A blank cell proliferation control experiment was also performed simultaneously.

[0090] The gel cell proliferation rate was measured using the MTT method. After 24 hours of incubation, 100 μL of MTT aqueous solution (concentration: 5 mg mL-1) was added to each well and the wells were placed in an incubator for 4 hours of incubation. The MTT solution was then removed, and 150 μL of dimethyl sulfoxide was added to dissolve the formazan crystals. The absorbance of the solution at a wavelength of 570 nm was measured using an ELISA reader to determine the degree of proliferation of L-929 cells in the gel. The cell proliferation rate was calculated according to the following formula: Cell growth rate (%) = (As / Ac) × 100%

[0091] Here, As is the absorbance at 570 nm of the sample solution, and Ac is the absorbance at 570 nm of the blank control.

[0092] The cell proliferation results are shown in Figure 10. As can be seen from the figure, the cell viability after 24 hours for gels C1 to C7 of the present invention was 137%, 136%, 135%, 136%, 137%, 139%, and 138%, respectively. All of these were significantly higher than the 100% cell viability of the blank control experiment in which no gel was added. The cell viability indicated that the gels of the present invention were not cytotoxic and that the porous structure of the gels had a certain promoting effect on cell proliferation.

[0093] Test Example 8: Detection of remaining active sites in two-site and three- or four-site multi-site cross-linked gels Approximately 2 g of ninhydrin was dissolved in 100 ml of purified water, mixed thoroughly, and stored at 2-8°C away from light for future use. 54.6 g of sodium acetate was dissolved in 20 ml of 1 mol / L acetic acid solution, then diluted to 500 ml with water for storage. Spermidine and spermine standards were placed in 10 ml measuring flasks, diluted to the mark with purified water, and shaken vigorously until homogenous to prepare 500 μg / ml spermidine and 500 μg / ml spermine standard working solutions. These were then diluted to 10 μg / ml, 50 μg / ml, and 100 μg / ml before use. The original 500 μg / ml standard working solution was used as a standard curve.

[0094] 1 mL of each of the cryogels before moist heat sterilization in Examples 1 to 7 and Comparative Examples 1 to 4 and the standard solutions of the spermine and spermidine series was taken, and 2.0 mL of the acetic acid-sodium acetate buffer solution and 2.0 mL of the ninhydrin solution were added in that order. The stoppers were sealed, mixed thoroughly, and heated in a water bath at 70°C for 30 minutes. The solution was then removed and rapidly cooled to room temperature. Purified water was added to dilute the solution to 25 mL and mixed uniformly. The mixed solution was taken and the absorbance of the amino group derivative at a wavelength of 565 nm was measured, and simultaneously the absorbance of the imino group derivative at a wavelength of 400 nm was detected. Blank correction was performed using purified water according to the same method.

[0095] Calculate the percentage of residual amino or imino groups according to the following formula: Residual amount ratio = (A0 - A i ) / A0×100% In the formula, A0 is the absorbance value measured in the control solution and Ai is the absorbance value measured in the sample.

[0096] Here, one spermidine molecule contains one imino group and two amino groups, and one spermine molecule contains two imino groups and two amino groups.

[0097] The test results are shown in Table 3: [Table 3]

[0098] As can be seen from the results in Table 3, gels C1 to C7 obtained in Examples 1 to 7 were three- or four-site multi-site cross-linked, and the residual amounts of amino and imino groups of spermidine or spermine were all less than 10%, indicating that the reaction efficiencies of amino and imino groups were both above 90%. This indicates that three-site cross-linking between spermidine and polyamino acids and four-site cross-linking between spermine and polyamino acids were formed through the pre-cross-linking and freeze-thaw cross-linking processes, which realizes efficient use of the cross-linking agent, is of great significance for rational control of the residual amount of cross-linking agent, and improves the safety of the product in clinical use. It also further proves the effectiveness of the three- or four-site multi-site cross-linking formed between the cross-linking agent and polyamino acids in the present invention. In Comparative Examples 1 and 2, the primary reactive group was the amino group due to the lack of a preliminary cross-linking step, and the reaction efficiency was approximately 85%. However, the residual amount of imino groups in spermidine or spermine was as high as 87% or more, indicating that very few imino groups were involved in the reaction. This is because the preliminary cross-linking treatment was not performed before freezing, the distance between the spermine or spermidine molecules and the polyamino acid molecular chain segments fixed by the ice crystals was large, and the probability of collision between the imino groups with low cross-linking reaction activity and the carboxyl groups of the molecular chains of the polyamino acid substance was extremely low, resulting in the efficiency of the cross-linking process. This is thought to be due to the decrease in crosslinking efficiency, and therefore the crosslinking reaction mainly involves the amino groups at both ends of the crosslinking agent, thereby forming two-site crosslinks, making it difficult to form three-site or four-site multi-site crosslinks, which have a more stable structure.Compared to Example 6, the gels obtained in Comparative Examples 3 and 4 had much higher residual amino and imino groups than Example 6, which also indicates that if the pre-crosslinking treatment is not carried out under appropriate conditions, the crosslinking efficiency in the subsequent freezing process will be significantly reduced, and the crosslinking reaction that mainly occurs is a two-site crosslinking reaction involving amino groups.

[0099] Finally, it should be noted that the above embodiments are only for illustrating the technical solutions of the present invention, but are not intended to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art may still modify the technical solutions described in the above embodiments or make equivalent substitutions for part or all of the technical features thereof, but these modifications or 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. 1. A polyamino acid gel material, comprising: The material is a polyamino acid gel obtained by subjecting endogenous polyamines and polyamino acids to preliminary crosslinking and freezing-thawing under the action of an active agent, and the crosslinking between the endogenous polyamines and the polyamino acids includes three-site crosslinking or four-site crosslinking.

2. 2. The gel material of claim 1, wherein the endogenous polyamines include spermidine and / or spermine.

3. 2. The gel material according to claim 1, wherein the polyamino acid is polyglutamic acid or / and polyaspartic acid.

4. 2. The gel material according to claim 1, wherein the pore size of the gel material is between 80 μm and 760 μm, and / or the porosity of the gel material is 95% or more, preferably 98% or more.

5. 2. The gel material of claim 1, wherein the activator comprises one or more of a water-soluble carbodiimide, a carbonium salt, and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride.

6. 1. A method for making a polyamino acid gel material, comprising: A method for producing a polyamino acid gel material, comprising: mixing a polyamino acid with an endogenous polyamine to obtain a mixed solution; adjusting the pH of the mixed solution to 4.00 to 7.40; adding an activator; stirring; pre-crosslinking at 25°C to 60°C for 5 to 60 minutes; then freeze-crosslinking; thawing the product after freeze-crosslinking; and repeating the freeze-thaw process 0 to 3 times to obtain the gel material.

7. The method according to claim 6, wherein the freeze-crosslinking temperature is −80° C. to −10° C. and the time is 2 hours to 48 hours, and the thawing temperature is 10° C. to 60° C. and the thawing time is 0.5 hours to 8 hours.

8. the endogenous polyamines include spermine and / or spermidine; Preferably, the activator comprises one or more of a water-soluble carbodiimide, a carbonium salt, and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride; Preferably, the polyamino acid comprises polyglutamic acid or / and polyaspartic acid; and / or the solvent of the mixed solution is one or a mixture of several selected from water, soluble alcohol, soluble ketone, DMF, DMA and DMSO, preferably the solvent is water.

9. the mass concentration of the polyamino acid in the mixed solution is 10 mg / mL to 160 mg / mL; When the endogenous polyamine is spermine, the amount of spermine added accounts for 1.0% to 62% of the molar amount of the polyamino acid structural unit; 7. The method according to claim 6, wherein when the endogenous polyamine is spermidine, the amount of spermidine added accounts for 0.5% to 52% of the molar amount of the polyamino acid structural unit.

10. the amount of the activator added accounts for 0.5% to 550%, preferably 50% to 500%, more preferably 50% to 300% of the molar amount of the polyamino acid structural unit; when the activator is a water-soluble carbodiimide, the method further comprises adding an auxiliary to the mixed solution; the auxiliary agent includes one or more of N-hydroxysuccinimide, sulfonated N-hydroxysuccinimide, tert-butanol, and 1-hydroxybenzotriazole; The method according to claim 9, wherein the amount of the auxiliary agent added is preferably 10% to 50% by mass of the carbodiimide.

11. 7. The method of claim 6, further comprising adding the thawed product to a phosphate buffer solution having a concentration of 10 mg / mL to 25 mg / mL, and then performing moist heat sterilization to obtain a gel material.

12. A gel material produced by the method of any one of claims 6 to 11.

13. Use of a gel material according to any one of claims 1 to 5, a gel material produced by a method according to any one of claims 6 to 11 or a gel material according to claim 12 in the manufacture of a medical, cosmetic or healthcare product, comprising: The medical and cosmetic products include one or more of soft tissue filling materials, cartilage repair materials, tissue implant materials, coating layers for biomaterial implants, scaffolding materials for tissue engineering, drug sustained release vehicles, drug targeting carriers, and wound dressings.

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