Injectable cross-linked collagen gel and preparation method thereof

Abstract

The invention relates to injectable cross-linked collagen gel and a preparation method thereof. Two crosslinking strategies of self-crosslinking and crosslinking agent crosslinking are fused together for the first time, and two types of amido bonds, namely amido bonds of a collagen-collagen structure (A) and amido bonds of a collagen-fatty amine-collagen structure (B), are respectively constructed through two crosslinking paths, namely catalysis of collagen polypeptide self-crosslinking and catalysis of fatty amine crosslinking, so that a hard / soft dual crosslinking network is obtained. The relative proportion of the structure A and the structure B in the final product is adjusted through a proper cross-linking process, so that the strength and the stability of the final product are adjusted and controlled, and the use requirements of different injection parts can be met.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials, specifically to an injectable cross-linked collagen gel and its preparation method. Background Technology

[0002] With the development of medical technology, the use of minimally invasive injectable fillers to correct skin wrinkles and depressions has gradually become accepted by the public. Clinically widely used fillers can be broadly categorized into sodium hyaluronate, collagen, and composite implants. Among these, collagen, a natural protein and a major component of skin tissue, is a relatively ideal material for correcting facial soft tissue defects. However, collagen suffers from immunogenicity, poor stability, and short retention time in the body, generally requiring chemical cross-linking to extend its lifespan.

[0003] Generally, there are two chemical cross-linking strategies for collagen materials: one is to not introduce other components and only cross-link the amino and carboxyl groups on the polypeptide chain; the other is to introduce a cross-linking agent and cross-link it with the amino or carboxyl sites on the polypeptide chain.

[0004] Peptide bonds in the polypeptide backbone possess some double bond properties, preventing arbitrary rotation. Therefore, polypeptide molecules are often rigid and susceptible to external environmental factors such as high temperature, high pressure, storage, and transportation, leading to easy chain breakage and poor product stability. In self-crosslinking strategies, whether crosslinking between polypeptide molecules or between amino and carboxyl groups, the resulting products, while exhibiting high modulus, lack flexibility and fail to meet the injectability requirements of hydrogels. Patent CN114470330B reports a collagen gel microparticle for tissue filling and its preparation method. This patent uses EDC as a crosslinking agent to achieve collagen self-crosslinking; however, a complex homogenization process is required to pulverize the crosslinked gel to achieve injectability. Furthermore, the system exhibits self-crosslinking of collagen, forming a strong rigid structure. Under high temperature or external force, this rigid structure is more prone to breakage, resulting in a rapid decrease in the gel's elastic modulus and ultimately weak product stability and support. Patent CN117582550A reports a lubricating injectable collagen gel and its preparation method. In this patent, to make the prepared hydrogel injectable, not only is homogenization and pulverization required, but also the addition of sodium hyaluronate solution for lubrication. Therefore, introducing a suitable crosslinking agent to balance the properties of the gel material may be a more effective modification strategy. Summary of the Invention

[0005] To address the shortcomings of current collagen crosslinking processes, this invention provides a novel crosslinking strategy (such as...) tailored to the characteristics of collagen materials. Figure 1As shown in the diagram, BHSPN (Bicontinuous Hard and Soft Phase Networks) is formed. This invention is the first to combine self-crosslinking and crosslinking agent crosslinking strategies. Through two crosslinking pathways—catalyzing collagen peptide self-crosslinking and catalyzing fatty amine crosslinking—two types of amide bonds are constructed: collagen-collagen and collagen-fatty amine-collagen amide bonds, resulting in a hard / soft dual crosslinked network. This invention utilizes the hard phase network formed by self-crosslinking as sacrificial bonds to dissipate external stress. During moist heat sterilization and product storage, it protects the soft phase network formed by crosslinking agent crosslinking, thereby maintaining the final product's support. The protected soft phase crosslinked network increases molecular chain length and winding pattern to improve the flexibility of the final product. This invention employs a fatty amine compound as a crosslinking agent. Under the action of a catalyst, and by optimizing the catalytic crosslinking reaction process, it achieves the construction of both collagen-collagen structure (A) and collagen-fatty amine-collagen structure (B) in one step. This maximizes the conversion rate of carboxyl groups on the polypeptide chain, reduces the disadvantage of low molecular weight of collagen polypeptide materials, and improves the retention time of gel products in vivo. Simultaneously, by adjusting the amount of catalyst and crosslinking agent, the relative proportions of structures A and B in the system can be controlled, thereby achieving control over the material's support, flexibility, and product stability.

[0006] In a first aspect, the present invention provides an injectable crosslinked collagen gel comprising collagen and a fatty amine crosslinking agent, which undergo a crosslinking reaction under the action of a catalyst. The collagen gel has a bicontinuous hard and soft phase network (BHSPN) formed by self-crosslinking and crosslinking with the crosslinking agent.

[0007] The collagen concentration in the collagen gel is 15 mg / mL to 60 mg / mL, for example, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL or 60 mg / mL, etc.

[0008] The particle size D50 of the injectable cross-linked collagen gel is 30–300 μm.

[0009] The particle size D90 of the injectable cross-linked collagen gel is 50–400 μm.

[0010] The collagen can be recombinant collagen or natural collagen.

[0011] The molecular weight of the collagen can be arbitrary, such as 1000 to 300000 kDa.

[0012] The natural collagen can be derived from horses, pigs, cattle, sheep, and chickens.

[0013] Preferably, the collagen is recombinant collagen; more preferably, it is recombinant human collagen or recombinant human collagen.

[0014] Preferably, the collagen is selected from type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type IX collagen, type X collagen, type XI collagen, type XII collagen, type XIII collagen, type XIV collagen, type XV collagen, type XVI collagen, type XVII collagen, and type XVIII collagen. More preferably, it is type I, type II, type III, or type XVII collagen.

[0015] The carbon chain length of the aliphatic amine crosslinking agent is 5 to 20, for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably 7 to 10.

[0016] The aliphatic amine crosslinking agent is a polyamine, preferably an endogenous polyamine, such as putrescine, spermine, spermidine, or their derivatives.

[0017] Preferably, the carboxyl group of collagen forms an amide bond with the amino group of the fatty amine crosslinking agent.

[0018] Preferably, the catalyst comprises one or more of carbodiimide, triphenylphosphine bromide, carbomonium salt, or 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM).

[0019] Catalysts can activate the carboxyl groups in collagen, causing them to react with their own amino groups to form a self-crosslinking structure, and / or cause them to react with the amino groups of fatty amine crosslinking agents to form a collagen-fatty amine-collagen structure.

[0020] More preferably, the molar ratio of the catalyst to the carboxyl groups in collagen is 1.0 to 3.0, such as 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, or 3.0, with 1.5 to 2.5 being the most preferred.

[0021] Preferably, the molar ratio of the carboxyl groups of the fatty amine crosslinking agent to collagen is 0.2 to 1.2, more preferably 0.3 to 1.

[0022] More preferably, the molar ratio of the catalyst to the aliphatic amine crosslinking agent is 1.5 to 5.0. For example, 1.5, 1.6, 2.0, 2.4, 2.5, 2.6, 2.8, 3.0, 3.3, 3.5, 3.7, 3.9, 4.0 or 5.0, etc.

[0023] In a system using an aliphatic polyamine crosslinking agent and a catalyst, collagen materials can achieve both continuous and dynamic crosslinking reactions. This is because the catalyst can activate carboxyl groups, initiating both the self-crosslinking reaction of collagen and catalyzing the crosslinking reaction of the crosslinking agent. Simultaneously, both collagen-collagen structure (A) and collagen-aliphatic amine-collagen structure (B) can be constructed. By controlling the amounts of crosslinking agent and catalyst, the A and B structures in the collagen gel can be in a suitable relative proportion, thereby comprehensively improving the performance of the gel material. This avoids the technical problems caused by differences in the crosslinking conditions of other crosslinking agents compared to the collagen self-crosslinking conditions (such as temperature and pH), resulting in low crosslinking efficiency, difficulty in controlling the crosslinking process, and the inability to obtain a material containing a gel with a suitable relative proportion of A and B structures in a single reaction.

[0024] The crosslinking reaction is carried out at a temperature of 20–40°C, such as 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, or 40°C, preferably at room temperature.

[0025] Preferably, the crosslinking reaction time is 1 to 20 hours, such as 1 hour, 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, or 20 hours, with 5 hours to 10 hours being the most preferred.

[0026] In some specific embodiments, the catalyst is a carbodiimide, such as dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

[0027] Preferably, when the catalyst is carbodiimide, the crosslinking reaction further includes an auxiliary agent selected from one or more of N-hydroxysuccinimide (NHS), sulfonated N-hydroxysuccinimide (Sulfo-NHS), tert-butanol, and 1-hydroxybenzotriazole (HOBt), with NHS being more preferred.

[0028] The mass ratio of the auxiliary agent to the catalyst is 0.01 to 0.5, for example, 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4 or 0.5, preferably 0.1 to 0.3.

[0029] In one embodiment of the present invention, spermidine is used as a crosslinking agent to form a collagen-collagen structure (A) and a collagen-speridine-collagen structure (B'), with the following structural formulas:

[0030]

[0031] In the collagen-collagen structure (A), Col represents collagen.

[0032]

[0033] In the collagen-spermidine-collagen structure (B'), Col represents collagen.

[0034] Preferably, the catalyst solution and the fatty amine crosslinking agent solution are added to the collagen solution to carry out a crosslinking reaction.

[0035] Preferably, the solvent in the fatty amine crosslinking agent solution can be water, physiological saline, etc.

[0036] Preferably, the fatty amine crosslinking agent is dissolved in a solvent, and the pH value is adjusted to obtain a fatty amine crosslinking agent solution. The pH value is 5.5 to 7.0, for example, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 7.0, etc., preferably 5.5 to 6.5.

[0037] The collagen solution is obtained by adding collagen to a solvent, adjusting the pH, and dissolving it.

[0038] Preferably, the collagen is added to a solvent to adjust the pH to 5.5-7.0, such as 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 7.0, etc., with 5.5-6.5 being the most preferred.

[0039] Preferably, when the catalyst is carbodiimide, the solution also contains an auxiliary agent, and the solvent can be water or physiological saline.

[0040] In some specific embodiments, a solution containing a catalyst and auxiliaries is first added to the collagen solution, followed by the addition of a fatty amine crosslinking agent solution.

[0041] The injectable cross-linked collagen gel has advantages such as good injectability, ease of injection, good support, high stability, and good safety, and can be used for subcutaneous injection filling.

[0042] The injectable cross-linked collagen gel of the present invention comprises a self-crosslinked collagen-collagen structure (A) and a collagen-fatty amine-collagen structure (B) formed by crosslinking with a crosslinking agent. At the molecular level, compared to structure B, structure A has shorter molecular chains and greater molecular tension, while the fatty carbon chains of the crosslinking agent in structure B impart greater flexibility to the molecules. The network formed by the aggregation of structures A is a rigid hard-phase network, while the network formed by the aggregation of structures B is a flexible soft-phase network. The two interpenetrate to form a hard / soft double crosslinked network structure; the hard-phase network provides a higher modulus, while the soft-phase network folds or coils and twists, hiding within the hard-phase network. In injectable cross-linked collagen gels, the ability of chain segments in the hard / soft dual cross-linked networks to resist external forces differs. When external force / energy is applied, due to the greater rigidity and brittleness of structure A, the rigid chain segments preferentially reach the stress limit and gradually break. Meanwhile, the flexible long chain segments of structure B, folded or coiled within the cross-linked network, do not break. Therefore, the covalent bonds in structure A can act as sacrificial bonds, protecting structure B and maintaining a high level of mechanical strength in the injectable cross-linked collagen gel. Furthermore, as the rigid chain segments of structure A gradually break, the external force / energy is consumed, releasing the long chain molecules in the folded or coiled structure B to resist the remaining external force / energy, significantly reducing the loss rate of structure B and fully utilizing the advantages of structure B (e.g., Figure 1 As shown in the figure, this allows the collagen gel material to have the dual characteristics of both A and B structures, while minimizing the disadvantages of each, resulting in a product with strong support, good stability, low elastic modulus loss rate, and longer storage time.

[0043] In a second aspect, the present invention provides a method for preparing the injectable cross-linked collagen gel, wherein the preparation method comprises collagen and a fatty amine cross-linking agent, and a cross-linking reaction is carried out under the action of a catalyst.

[0044] The crosslinking includes self-crosslinking and crosslinking agent crosslinking.

[0045] Preferably, the preparation method includes:

[0046] a) Prepare a collagen solution;

[0047] b) Prepare a solution of aliphatic amine crosslinking agent;

[0048] c) Prepare a solution containing the catalyst;

[0049] d) Add the solutions from step b) and c) to the collagen solution from step a) to carry out a cross-linking reaction.

[0050] Step a) preparing the collagen solution includes adding collagen to a solvent, adjusting the pH, and dissolving it.

[0051] Preferably, the collagen is added to a solvent to adjust the pH to 5.5-7.0, such as 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 7.0, etc., with 5.5-6.5 being the most preferred.

[0052] Preferably, the dissolution is carried out under magnetic stirring conditions.

[0053] Preferably, the dissolution temperature in step a) is 20-40℃, such as 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃ or 40℃, and preferably at room temperature.

[0054] Preferably, the dissolution time is 10 to 50 minutes, more preferably 20 to 30 minutes.

[0055] Preferably, the concentration of collagen in the collagen solution in step a) is 70 mg / ml to 150 mg / ml.

[0056] Preferably, step b) preparing the fatty amine crosslinking agent solution includes dissolving the fatty amine crosslinking agent in a solvent and adjusting the pH to 5.5-7.0, such as 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 7.0, etc., preferably 5.5-6.5.

[0057] Preferably, the solution in step c) further includes an auxiliary agent.

[0058] Preferably, the volume of the solution in step b) is equal to that in step c).

[0059] Preferably, steps b) and c) are performed at room temperature.

[0060] Preferably, the molar ratio of the catalyst to the carboxyl groups in the collagen during the cross-linking reaction is 1.0 to 3.0, such as 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5 or 3.0, with 1.5 to 2.5 being the most preferred.

[0061] The molar ratio of the fatty amine crosslinking agent to the carboxyl group of collagen is 0.2 to 1.2. For example, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 or 1.2; preferably 0.3 to 1.

[0062] Preferably, the molar ratio of the catalyst to the aliphatic amine crosslinking agent is 1.5 to 5.0, for example, 1.5, 1.6, 2.0, 2.4, 2.5, 2.6, 2.8, 3.0, 3.3, 3.5, 3.7, 3.9, 4.0 or 5.0.

[0063] The mass ratio of the auxiliary agent to the catalyst is 0.01 to 0.5, for example, 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4 or 0.5, preferably 0.1 to 0.3.

[0064] Preferably, the solvent in steps a), b), and c) can be water, physiological saline, etc.

[0065] This invention optimizes the relative proportions of collagen-collagen structures (A) formed by collagen self-crosslinking and collagen-fatty amine-collagen structures (B) formed by crosslinking agent by adjusting the amount of crosslinking agent added. In the crosslinking system, if too much crosslinking agent is used, the crosslinking reaction is emphasized, resulting in an excess of B structures and a deficiency of A structures in the crosslinked gel, which is detrimental to the protection of B structures and thus leads to poor gel support. Conversely, if too much catalyst is used, the self-crosslinking reaction of collagen is emphasized, resulting in an excess of A structures and a deficiency of B structures in the crosslinked gel. The rigid A structures are prone to breakage, leading to poor gel stability and a hard gel unsuitable for injection. Furthermore, excessive introduction of catalysts and crosslinking agents complicates subsequent purification and impurity removal processes. Therefore, it is necessary to consider not only the amount of catalyst and crosslinking agent but also their ratio. The inventors discovered that the catalyst... When the molar ratio of catalyst to crosslinking agent is in the range of 1.5 to 5.0, the A / B structure ratio in the hard / soft double crosslinking network structure is most suitable. At this point, the rigid A structure can fully exert the sacrificial bond effect to protect the flexible B structure, resulting in high strength and stability of the gel product. If the molar ratio of catalyst to crosslinking agent exceeds 5.0, the relative proportion of B structure is too high, and the rigid A structure is relatively reduced, failing to provide sufficient sacrificial sites to protect the B structure. Therefore, the support or strength of the gel material decreases. If the relative proportion of B structure is too low, although the gel flexibility and elasticity are improved, the resistance to external forces decreases, and the resistance to damp heat sterilization and enzymatic degradation is poor.

[0066] In the cross-linking reaction, the concentration of the collagen solution in step a) is 70 mg / mL to 150 mg / mL, for example, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL or 150 mg / mL, etc.

[0067] Preferably, step d) includes adding the solution from step b) to the solution from step a), and then adding the solution from step c) to carry out a crosslinking reaction; or adding the solution from step c) to the solution from step a), and then adding the solution from step b) to carry out a crosslinking reaction; or simultaneously adding the solutions from steps b) and c) to the solution from step a) to carry out a crosslinking reaction.

[0068] Preferably, the solution from step c) is added to the collagen solution from step a) first, and then the solution from step b) is added.

[0069] This invention employs a crosslinking agent + catalyst system, resulting in a dual dynamic and continuous crosslinking reaction involving both self-crosslinking of collagen and crosslinking by the crosslinking agent. Simultaneously, this invention optimizes the order of addition of the crosslinking agent and catalyst. By adding the catalyst first and then the crosslinking agent, the instantaneous formation rate of structures A and B is optimized. At the molecular level, adding the catalyst solution first and then the fatty amine crosslinking agent solution to the collagen solution preferentially forms the shorter self-crosslinked A structure, which has better rigidity. This facilitates the insertion and entanglement of the longer, more flexible B structure formed by the crosslinking agent, resulting in a denser and more stable dual network structure, thus improving the elastic modulus and support strength of the gel product.

[0070] Furthermore, the solution in step c) and the solution in step b) can be added alternately in steps.

[0071] Specifically, first add a portion (e.g., 1 / 10 to 1 / 3 volume) of the solution from step c) to the collagen solution from step a), then add an equal volume of the solution from step b), and repeat the above steps, adding equal volumes of the solution from step c) and the solution from step b) alternately to the mixed solution until all the solutions from steps b) and c) have been added.

[0072] This invention achieves dynamic cross-linking of collagen by controlling the alternating addition of cross-linking agents and catalyst solutions. This ensures sufficient contact between the cross-linking agent, catalyst, and collagen, facilitating both self-cross-linking and cross-linking reactions. This results in a more uniform hard / soft dual cross-linked network formed by structures A and B, improving the efficiency of the dual cross-linking reaction. Furthermore, the alternating addition method allows for tighter interpenetration of the in-situ formed A and B structures. By alternately adding catalyst and cross-linking agent, each B structure is "surrounded" by an A structure. The flexible B structure can tightly interpenetrate and entwine with the rigid A structure. This effectively utilizes the sacrificial bonds of the A structure to protect each B structure from external damage, achieving in-situ protection of the B structure and thus improving gel strength and stability.

[0073] Preferably, in step d), the solution from step c) and the solution from step b) are added to the collagen solution from step a) by dripping or using a syringe pump.

[0074] The drop rate is 0.01 ml / min to 1 ml / min, more preferably 0.03 ml / min to 0.08 ml / min.

[0075] Preferably, in step d), during the mixing process of the solution in step c) and the solution in step b), the addition rates of the solution in step c) and the solution in step b) are the same.

[0076] The crosslinking reaction is carried out at a temperature of 20–40°C, such as 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, or 40°C, preferably at room temperature.

[0077] Preferably, the crosslinking reaction time is 1 to 20 hours, such as 1 hour, 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, or 20 hours, with 5 hours to 10 hours being the most preferred.

[0078] The preparation method may further include: e) pulverizing, washing and drying the crosslinking reaction product of step d).

[0079] The pulverizing process is preferably performed by cutting with scissors or by using a homogenizer, wherein the homogenizer rotates at a speed of 2000–8000 rpm.

[0080] The preferred pulverization time is 1 min to 10 min.

[0081] The cleaning is preferably performed using solvent cleaning or dialysis, such as anhydrous ethanol.

[0082] During the anhydrous ethanol cleaning process, the pulverized gel is first dispersed in anhydrous ethanol at a volume ratio of 1:95 to 1:100, and then washed with anhydrous ethanol in small amounts multiple times.

[0083] During the dialysis cleaning process, the pulverized gel is transferred to the dialysis bag, and the dialysate is physiological saline, purified water, or phosphate buffer solution.

[0084] The cleaning frequency is preferably 1 to 20 times.

[0085] The drying process can be vacuum drying or freeze drying.

[0086] The drying time can be 20h to 100h, preferably 40h to 50h.

[0087] The drying temperature can be from 20℃ to 80℃.

[0088] The preparation method may further include: f) reconstitute the dried material and perform moist heat sterilization.

[0089] The diluent used for reconstitution includes one or more of the following: water (purified water, distilled water, etc.), physiological saline, phosphate equilibrium solution, or phosphate buffer solution.

[0090] Preferably, the diluted mother liquor further includes one or more of lidocaine hydrochloride, mannitol, and / or glycerol.

[0091] Preferably, the pH of the diluted mother liquor is 6.5 to 8.0, such as 6.5, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0, with 7.5 to 8.0 being the most preferred.

[0092] Preferably, the glycerol content is 0.5% to 2.0% by mass.

[0093] Preferably, the concentration of lidocaine hydrochloride is 1 mg / ml to 5 mg / ml.

[0094] The temperature for moist heat sterilization can be 100℃ to 150℃, such as 110℃, 120℃, 130℃, 140℃ or 150℃, preferably 116℃ to 121℃.

[0095] The time for moist heat sterilization can be 10 min to 30 min, preferably 15 min to 30 min.

[0096] It is preferable to perform moist heat sterilization after filling.

[0097] The filling process involves dissolving the cross-linking reaction product in a diluted mother liquor, swelling it, stirring and mixing it, and then filling it into a syringe.

[0098] The swelling time can be 20h to 50h.

[0099] The cross-linking reaction product is dissolved in the diluted mother liquor to achieve a concentration of 25 mg / ml to 60 mg / ml.

[0100] In some specific embodiments, the preparation method includes:

[0101] a) After dissolving collagen in water, adjust the pH to 5.5–7.0, and dissolve at 20℃–40℃ for 10–50 min to obtain a collagen solution with a concentration of 70 mg / ml–150 mg / ml;

[0102] b) After dissolving the fatty amine crosslinking agent in water, adjust the pH to 5.5–7.0;

[0103] c) The catalyst and auxiliaries are dissolved in water to obtain a solution containing the catalyst and auxiliaries;

[0104] In this process, the molar ratio of the catalyst to the carboxyl groups in collagen is 1.0–3.0, the molar ratio of the cross-linking agent to the carboxyl groups in collagen is 0.2–1.2, the molar ratio of the catalyst to the cross-linking agent is 1.5–5.0, and the mass ratio of the auxiliary agent to the catalyst is 0.01–0.5. The solution in step b) has the same volume as the solution in step c). The catalyst is carbodiimide, and the auxiliary agent is NHS.

[0105] d) Add the solution containing the catalyst and auxiliaries from step c) to the collagen solution from step a), and then add the fatty amine crosslinking agent solution from step b) to obtain a mixed solution, wherein the collagen concentration is 65 mg / mL to 130 mg / mL. The mixed solution is placed at 20 to 40 °C for a crosslinking reaction for 1 to 20 h.

[0106] In a third aspect, the present invention provides an injectable cross-linked collagen gel obtained by the above preparation method.

[0107] In a fourth aspect, the present invention provides an application of the above-described injectable cross-linked collagen gel or the injectable cross-linked collagen gel obtained by the preparation method in the preparation of medical materials, cosmetics or drug carriers.

[0108] Preferably, the medical materials include, but are not limited to, one or more of the following: surgical sutures, medical sponges, medical dressings (e.g., wound dressings, healing dressings, or repair dressings), hemostatic materials (for intraoperative and postoperative venous hemostasis), artificial organs (e.g., artificial skin, artificial bones, artificial cartilage, artificial blood vessels, artificial tendons, etc.), filling materials (e.g., skin filling materials or tissue filling materials), repair materials (e.g., skin repair materials, tissue repair materials), or biological scaffolds.

[0109] The skin filler material can be used to eliminate wrinkles (such as wrinkles around the eyes, forehead wrinkles, frown lines, perioral wrinkles, nasolabial folds, tear troughs, nasolabial folds, neck wrinkles, hand wrinkles, stretch marks, etc.) and for anti-aging purposes.

[0110] Preferably, the tissue repair material can be bone tissue repair material, cartilage tissue repair material, corneal tissue repair material, cardiovascular tissue repair material, liver tissue repair material, scar removal material, wound repair material, etc.

[0111] Preferably, the injectable cross-linked collagen gel can be used as a drug carrier to achieve purposes such as sustained release, controlled release, and targeted drug delivery.

[0112] A fifth aspect of the present invention provides a cosmetic method comprising injecting an injectable cross-linked collagen gel prepared according to the first or second aspect into the skin or tissue to be filled.

[0113] The beauty treatments mentioned can include wrinkle removal (such as wrinkles around the eyes, forehead wrinkles, frown lines, perioral wrinkles, nasolabial folds, tear troughs, nasolabial folds, neck wrinkles, hand wrinkles, stretch marks, etc.), anti-aging, scar removal, and wound repair.

[0114] A sixth aspect of the present invention provides a method for skin or tissue repair, the method comprising injecting an injectable cross-linked collagen gel prepared in the first or second aspect into the skin or tissue to be repaired.

[0115] The tissue repair can be bone tissue repair, cartilage tissue repair, corneal tissue repair materials, cardiovascular tissue repair, liver tissue repair, etc.

[0116] The skin repair mentioned can be wound repair or scar removal, etc.

[0117] The skin or tissue repair methods described can be for therapeutic or non-therapeutic purposes.

[0118] A seventh aspect of the present invention provides a method for treating a disease, the method comprising injecting an injectable cross-linked collagen gel prepared according to the first or second aspect into a subject in need.

[0119] The disease mentioned can be inflammation, burns, etc.

[0120] Preferably, the inflammation includes degenerative inflammation, exudative inflammation (e.g., one or more of serous inflammation, fibrinous inflammation, purulent inflammation, hemorrhagic inflammation, necrotizing inflammation, catarrhal inflammation), proliferative inflammation, or specific inflammation (e.g., one or more of tuberculosis, syphilis, leprosy, or lymphogranuloma).

[0121] The term "treatment" as used in this invention refers to delaying, terminating, blocking, controlling, stopping, reducing, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

[0122] The beneficial effects of this invention are:

[0123] 1. This invention provides an effective cross-linking strategy targeting the structural characteristics of collagen materials. Under the action of a catalyst and a cross-linking agent, collagen dynamically and continuously undergoes self-cross-linking and cross-linking reactions with the cross-linking agent, forming a collagen-collagen structure (A) formed by self-cross-linking and a collagen-fatty amine-collagen structure (B) formed by cross-linking agent, resulting in a collagen gel with BHSPN hard / soft dual cross-linking. Under the action of the catalyst, not only can the carboxyl groups of collagen be activated, enabling the cross-linking agent to form a collagen-fatty amine-collagen structure (B), but also the free carboxyl and amino groups of the collagen polypeptide chain can undergo self-cross-linking to form a collagen-collagen structure (A). On the one hand, the cross-linking network formed by the self-cross-linking structure has stress concentration and high rigidity, providing the cross-linked gel with high modulus and strength; on the other hand, during the cross-linking process of the cross-linking agent, the long-chain structure of the C-C bonds in the fatty amine cross-linking agent endows the cross-linking network structure with more flexibility, which can balance the rigidity of the collagen self-cross-linked polypeptide structure, making the cross-linked gel have excellent elasticity and injectability, providing a more valuable cross-linking structure for collagen materials.

[0124] 2. This invention utilizes a single self-crosslinking process to investigate the optimal amount of catalyst, maximizing the conversion rate of free carboxyl groups in collagen polypeptide chains and reducing the disadvantages of low molecular weight and rapid degradation of collagen raw materials. Furthermore, by screening the amount of crosslinking agent, the relative proportions of self-crosslinking and crosslinking agent-crosslinked structures in the crosslinking system can be dynamically adjusted. Within a suitable range of catalyst and crosslinking agent dosages, a certain proportion of hard phase network (rigid A structure) and soft phase network (flexible B structure) are formed. The rigid A structure fully utilizes its energy dissipation function during moist heat sterilization and product storage, providing sacrificial bonds to protect the flexible B structure. The released B structure allows the hydrogel to retain a certain degree of support while exhibiting good injectability, extensibility, and stability, meeting the clinical needs of different injection sites.

[0125] 3. In the crosslinking reaction process, the present invention optimizes the order of adding crosslinking agent and catalyst. By adding catalyst first and then crosslinking agent, it is beneficial to preferentially form rigid short chain segment A structure, making it easier for flexible long chain segment B structure to be inserted into A structure; making the in-situ formed double network structure more compact and stable, effectively improving the strength and stability of the final product, and the gel product has small modulus loss and good storage stability. Attached Figure Description

[0126] Figure 1 The diagram shows the molecular chain segment breakage of structures A and B' in cross-linked collagen gel under external force.

[0127] Figure 2 The following is a sample of the collagen sequence and sequence analysis report used in the examples.

[0128] Figure 3 The diagram shows the cross-linking mechanism of amide bonds formed between carboxyl and amino groups in EDC / NHS catalysis.

[0129] Figure 4 The image shows the infrared spectrum (2000 cm⁻¹) of collagen raw material and the single self-crosslinking product (before sterilization) of Example 1-b. -1 ~1000cm -1 Local wavenumber comparison chart.

[0130] Figure 5 The image shows a comparison of the infrared spectra of a single self-crosslinking product (before sterilization) and a product crosslinked by a self-crosslinking agent and a crosslinking agent (before sterilization).

[0131] Figure 6 The images shown are photos of gel samples injected in the examples and comparative examples, where a is a photo of gel sample injected in Example 2 and b is a photo of gel sample injected in Comparative Example 1. Detailed Implementation

[0132] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments and accompanying drawings. The described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0133] It should be noted that, unless otherwise specified, the experimental methods and reagents used in the embodiments of the present invention are all conventional experimental methods and reagents in the art.

[0134] The collagen used in the examples is recombinant collagen, derived from human type III collagen (SEQ ID NO: 1: QYDSYDVKSG VAVGGERGAP GFRGPAGPNG IPGEKGPAGERGAPGERGAP GFRGPAGPNGIPGEKGPAGE RGAPGERGAP GFRGPAGPNGIPGEKGPAGE RGAPGERGAP GFRGPAGPNG IPGEKGPAGE RGAPGERGAP GFRGPAGPNG IPGEKGPAGE RGAPGERGAP GFRGPAGPNG IPGEKGPAGE RGAPGERGAPGFRGPAGPNG IPGEKGPAGE RGAPGERGAP GFRGPAGPNG IPGEKGPAGE RGAPGERGAP GFRGPAGPNGIPGEKGPAGE RGAPGERGAP GFRGPAGPNG IPGEKGPAGE RGAPCGGVGA AAIAGIGGEK AGGFAPYYG), with a molecular weight of 32 kDa and a total of 339 amino acids, of which 10% contain free carboxyl groups (see...). Figure 2 ).

[0135] Example 1: Determining the amount of catalyst used

[0136] Weigh 1.0g of collagen and dissolve it in purified water, adjust the pH to 6.0, and dissolve at room temperature for 20 minutes. Then add a certain amount of EDC and NHS, mix well, and crosslink at room temperature for 6 hours. The EDC / NHS catalytic mechanism is described in [link to EDC / NHS catalytic mechanism description]. Figure 3 .

[0137] The amount of catalyst EDC used is determined by the free carboxyl group content of the collagen polypeptide chain. The amount of free carboxyl groups in collagen can be obtained from the amino acid sequence of the polypeptide, or calculated from the glutamic acid and aspartic acid content in the amino acid analysis report provided by the manufacturer.

[0138] The self-crosslinking of collagen was investigated when the molar ratio of catalyst EDC to collagen carboxyl groups was 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, and 3.5, with the mass ratio of auxiliary agent NHS to EDC being 0.2. The infrared spectra of the crosslinked gel samples were analyzed using different catalyst dosages, based on the presence of carboxyl groups in the samples (1403 cm⁻¹). -1 The signal peak and the amide I band at 1656 cm⁻¹ -1 The ratio of signal peak intensities is used to characterize the amount of unreacted free carboxyl groups in the crosslinked system. Taking Example 1-b as an example, the infrared spectral results are as follows: Figure 4 As shown, the test results of each sample in Example 1 are shown in Table 1.

[0139] Table 1. Results of EDC Dosage Screening for Catalysts

[0140]

[0141] Note: 1403cm -1 The signal peak is weak, making it difficult for instruments to identify.

[0142] As shown in Table 1, when the molar ratio of EDC to carboxyl groups in collagen is within the range of 0.5-2.5 (Examples 1-a to 1-e), as the amount of EDC gradually increases, the crosslinked product shows an increase in the infrared spectrum at 1403 cm⁻¹. -1 With 1656cm -1 The ratio of signal peak intensity decreased from 0.31 to 0.14. When the molar ratio of catalyst to collagen carboxyl groups was 0.5 (Example 1-a), the peak intensity was 1403 cm⁻¹. -1 With 1656cm -1 The signal peak intensity ratio was relatively high (0.31). When the catalyst dosage was increased to a molar ratio of 1.0 to the carboxyl group of collagen, the peak intensity was 1403 cm⁻¹. -1 With 1656cm -1 The ratio of signal peak intensities decreased significantly (0.20), indicating a significant reduction in the amount of free carboxyl groups in the crosslinking system. This suggests that most of the carboxyl groups in collagen underwent self-crosslinking. As the catalyst dosage was further increased, the decrease in the amount of free carboxyl groups diminished until it ceased to change. When the catalyst dosage was further increased, and the molar ratio of EDC to collagen carboxyl groups was 3.5 (Example 1-g), the infrared spectrum of the product at 1403 cm⁻¹... -1 The signal peak disappeared or its relationship with 1656 cm⁻¹ -1 The ratio of signal peak intensities was reduced to a minimum. Although a higher catalyst dosage leads to higher carboxyl conversion efficiency, it also places higher demands on the catalyst removal process, increasing product safety risks. Furthermore, excessively high self-crosslinking can cause rigid structures to break easily, resulting in a high elastic modulus loss rate. For example, in Comparative Example 1, the self-crosslinked gel sample, after moist heat sterilization, showed an elastic modulus loss rate exceeding 70%, failing to meet clinical use and product storage requirements. Therefore, this invention selects a catalyst-to-carboxyl molar ratio of 1.0–3.0, preferably within the range of 1.5–2.5, which is more suitable.

[0143] Example 2: Preparation of an injectable cross-linked collagen gel

[0144] a) Weigh 1.0g of collagen and dissolve it in 10ml of purified water. Adjust the pH to 6.0 with hydrochloric acid solution, add a magnetic stir bar, and stir at room temperature for 20min to dissolve the collagen solution for later use.

[0145] b) Weigh 0.05g of spermidine (speridine / collagen carboxyl molar ratio of 0.32) and dissolve it in 0.5ml of purified water. Adjust the pH to 6.0 with hydrochloric acid solution and set aside.

[0146] c) Weigh 0.31g EDC (the molar ratio of EDC to carboxyl groups in collagen is 1.5, and the molar ratio of EDC to cross-linking agent spermidine is 4.7) and 0.06g NHS, dissolve them in 0.5ml of purified water, and set aside.

[0147] d) First, add the solution containing EDC and NHS from step c) to the collagen solution from step a). After the addition is complete, continue to add the spermidine solution from step b) to obtain a mixed solution, and then place it at room temperature for crosslinking for 6 hours.

[0148] e) The cross-linked gel was homogenized at 5000 rpm for 5 min, then transferred to 100 ml of anhydrous ethanol, filtered, and the solid on the filter membrane was washed 10 times with 500 ml of anhydrous ethanol. Finally, the solid was sent to a vacuum drying oven at 40 ℃ for 40 h.

[0149] f) Prepare a diluted stock solution using physiological saline, purified water, glycerol, and lidocaine hydrochloride, wherein the glycerol content is 1% and the lidocaine hydrochloride content is 3 mg / ml, and adjust the pH of the diluted stock solution to 7.5.

[0150] The dried solid obtained in step e) is dispersed into the previously prepared diluted mother liquor to make the cross-linked collagen concentration 35 mg / ml. After swelling, it is stirred and mixed for 3 hours (i.e., the sample before sterilization). After being sterilized by moist heat at 121°C for 15 minutes, the injectable cross-linked collagen gel (i.e., the sample after sterilization) is obtained.

[0151] Example 3

[0152] Only the amount of spermidine, the cross-linking agent, was changed in step b), using 0.08 g of spermidine (spermidine / collagen carboxyl molar ratio of 0.51, EDC / cross-linking agent molar ratio of 2.94). All other steps were the same as in Example 2. The final product was an injectable cross-linked collagen gel.

[0153] Example 4

[0154] Only the amount of spermidine, the cross-linking agent, was changed in step b), using 0.15g of spermidine (spermidine / collagen carboxyl molar ratio of 0.96, EDC / cross-linking agent molar ratio of 1.6). All other steps were the same as in Example 2. The final product was an injectable cross-linked collagen gel.

[0155] Example 5

[0156] Only the type and amount of crosslinking agent in step b) were changed, using 0.21g of spermine (spermine / collagen carboxyl molar ratio of 0.96, EDC / crosslinking agent molar ratio of 1.6), while the other steps were the same as in Example 2. The final product was an injectable crosslinked collagen gel.

[0157] Example 6

[0158] Only the process parameters in steps b) and d) are changed; all other steps are the same as in Example 4.

[0159] b) Weigh 0.15g of spermidine and dissolve it in 0.5ml of purified water. Adjust the pH to 6.0 with hydrochloric acid solution and set aside.

[0160] d) The solution containing EDC and NHS from step c) and the spermidine solution from step b) are simultaneously added to the collagen solution from step a) to obtain a mixed solution, which is then crosslinked at room temperature for 6 hours. The final product is an injectable crosslinked collagen gel.

[0161] Example 7

[0162] Only the process parameters in steps b) and d) are changed; all other steps are the same as in Example 4.

[0163] b) Weigh 0.15g of spermidine and dissolve it in 0.5ml of purified water. Adjust the pH to 6.0 with hydrochloric acid solution and set aside.

[0164] d) First, add the spermidine-containing solution from step b) to the collagen solution from step a). After the addition is complete, continue adding the EDC and NHS solution from step c) to obtain a mixed solution, and then crosslink it at room temperature for 6 hours. Finally, an injectable crosslinked collagen gel is obtained.

[0165] Example 8

[0166] a) Weigh 1.0g of collagen and dissolve it in 10ml of purified water. Adjust the pH to 6.0 with hydrochloric acid solution, add a magnetic stir bar, and stir at room temperature for 20min to dissolve the collagen solution for later use.

[0167] b) Weigh 0.15g of spermidine and dissolve it in 0.5ml of purified water. Adjust the pH to 6.0 with hydrochloric acid solution, then install it in a syringe pump and set the flow rate to 0.05ml / min.

[0168] c) Weigh 0.31g EDC (the molar ratio of EDC to carboxyl groups in collagen is 1.5, and the molar ratio of EDC to cross-linking agent spermidine is 1.6) and 0.06g NHS, dissolve them in 0.5ml of purified water, then install them in a syringe pump and set the flow rate to 0.05ml / min for later use.

[0169] d) Place the two syringe pump tubing into the collagen solution. First, turn on the syringe pump containing the EDC / NHS solution to inject the EDC / NHS into the collagen solution. After 1 minute, turn on the syringe pump containing the spermidine solution to inject the spermidine solution into the collagen solution. This allows the droplets containing EDC and the droplets containing spermidine to be added to the collagen solution alternately until both solutions b) and c) are completely added. Then, place the mixed solution at room temperature for crosslinking for 6 hours.

[0170] All other steps are the same as in Example 4. The final product is an injectable cross-linked collagen gel.

[0171] Comparative Example 1

[0172] a) Weigh 1.0g of collagen and dissolve it in 10ml of purified water. Adjust the pH to 6.0, add a magnetic stir bar, and stir at room temperature for 20 minutes to dissolve the collagen solution for later use.

[0173] b) Weigh 0.31g EDC and 0.06g NHS and dissolve them in 0.5ml purified water for later use.

[0174] c) Add the solution from step b) to the collagen solution from step a), and crosslink at room temperature for 6 hours after the addition is complete.

[0175] The subsequent processes are the same as steps e)-f) in Example 2.

[0176] Comparative Example 2

[0177] a) Weigh 1.0g of collagen and dissolve it in 10ml of purified water. Adjust the pH to 6.0 with hydrochloric acid solution, add a magnetic stir bar, and stir at room temperature for 20min to dissolve the collagen solution for later use.

[0178] b) Weigh 0.04g of spermidine (speridine / collagen carboxyl molar ratio of 0.26) and dissolve it in 0.5ml of purified water. Adjust the pH to 6.0 with hydrochloric acid solution and set aside.

[0179] c) Weigh 0.31g EDC (the molar ratio of EDC to carboxyl groups in collagen is 1.5, and the molar ratio of EDC to crosslinking agent is 5.9) and 0.06g NHS, dissolve them in 0.5ml of purified water, and set aside.

[0180] Steps d)-f) are the same as in Example 2.

[0181] Comparative Example 3

[0182] a) Weigh 1.0g of collagen and dissolve it in 10ml of purified water. Adjust the pH to 6.0 with hydrochloric acid solution, add a magnetic stir bar, and stir at room temperature for 20min to dissolve the collagen solution for later use.

[0183] b) Weigh 0.17g of spermidine (speridine / collagen carboxyl molar ratio of 1.09) and dissolve it in 0.5ml of purified water. Adjust the pH to 6.0 with hydrochloric acid solution and set aside.

[0184] c) Weigh 0.31g EDC (the molar ratio of EDC to carboxyl groups in collagen is 1.5, and the molar ratio of EDC to cross-linking agent is 1.4) and 0.06g NHS, dissolve them in 0.5ml of purified water, and set aside.

[0185] Steps d)-f) are the same as in Example 2.

[0186] Performance testing 1 Infrared spectroscopy test

[0187] Take appropriate amounts of the samples from Examples 2-8 and Comparative Examples 1-3 before sterilization, dry them using a low-temperature freeze-drying method, compress the dried samples into tablets using potassium bromide, and determine the infrared spectra according to the method specified in General Chapter 0402 of Part IV of the Pharmacopoeia of the People's Republic of China (2020 Edition). The results are shown in Table 2 and exemplary results. Figure 5 .

[0188] Compared with single self-crosslinking (as in Comparative Example 1), the system using aliphatic amine crosslinking agent + catalyst (as in Example 2) forms collagen-collagen structure (A) and collagen-liphatic amine-collagen structure (B). Therefore, the relative content of structure B in crosslinked collagen can be characterized by the CN stretching vibration peak of secondary amine in aliphatic amine by infrared spectroscopy and the signal intensity ratio with the characteristic peak of amide bond.

[0189] like Figure 5 As shown, in the cross-linked collagen sample, the infrared spectrum at 1383 cm⁻¹... -1 This is the CN stretching vibration peak of secondary amines in aliphatic amines, at 1656 cm⁻¹. -1 The amide I group, which forms an amide bond, has a characteristic peak and can be identified using a 1383 cm⁻¹. -1 With 1656cm -1 The ratio of signal peak intensities reflects the relative content of collagen-fatty amine-collagen structure (B) in the cross-linked sample.

[0190] Performance testing 2. Extensibility test

[0191] A 27G injection syringe was used to push the gel samples of Example 2 and Comparative Example 1. The gel sample of Comparative Example 1 could not be pushed out, resulting in needle blockage, indicating that the self-crosslinked gel sample had poor injectability. In contrast, the sample of Example 2 showed good push continuity, and the gel product could be presented as a smooth thread without breaking. It had good extensibility and was injectable. Figure 6(As shown in a). Furthermore, using a 23G injection needle with a larger outer diameter to perform a push-in test on the gel sample of Comparative Example 1, it was found that the sample of Comparative Example 1 was pushed out in a "droplet" pattern; the gel push was discontinuous, with a strong sense of pause (as shown in a figure). Figure 6 As shown in b), this indicates that Comparative Example 1 only uses a self-crosslinking process, resulting in an excessively rigid structure that cannot achieve continuous and uniform injection, thus making it unsuitable for clinical injection use.

[0192] Performance testing 3: Viscoelasticity test

[0193] The elastic modulus of gel samples from Examples 2-8 and Comparative Examples 1-3 before and after sterilization was tested using a rheometer: 2 ml of each sample from the examples and comparative examples before and after sterilization was taken, and the elastic modulus (G') of the gel was measured using a rheometer, and the elastic modulus loss rate G' was calculated.

[0194] G' change rate = (G' before sterilization - G' after sterilization) / G' before sterilization;

[0195] The rheometer parameters were as follows: operating gap: 45000 μm, loading gap: 1000 μm, operating temperature: 37℃, deformation: 0.1%, frequency: 1 Hz, and running time: 60 s. The test results of the elastic modulus of the samples are shown in Table 2.

[0196] Table 2 Summary of performance test data for both examples and comparative examples

[0197]

[0198]

[0199] As shown by the infrared spectral data in Table 2, in Examples 2-4, with the increase of the crosslinking agent spermidine, the relative proportion of collagen-speridine-collagen structure (B') in the crosslinked samples gradually increased from 0.22 to 0.48, while the relative proportion of collagen-collagen structure (A) gradually decreased. This matches the trend that the increased amount of crosslinking agent leads to an increase in the products of the crosslinking reaction.

[0200] From the data of collagen samples in Examples 2-4 and Comparative Example 1, it can be seen that the modulus of the samples before sterilization was all within the range of 500-710 Pa, with slight differences. The collagen gel sample in Comparative Example 1 had the highest elastic modulus (701 Pa), which is because only self-crosslinking occurred in Comparative Example 1, and only rigid collagen-collagen structures (A) were formed in the gel network. However, after moist heat sterilization, the molecular chain segments of the rigid structure of the gel in Comparative Example 1 were more likely to break under high temperature or external force, resulting in a rapid decrease in the elastic modulus of the gel. Therefore, the elastic modulus of the sample dropped sharply to 195 Pa after sterilization, with a loss rate of up to 72.2%.

[0201] The elastic modulus loss rates of samples in Examples 2-4 after sterilization were all below 30%, significantly lower than that of Comparative Example 1. This is because cross-linking agents were introduced in Examples 2-4, which combined the self-cross-linking of collagen and the cross-linking by the cross-linking agent into a cross-linking system. The collagen gel samples, possessing both collagen-collagen structure (A) and collagen-spermidine-collagen structure (B'), formed a hard / soft dual cross-linking network. The introduction of the B' structure reduced the relative proportion of the A structure, minimizing damage or loss to the gel sample from external forces or moist heat sterilization, significantly improving sample stability and enhancing the gel sample's resistance to moist heat sterilization. Simultaneously, the soft phase network formed by the flexible B' structure endowed the collagen product with good extensibility and flexibility, meeting practical application requirements (such as...). Figure 6 (As shown).

[0202] Data from Examples 2-5 and Comparative Example 2 show that, due to the low amount of crosslinking agent in Comparative Example 2, the molar ratio of catalyst EDC to crosslinking agent in the system was 5.9 (higher than the upper limit of 5). This resulted in an excessive proportion of rigid A structures in the collagen gel network, making it susceptible to external forces (such as homogenization and washing processes), leading to molecular chain breakage. Therefore, the elastic modulus of the gel sample in Comparative Example 2 was slightly lower than that in Example 2 before sterilization. Furthermore, during moist heat sterilization, the external forces were more pronounced, resulting in a significant decrease in the elastic modulus of the gel sample. Its elastic modulus loss rate (41.5%) was also significantly higher than that of Example 2. The product stability of Comparative Example 2 was poor and could not meet usage requirements. In addition, a high proportion of rigid structures could also lead to problems such as difficulty in injection and easy needle clogging.

[0203] Data from Examples 2-5 and Comparative Example 3 show that the amount of spermidine, the crosslinking agent, was too high in Comparative Example 3. The molar ratio of catalyst to crosslinking agent in the system was 1.4 (below the lower limit of 1.5). Infrared spectral data showed that the relative proportion of collagen-speridine-collagen structure (B') was also increased compared to Example 4 (as shown in Table 1). However, the elastic modulus of the samples before and after sterilization was lower than that of Example 4, showing a significant downward trend. This indicates that if too much crosslinking agent is added, the ratio of catalyst to crosslinking agent in the system is too low, and the relative proportion of rigid A structure in the crosslinked gel is too small, its protective effect as a sacrificial bond is limited. Under moist heat sterilization or external force, a large number of B' structures are still destroyed, the gel network structure is not stable enough, and the sample's support is insufficient. In addition, with excessive crosslinking agent, the total carboxyl group content of collagen in the system is much lower than the amino group content of spermidine, resulting in unilateral crosslinking of carboxyl groups and spermidine rather than bilateral crosslinking. As a result, the elastic modulus of the gel sample is actually lower than that of Example 2, which used less crosslinking agent. Furthermore, excessive cross-linking agents also increase the burden on the post-processing of removal and purification. This also illustrates that, under the catalytic system of this invention, simply increasing the amount of cross-linking agent added will not yield a gel product with higher elastic modulus. It is necessary to comprehensively consider the occurrence of collagen self-crosslinking and cross-linking by the cross-linking agent. Therefore, it is necessary to control the amount and ratio of cross-linking agent and catalyst, so that the catalyst / cross-linking agent molar ratio is more suitable. In the process of dual cross-linking reaction, the degree of occurrence of self-crosslinking and cross-linking by the cross-linking agent can be fully balanced, and the collagen gel with the advantages of high elastic modulus and good stability can be obtained.

[0204] Examples 4, 6, and 7 show that different order of addition of the catalyst and crosslinking agent affects the elastic modulus and stability of the gel product. Example 4, by adding the catalyst first and then the crosslinking agent, allows the flexible B structure to more easily interpenetrate within the preferentially formed rigid A structure, effectively reinforcing the hard / soft dual crosslinking network. This results in a more stable gel network structure and thus the highest elastic modulus before sterilization. Furthermore, the effective interpenetration between the A and B structures improves the efficiency of the dual crosslinking, leading to a denser crosslinking network. Consequently, the gel exhibits stronger resistance to moist heat sterilization and better stability. The addition methods in Examples 6 and 7 can also form a hard / soft dual crosslinked network to meet the application requirements. However, Example 6 adds both the catalyst and the crosslinking agent, causing self-crosslinking and crosslinking by the crosslinking agent to occur simultaneously. There is a certain degree of competition in the crosslinking system, so the efficiency of the dual crosslinking reaction is slightly reduced. Example 7 uses the order of adding the crosslinking agent first and then the catalyst. Without the catalyst, collagen and the crosslinking agent do not react. When the catalyst is introduced, both undergo self-crosslinking and crosslinking by the crosslinking agent simultaneously. At this time, there is also competition in the dual crosslinking reaction and the reaction process is uncontrollable. Therefore, the crosslinking reaction is also reduced. As a result, the elastic modulus of the gel samples of Examples 6 and 7 before and after sterilization is slightly lower than that of the gel sample of Example 4, and the resistance to moist heat sterilization is also slightly reduced.

[0205] Furthermore, in Example 8, the catalyst and crosslinking agent were added alternately, allowing the crosslinking agent and catalyst to fully contact the collagen, which improved the uniformity, continuity, and efficiency of the double crosslinking reaction, resulting in a more tightly interwoven A and B structures formed in situ. Therefore, the elastic modulus was improved before and after sterilization. Moreover, the alternating addition of the catalyst and crosslinking agent could present a rhythm of "one drop of EDC solution followed by one drop of spermidine solution," so that each B structure was "surrounded" by the A structure, effectively protecting each B structure from external damage and achieving in-situ fixed-point protection of the B structure. Therefore, the elastic modulus loss rate of the gel product was low, and the stability was further improved.

[0206] Performance Test 4: High Temperature Stability Assessment

[0207] The sterilized gel products of Examples 2-7 and Comparative Examples 1-3 were placed in an environment of 40°C. The elastic modulus of the samples was tested at 0 months, 1 month, 2 months and 3 months respectively. The modulus reduction rate of the formulation samples was calculated by comparing with the data at 0 months to reflect the stability of the formulation.

[0208] The results are shown in Table 3. Through the samples of the examples and comparative examples, it can be seen from the trend of elastic modulus change after storage at different times that the gel samples of Examples 2 to 7 had a low rate of change in elastic modulus and a loss rate of less than 10% after being placed at high temperature for 3 months, which means that the product has high stability. In Comparative Example 1, the self-crosslinking product only contains rigid collagen-collagen structures (A). Therefore, during high-temperature storage, it is easily affected by external conditions, resulting in molecular chain segment breakage and damage to the gel network structure. Consequently, the elastic modulus loss rate of the sample after 3 months of high-temperature storage is close to 50%. In Comparative Example 2, the molar ratio of catalyst EDC to crosslinking agent is 5.9 (higher than the upper limit of 5), resulting in more collagen-collagen structures (A) remaining in the final gel. Therefore, these structures are prone to breakage under high temperature or external force, affecting the product's storage stability, and the elastic modulus change rate is close to 32%. In Comparative Example 3, the ratio of catalyst to crosslinking agent in the crosslinking system is too low, resulting in unilateral crosslinking of collagen and spermidine instead of bilateral crosslinking. This not only reduces the elastic modulus of the gel but also leads to poor stability of the unilateral crosslinked product, with molecular chain segments easily breaking, resulting in a high elastic modulus loss rate. In summary, the crosslinking system of the present invention can produce injectable crosslinked collagen gels with advantages such as good support, good stability, good extensibility, and long storage time.

[0209] Table 3. Rate of change of elastic modulus in high-temperature stability studies of examples and comparative examples

[0210] Examine the samples 1 month 2 months 3 months Example 2 4.25％ 7.24％ 10.07％ Example 3 3.12％ 5.83％ 9.10％ Example 4 1.72％ 3.80％ 5.08％ Example 5 1.99％ 4.01％ 5.21％ Example 6 2.91％ 4.61％ 6.25％ Example 7 3.00％ 5.23％ 7.53％ Comparative Example 1 22.05％ 35.90％ 48.72％ Comparative Example 2 13.99％ 19.45％ 31.74％ Comparative Example 3 11.60％ 16.26％ 18.02％

[0211] Rate of change of elastic modulus = (elastic modulus at 0 months - elastic modulus at x months) / elastic modulus at 0 months * 100%

[0212] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. An injectable cross-linked collagen gel, characterized in that, The collagen gel comprises collagen and a fatty amine cross-linking agent, and the cross-linking reaction is carried out under the action of a catalyst, wherein the collagen gel has a hard / soft double cross-linking network formed by self-cross-linking and cross-linking of the cross-linking agent.

2. The injectable cross-linked collagen gel according to claim 1, wherein, The fatty amine cross-linking agent is a polyamine; preferably, the polyamine is putrescine, spermine or spermidine. The molar ratio of the fatty amine cross-linking agent to the carboxyl group of the collagen is 0.2-1.

2.

3. The injectable cross-linked collagen gel of claim 1, wherein, The catalyst is selected from one or more of carbodiimide, triphenylphosphine and bromide to form bromide salt, carbonium salt or 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium chloride (DMTMM). Preferably, the catalyst is carbodiimide, such as dicyclohexyl carbodiimide, N,N'-diisopropyl carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The molar ratio of the catalyst to the carboxyl group of the collagen is 1.0-3.0, preferably 1.5-2.

5.

4. The injectable cross-linked collagen gel according to claim 3, wherein, The molar ratio of the catalyst to the fatty amine cross-linking agent is 1.5-5.

0.

5. A method of preparing the injectable cross-linked collagen gel according to any one of claims 1 to 4, characterized in that, The preparation method comprises: a) preparing a collagen solution; b) preparing a fatty amine cross-linking agent solution; c) preparing a solution containing a catalyst; d) adding the solution of step b) and the solution of step c) to the collagen solution of step a) to carry out a cross-linking reaction.

6. The preparation method according to claim 5, characterized in that, The step a) of preparing a collagen solution comprises adding collagen to a solvent and adjusting pH to dissolve; Preferably, the collagen is added to the solvent to adjust the pH to 5.5-7.0; The concentration of the collagen solution is 70 mg / mL-150 mg / mL; The collagen is selected from recombinant collagen or natural collagen.

7. The preparation method according to claim 5, characterized in that, The step b) of preparing a fatty amine cross-linking agent solution comprises dissolving the fatty amine cross-linking agent in a solvent and adjusting the pH to 5.5-7.0, preferably 5.5-6.

5.

8. The preparation method according to claim 5, characterized in that, The step d) comprises: first adding the solution of step c) to the collagen solution of step a), and then adding the solution of step b).

9. The preparation method according to claim 5, characterized in that, The temperature of the cross-linking reaction of step d) is 20-40℃; Preferably, the time of the cross-linking reaction is 1-20 h.

10. Use of the injectable cross-linked collagen gel of any one of claims 1-4 or the injectable cross-linked collagen gel obtained by the preparation method of any one of claims 5-9 in the preparation of a medical material, a cosmetic or a drug carrier. Preferably, the medical material comprises one or two or more of a surgical suture, a medical sponge, a medical dressing, a hemostatic material, an artificial organ, a filling material, a repair material or a biological scaffold.

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