Cross-linked polyamide hydrogel material and preparation method therefor

Abstract

Disclosed in the present invention are a cross-linked polyamide hydrogel material and a preparation method therefor. The hydrogel is obtained by means of a cross-linking reaction between a water-soluble polyamide and a cross-linking agent. The water-soluble polyamide comprises poly(malic acid-spermidine), poly(malic acid-spermine), poly(succinic acid-spermidine), or poly(succinic acid-spermine). The cross-linking agent is an endogenous dibasic acid. The cross-linking reaction means that after being activated by a catalyst, a carboxyl group on the endogenous dibasic acid undergoes a two-step activation cross-linking reaction with an imine group on the backbone of the polyamide to obtain the hydrogel. The present invention prepares the hydrogel by means of a two-step activation cross-linking technique. By means of the cross-linking technique, the amounts of the catalyst and the cross-linking agent can be reduced, and hydrogels having different properties can also be prepared by controlling the type of the cross-linking agent and cross-linking conditions, thereby improving the cross-linking reaction efficiency and achieving the control of the degradation rate of the hydrogel and the release rate of an active monomer, exhibiting broad application prospects.

Description

A cross-linked polyamide hydrogel material and its preparation method Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a cross-linked polyamide hydrogel material and its preparation method. Background Technology

[0002] Hydrogels are hydrophilic three-dimensional mesh structures that can rapidly absorb water and swell, storing moisture within the hydrogel mesh. Due to these properties, they can be used for wound repair, tissue filling, cell culture, and sustained drug release.

[0003] Hydrogels are typically formed by physical or chemical crosslinking of hydrophilic polymers such as hyaluronic acid, collagen, polyamide, polyvinyl alcohol, or polyacrylic acid. Patent document CN101918474A reports a hydroxyl polyamide gelling agent, which, unlike conventional polyamides (such as nylon 66), possesses excellent hydrophilicity and can be directly mixed with water to form a gelling agent. However, its monomers lack biological activity, limiting its application. Patent document CN113896915A reports a hydrogel prepared by crosslinking hyaluronic acid and spermidine, with spermidine acting as the crosslinking agent. Although this document describes that the hydrogel can generate biological activity by releasing spermidine through degradation, the content of spermidine as the crosslinking agent in this hydrogel is low, resulting in a lower concentration of spermidine released during degradation and limited biological activity. Therefore, this invention uses biologically active spermidine and spermidine as polymer monomers, which can effectively increase the release concentration during polymer degradation and facilitate the activation of biological activity.

[0004] Currently, there are no reports on the crosslinking reaction conditions and crosslinked hydrogel properties of novel hydrophilic polyamides (such as poly(malic acid-spermine), poly(malic acid-spermine), poly(succinic acid-spermine), and poly(succinic acid-spermine)) with endogenous polyacids. Because the monomers spermine and spermidine in hydrophilic polyamides are easily oxidized and hydrolyzed under acidic conditions, and easily cyclized and oxidized under high temperatures, traditional polyamide crosslinking reactions easily damage the monomers, thus affecting the release efficiency of the polymer monomers and limiting the effects of spermine, spermidine, and other monomers on stimulating biological activity and reversing aging.

[0005] While existing technologies employ one-step catalysis with EDCl or dehydration of acid anhydrides to form crosslinked polyamides, these methods are not suitable for the crosslinking reactions of hydrophilic polyamides (e.g., poly(malic acid-spermine), poly(malic acid-spermine), poly(succinic acid-spermine), and poly(succinic acid-spermine)) with endogenous polyacids. For example, CN10361991A reports the use of organic polyacids as crosslinking agents to prepare polyamide hydrogels via amide crosslinking with α-primary amines of poly-ε-lysine under EDCl and HONSU activation catalysis. However, this patent only targets the crosslinking reaction of polyamides with highly active primary amine groups, and is not applicable to reactions where the main chain contains only less active imine groups. This is because imine groups have high steric hindrance in crosslinking and low degrees of freedom on the main chain, making effective crosslinking difficult using this method. CN102350226A and CN1160826... 46A reports a process of dehydrating and crosslinking polyamides using dianhydrides or organic polyacids under concentrated acid (concentrated sulfuric acid or hydrochloric acid) or high temperature (200-360℃) conditions. Acid anhydrides are easily hydrolyzed, so a high temperature or concentrated acid system with relatively harsh reaction conditions must be used. However, under concentrated acid and high temperature systems, the stability of active monomers (malic acid, spermine or spermidine, etc.) is easily destroyed, which is not conducive to the release of monomers and the exertion of anti-aging effects. Therefore, the above-mentioned traditional crosslinking methods are not suitable for the crosslinking reaction of hydrophilic polyamides containing endogenous monomers and whose main chain does not contain primary amine groups.

[0006] To address this technical problem, it is necessary to develop a specific cross-linking reaction system that can not only ensure the efficiency of the cross-linking reaction but also protect the stability of endogenous monomers from being destroyed, and exert corresponding bioactive effects during the hydrogel degradation process. Summary of the Invention

[0007] To overcome the shortcomings of the prior art, the present invention provides a cross-linked polyamide hydrogel material and its preparation method.

[0008] This invention uses a novel hydrophilic polyamide, such as poly(malic acid-spermine), poly(malic acid-spermine), poly(succinic acid-spermine), and poly(succinic acid-spermine), in the preparation of hydrogels. Its polymer monomers include endogenous polyacids such as malic acid and succinic acid, as well as endogenous polyamines such as spermine and spermine, which have good bioactivity. The above-mentioned hydrophilic polyamide is crosslinked with endogenous diacids to obtain a bioactive polyamide hydrogel.

[0009] The active group of the hydrophilic polyamide polymer backbone of this invention is an imine group, which has low reactivity. In addition, the large steric hindrance on the backbone makes it even more difficult to react. It is difficult to form effective crosslinking using conventional catalysts such as EDCl. This is because although the carboxyl group activated by EDCl can react with the imine group on the backbone to form an amide bond, after one carboxyl group in the crosslinking agent diacid molecule reacts and connects with the backbone, the other carboxyl group will be affected by the steric hindrance of the surrounding polymer backbone, which restricts the movement of the second carboxyl group. Therefore, even if the second carboxyl group can be activated, it is difficult to form an effective collision with the imine group within the activation time window. As a result, the crosslinking efficiency is low, and the amount of crosslinking agent consumed is too high. It is necessary to remove the adjuvant, which requires additional purification, impurity removal and other post-processing processes, which increases the cost and limits the application of medical materials. Therefore, this invention develops a two-step activated crosslinking technology (TACT) to prepare hydrogels, which can effectively improve the crosslinking reaction efficiency of water-soluble polyamides, reduce the amount of catalyst and crosslinking agent used, and also prepare hydrogels with different properties by controlling the type of crosslinking agent and crosslinking conditions. It can also control the degradation rate of hydrogels and the release rate of active monomers, thereby exerting physiological activities such as anti-inflammatory, antioxidant, wound healing promotion and anti-aging.

[0010] This invention prepares water-soluble polyamide from solution form into hydrogel form. The main chain has a high content of spermine and spermidine active monomers. During the degradation and release process of the small polymer monomer molecules, a high-concentration microenvironment can be generated, resulting in significant biological activity. It has excellent anti-inflammatory and antioxidant effects, making it more suitable for filling and dressings, and has considerable application prospects in the field of medical materials.

[0011] In a first aspect, the present invention provides a crosslinked polyamide hydrogel, said hydrogel being obtained by a crosslinking reaction of a water-soluble polyamide and a crosslinking agent.

[0012] Further, the water-soluble polyamide includes poly(malic acid-spermidine), poly(malic acid-spermidine), poly(succinic acid-spermidine), or poly(succinic acid-spermidine).

[0013] Furthermore, the polyamide is a linear polymer.

[0014] In some embodiments of the present invention, the polyamide comprises a structure selected from the following:

[0015] Where n is the degree of polymerization, which is related to the molecular weight.

[0016] The water-soluble polyamide was obtained using the preparation method described in patent CN202411042223.4.

[0017] Furthermore, the crosslinking agent is an endogenous dicarboxylic acid, selected from one or more of malic acid, succinic acid, itaconic acid, fumaric acid and α-ketoglutaric acid, preferably malic acid and / or succinic acid.

[0018] Furthermore, the cross-linking reaction refers to a two-step activation cross-linking reaction between an endogenous dicarboxylic acid activated by a catalyst and a water-soluble polyamide to obtain a hydrogel.

[0019] Furthermore, the cross-linking reaction refers to a two-step activation cross-linking reaction in which the carboxyl group on the endogenous dicarboxylic acid is activated by a catalyst and then reacts with the imine group on the main chain of the water-soluble polyamide to obtain a hydrogel.

[0020] Furthermore, the catalyst includes a first catalyst and a second catalyst, which are used for the two-step activation crosslinking reaction, respectively.

[0021] Furthermore, the first catalyst is a carboxyl activator selected from: carbodiimide, N,N'-carbonyldiimidazole (CDI), 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM), 4-dimethylaminopyridine (DMAP), or a salt solution thereof.

[0022] Further, the carbodiimide is selected from: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide, 1-cyclohexyl-3-(2-N-morpholinylethyl)carbodiimide, and 1,3-bis[bis(methoxymethyl)methyl]carbodiimide.

[0023] Furthermore, an auxiliary agent may be added to the activation crosslinking reaction, which is selected from: N-hydroxysuccinimide (NHS), N-hydroxysulfosuccinimide (Sulfo-NHS), tert-butanol, and 1-hydroxybenzotriazole (HOBT).

[0024] Furthermore, the second catalyst is succinic anhydride.

[0025] Furthermore, the two-step activation crosslinking reaction refers to the following: in the first activation crosslinking reaction, a carboxyl group on the endogenous diacid is activated by the first catalyst and then crosslinks with an imine group on the water-soluble polyamide backbone to obtain a polyamide with a carboxyl group; in the second activation crosslinking reaction, another carboxyl group on the endogenous diacid is activated by the second catalyst and then crosslinks with an imine group on the water-soluble polyamide backbone to obtain a crosslinked polyamide hydrogel.

[0026] A second aspect of the present invention provides a method for preparing a cross-linked polyamide hydrogel, the method comprising the following steps:

[0027] 1) Add a crosslinking agent to a water-soluble polyamide solution, adjust the pH of the system, add the first catalyst, and carry out the first activation crosslinking reaction;

[0028] 2) Adjust the pH value of the product from step 1), add the second catalyst, and carry out the second activation crosslinking reaction to obtain the hydrogel.

[0029] Furthermore, the water-soluble polyamide has the definition described in the first aspect of the present invention, including poly(malic acid-spermidine), poly(malic acid-spermidine), poly(succinic acid-spermidine), or poly(succinic acid-spermidine).

[0030] In some embodiments of the present invention, the water-soluble polyamide solution is an aqueous solution of water-soluble polyamide.

[0031] Further, the mass concentration of the water-soluble polyamide solution is 15-300 mg / mL, specifically 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300 mg / mL, preferably 60-120 mg / mL.

[0032] Furthermore, the crosslinking agent is an endogenous dicarboxylic acid, selected from one or more of malic acid, succinic acid, itaconic acid, fumaric acid and α-ketoglutaric acid, preferably malic acid and / or succinic acid.

[0033] Further, the molar number of the crosslinking agent is 5-60% of the molar number of imine groups in the water-soluble polyamide, specifically such as 5, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60%, preferably 10-60%, and more preferably 15-60%.

[0034] Furthermore, when using a composite crosslinking agent, the release rate of polyamide monomers can be controlled by limiting the molar ratio of the crosslinking agent.

[0035] In one embodiment of the present invention, a composite crosslinking agent, malic acid, and succinic acid are used, with a molar ratio of 1:(0.5-2), specifically such as 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.0, preferably 1:1.

[0036] Further, in step 1), the first catalyst is a carboxyl activator selected from: carbodiimide, N,N'-carbonyldiimidazole (CDI), 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM), 4-dimethylaminopyridine (DMAP), or a salt solution thereof.

[0037] Further, the carbodiimide is selected from: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide, 1-cyclohexyl-3-(2-N-morpholinylethyl)carbodiimide, and 1,3-bis[bis(methoxymethyl)methyl]carbodiimide.

[0038] Furthermore, in the first activation crosslinking reaction of step 1), an auxiliary agent may be added, which is selected from: N-hydroxysuccinimide (NHS), N-hydroxysulfosuccinimide (Sulfo-NHS), tert-butanol, and 1-hydroxybenzotriazole (HOBT).

[0039] Furthermore, the number of moles of the first catalyst is 1.1-1.5 times the number of moles of the crosslinking agent, specifically 1.1, 1.2, 1.3, 1.4, or 1.5 times, which can ensure that one carboxyl group of the endogenous dicarboxylic acid in the crosslinking agent is activated.

[0040] Furthermore, the molar number of the additive is 1.1-1.5 times the molar number of the crosslinking agent, specifically 1.1, 1.2, 1.3, 1.4, or 1.5 times.

[0041] In some embodiments of the present invention, the molar ratio of the first catalyst to the auxiliary agent is 1:(0.5-2), specifically such as 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.0, preferably 1:1.

[0042] Further, in step 1), the pH value is 5.0-6.5, specifically such as 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5, preferably 5.0-6.0. The solvent used to adjust the pH value of the system in this invention is a 3-6 mol / L hydrochloric acid solution.

[0043] Further, in step 1), the first activation crosslinking temperature is 25-60℃ (specifically 25, 30, 35, 40, 45, 50, 55, 60℃), and the time is 10-20h (10, 12, 14, 16, 18, 20h).

[0044] In the first activation crosslinking reaction, a carboxyl group on the endogenous dicarboxylic acid is activated by the first catalyst and then crosslinks with an imine group on the main chain of the water-soluble polyamide to obtain a water-soluble polyamide with a carboxyl group.

[0045] Since the water-soluble polyamide backbone of this invention has no primary amine groups and only has imine groups with weak reactivity, by controlling process parameters such as catalyst, auxiliaries, crosslinking agents, pH value, reaction temperature and time in the system, it is possible to activate only one carboxyl group of the crosslinking agent diacid and couple it to the polyamide backbone in a single grafting manner to form a water-soluble polyamide with a carboxyl group, thereby facilitating the second-step activation crosslinking reaction.

[0046] Further, in step 2), the second catalyst is succinic anhydride.

[0047] Further, in step 2), the number of moles of the second catalyst (succinic anhydride) is 0.7-1.3 times the number of moles of the crosslinking agent, specifically 0.7, 0.8, 1, 1.2, or 1.3 times.

[0048] Further, in step 2), the pH value is 4-5.2, specifically such as 4, 4.2, 4.5, 4.7, 5, 5.1, 5.2, preferably 4.6-5.2.

[0049] Further, in step 2), the second activation crosslinking temperature is 5-25℃ (specifically 5, 10, 15, 20, 21, 22, 23, 24, 25℃), and the time is 1-4h (specifically 1, 2, 3, 4h).

[0050] During the second activation crosslinking reaction, another carboxyl group on the endogenous dicarboxylic acid is activated by the second catalyst and undergoes a crosslinking reaction with the imine group on the water-soluble polyamide backbone to obtain the target product, namely crosslinked polyamide hydrogel.

[0051] The second step of the activation crosslinking process in this invention uses succinic anhydride. Activating the second carboxyl group in the diacid crosslinking agent has the following advantages: Firstly, the anhydride can activate the second carboxyl group of the crosslinking agent. Succinic anhydride reacts with the single carboxyl group on the polymer to form a carboxyl-containing active intermediate, mixed anhydride. The anhydride bond at both ends of the active intermediate has hydrophobic alkyl chains, which weakens the attack of water on the anhydride bond in the reaction system. Therefore, the activated anhydride bond is relatively stable, thereby extending the activation reaction time of the second carboxyl group and effectively improving the reaction efficiency. Secondly, the active intermediate forms ion pairs with the imine group in the nearby polyamide. Through the electrostatic interaction between the carboxyl group and the imine group, the distance between them is shortened, thereby forming an effective collision to generate an amide bond. This allows the activated carboxyl group to undergo second amide bond crosslinking with the imine group, completing the crosslinking reaction. In addition, the succinic acid obtained after hydrolysis of succinic anhydride can correspond to the endogenous polyacid part of the monomer in the polyamide molecule. Therefore, using a homologous monoacid anhydride as the catalyst for the second activation crosslinking has good biocompatibility and eliminates the need for additional post-processing such as impurity removal.

[0052] In one embodiment of the present invention, taking the process of succinic acid crosslinking poly(malic acid-spermidine) hydrogel as an example, the mechanism of the second step of succinic anhydride crosslinking is as follows: After the first crosslinking reaction, a small amount of catalyst ensures that one carboxyl group of the crosslinking agent succinic acid is activated and reacts with the imine group; then, succinic anhydride is added in the second step of activation crosslinking. The succinic anhydride activates the second carboxyl group of the crosslinking agent and forms a mixed anhydride with the active intermediate with a carboxyl group attached to the polymer. With the help of the hydrophobic alkyl chains at both ends of the anhydride bond of the active intermediate, the attack of water on the anhydride bond in the reaction system is weakened. Therefore, the formed anhydride bond is relatively stable, prolonging the activation reaction time of the second carboxyl group. This solves the technical problem of easy hydrolysis and deactivation of anhydride and short reaction window period, effectively improving the crosslinking reaction efficiency; on the other hand, the active intermediate and the imine group in the polyamide form an ion pair structure. Through the electrostatic interaction between the two, the distance between the imine group and the activated carboxyl group is shortened, thereby causing effective collision and coupling, so that the activated carboxyl group reacts with the imine group to generate an amide bond, completing the second step of activation crosslinking reaction. At the same time, it generates endogenous succinic acid, and the hydrogel system has good biocompatibility, saving complicated post-processing steps.

[0053] Furthermore, the preparation method also includes a post-processing step, which includes an immersion purification step.

[0054] In one embodiment of the present invention, the cross-linked hydrogel is immersed in PBS (pH=7.4, 0.01M) to purify and remove the catalyst and unreacted cross-linking agent, then pulverized and sterilized to obtain a sterile hydrogel.

[0055] Furthermore, the solvent used for the soaking purification is phosphate buffered saline (PBS). The purpose of soaking purification in this invention is to remove the catalyst and uncrosslinked endogenous dicarboxylic acids.

[0056] Furthermore, the volume of the solvent used for the soaking and purification is 50-200 times that of the hydrogel, specifically 50, 60, 70, 80, 90, 100, 120, 150, 180, or 200 times, preferably 50-100 times.

[0057] Furthermore, the number of soaking and purification cycles is 5-12, specifically 5, 6, 7, 8, 9, 10, 11, or 12, preferably 5-8.

[0058] Furthermore, the soaking and purification time is 1-12 hours, specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours, preferably 5-6 hours.

[0059] Furthermore, the soaking and purification process also includes drying and sterilization steps.

[0060] Furthermore, the soaking and purification process may also include a pulverizing step.

[0061] Furthermore, the soaking and purification process may also include a compounding step.

[0062] Furthermore, the sterilization method is irradiation sterilization or moist heat sterilization.

[0063] Furthermore, the irradiation dose for the irradiation sterilization is 10-30 kJ, specifically 10, 15, 20, 25, or 30 kJ. In some embodiments of the present invention, the irradiation dose is 15 kJ.

[0064] Further, the temperature for moist heat sterilization is 120-130℃, specifically 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, or 130℃; the time for moist heat sterilization is 10-45 minutes, specifically 10, 12, 15, 18, 20, 22, 25, 28, 30, 32, 35, 38, 40, 42, or 45 minutes. In some embodiments of the present invention, the temperature for moist heat sterilization is 121℃, and the time is 15 minutes.

[0065] Furthermore, the drying process is vacuum drying or freeze drying. In some embodiments of the present invention, the drying process is freeze drying. Freeze drying can be performed under conditions commonly used in the art, such as -10 to -30°C for 10-24 hours.

[0066] Furthermore, after pulverization, the particle size of the hydrogel is 100-500 μm.

[0067] Furthermore, the solvent used in the compounding process is physiological saline or phosphate-buffered saline (PBS), preferably phosphate-buffered saline. The phosphate-buffered saline has a concentration of 0.01 M and a pH of 7.4.

[0068] Furthermore, after compounding, the mass concentration of the hydrogel is 10-50 mg / mL, specifically 10, 15, 20, 25, 30, 35, 40, 45, and 50 mg / mL. In some embodiments of the present invention, the mass concentration of the hydrogel after compounding is 20 mg / mL.

[0069] A third aspect of the present invention provides the application of the cross-linked polyamide hydrogel described in the first aspect of the present invention or the cross-linked polyamide hydrogel prepared in the second aspect of the present invention in the preparation of tissue engineering materials.

[0070] Furthermore, the tissue engineering materials can be used to fill and repair soft tissue, promote wound healing, etc., including but not limited to wound dressings, biological patches, tissue adhesives, tissue engineering scaffolds, and tissue fillers (such as soft tissue injectable fillers for eliminating facial skin wrinkles).

[0071] In a fourth aspect, the present invention provides the application of the cross-linked polyamide hydrogel described in the first aspect of the present invention or the cross-linked polyamide hydrogel prepared in the second aspect of the present invention in the preparation of a drug carrier.

[0072] A fifth aspect of the present invention provides the use of the cross-linked polyamide hydrogel described in the first aspect of the present invention or the cross-linked polyamide hydrogel prepared in the second aspect of the present invention in the preparation of products for medical aesthetic and / or cosmetic purposes.

[0073] Furthermore, the product used in the cosmetic application is a cosmetic product.

[0074] In a sixth aspect, the present invention provides the application of the cross-linked polyamide hydrogel described in the first aspect of the present invention or the cross-linked polyamide hydrogel prepared in the second aspect of the present invention in filling and repairing soft tissue, promoting wound healing, and eliminating facial skin wrinkles.

[0075] A seventh aspect of the present invention provides a tissue engineering material (e.g., wound dressing, biological patch, tissue adhesive, tissue engineering scaffold, tissue filler) comprising the cross-linked polyamide hydrogel described in the first aspect of the present invention or the cross-linked polyamide hydrogel prepared in the second aspect of the present invention.

[0076] In an eighth aspect, the present invention provides a drug carrier comprising the cross-linked polyamide hydrogel described in the first aspect of the present invention or the cross-linked polyamide hydrogel prepared in the second aspect of the present invention.

[0077] A ninth aspect of the present invention provides a product for medical aesthetic and / or cosmetic use, comprising the cross-linked polyamide hydrogel described in the first aspect of the present invention or the cross-linked polyamide hydrogel prepared in the second aspect of the present invention.

[0078] A tenth aspect of the present invention provides a method for preparing tissue engineering materials, drug carriers, or products for medical aesthetic and / or cosmetic purposes (as described in aspects three, four, five, seven, eight, and nine of the present invention), comprising the step of using the cross-linked polyamide hydrogel described in the first aspect of the present invention or the cross-linked polyamide hydrogel prepared in the second aspect of the present invention.

[0079] The eleventh aspect of the present invention provides a method for filling and repairing soft tissue, promoting wound healing, and eliminating facial skin wrinkles, comprising the step of applying the cross-linked polyamide hydrogel described in the first aspect of the present invention or the cross-linked polyamide hydrogel prepared in the second aspect of the present invention.

[0080] The present invention has the following beneficial effects:

[0081] 1. This invention prepares a hydrogel from a solution using a two-step activated crosslinking (TACT) technique, which involves polyamide containing endogenous polyamines and polyacid monomers. Under the action of a dual-catalytic system, the spermine and spermidine in the main chain are not easily oxidized, acidified, or hydrolyzed, resulting in good stability. During the degradation and release of small polymer monomer molecules, a high-concentration microenvironment with significant biological activity can be generated. The degradation can exert excellent effects at the application site, showing considerable application prospects in the field of medical materials.

[0082] 2. This invention employs a two-step activation crosslinking process. In the first step of the activation crosslinking reaction, a small amount of carboxyl catalyst is used to activate one carboxyl group in the diacid crosslinking agent, which then undergoes a single-link crosslinking reaction with the imine group of the polyamide. In the second step, succinic anhydride activation crosslinking activates the other carboxyl group in the crosslinking agent, forming an active intermediate. The hydrophobic segments formed at both ends improve the stability of the anhydride bond, extend the window time for the second carboxyl group to undergo crosslinking, and improve the effectiveness of the crosslinking reaction. At the same time, the formed intermediate forms ion pairs with the imine group in the free polymer monomer, using electrostatic interaction to bring the two closer together, increasing the collision probability and significantly improving the efficiency of the crosslinking reaction.

[0083] 3. This invention employs a two-step activation crosslinking process, which greatly reduces the amount of catalyst and crosslinking agent required. It can efficiently prepare polyamide hydrogel products with active monomers, and the crosslinking efficiency is high, which helps to improve the economy of the reaction and makes the reaction more green and environmentally friendly.

[0084] 4. The two-step activation crosslinking process of the present invention can be extended to the crosslinking reaction process of polyamides whose main chain contains only imine groups and / or whose monomers have high activity, thus pioneering a new crosslinking process and developing a more efficient crosslinking system for hydrogel products of water-soluble polyamides.

[0085] 5. By adjusting the activity of the crosslinking agent during the crosslinking reaction, this invention can control the degradation rate of the hydrogel, thereby controlling the release rate of the active monomers. The release rate of the monomers can be adjusted as needed, resulting in a more intelligent hydrogel that can be matched with more application scenarios.

[0086] 6. The cross-linked polyamide hydrogels obtained by the preparation method of the present invention are of many types and can be arbitrarily combined to obtain hydrogels with different properties, which can meet the application needs of most scenarios.

[0087] 7. This invention improves the usability of hydrogels in soft tissue filling, soft tissue repair, medical aesthetics and other fields, making them suitable for widespread application. Attached Figure Description

[0088] Figure 1 shows a photograph of the cross-linked polyamide hydrogel after sterilization. From left to right, the final products obtained in Examples 1-5 are polyamide hydrogels.

[0089] Figure 2 shows photographs of the tilted products of Example 1 (left) and Comparative Example 1-A (right).

[0090] Figure 3 shows microscopic photographs of the cell experiments in Test Example 3. The left image is the blank group, and the right image is the experimental group photograph of Example 5.

[0091] Figure 4 shows the H&E staining of the animal experiment in Test Example 4 (the substance indicated by the arrow "↑" is the newly formed blood vessel).

[0092] Figure 5 shows the Masson staining pattern of the animal experiment in Test Example 4 (the substance indicated by the arrow "↑" is intermediate-sized new collagen). Detailed Implementation

[0093] Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0094] All publications, patents, and published patent specifications cited in this article are incorporated herein in their entirety through citation.

[0095] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0096] The water-soluble polyamide poly(malic acid-spermidine) used in Examples 1, 3, and Comparative Example 1 was prepared according to Example 2 of patent CN202411042223.4 (A biocompatible polyamide material and its preparation method and application).

[0097] The water-soluble polyamide poly(succinic acid-spermine) used in Examples 2, 4-5, and Comparative Examples 2-4 was prepared according to Example 6 of Patent CN202411042223.4 (A biocompatible polyamide material and its preparation method and application); the poly(succinic acid-spermine) in Example 6 was also prepared by the method of this patent.

[0098] The "dicarboxylic acid" mentioned in this invention refers to a dicarboxylic acid comprising two carboxyl groups.

[0099] Example 1: Preparation of malic acid crosslinked poly(malic acid-spermidine) hydrogel

[0100] 1) Weigh 1.0g of poly(malic acid-spermidine) polyamide material (3.83 mmol of imine moles), then add 10mL of purified water. After complete dissolution, the concentration of poly(malic acid-spermidine) is 100mg / mL. Add 77mg of malic acid (0.57 mmol of moles, the crosslinking agent accounts for about 15% of the imine moles in the polyamide) to the solution. After thorough dissolution, mix evenly. Then adjust the pH of the polyamide solution to about 5.2 with 6mol / L hydrochloric acid solution. Subsequently, add 0.68mmol of the first catalyst EDC and 0.68mmol of the auxiliary agent NHS and continue stirring evenly. Seal and place in a 30℃ forced-air drying oven for 14h.

[0101] 2) Take out the first cross-linking reaction product from step 1), adjust the pH of the system to 4.7, and place it in a constant temperature oven at 25°C. Then weigh 59.4 mg of the second catalyst, succinic anhydride (0.59 mmol), add it to the reaction system, stir evenly, and then place it in a 25°C oven for 2 hours to obtain polyamide hydrogel.

[0102] 3) The obtained polyamide hydrogel was purified by immersing it in 50 times PBS (pH=7.40, 0.01M) water. The solution was changed every 1 hour for a total of 6 times to remove unreacted crosslinking agents and catalysts. The hydrogel was then freeze-dried (i.e., before sterilization) and then terminally sterilized with an irradiation dose of 15KGy (i.e., after sterilization) to obtain the final product. The final product was stored at -20℃ for later use.

[0103] Example 2: Preparation of malic acid crosslinked poly(succinic acid-spermidine) hydrogel

[0104] 1) Weigh 1.0g of poly(succinic acid-spermidine) polyamide material (4.06 mmol of imine moles), then add 10mL of purified water. After complete dissolution, the concentration of poly(succinic acid-spermidine) is 100mg / mL. Add 109mg of malic acid (0.82 mmol of moles, the crosslinking agent accounts for about 20% of the imine moles in the polyamide) to the solution. After thorough dissolution, mix evenly. Then adjust the pH of the polyamide solution to about 5.4 with 6mol / L hydrochloric acid solution. Subsequently, add 0.98mmol of the first catalyst EDC and 0.98mmol of the auxiliary agent NHS and continue stirring evenly. Seal and place in a 30℃ forced-air drying oven for 14h.

[0105] 2) Take out the first crosslinking reaction product from step 1), adjust the pH of the system to 5.0, and place it in a 25°C constant temperature oven to cool down. Then weigh 90 mg of the second catalyst, succinic anhydride (0.9 mmol), add it to the reaction system, stir evenly, and then place it in a 25°C oven to react for 1 h to obtain polyamide hydrogel.

[0106] 3) The obtained hydrogel was immersed in 60 volumes of PBS (pH=7.40, 0.01M) for purification. The solution was changed every 1 hour for a total of 5 times to remove unreacted cross-linking agents and catalysts. The obtained hydrogel was then freeze-dried (i.e., before sterilization) and then sterilized at 121℃ for 15 minutes by moist heat sterilization (i.e., after sterilization) to obtain the final product. The final product was stored at -20℃ for later use.

[0107] Example 3: Preparation of succinic acid crosslinked poly(malic acid-spermidine) hydrogel

[0108] 1) Weigh 1.0g of poly(malic acid-spermidine) polyamide material (3.83 mmol of imine moles), then add 10mL of purified water. After complete dissolution, the concentration of poly(malic acid-spermidine) is 100mg / mL. Add 126mg of succinic acid (1.07 mmol of moles, the crosslinking agent molar ratio accounts for about 28% of the imine moles in the polyamide) to the solution. After complete dissolution, mix evenly. Then adjust the pH of the polyamide solution to about 6.0 with 6mol / L hydrochloric acid solution. Subsequently, add 1.28mmol of the first catalyst EDC and 1.28mmol of the auxiliary agent NHS and continue stirring evenly. Seal and place in a 45℃ forced-air drying oven for 10h.

[0109] 2) Take out the first cross-linking reaction product from step 1), adjust the pH of the system to 4.0, and place it in a 20℃ constant temperature oven. Then weigh 128 mg of the second catalyst, succinic anhydride (1.28 mmol), add it to the reaction system, stir evenly, and then place it in a 20℃ oven for 4 h to obtain polyamide hydrogel.

[0110] 3) The obtained hydrogel was immersed in 100 times PBS (pH=7.40, 0.01M) for purification. The solution was changed every 1 hour for a total of 6 times to remove unreacted cross-linking agents and catalysts. The obtained hydrogel was then freeze-dried (i.e., before sterilization) and then terminally sterilized with an irradiation dose of 15KGy (i.e., after sterilization) to obtain the final product. The final product was stored at -20℃ for later use.

[0111] Example 4: Preparation of succinic acid crosslinked poly(succinic acid-spermidine) hydrogel

[0112] 1) Weigh 1.0g of poly(succinic acid-spermidine) polyamide material (4.06 mmol of imine moles), then add 10mL of purified water. After complete dissolution, the concentration of poly(succinic acid-spermidine) is 100mg / mL. Add 96.8mg of succinic acid (0.82 mmol of moles, the crosslinking agent accounts for about 20% of the imine moles in the polyamide) to the solution. After complete dissolution, mix evenly. Then adjust the pH of the polyamide solution to about 6.0 with 6mol / L hydrochloric acid solution. Subsequently, add 0.98mmol of the first catalyst EDC and 0.98mmol of the auxiliary agent NHS and continue stirring evenly. Seal and place in a 45℃ forced-air drying oven for 10h.

[0113] 2) Take out the first cross-linking reaction product from step 1), adjust the pH of the system to 4.7, and place it in a 20℃ constant temperature oven. Then weigh 98 mg of the second catalyst, succinic anhydride (0.98 mmol), add it to the reaction system, stir evenly, and then place it in a 20℃ oven for 4 h to obtain polyamide hydrogel.

[0114] 3) The obtained hydrogel was immersed in 100 times PBS (pH=7.40, 0.01M) for purification. The solution was changed every 1 hour for a total of 6 times to remove unreacted cross-linking agents and catalysts. The hydrogel particles were then pulverized using a high-shear disperser. After pulverization, the hydrogel was reconstituted with 10 mg / mL pH7.4 phosphate buffer to 20 mg / mL (i.e., before sterilization). The hydrogel was then filled into pre-filled syringes and sterilized by moist heat at 121℃ for 15 min (i.e., after sterilization) to obtain the final product hydrogel.

[0115] Example 5: Preparation of cross-linked poly(succinic acid-spermidine) hydrogel using a mixed cross-linking agent (malic acid:succinic acid = 1:1)

[0116] 1) Weigh 1.0g of poly(succinic acid-spermidine) polyamide material (4.06 mmol of imine moles), then add 10mL of purified water. After complete dissolution, the concentration of poly(succinic acid-spermidine) is 100mg / mL. Add malic acid (81.7mg) and succinic acid (72mg) to the solution, totaling 1.22mmol. The total crosslinking agent moles account for about 30% of the imine moles in the polyamide. The molar ratio of malic acid to succinic acid is 1:1. Adjust the pH of the polyamide solution to about 5.2 with 6mol / L hydrochloric acid solution, then add and stir well. Add 1.46mmol of the first catalyst EDC and 1.46mmol of the auxiliary agent NHS and continue stirring until homogeneous. Seal and place in a 30℃ forced-air drying oven for 14h.

[0117] 2) Take out the first cross-linking reaction product from step 1), adjust the pH of the system to 4.8, and place it in a 20℃ constant temperature oven. Then weigh 98 mg of the second catalyst, succinic anhydride (0.98 mmol), add it to the reaction system, stir evenly, and then place it in a 20℃ oven for 4 h to obtain polyamide hydrogel.

[0118] 3) After the reaction, the obtained hydrogel was immersed in 100 times PBS (pH=7.40, 0.01M) for purification. The solution was changed every 1 hour for a total of 6 times to remove unreacted cross-linking agents and catalysts. The hydrogel particles were then pulverized using a high-shear disperser. After pulverization, the hydrogel was reconstituted with 10 mg / mL pH 7.4 phosphate buffer to a concentration of 20 mg / mL (i.e., before sterilization). The hydrogel was then filled into pre-filled syringes and sterilized by moist heat at 121℃ for 15 min (i.e., after sterilization) to obtain the final product hydrogel.

[0119] Example 6: Preparation of polyamide hydrogel by crosslinking with fumaric acid

[0120] 1) Weigh 1.0 g of poly(succinic acid-spermine) polyamide material (3.64 mmol imine moles), then add 10 mL of purified water. After complete dissolution, the concentration of poly(succinic acid-spermine) is 100 mg / mL. Add 254 mg of fumaric acid (2.19 mmol moles, the crosslinking agent accounts for about 60% of the imine moles in the polyamide) to the solution. Adjust the pH of the polyamide solution to about 5.0 with 6 mol / L hydrochloric acid solution, then add and stir well. Add 3.28 mmol of the first catalyst EDC and 3.28 mmol of the auxiliary agent NHS and continue stirring until uniform. Seal and place in a 30℃ forced-air drying oven for 14 h.

[0121] 2) Take out the first cross-linking reaction product from step 1), adjust the pH of the system to 4.8, and place it in a 20℃ constant temperature oven. Then weigh 262 mg of the second catalyst, succinic anhydride (2.62 mmol), add it to the reaction system, stir evenly, and then place it in a 20℃ oven for 4 h to obtain polyamide hydrogel.

[0122] 3) After the reaction, the obtained hydrogel was immersed in 100 times PBS (pH=7.40, 0.01M) for purification. The solution was changed every 1 hour for a total of 6 times to remove unreacted cross-linking agents and catalysts. The hydrogel particles were then pulverized using a high-shear disperser. After pulverization, the hydrogel was reconstituted with 10 mg / mL pH 7.4 phosphate buffer to a concentration of 20 mg / mL (i.e., before sterilization). The hydrogel was then filled into pre-filled syringes and sterilized by moist heat at 121℃ for 15 min (i.e., after sterilization) to obtain the final product hydrogel.

[0123] Comparative Example 1-A: One-step malic acid cross-linking poly(malic acid-spermidine)

[0124] Weigh 1.0 g of poly(malic acid-spermidine) polyamide material (3.83 mmol imine moles) and add 10 mL of purified water. After complete dissolution, the concentration of poly(malic acid-spermidine) is 100 mg / mL. Add 143 mg of malic acid (1.07 mmol moles, the crosslinking agent mole accounts for approximately 28% of the imine moles in the polyamide) to the solution, dissolve thoroughly, and mix well. Then adjust the pH of the polyamide solution to approximately 5.2 with 6 mol / L hydrochloric acid solution. Subsequently, add 3.21 mmol EDC catalyst and 3.21 mmol NHS additive and continue stirring until homogeneous. Seal and place in a 30°C forced-air drying oven for 14 h. Afterward, remove the system and observe that it remains in a solution-like, non-gel state, indicating a low degree of crosslinking.

[0125] Comparative Example 1-B: One-step malic acid cross-linking poly(malic acid-spermidine)

[0126] 1) Weigh 1.0g of poly(malic acid-spermidine) polyamide material (3.83 mmol of imine moles), then add 10mL of purified water. After complete dissolution, the concentration of poly(malic acid-spermidine) is 100mg / mL. Add 143mg of malic acid (1.07 mmol of moles, the crosslinking agent accounts for about 28% of the imine moles in the polyamide) to the solution. After thorough dissolution, mix evenly. Then adjust the pH of the polyamide solution to about 5.2 with 6mol / L hydrochloric acid solution. Subsequently, add 5.3mmol of EDC catalyst and 5.3mmol of NHS additive and continue stirring evenly. Seal and place in a 30℃ forced-air drying oven for 14h.

[0127] Step 2) is the same as step 3) in Example 1.

[0128] Comparative Example 2: High-Temperature Dehydration Method

[0129] Weigh 1.0 g of poly(succinic acid-spermine)polyamide material (4.06 mmol of imine moles) into a round-bottom flask, then add 164 mg of malic acid (1.07 mmol of moles, the crosslinking agent accounts for about 26% of the imine moles in the polyamide), mix thoroughly, and heat to 180 °C under nitrogen protection. The system turns brownish-yellow. After cooling, dissolve the product in water to obtain a still flowing solution, indicating that the crosslinking reaction did not occur and polyamide hydrogel could not be obtained.

[0130] Comparative Example 3: Concentrated Sulfuric Acid Catalytic Method

[0131] Weigh 1.0 g of poly(succinic acid-spermine) polyamide material (4.06 mmol of imine moles) into a round-bottom flask, then add 164 mg of malic acid (1.07 mmol of moles, the crosslinking agent accounts for about 26% of the imine moles in the polyamide), mix thoroughly, add 150 mg of concentrated sulfuric acid, react at room temperature for 2 h, then add sodium hydroxide aqueous solution to adjust the pH to neutral. It was observed that the system was still a flowing solution, indicating that no crosslinking reaction occurred and polyamide hydrogel could not be obtained.

[0132] Comparative Example 4: Reaction catalyzed by succinic anhydride

[0133] Weigh 1.0 g of poly(succinic acid-spermine) polyamide material (4.06 mmol of imine moles) into a round-bottom flask, then add 164 mg of malic acid (1.07 mmol of moles, the crosslinking agent accounts for about 26% of the imine moles in the polyamide), mix thoroughly, adjust the pH of the system to 4.0, then weigh 321 mg of succinic anhydride (3.21 mmol of moles) and add it to the reaction system for catalytic crosslinking, stir evenly, and then place it in a 20°C oven for 4 h. The system is still in solution state, the degree of crosslinking is too low, and it cannot form a hydrogel.

[0134] Test Example 1: Rheological property testing of cross-linked polyamide hydrogels

[0135] The polyamide hydrogels obtained in Examples 1-6 and Comparative Example 1-B were divided into two types: pre-sterilization and post-sterilization. 2.0 mL samples were taken from each type, and the elastic modulus (G') of the hydrogels was measured using a TA DHR-2 plate rheometer. The elastic modulus loss rate was calculated. The G' loss rate was calculated using the following formula:

[0136] G' change rate = (G' after sterilization - G' before sterilization) / G' before sterilization;

[0137] The rheometer parameters were as follows: operating gap: 1000 μm, loading gap: 45000 μm, operating temperature: 37℃, deformation: 1%, frequency: 0.9 Hz, and running time: 60 s. The rheological data of the product samples prepared in each embodiment and comparative example are shown in Table 1.

[0138] Table 1: Rheological data of hydrogels

[0139] The G' change rate can be considered an indicator of the change in the degree of cross-linking of the hydrogel during sterilization, and it reflects the stability of the hydrogel during sterilization. As can be seen from the data in Table 1, the G' change rate is related to the polyamide matrix, the cross-linking agent, and the sterilization method.

[0140] The samples in Examples 1-6 all had high elastic moduli before and after sterilization, indicating that the water-soluble polyamide had undergone an effective cross-linking reaction and formed a gelled polyamide material.

[0141] As can be seen from Example 1 and Comparative Example 1, the hydrogel product of Example 1 has a significantly higher elastic modulus than that of Comparative Example 1-B before sterilization. This is because Example 1 uses a dual-catalytic crosslinking system of EDC and succinic anhydride, which can not only effectively activate the carboxyl groups but also improve the reactivity of the imine groups, significantly improving the crosslinking efficiency of the polyamide. The elastic modulus of the hydrogel product is as high as 1256.6 Pa. In contrast, in Comparative Example 1, the crosslinking efficiency is low due to the use of only a single catalyst system. In Comparative Example 1-B, only EDC is used for crosslinking. Even if the amount of catalyst is increased to 7 times that of Example 1, the crosslinking efficiency is still low because the poly(malic acid-spermidine) backbone only has imine groups with weak reactivity. Therefore, the elastic modulus of the hydrogel product is low (789.2 Pa). Moreover, due to the addition of a large amount of catalyst EDC and auxiliary agent NHS, there are too many auxiliary agents in the system, making it difficult to purify the resulting gel. A more complex post-treatment removal process is required, which increases the purification process cost. The residual auxiliary agents are detrimental to biological applications and have biotoxicity. The residual auxiliary agents in the resulting gel will affect its biological application.

[0142] Furthermore, the gels of Examples 1, 2, and Comparative Example 1-B were purified and sterilized using the same method, and their cytotoxicity was tested (the detection method was the same as the cell proliferation assay in Test Example 3). The data are shown in Table 2. It can be seen that after the same soaking and purification treatment, the polyamide hydrogels of Examples 1 and 2 were free of cytotoxicity, while the purified gel in Comparative Example 1-B still had high cytotoxicity (cell proliferation rate was only 11.5%). Further, after extracting the gels obtained in Examples 1 and Comparative Example 1-B, the absorbance of NHS at 260 nm was tested using spectrophotometry. The NHS concentration in the gel of Example 1 was determined to be 1 μg / ml, while the residual amount in the gel of Comparative Example 1-B was 46 μg / ml. Therefore, it can be determined that the residue of the additives caused cytotoxicity. The residue of additives is detrimental to biological applications and has biotoxicity; the residual additives in the resulting gel will affect its biological application.

[0143] Table 2: Cytotoxicity test results

[0144] Meanwhile, after sterilization, due to the low crosslinking efficiency of Comparative Example 1-B, there is a lot of free polyamide in the system. Under the influence of a large number of catalysts, additives and external factors such as dialysis to remove impurities, the monomers are more easily destroyed, resulting in a significant reduction in elastic modulus and a loss rate as high as 64%.

[0145] In Comparative Example 1-A, which also uses a single catalytic system, the amount of crosslinking agent is almost twice that of Example 1, and the amount of EDC catalyst is almost five times that of the catalyst in Example 1. However, after the reaction, it is still in a solution state rather than a hydrogel state. This is because the imine groups in poly(malic acid-spermidine) have low activity, large crosslinking steric hindrance, and low degree of freedom on the main chain, making it difficult to achieve effective crosslinking using this method. This also shows that even if the amount of crosslinking agent and EDC catalyst is increased (such as in Comparative Example 1-A and Comparative Example 1-B), it is still impossible to obtain a hydrogel product with a high degree of crosslinking (effective crosslinking of the carboxyl double arms) and good stability in a one-step crosslinking system.

[0146] Figure 2 further shows photographs of the tilted state of the final products of Example 1 (left) and Comparative Example 1-A (right). It can be clearly seen that Example 1 on the left is in a gel state, with a certain degree of opacity and obvious gel-like adhesion; while the product of Comparative Example 1-A on the right is in a transparent solution state, with obvious fluidity and light transmittance, which also indicates that the comparative example did not form a polyamide gel. This is mainly due to the weak reactivity of imine groups and the failure to undergo effective cross-linking.

[0147] Comparative Examples 2 and 3 employed high-temperature and concentrated sulfuric acid dehydration crosslinking processes, respectively, but neither yielded polyamide hydrogels. This is because the main chain monomer of poly(succinic acid-spermidine) is quite sensitive and is prone to breakage of the polyamide main chain under harsh reaction conditions. As a result, the product exhibits a flowing, non-crosslinked solution state, and this process cannot form crosslinked hydrogel products.

[0148] In Comparative Example 4, a mild succinic anhydride was used for a one-step crosslinking reaction. Because the anhydride is prone to rapid hydrolysis and has a short reaction window, and given the low activity of the imine groups in the polyamide, the anhydride cannot quickly and effectively activate the carboxyl functional groups in the crosslinking agent. This results in the carboxyl groups not being effectively activated, or failing to form amide bonds with the imine groups before the catalyst, succinic anhydride, hydrolyzes and becomes deactivated, preventing an effective crosslinking reaction to form the polyamide gel backbone. Therefore, the product also fails to form a crosslinked gel and remains a flowing solution.

[0149] This also explains that the first step of the present invention, activation crosslinking, cannot directly use succinic anhydride, but must first use EDC with a longer activation carboxyl window for catalysis. This can effectively avoid the problem of anhydride hydrolysis and deactivation, which would lead to the loss of catalytic performance. Furthermore, in the second step of catalytic crosslinking, succinic anhydride catalysis is used to form an intermediate active body with the crosslinking product of the first step, which can improve the stability of the anhydride bond and delay hydrolysis. This ensures that the product can exert its catalytic activity. Moreover, the product after the crosslinking reaction still contains biologically active endogenous succinic acid, which will not have a negative impact or additional burden on the monomer stability, impurity removal and other processes in the system.

[0150] The effectiveness of the two-step crosslinking process of the present invention can be verified by Examples 1-2 and Comparative Examples 1-4. Furthermore, as can be seen from Table 1, the present invention can intelligently control the monomer release rate of polyamide hydrogel by adjusting the type of crosslinking agent of endogenous diacid and polyamide monomer according to their different reactivity, thereby adapting to the application needs of various scenarios, enriching the application range of polyamide hydrogel, and facilitating the formation of diversified polyamide hydrogel products with different properties.

[0151] As can be seen from Examples 1 and 3, when containing the same highly reactive monomer polyamide molecule (malic acid-spermidine), the hydrogel of Example 1, crosslinked with a highly reactive crosslinking agent (malic acid), has a significantly higher elastic modulus (1256.6 Pa) than the hydrogel of Example 3, which uses a moderately reactive crosslinking agent (succinic acid) (987.3 Pa). Even with an increased proportion of crosslinking agent, the elastic modulus is still lower than that of Example 1. This is because malic acid has relatively high activity and more hydrophilic groups. The hydrogel of Example 1 contains a large number of amide structures and hydroxyl groups derived from the side groups of malic acid, which allows the hydrogel to lock in more water and has good water-locking properties. Therefore, the water in the network structure of this hydrogel makes it harder and has a higher elastic modulus. However, the G' loss of this hydrogel during the sterilization process is greater (53%), which is higher than the loss rate of Example 3 (33%). This is because the relatively reactive malic acid monomer is more easily transformed by external light, electricity, heat, and enzymes, resulting in a significant decrease in elastic modulus after sterilization.

[0152] Comparing Examples 1 and 2, it can be found that both use malic acid, a highly active crosslinking agent, to create polyamide crosslinked hydrogels. Compared with Example 1, the polyamide backbone in Example 2 has fewer hydrophilic structures, resulting in reduced water-locking ability and a lower elastic modulus and a softer hydrogel (even with an increased amount of crosslinking agent). However, the activity of succinic acid monomer in the polyamide is lower than that of malic acid in Example 1. Therefore, during high-temperature sterilization, it is less susceptible to light, electricity, heat, and enzymes, reducing the risk of chemical bond breakage in the polyamide backbone. Consequently, the polymer is less prone to depolymerization and release of active monomers, resulting in a lower elastic modulus loss rate and enhanced hydrogel stability.

[0153] In summary, this invention allows for the intelligent design of gels by adjusting the types of crosslinking agents and polyamide active monomers to obtain gel materials with different properties. Specifically, polyamide hydrogels with a larger G' change rate degrade faster and are suitable for wound repair, releasing monomers quickly to achieve anti-inflammatory effects and promote cell function and wound healing. Polyamide hydrogels with a smaller G' change rate exhibit high stability and slower degradation, making them suitable for tissue filling. By slowly releasing monomers, they improve the state of cells at the filling site, thereby promoting collagen production and improving skin elasticity.

[0154] In Example 4, the polyamide contains a moderately active polyacid monomer polyamide host molecule (succinic acid), which is further cross-linked using a moderately active cross-linking agent succinic acid. The resulting hydrogel has strong stability, with an elastic modulus loss rate of only 8% after sterilization. The polyamide hydrogel in Example 6 has an elastic modulus loss rate of 15% after sterilization. This type of polyamide hydrogel material with high elastic modulus, high hardness, and high stability is more suitable for deep, long-lasting soft tissue filling.

[0155] Furthermore, in Example 5, a combination of malic acid and succinic acid, two monomers with medium and high activity, was used to crosslink the polyamide. Compared with Example 1, although the elastic modulus of the hydrogel product was slightly lower before sterilization due to the monomer activity, the stability of the hydrogel was significantly enhanced, and the loss rate was reduced to 25%. This also proves that the present invention, by using two crosslinking agents with different activities, can meet the needs of different application sites, regulate the degradation performance of the hydrogel, and program the hydrogel according to the application requirements to prepare intelligent hydrogels that meet the requirements of application scenarios.

[0156] In addition, to fully illustrate the influence of different raw material monomers and crosslinking agent types on the performance of polyamide hydrogel products, the present invention also prepared the following samples and tested the mechanical properties and sterilization stability of the gel products according to the conditions of Test Example 1. The results are shown in Table 3.

[0157] Sample 1: The other steps are the same as in Example 4, except that the crosslinking agent is 0.82 mmol of malic acid (accounting for 20% of the molar number of imine groups in the polyamide).

[0158] Sample 2: The other steps are the same as in Example 4, except that: the raw material is 1g of poly(malic acid-spermine) polyamide material (3.83 mmol of imine moles), the amount of crosslinking agent malic acid is 0.77 mmol (accounting for 20% of the imine moles in the polyamide), the amount of the first catalyst EDC is 0.916 mmol, the amount of the auxiliary agent NHS is 0.916 mmol, and the amount of the second catalyst succinic anhydride is 0.916 mmol.

[0159] Table 3: Variation of Elastic Modulus with Different Raw Materials and Crosslinking Agents

[0160] It can be seen that, with other conditions remaining unchanged, the effect of changing only the type of polyamide and crosslinking agent on the mechanical properties and stability of the hydrogel product is consistent with the conclusions in Test Example 1. That is, when the polyamide is the same, the hydrogel prepared in Sample 1, which uses a highly active crosslinking agent and is rich in hydrophilic groups, has a higher elastic modulus than the hydrogel prepared in Example 4 using the moderately active crosslinking agent, succinic acid. However, due to the high activity of malic acid, it is prone to transformation during sterilization, resulting in a higher rate of change in G'. When the type of crosslinking agent is the same, compared with Sample 2, Sample 1 contains poly(malic acid-spermidine), which has high monomer activity and is rich in hydrophilic groups. The hydrogel prepared in Sample 1 has a higher elastic modulus, but poorer stability, and a slightly higher rate of change in G'.

[0161] Test Example 2: Degradation Experiment of Crosslinked Polyamide Hydrogel

[0162] The purpose of this experiment is to simulate the degradation process of cross-linked polyamide hydrogel in vivo and explore the changes in monomer release after implantation. This experiment can also prove that the cross-linked hydrogel in this invention can be controlled to degrade in vivo, continuously release active molecules, and produce active effects.

[0163] Take 2 mL of each of the finished hydrogel samples from Examples 1, 4, and 5, seal them in vials, and place them in physiological saline at 37°C in a forced-air drying oven for hydrolysis experiments (simulating in vivo degradation experiments). In the early stage, take 3 parallel samples every 3 days, and in the later stage, take 3 parallel samples every 5 days. After cooling to about 25°C, test their elastic modulus (test method is the same as in Example 1). The results are shown in Table 4.

[0164] As can be seen from Table 4, the degradation rates of the hydrogels obtained in the three examples are consistent with those before and after sterilization in Test Example 1. The hydrogel made of polyamide with malic acid as the main chain and crosslinking agent in Example 1 has the fastest degradation rate due to the high activity of malic acid. The hydrogel made of polyamide with succinic acid as the main chain and crosslinking agent in Example 4 has the slowest degradation rate due to the slightly lower activity of succinic acid.

[0165] In Example 5, the degradation rate of the hydrogel can be adjusted by using a mixture of two crosslinking agents compared to Example 1, and the retention rate of the hydrogel's elastic modulus after 30 days is between that of Example 1 and Example 4.

[0166] Table 4: Degradation results of cross-linked polyamide hydrogels (G' change)

[0167] Another hydrogel sample obtained in Example 5 was used to monitor the release concentration of small molecules (malic acid, spermidine, and succinic acid) during the hydrogel degradation process. The results are shown in Table 5. Monitoring the release of small molecules from the hydrogel in Example 5 revealed a continuous release of small molecules during degradation, thus demonstrating its bioactivity. This further proves that the hydrogel of this invention, prepared using biocompatible polyamide and endogenous dicarboxylic acid, is a cross-linked polyamide hydrogel capable of releasing active small molecules. Furthermore, a suitable preparation method allows for intelligent control and design of the hydrogel's degradation rate, thereby achieving effective control over the hydrogel's bioactivity.

[0168] Table 5: Results of cross-linked hydrogel monomer release monitoring experiment in Example 5

[0169] Test Example 3: Cross-linked polyamide hydrogel cell culture experiment

[0170] The hydrogel from Example 5 was used for cellular bioactivity testing: the sterilized final product of the hydrogel from Example 5 was diluted 1:5 with DMEM complete medium to form the test sample group, and the DMEM complete medium group from the same day served as the blank control. Each group had 6 wells, and the plates were placed in a 37°C, 5% CO2 saturated humidity incubator to prepare three 96-well plates, which were cultured for 24, 48, and 72 hours, respectively. After culture, the cell culture plates were removed, and the cell state was observed and recorded using a microscope (as shown in Figure 3). The medium was then discarded, and 100 mL of CCK-8 detection solution was added to each well. The plates were then placed in an incubator and cultured for another 2 hours. The absorbance (OD value) at 450 nm was measured using a microplate reader, and the cell viability at different culture days was calculated (as shown in Table 6). As shown in Figure 3, the cell experiment micrographs show that the experimental group (Example 5) has a denser cell number and more regular cell shape compared to the control group. This indicates that the polyamide hydrogel can improve cell state and enhance cell viability. Meanwhile, the CCK-8 test data in Table 6 also proves that the hydrogel has the effect of promoting cell proliferation, indicating that the polyamide of the present invention has good biological activity.

[0171] Table 6: Results of Cell Proliferation Activity Detection

[0172] Test Example 4: Animal Experiment

[0173] Four male Japanese White rabbits, aged 6 months, were implanted with the sterilized gel from Example 4. After a week of acclimatization, and once no obvious abnormalities or signs of disease were observed, the experiment was conducted. The rabbits were fasted for one day before the procedure but allowed free water. Before the procedure, the animals were anesthetized and, once the anesthesia was stable, were fixed in a prone position and continuously anesthetized with a gas anesthesia machine. Hair in the implantation area was removed, and the surgical area was thoroughly disinfected. Four injection sites were selected on each rabbit's back, and the gel was injected evenly from these sites, with an injection volume of 2 mL per animal. Two and four weeks after injection, tissue samples were dissected from the implantation area. The tissue was fixed in formaldehyde, decalcified, embedded in paraffin, and routinely sectioned. H&E staining and Masson's trichrome staining were performed to observe the tissue reaction and collagen production in the implantation area.

[0174] As shown in Figure 4, significant cell ingrowth occurred after implantation of the polyamide gel, and numerous new blood vessels (indicated by the arrow ↑) were observed within the implant. The role of angiogenesis includes promoting tissue repair and regeneration, improving the biocompatibility of the material, and enhancing its functionality. As shown in Figure 5, moderate amounts of newly formed collagen were visible within the polyamide gel implant (indicated by the arrow ↑, gray area). These animal implantation data demonstrate both the safety of the material and its effective promotion of tissue repair, fostering the formation of new collagen and blood vessels.

[0175] In summary, this invention develops a novel crosslinking system for novel hydrophilic polyamide materials. The polyamide backbone is composed of endogenous polyamines (such as spermine and spermine) and endogenous polyacids (such as malic acid and succinic acid) as basic units. Compared to traditional polyamides containing primary amine groups (-NH2), the active groups in this polymer backbone are less reactive imine groups (-NH-), and the polymer backbone has greater steric hindrance, making the crosslinking reaction of this novel polyamide material difficult and hindering gel formation. Furthermore, traditional polyamide crosslinking technologies suffer from harsh reaction conditions (such as high temperature, high pressure, concentrated sulfuric acid, etc.) and short reaction windows, which can lead to the oxidation and decomposition of endogenous polyacid monomers in the polyamide backbone, resulting in chain breakage, easy degradation of the gel product, and inability to maintain stability.

[0176] The "two-step activated crosslinking technology" (TACT) of this invention is particularly suitable for this type of novel endogenous polyamide. By using endogenous diacids (such as malic acid and succinic acid) as crosslinking agents, combined with carbodiimide / succinic anhydride stepwise catalysis, an activated crosslinking system is formed. This achieves highly efficient crosslinking of polyamides under mild conditions (avoiding high temperatures and concentrated sulfuric acid), significantly improving crosslinking efficiency, reducing the amount of catalyst and crosslinking agent used, and allowing for precise control of hydrogel properties (such as degradation rate and release of active monomers) by adjusting the type and conditions of the crosslinking agent. This enables the hydrogel to exert physiological activities such as anti-inflammatory, antioxidant, wound healing promotion, and anti-aging effects, thereby meeting diverse clinical needs. Furthermore, the raw materials, monomers, and crosslinking agents of this invention are all endogenous polyamines or endogenous carboxylic acids; therefore, the resulting polyamide hydrogel exhibits excellent biocompatibility and safety.

[0177] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0178] The foregoing embodiments and methods described in this invention may vary based on the capabilities, experience, and preferences of those skilled in the art.

[0179] The fact that the steps of the method are listed in a certain order in this invention does not constitute any restriction on the order of the method steps.

Claims

1. A cross-linked polyamide hydrogel, characterized in that, The hydrogel is obtained by crosslinking water-soluble polyamide and crosslinking agent; the water-soluble polyamide includes poly(malic acid-spermine), poly(malic acid-spermine), poly(succinic acid-spermine), or poly(succinic acid-spermine); The crosslinking agent is an endogenous dicarboxylic acid, selected from one or more of malic acid, succinic acid, itaconic acid, fumaric acid, and α-ketoglutaric acid.

2. The cross-linked polyamide hydrogel according to claim 1, characterized in that, The crosslinking reaction refers to a two-step activation crosslinking reaction between the crosslinking agent, activated by a catalyst, and the water-soluble polyamide to obtain a hydrogel.

3. The cross-linked polyamide hydrogel according to claim 2, characterized in that, The catalyst includes a first catalyst and a second catalyst; The first catalyst is selected from: carbodiimide, N,N'-carbonyldiimidazole, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, 4-dimethylaminopyridine, or a salt solution thereof; The second catalyst is succinic anhydride.

4. A method for preparing a cross-linked polyamide hydrogel, characterized in that, The preparation method includes the following steps: 1) Add a crosslinking agent to a water-soluble polyamide solution, adjust the pH of the system, add the first catalyst, and carry out the first activation crosslinking reaction; 2) Adjust the pH value of the product from step 1), add the second catalyst, and carry out the second activation crosslinking reaction to obtain the hydrogel; The water-soluble polyamide includes poly(malic acid-spermidine), poly(malic acid-spermidine), poly(succinic acid-spermidine), or poly(succinic acid-spermidine); The crosslinking agent is an endogenous dicarboxylic acid, selected from one or more of malic acid, succinic acid, itaconic acid, fumaric acid, and α-ketoglutaric acid.

5. The preparation method according to claim 4, characterized in that, The water-soluble polyamide solution has a mass concentration of 15-300 mg / mL, preferably 60-120 mg / mL; The molar number of the crosslinking agent is 5-60% of the molar number of imine groups in the water-soluble polyamide.

6. The preparation method according to claim 4, characterized in that, The first catalyst is selected from: carbodiimide, N,N'-carbonyldiimidazole, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, 4-dimethylaminopyridine, or a salt solution thereof; The number of moles of the first catalyst is 1.1 to 1.5 times the number of moles of the crosslinking agent.

7. The preparation method according to claim 6, characterized in that, In step 1), the pH value is 5.0-6.5, preferably 5.0-6.0; In step 1), the first activation crosslinking temperature is 25-60℃ and the time is 10-20h.

8. The preparation method according to claim 6 or 7, characterized in that, In the first activation crosslinking reaction of step 1), an auxiliary agent is also added, selected from: N-hydroxysuccinimide, N-hydroxysulfosuccinimide, tert-butanol, and 1-hydroxybenzotriazole; The molar number of the auxiliary agent is 1.1-1.5 times the molar number of the crosslinking agent; Preferably, the molar ratio of the first catalyst to the auxiliary agent is 1:(0.5-2).

9. The preparation method according to claim 4, characterized in that, The second catalyst is succinic anhydride; In step 2), the number of moles of the second catalyst is 0.7-1.3 times the number of moles of the crosslinking agent; Preferably, in step 2), the pH value is 4-5.2, more preferably 4.6-5.2; Preferably, in step 2), the second activation crosslinking temperature is 5-25°C and the time is 1-4 hours.

10. The use of the cross-linked polyamide hydrogel according to any one of claims 1-3 or the cross-linked polyamide hydrogel prepared by the preparation method according to any one of claims 4-9 in the preparation of tissue engineering materials, drug carriers, or products for medical aesthetic and / or cosmetic purposes; Preferably, the tissue engineering material includes wound dressings, biological patches, tissue adhesives, tissue engineering scaffolds, or tissue fillers.

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