High biocompatibility hydrogel based on enzyme catalyzed crosslinking and preparation method thereof

Abstract

The application relates to the technical field of biomaterials, and discloses a high-biocompatibility hydrogel based on enzyme catalysis cross-linking and a preparation method thereof, which comprises the following steps: mixing a polypeptide mother liquor containing a tyrosine residue, functional nanoparticle tyrosinase and an MES buffer solution to obtain precursor solution A; mixing gelatin, recombinant elastin and a phosphate buffered saline solution to obtain a polymer mother liquor, and adding microbial transglutaminase into the polymer mother liquor to obtain precursor solution B; and mixing and reacting the precursor solution A and the precursor solution B to obtain the high-biocompatibility hydrogel based on enzyme catalysis cross-linking. In the application, the interpenetrating network constructed by the tyrosinase and the microbial transglutaminase sequentially catalyzes a significant synergistic enhancement effect. In a hyperglycemic environment, a mesoporous silica nanoparticle built-in level connection system is automatically activated, realizing multiple effects of antibiosis, anti-inflammation and promotion of angiogenesis.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and more specifically, to a highly biocompatible hydrogel based on enzyme-catalyzed crosslinking and its preparation method. Background Technology

[0002] Enzyme-catalyzed cross-linked hydrogels have become promising advanced biomaterials in fields such as tissue engineering, drug delivery, and regenerative medicine due to their mild reaction conditions (room temperature, near-neutral pH), controllable cross-linking process, and excellent biocompatibility. Among them, enzyme catalytic systems represented by tyrosinase and microbial transglutaminase (mTGase) have attracted much attention.

[0003] Tyrosinase catalytic system: This system specifically oxidizes tyrosine residues in polypeptide chains to generate highly reactive quinone intermediates, which then undergo intermolecular coupling to form strong carbon-carbon or carbon-oxygen bonds. This reaction requires no toxic chemical cross-linking agents, and the reaction rate can be adjusted by enzyme concentration and dissolved oxygen levels, making it ideal for constructing injectable, in-situ moldable hydrogels.

[0004] Microbial transglutaminase catalytic system: This enzyme catalyzes an acyl transfer reaction between the γ-amide group of glutamine residues and the ε-amino group of lysine residues in proteins or peptides, forming a covalent ε-(γ-glutamyl)lysine isopeptide bond. This reaction exhibits high substrate specificity and bioorthogonality, and can be used to construct biomimetic matrices based on natural proteins (such as gelatin and elastin).

[0005] In recent years, research has shifted from single-enzyme catalytic systems to multi-enzyme synergistic or cascade strategies. For example, the sequential catalysis of tyrosinase and transglutaminase has led to the construction of hydrogels with interpenetrating or dual-network structures, aiming to synergistically enhance the mechanical properties and biological functions of materials. Simultaneously, combining inorganic nanoparticles (such as mesoporous silica nanoparticles, MSNs) with enzyme-catalyzed hydrogels to endow materials with drug loading / controlled release, enhanced mechanical properties, or the introduction of stimulus responsiveness is a significant current trend in this field. These "nanocomposite materials" demonstrate immense application potential in constructing intelligent therapeutic platforms, such as drug release carriers that respond to the disease microenvironment.

[0006] Despite significant advancements in existing technologies, the following key challenges remain when applying them to demanding biomedical applications such as chronic wound healing and tissue defect filling: The materials have limited functionality and lack synergistic therapeutic capabilities: Most research focuses on the structural construction of hydrogels or the loading of single drugs, resulting in relatively singular functions. Existing technologies struggle to integrate multiple functions simultaneously within a single system, such as structural support, long-term drug release, and dynamic response to the disease microenvironment. For example, while simple enzyme-crosslinked hydrogels exhibit good biocompatibility, they lack the ability to actively regulate the specific environments of high oxidative stress and hyperglycemia, such as in diabetic wounds.

[0007] Balancing mechanical properties, degradation rate, and bioactivity is challenging: single-network hydrogels often struggle to simultaneously achieve suitable mechanical strength, adjustable degradation rate, and cell-growth-promoting bioactivity. While dual-network hydrogels can improve mechanical properties, the bonding between networks (such as simple physical interpenetration) may not be robust enough, leading to preferential degradation in long-term in vivo environments and premature structural collapse. Furthermore, exogenously added free therapeutic enzymes (such as glucose oxidase) are easily lost within the gel network, resulting in unsustainable activity.

[0008] Integration strategies for nanocomposites need optimization: When introducing functional nanoparticles into hydrogels, common simple blending methods may lead to nanoparticle aggregation or leakage, affecting the uniformity, long-term stability, and functional reliability of the gel. Achieving stable and uniform dispersion of nanoparticles in hydrogel networks, and ensuring that their functional molecules (such as enzymes and catalysts) maintain high activity in the complex gel environment, are current technical challenges. Summary of the Invention

[0009] In view of this, the present invention proposes a highly biocompatible hydrogel based on enzyme-catalyzed crosslinking and its preparation method, aiming to solve the problems in the current technology of easy loss of functional components and difficulty in maintaining activity, insufficient stability and performance adjustability of dual network structure, and limited responsiveness to complex pathological microenvironments.

[0010] This invention proposes a method for preparing a highly biocompatible hydrogel based on enzyme-catalyzed crosslinking, comprising the following steps: The peptide containing tyrosine residues was mixed with 2-morpholine ethanesulfonic acid buffer to obtain the peptide stock solution; Functional nanoparticles were obtained by mixing mesoporous silica nanoparticles, copper nanoparticles, glucose oxidase and phosphate buffer solution for adsorption reaction, and then resuspended in phosphate buffer solution to obtain nanoparticle suspension. Precursor solution A was obtained by mixing the polypeptide stock solution, nanoparticle suspension, tyrosinase and MES buffer. Gelatin, recombinant elastin and phosphate buffer solution were mixed to obtain polymer mother liquor. Microbial transglutaminase was added to polymer mother liquor to obtain precursor solution B. Precursor solution A and precursor solution B were mixed and reacted to obtain a highly biocompatible hydrogel based on enzyme-catalyzed crosslinking.

[0011] Preferably, the polypeptide containing tyrosine residues is prepared by solid-phase peptide synthesis or by protein hydrolysis. The concentration of peptides containing tyrosine residues in the peptide mother liquor is 10~50 mg / mL.

[0012] Preferably, the method for preparing the mesoporous silica nanoparticles is as follows: Hexadecyltrimethylammonium bromide was dissolved in a mixed solvent, and then ammonia was added dropwise while stirring to obtain a micelle template; Then, the tetraethyl orthosilicate solution was added dropwise to the micelle template and stirred to react and obtain a gel; The gel was subjected to static aging, washing, drying, and calcination to obtain mesoporous silica nanoparticles.

[0013] Preferably, the mixing ratio of the mesoporous silica nanoparticles, copper nanoparticles, glucose oxidase and phosphate buffer solution is 2~3 mg: 0.8~1.5 mg: 4~6 mg: 0.8~1.2 mL; The adsorption reaction is carried out at a temperature of 0~5℃ for 20~28h. The concentration of the nanoparticle suspension is 2~3 mg / mL.

[0014] Preferably, the precursor solution A contains a polypeptide concentration of 5-25 mg / mL, a tyrosinase activity of 50-200 U / mL, and a nanoparticle concentration of 0.1-0.5 mg / mL.

[0015] Preferably, the concentration of gelatin in the precursor solution B is 10-20 mg / mL, the concentration of recombinant elastin is 10-20 mg / mL, and the activity of microbial transglutaminase is 10-50 U / mL.

[0016] Preferably, the precursor solution A further includes a pre-crosslinking step before mixing with the precursor solution B, wherein the pre-crosslinking temperature is 20~25℃ and the time is 10~20min.

[0017] Preferably, the mixing ratio of precursor solution A to precursor solution B is 1:1~2; The reaction temperature after mixing the two is 35~37℃ and the time is 10~60min.

[0018] This invention provides a highly biocompatible hydrogel prepared by the above-described method.

[0019] This invention provides an application of the highly biocompatible hydrogel in the preparation of drug release carriers and bioactive substance delivery carriers.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: In this invention, the interpenetrating network constructed by the sequential catalysis of tyrosinase and microbial transglutaminase produces a significant synergistic enhancement effect. The covalently bonded network formed by tyrosinase provides strength, while the protein network formed by microbial transglutaminase provides toughness, resulting in a hydrogel with a compressive modulus, elongation at break, and fatigue resistance far exceeding that of a single-network gel. Precise control of mechanical properties can be achieved by independently adjusting the concentrations or reaction parameters of the two enzymes. The two networks are generated in situ through chemical bonds and intertwine tightly, effectively avoiding the overall structural collapse problem caused by preferential degradation of one phase, which is common in traditional physical interpenetrating networks. By adjusting the network density and protein composition, different tissue regeneration cycles can be matched.

[0021] By co-localizing glucose oxidase and copper nanoparticles within mesoporous silica nanoparticles, the enzyme and catalyst are firmly confined, preventing leakage. The microenvironment of the mesoporous silica nanoparticles provides protection for the enzyme. The close proximity of functional components at the nanoscale significantly enhances the efficiency of the cascade reaction of "glucose oxidation → hydrogen peroxide generation → hydroxyl radical generation." This hydrogel transforms from a "passive" material into an "active" therapeutic platform. In a hyperglycemic environment, its built-in cascade system is automatically activated, achieving multiple effects including antibacterial, anti-inflammatory, and angiogenesis promotion.

[0022] Furthermore, the precursor solution A and precursor solution B described in this invention can be mixed and injected in situ at the lesion site using a dual-barrel syringe, and the final cross-linking is rapidly completed at body temperature, perfectly conforming to irregular wound surfaces and achieving minimally invasive implantation. All core components exhibit good biocompatibility. The dual-network structure highly mimics the fibrous interwoven network of the natural extracellular matrix, providing cells with an ideal biomimetic microenvironment.

[0023] During the preparation process, various drugs can be introduced into the nanoparticles, and the mesoporous structure of the mesoporous silica nanoparticles can efficiently load various drugs. The gel network can provide physical controlled release, while the cascade reaction triggered by the pathological microenvironment can further achieve stimulus-responsive drug release. In addition, the biomimetic and mild molding conditions of the hydrogel described in this application can be used to encapsulate living cells, forming a "cell-hydrogel" composite construct that can be used for tissue regeneration. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0025] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] This invention proposes a method for preparing a highly biocompatible hydrogel based on enzyme-catalyzed crosslinking, comprising the following steps: The peptide containing tyrosine residues was mixed with 2-morpholine ethanesulfonic acid buffer to obtain the peptide stock solution; Functional nanoparticles were obtained by mixing mesoporous silica nanoparticles, copper nanoparticles, glucose oxidase and phosphate buffer solution for adsorption reaction, and then resuspended in phosphate buffer solution to obtain nanoparticle suspension. Precursor solution A was obtained by mixing the polypeptide stock solution, nanoparticle suspension, tyrosinase and MES buffer. Gelatin, recombinant elastin and phosphate buffer solution were mixed to obtain polymer mother liquor. Microbial transglutaminase was added to polymer mother liquor to obtain precursor solution B. Precursor solution A and precursor solution B were mixed and reacted to obtain a highly biocompatible hydrogel based on enzyme-catalyzed crosslinking.

[0030] In this invention, the polypeptide containing tyrosine residues is prepared by solid-phase peptide synthesis or by protein hydrolysis.

[0031] In this invention, the concentration of peptides containing tyrosine residues in the peptide mother liquor is 10-50 mg / mL, preferably 20-40 mg / mL, more preferably 25-35 mg / mL, and even more preferably 30 mg / mL. Obtaining a peptide mother liquor with a stable concentration provides sufficient and controllable crosslinking sites for subsequent crosslinking. A peptide mother liquor within this concentration range can balance solution viscosity and crosslinking efficiency, avoiding both insufficient crosslinking due to excessively low concentration and excessively high concentration that makes the solution too viscous and difficult to mix.

[0032] Solid-phase peptide synthesis: It allows for precise control of the peptide sequence and the position and content of tyrosine residues, making it suitable for preparing customized peptides with well-defined structures.

[0033] Hydrolyzed protein method: Using natural proteins (such as soy protein and gelatin) as raw materials, mixed polypeptides containing tyrosine residues are obtained through enzymatic hydrolysis or acid hydrolysis. It is low in cost and widely available.

[0034] In this invention, the method for preparing the mesoporous silica nanoparticles is as follows: Hexadecyltrimethylammonium bromide was dissolved in a mixed solvent, and then ammonia was added dropwise while stirring to obtain a micelle template; Then, the tetraethyl orthosilicate solution was added dropwise to the micelle template and stirred to react and obtain a gel; The gel was subjected to static aging, washing, drying, and calcination to obtain mesoporous silica nanoparticles.

[0035] In this invention, the mixed solvent is a mixture of ethanol and water, with a volume ratio of ethanol to water of 1:4. The concentration of hexadecyltrimethylammonium bromide in the mixed solvent is 0.05~0.15 mol / L, preferably 0.08~0.12 mol / L, more preferably 0.09~0.11 mol / L, and even more preferably 0.1 mol / L. The amount of ammonia added is 5% to 10% of the volume of the mixed solvent, preferably 6% to 8%, more preferably 7%; the mass concentration of ammonia is 5% to 8%; the micelles formed by hexadecyltrimethylammonium bromide provide a template for the mesoporous structure of the mesoporous silica nanoparticles; The concentration of tetraethyl orthosilicate in the tetraethyl orthosilicate solution is 0.5~1.5 mol / L, preferably 0.8~1.2 mol / L, more preferably 0.9~1.1 mol / L, and even more preferably 1.0 mol / L; the solvent is anhydrous ethanol; The ratio of tetraethyl orthosilicate to hexadecyltrimethylammonium bromide is 2-4:1, preferably 2.5-3.5:1, more preferably 2.8-3.2:1, and even more preferably 3:1; the tetraethyl orthosilicate hydrolyzes and condenses to form a siloxane skeleton, which encapsulates hexadecyltrimethylammonium bromide micelles to obtain a gel with a regular mesoporous structure and a large specific surface area. The aging temperature is 25~35℃, preferably 28~32℃, more preferably 30℃; the time is 12~24h, preferably 16~20h, more preferably 18h. Aging can stabilize the gel structure and reduce mesopore collapse. The calcination temperature is 500~600℃, preferably 520~580℃, more preferably 540~560℃, and even more preferably 550℃; the time is 4~6h, preferably 4.5~5.5h, and even more preferably 5h. The purpose of calcination is to completely remove the template agent and form open mesopores.

[0036] In this invention, the mixing ratio of the mesoporous silica nanoparticles, copper nanoparticles, glucose oxidase, and phosphate buffer solution is 2-3 mg: 0.8-1.5 mg: 4-6 mg: 0.8-1.2 mL, preferably 2.2-2.8 mg: 1.0-1.3 mg: 4.5-5.5 mg, more preferably 2.4-2.6 mg: 1.1-1.2 mg: 4.8-5.2 mg: 0.95-1.05 mL, and even more preferably 2.5 mg: 1.15 mg: 5 mg: 1 mL; In this invention, the adsorption reaction temperature is 0~5℃, preferably 1~4℃, more preferably 2~3℃, and even more preferably 4℃; the time is 20~28h, preferably 22~26h, more preferably 23~25h, and even more preferably 24h. The high specific surface area of ​​mesoporous silica nanoparticles is used to adsorb copper nanoparticles and glucose oxidase, thereby achieving the loading of functional components.

[0037] In this invention, the concentration of the nanoparticle suspension is 2~3 mg / mL, preferably 2.2~2.8 mg / mL, more preferably 2.4~2.6 mg / mL, and even more preferably 2.5 mg / mL.

[0038] In this invention, the concentration of the polypeptide in the precursor solution A is 5-25 mg / mL, preferably 10-20 mg / mL, more preferably 12-18 mg / mL, and even more preferably 15 mg / mL; the activity of the tyrosinase is 50-200 U / mL, preferably 80-160 U / mL, more preferably 100-140 U / mL, and even more preferably 120 U / mL; and the concentration of the nanoparticles is 0.1-0.5 mg / mL, preferably 0.2-0.4 mg / mL, more preferably 0.25-0.35 mg / mL, and even more preferably 0.3 mg / mL.

[0039] In this invention, the concentration of gelatin in the precursor solution B is 10-20 mg / mL, preferably 12-18 mg / mL, more preferably 14-16 mg / mL, and even more preferably 15 mg / mL; the concentration of recombinant elastin is 10-20 mg / mL, preferably 12-18 mg / mL, more preferably 14-16 mg / mL, and even more preferably 15 mg / mL; and the activity of microbial transglutaminase is 10-50 U / mL, preferably 20-40 U / mL, more preferably 25-35 U / mL, and even more preferably 30 U / mL.

[0040] In this invention, the precursor solution A is further included in the pre-crosslinking step before being mixed with the precursor solution B. The pre-crosslinking temperature is 20~25℃, preferably 21~24℃, more preferably 22~23℃, and even more preferably 23℃; the time is 10~20min, preferably 12~18min, more preferably 14~16min, and even more preferably 15min.

[0041] In this invention, the mixing ratio of precursor solution A to precursor solution B is 1:1 to 2, preferably 1:1.2 to 1.8, more preferably 1:1.4 to 1.6, and even more preferably 1:1.5; The reaction temperature after mixing the two is 35~37℃, preferably 35.5~36.5℃, more preferably 37℃; the reaction time is 10~60min, preferably 20~40min, more preferably 25~35min, more preferably 30min.

[0042] This invention provides a highly biocompatible hydrogel prepared by the above-described method.

[0043] This invention provides an application of the highly biocompatible hydrogel in the preparation of drug release carriers and bioactive substance delivery carriers.

[0044] Example 1 (1) Preparation of polypeptides containing tyrosine residues: Solid support: Rink Amide MBHA resin, degree of substitution 0.5 mmol / g, amount used 0.2 g (loading capacity 0.1 mmol); Protected amino acids: Fmoc-Tyr(tBu)-OH (tyrosine side chain phenolic hydroxyl group is protected with tert-butyl), Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Gly-OH (glycine has no side chain and does not require protection); Coupling reagent: HBTU (O-benzotriazole-tetramethylurea hexafluorophosphate) 0.38g; Activated base: NMM (N-methylmorpholine); Deprotection agent: 20% piperidine in DMF solution (v / v); Solvents: DMF (N,N-dimethylformamide, peptide synthesis grade), DCM (dichloromethane, analytical grade), methanol (analytical grade), diethyl ether (anhydrous, analytical grade); Cutting reagents: trifluoroacetic acid (TFA, ≥99.5%), triisopropylsilane (TIS, ≥99%), water (HPLC grade); Place 0.20 g of Rink Amide resin in a reaction column, add 5 mL of DMF, shake at room temperature to swell for 30 minutes, and drain. Add 5 mL of 20% piperidine, shake for 5 minutes, and drain. Repeat once. Then wash the resin 5 times with DMF (5 mL each time, shake for 1 minute each time and drain). Take several resin particles for Kaiser Test. A positive result (blue) indicates that Fmoc has been completely removed and free amino groups are present.

[0045] Weigh Fmoc-Leu-OH (4 eq, 0.4 mmol, ~141 mg) and HBTU (4 eq, 0.4 mmol, ~152 mg) into a vial, add 4 mL of DMF to dissolve them, add DIPEA (8 eq, 0.8 mmol, ~139 μL), vortex mix for 15 seconds, and immediately add this activation solution to the resin. Incubate at room temperature with shaking for 35 minutes, drain, and wash the resin three times with DMF. Repeat the above steps to couple the next Fmoc protected amino acid sequentially, in the following order: Leu → Phe → Gly → Gly → Tyr(tBu). Perform ninhydrin detection after each coupling. A negative result (pale yellow or colorless resin beads) indicates complete coupling; a positive result (blue) requires a second coupling.

[0046] After Tyr(tBu) coupling and washing, a final Fmoc deprotection was performed (5 mL of 20% piperidine / DMF was added, shaken for 5 minutes, drained, and repeated once, followed by washing the resin with DMF 5 times), to obtain the fully deprotected peptide chain H-Tyr(tBu)-Gly-Gly-Phe-Leu-resin, which is still attached to the resin side.

[0047] Wash the resin three times with DCM and then dry it until it is loose.

[0048] Prepare the cutting fluid according to the volume ratio of TFA:TIS:H2O = 95:2.5:2.5. Add the cutting fluid to the resin, ensuring that the resin is completely submerged, and react with shaking at room temperature for 3 hours.

[0049] Filter the reaction solution into centrifuge tubes. Wash the resin once with 1 mL of TFA and combine the filtrates. Slowly add about 40 mL of pre-cooled anhydrous ether (in an ice bath) to the TFA filtrate containing the peptide. The peptide will form a white precipitate. Centrifuge at 4000 rpm for 5 min at 4 °C. Carefully decant the supernatant. Wash the precipitate three times with cold ether (about 20 mL), vortexing and centrifuging after each wash. Air dry the precipitate in a fume hood for 10 min, and then dry it overnight in a vacuum drying oven to obtain the peptide containing tyrosine residues.

[0050] (2) Preparation of functional nanoparticles: First, hexadecyltrimethylammonium bromide was dissolved in a 25% (v / L) aqueous ethanol solution, resulting in a concentration of 0.1 mol / L; then, 10% (v / L) of ammonia solution with a mass concentration of 8% was added dropwise to form a micelle template. A 1 mol / L tetraethyl orthosilicate solution was added dropwise to a micelle template, with a molar ratio of tetraethyl orthosilicate to hexadecyltrimethylammonium bromide of 3:1. The mixture was stirred at 500 r / min until it became gel-like. It was then aged at 30 °C for 20 h and transferred to a tube furnace for calcination at 500 °C for 5 h in an oxygen-free environment to obtain mesoporous silica nanoparticles.

[0051] Mesoporous silica nanoparticles, copper nanoparticles, glucose oxidase, and phosphate buffer solution were mixed in a ratio of 2 mg: 1 mg: 5 mg: 1 mL and subjected to an adsorption reaction under ice-water bath conditions. After 24 h of reaction, functional nanoparticles were obtained. The mixture was centrifuged at 8000 r / min for 10 min to separate the functional nanoparticles, and then washed with water 3 times for later use.

[0052] (3) Preparation of highly biocompatible hydrogels: The above-mentioned peptide containing tyrosine residues was mixed with 2-morpholine ethanesulfonic acid buffer (MES-HCl, pH=5.8) to obtain a peptide stock solution with a concentration of 35 mg / mL; The above functional nanoparticles were resuspended in phosphate buffer solution to obtain a nanoparticle suspension with a concentration of 3 mg / mL. The precursor solution A was prepared by mixing the peptide stock solution, nanoparticle suspension, tyrosinase and MES buffer. The peptide concentration in precursor solution A was 20 mg / mL, the tyrosinase activity was 120 U / mL, and the nanoparticle concentration was 0.3 mg / mL.

[0053] Gelatin, recombinant elastin, and phosphate buffer solution were mixed to obtain a polymer mother liquor. Microbial transglutaminase was added to the polymer mother liquor to obtain precursor solution B. The concentration of gelatin in precursor solution B was 10 mg / mL, the concentration of recombinant elastin was 20 mg / mL, and the activity of microbial transglutaminase was 50 U / mL.

[0054] Precursor solution A and precursor solution B were mixed at a volume ratio of 1:1 and reacted at 37°C for 30 min to obtain a highly biocompatible hydrogel.

[0055] Example 2 (1) Preparation of polypeptides containing tyrosine residues: Solid support: Wang resin, degree of substitution 0.8 mmol / g, amount used 0.125g (loading capacity 0.1 mmol); Protected amino acids: Fmoc-Tyr(2,6-DiCl-Bzl)-OH (tyrosine side chain phenolic hydroxyl group is protected with 2,6-dichlorobenzyl), Fmoc-Ile-OH, Fmoc-Trp-OH, Fmoc-Gly-OH; Coupling reagent: HATU (O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate) 0.15g; Activated base: DIPEA (N,N-diisopropylethylamine); Deprotection agent: 20% piperidine in DMF solution (v / v); Solvents: DMF (peptide synthesis grade), DCM (analytical grade), methanol (analytical grade), diethyl ether (anhydrous, analytical grade); Cutting reagents: trifluoroacetic acid (TFA, ≥99.5%), anisole (PhSMe, ≥99%), water (HPLC grade); Place 0.125 g of Wang resin in a reaction column, add 5 mL of DMF, shake at room temperature for 40 minutes to allow swelling, and drain. Add 5 mL of 20% piperidine / DMF, shake for 5 minutes, drain, and repeat once. Then wash the resin 5 times with DMF (5 mL each time, shake for 1 minute and drain). Take several resins for a Kaiser Test; a blue (positive) color indicates complete removal of Fmoc.

[0056] Weigh Fmoc-Ile-OH (4 eq, 0.4 mmol, ~141 mg) and HATU (4 eq, 0.4 mmol, ~152 mg) into a vial, dissolve in 4 mL of DMF, add DIPEA (8 eq, 0.8 mmol, ~139 μL), vortex for 20 seconds, then immediately add to the resin. Incubate at room temperature with shaking for 40 minutes, drain, and wash three times with DMF. Repeat the above steps, coupling and protecting amino acids sequentially in the order of "Ile→Trp→Gly→Gly→Tyr(2,6-DiCl-Bzl)". Perform ninhydrin detection after each coupling; a pale yellow resin bead (negative) indicates complete coupling, while a positive result indicates a second coupling.

[0057] After Tyr(2,6-DiCl-Bzl) coupling washing was completed, a final Fmoc deprotection was performed (5 mL 20% piperidine / DMF, shaken for 5 minutes × 2 times, followed by DMF washing 5 times) to obtain H-Tyr(2,6-DiCl-Bzl)-Gly-Gly-Trp-Ile- resin.

[0058] The resin was washed three times with DCM and dried until loose. A cutting buffer was prepared at a volume ratio of TFA:PhSMe:H2O = 90:5:5. The resin was submerged, and the reaction mixture was shaken at room temperature for 3.5 hours. The reaction mixture was collected by filtration. The resin was washed once with 1 mL of TFA, and the filtrates were combined. The filtrate was slowly added dropwise to 40 mL of pre-cooled anhydrous ether (ice bath), resulting in a white precipitate. The precipitate was centrifuged at 4000 rpm for 5 min at 4 °C, the supernatant was discarded, and the precipitate was washed three times with cold ether (20 mL each time). After air-drying in a fume hood for 15 minutes, the precipitate was vacuum-dried overnight to obtain the target peptide.

[0059] (2) Preparation of functional nanoparticles: Hexadecyltrimethylammonium bromide was dissolved in a 20% aqueous ethanol solution with a concentration of 0.12 mol / L; ammonia solution with a mass concentration of 10% (8% of the total volume of the solution) was added dropwise to form a micelle template. A 1 mol / L tetraethyl orthosilicate solution was dropped into the template, and the molar ratio of tetraethyl orthosilicate to hexadecyltrimethylammonium bromide was controlled at 4:1. The mixture was stirred at 600 r / min until it reached a gel state, aged at 35 °C for 18 h, and calcined in a tube furnace at 600 °C for 4 h in an oxygen-free environment to obtain mesoporous silica nanoparticles.

[0060] Mix mesoporous silica nanoparticles, copper nanoparticles, glucose oxidase, and phosphate buffer in a ratio of 3 mg: 1 mg: 4 mg: 1 mL. Incubate the mixture in an ice-water bath for 22 hours to allow it to adsorb and react. Separate the particles by centrifugation at 8000 rpm for 10 minutes. Wash the particles three times with water before use.

[0061] (3) Preparation of highly biocompatible hydrogels: The target peptide was mixed with MES-HCl buffer (pH=5.8) to prepare a peptide stock solution with a concentration of 40 mg / mL; The functional nanoparticles were resuspended in phosphate buffer to obtain a suspension with a concentration of 4 mg / mL. Precursor solution A was prepared by mixing the polypeptide stock solution, nanoparticle suspension, tyrosinase and MES buffer: polypeptide concentration 22 mg / mL, tyrosinase activity 130 U / mL, and nanoparticle concentration 0.4 mg / mL.

[0062] Gelatin and recombinant elastin were dissolved in phosphate buffer to obtain a polymer stock solution. Microbial transglutaminase was added to prepare precursor solution B: gelatin concentration 12 mg / mL, recombinant elastin concentration 18 mg / mL, enzyme activity 60 U / mL.

[0063] Solutions A and B were mixed in a 1:1 volume ratio and reacted at 37°C for 25 minutes to obtain a highly biocompatible hydrogel.

[0064] Example 3 (1) Preparation of polypeptides containing tyrosine residues: Solid support: Rink Amide MBHA resin, degree of substitution 0.6 mmol / g, amount used 0.167g (loading 0.1 mmol). Protected amino acids: Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-Tyr(tBu)-OH (double tyrosine sequence), Fmoc-Ala-OH, Fmoc-Gly-OH; Coupling reagent: PyBOP (benzotriazol-1-yl-oxytripyrrolidinyl phosphate hexafluorophosphate) 0.21 g; Activated base: NMM (N-methylmorpholine); Deprotection agent: 20% piperidine in DMF solution (v / v); Solvents: DMF (peptide synthesis grade), DCM (analytical grade), methanol (analytical grade), diethyl ether (anhydrous, analytical grade); Cutting reagents: TFA, TIS, H2O (volume ratio 92.5:5:2.5); Place 0.167 g of Rink Amide MBHA resin in the reaction column, add 5 mL of DCM, shake to swell for 30 minutes, and drain. Add 5 mL of 20% piperidine / DMF, shake for 5 minutes twice, and wash 5 times with DMF (5 mL each time). A blue Kaiser test indicates complete removal of Fmoc.

[0065] Weigh Fmoc-Val-OH (4 eq, 0.4 mmol, ~128 mg) and PyBOP (4 eq, 0.4 mmol, ~210 mg) into a vial, dissolve in 4 mL of DMF, add NMM (8 eq, 0.8 mmol, ~88 μL), vortex for 15 seconds, then add the resin. React at room temperature with shaking for 40 minutes, drain, and wash three times with DMF. Couple in the order of "Val→Tyr(tBu)→Ala→Gly→Tyr(tBu)", extending the reaction time to 60 minutes for double tyrosine residue coupling. Complete coupling is indicated by a negative ninhydrin test after each coupling step.

[0066] After the final Fmoc deprotection, the resin was washed three times with DCM and dried. A cutting buffer was prepared to immerse the resin, and the reaction was carried out at room temperature with shaking for 3 hours. The filtrate was collected by filtration, and the resin was washed once with 1 mL of TFA. 40 mL of pre-cooled diethyl ether was added dropwise to the filtrate, and the precipitate was centrifuged, washed, and vacuum dried overnight to obtain a polypeptide with two tyrosine residues.

[0067] (2) Preparation of functional nanoparticles: Sodium dodecyl sulfate was dissolved in a 30% ethanol aqueous solution to a concentration of 0.08 mol / L; ammonia solution with a mass concentration of 8% and a total volume of 12% of the solution was added dropwise to form a micelle template. Add 1 mol / L tetraethyl orthosilicate solution, control the molar ratio of tetraethyl orthosilicate to sodium dodecyl sulfate to be 3.5:1, stir at 550 r / min until gel-like, age at 28℃ for 24 h, and calcine in a tube furnace at 550℃ for 5 h in an oxygen-free environment to obtain mesoporous silica nanoparticles.

[0068] Mix mesoporous silica nanoparticles, copper nanoparticles, glucose oxidase, and phosphate buffer at a ratio of 2.5 mg: 1 mg: 5 mg: 1 mL, adsorb in an ice-water bath for 26 hours, centrifuge at 8000 rpm for 10 minutes, and wash three times with water before use.

[0069] (3) Preparation of highly biocompatible hydrogels: The target peptide was mixed with MES-HCl buffer (pH=5.8) to prepare a peptide stock solution of 38 mg / mL; The nanoparticles were resuspended in phosphate buffer to obtain a suspension of 3.5 mg / mL. The precursor solution A was prepared by mixing: peptide concentration 24 mg / mL, tyrosinase activity 125 U / mL, and nanoparticle concentration 0.35 mg / mL.

[0070] Gelatin, recombinant elastin, and hyaluronic acid were dissolved in phosphate buffer to obtain a polymer stock solution. Microbial transglutaminase was added to prepare precursor solution B: gelatin 11 mg / mL, recombinant elastin 19 mg / mL, hyaluronic acid 2 mg / mL, enzyme activity 55 U / mL.

[0071] Solutions A and B are mixed in a 1:1 volume ratio and reacted at 37°C for 28 minutes to obtain a highly biocompatible hydrogel.

[0072] The properties of the highly biocompatible hydrogels prepared in Examples 1-3 were tested using the following methods: 1. Biocompatibility testing (1) Cytotoxicity test Preparation of gel extract: After sterilization, the three gels were immersed in DMEM medium at a ratio of 1g / 10mL, incubated at 37℃ for 24h, and then filtered to remove bacteria. Cell seeding: L929 fibroblasts were seeded in 96-well plates at a density of 5 × 10⁶ cells / well. 3 Cells / well, incubate for 24 hours; Group treatment: Different concentrations of extract (25%, 50%, 100%) were added, and blank culture medium was set as the control group. The culture was continued for 24h, 48h, and 72h. Assay: Add 20 μL of MTT solution (5 mg / mL) to each well, incubate for 4 h, discard the supernatant, add 150 μL of DMSO, shake to dissolve, and then measure the OD. 490 value; Cell viability = (OD value of experimental group / OD value of control group) × 100%, toxicity level (0-4).

[0073] (2) Blood compatibility test A gel sample (0.1 g) was mixed with fresh rabbit blood (with anticoagulant added) in a specific ratio, incubated at 37°C for 1 h, and then centrifuged to measure the OD. 540 value; Hemolysis rate = (sample OD value - negative control OD value) / (positive control OD value - negative control OD value) × 100%, hemolysis rate < 5% is acceptable; Coagulation function test: Prothrombin time (PT) and activated partial thromboplastin time (APTT) were used for testing. The deviation from the blank control group was less than 10% to be considered qualified.

[0074] 2. Mechanical property testing Sample preparation: The three gels were prepared into cylindrical samples with a diameter of 8 mm and a height of 4 mm; Compression performance test: Using a universal testing machine, the compression rate is 1 mm / min, and the material is compressed to 50% of its original height. The stress-strain curve is then recorded. Fatigue resistance test: After soaking in PBS at 37℃ and pH 7.4 for 24 hours, a cyclic compression test was performed (compression rate 30%, number of cycles 50, frequency 0.5Hz), and the maximum stress change in each cycle was recorded.

[0075] The compressive modulus, fracture strength, elongation at break, and fatigue retention rate of the material are calculated based on the test results (maximum stress in the 50th cycle / maximum stress in the 1st cycle × 100%).

[0076] 3. Degradation rate test Sample preparation: All three types of gels were made into discs with a diameter of 10 mm and a height of 2 mm, and weighed accurately (W0). Degradation conditions: Place in PBS buffer (containing 1 mg / mL proteinase K, simulating the in vivo enzymatic digestion environment) at 37℃ and pH 7.4, with a bath ratio of 1:50 (g / mL), and change the buffer regularly; Testing cycle: Samples were collected at 1d, 3d, 7d, 14d, 21d, and 28d respectively, freeze-dried, and accurately weighed (W). t ).

[0077] Degradation rate = (W0 - W) t ) / W0×100%.

[0078] 4. Drug release rate test after encapsulation When preparing the hydrogel precursor solution, the drug is added in a set ratio and stirred evenly, and then the drug-loaded gel is prepared according to the original cross-linking process. Drug selection: small molecule drugs (ibuprofen) and large molecule drugs (bovine serum albumin, BSA).

[0079] Release experiment: Place the drug-loaded gel (containing approximately 1 mg of drug) in a dialysis bag and immerse it in 30 mL of PBS buffer (pH 7.4, 37°C, magnetic stirring speed 100 r / min). Control group (no glucose) and high glucose group (20 mmol / L glucose) had their release medium changed regularly. Samples were taken at set time points (0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 48h, 72h), and drug concentration was determined using a UV-Vis spectrophotometer (ibuprofen, λ=220nm) or the BCA method (BSA). The cumulative release rate and release half-life were determined and calculated.

[0080] The test results are shown in Table 1.

[0081] Table 1. Performance test results of the highly biocompatible hydrogels prepared in Examples 1-3

[0082] All data in Table 1 are expressed as mean ± standard deviation. Three parallel samples were set up for each test group, and the data were compiled after statistical analysis.

[0083] All three gels meet the requirements for biomedical applications (cell survival rate >80%, hemolysis rate <5%, coagulation function deviation <10%). Example 3 has better biocompatibility due to the synergistic effect of the addition of hyaluronic acid and bistyrosine peptide. Mechanical properties: The bistyrosine peptide in Example 3 increases the crosslinking density and hyaluronic acid enhances the network toughness, thus the compressive modulus, fracture strength and fatigue resistance retention rate are all optimal. Degradation rate: The dual-network structure of Example 3 is more stable, and hyaluronic acid delays enzymatic hydrolysis, resulting in the slowest degradation rate, which is more suitable for long-term tissue regeneration needs. Drug release: Small molecule drugs (ibuprofen) have a faster release rate than large molecule drugs (BSA), which is consistent with differences in diffusion mechanisms; The high-glucose group had a higher cumulative release rate and a shorter release half-life than the control group due to the disruption of the gel network caused by the glucose-responsive cascade reaction. The dense network structure of Example 3 significantly hinders drug diffusion, resulting in the lowest cumulative release rate, the longest release half-life, and the best controlled release effect.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a highly biocompatible hydrogel based on enzyme-catalyzed crosslinking, characterized in that, Includes the following steps: The peptide containing tyrosine residues was mixed with 2-morpholine ethanesulfonic acid buffer to obtain the peptide stock solution; Functional nanoparticles were obtained by mixing mesoporous silica nanoparticles, copper nanoparticles, glucose oxidase and phosphate buffer solution for adsorption reaction, and then resuspended in phosphate buffer solution to obtain nanoparticle suspension. Precursor solution A was obtained by mixing the polypeptide stock solution, nanoparticle suspension, tyrosinase and MES buffer. Gelatin, recombinant elastin and phosphate buffer solution were mixed to obtain polymer mother liquor. Microbial transglutaminase was added to polymer mother liquor to obtain precursor solution B. Precursor solution A and precursor solution B were mixed and reacted to obtain a highly biocompatible hydrogel based on enzyme-catalyzed crosslinking.

2. The method for preparing a highly biocompatible hydrogel based on enzyme-catalyzed crosslinking according to claim 1, characterized in that, The polypeptide containing tyrosine residues is prepared by solid-phase peptide synthesis or by protein hydrolysis. The concentration of peptides containing tyrosine residues in the peptide mother liquor is 10~50 mg / mL.

3. The method for preparing a highly biocompatible hydrogel based on enzyme-catalyzed crosslinking according to claim 1, characterized in that, The method for preparing the mesoporous silica nanoparticles is as follows: Hexadecyltrimethylammonium bromide was dissolved in a mixed solvent, and then ammonia was added dropwise while stirring to obtain a micelle template; Then, the tetraethyl orthosilicate solution was added dropwise to the micelle template and stirred to react and obtain a gel; The gel was subjected to static aging, washing, drying, and calcination to obtain mesoporous silica nanoparticles.

4. The method for preparing a highly biocompatible hydrogel based on enzyme-catalyzed crosslinking according to claim 1, characterized in that, The mixing ratio of the mesoporous silica nanoparticles, copper nanoparticles, glucose oxidase, and phosphate buffer solution is 2~3 mg: 0.8~1.5 mg: 4~6 mg: 0.8~1.2 mL; The adsorption reaction is carried out at a temperature of 0~5℃ for 20~28h. The concentration of the nanoparticle suspension is 2~3 mg / mL.

5. The method for preparing a highly biocompatible hydrogel based on enzyme-catalyzed crosslinking according to claim 1, characterized in that, The precursor solution A contains a polypeptide concentration of 5-25 mg / mL, a tyrosinase activity of 50-200 U / mL, and a nanoparticle concentration of 0.1-0.5 mg / mL.

6. The method for preparing a highly biocompatible hydrogel based on enzyme-catalyzed crosslinking according to claim 1, characterized in that, The precursor solution B contains gelatin at a concentration of 10-20 mg / mL, recombinant elastin at a concentration of 10-20 mg / mL, and microbial transglutaminase at an activity of 10-50 U / mL.

7. The method for preparing a highly biocompatible hydrogel based on enzyme-catalyzed crosslinking according to claim 1, characterized in that, The precursor solution A further includes a pre-crosslinking step before being mixed with the precursor solution B, wherein the pre-crosslinking temperature is 20~25℃ and the time is 10~20min.

8. The method for preparing a highly biocompatible hydrogel based on enzyme-catalyzed crosslinking according to claim 1, characterized in that, The ratio of precursor solution A to precursor solution B is 1:1~2; The reaction temperature after mixing the two is 35~37℃ and the time is 10~60min.

9. The highly biocompatible hydrogel prepared by the method for preparing a highly biocompatible hydrogel based on enzyme-catalyzed crosslinking as described in any one of claims 1 to 8.

10. The application of the highly biocompatible hydrogel of claim 9 in the preparation of drug release carriers and bioactive substance delivery carriers.

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