Acid-triggered step-by-step bonding macromolecular tissue adhesive as well as preparation method and application thereof

Abstract

The invention discloses a macromolecular tissue adhesive for acid-triggered step-by-step adhesion as well as a preparation method and application thereof, and belongs to the technical field of medical macromolecular materials. The polymer tissue adhesive of the present invention comprises an ionized polymer, the ionized polymer is prepared by free radical polymerization of a monomer component in an alkaline aqueous phase, and the monomer component comprises an anhydride-terminated cationic-pi-type amino acid vinyl monomer and a hydroxyalkyl acrylamide monomer. The polymer tissue adhesive disclosed by the invention is dissolved in a water-phase medium to form a precursor solution with excellent flowability, and has endoscope in-situ injection delivery performance; meanwhile, when the precursor solution is in contact with gastric acid, solution-gel conversion can be generated, interface drainage and rapid bonding effects can be achieved, the bonding strength on gastric ulcer wounds can be effectively improved, adjacent cation-pi adhesion units can be formed, long-term stable sealing of ulcer areas is achieved, and the sealing effect is good. And the time of sealing the gastric ulcer wound by the tissue adhesive is prolonged to more than 96 hours.

Description

Technical Field

[0001] This application belongs to the field of medical polymer materials technology, and specifically relates to an acid-triggered stepwise bonding polymer tissue adhesive, its preparation method and application. Background Technology

[0002] Endoscopic resection is a common clinical treatment for early-stage gastric cancer, but postoperative ulceration is prone to bleeding, perforation, and poor healing. Traditionally, metal clips are used to close the wound, combined with proton pump inhibitors, but this can easily lead to secondary injuries and potential small bowel damage. Therefore, developing safe and effective postoperative ulcer management protocols for endoscopic resection is of great significance.

[0003] The continuous stimulation of gastric acid and pepsin is a key factor contributing to the difficulty in healing ulcer wounds. Currently, a polymeric hydrogel has been successfully developed for the treatment of gastric ulcer wounds. It possesses a biomimetic structure similar to the extracellular matrix, high water content, strong tissue adaptability, high biocompatibility, and mechanical support. It can effectively inhibit bleeding, seal tissue, and plug irregular wounds, while also blocking irritation and promoting wound healing, making it an excellent adhesive for repairing tissue damage in vivo.

[0004] Although polymeric hydrogels have been used to treat gastric ulcer wounds, their clinical translation still faces the following challenges: First, traditional polymeric hydrogels cannot be delivered to the ulcer in situ under endoscopic guidance and effectively seal the wound. Second, the highly acidic and moist environment of the stomach can easily disrupt the intermolecular forces at the interface between the hydrogel and the ulcer tissue, weakening its mechanical properties and structural stability, making it difficult to establish a strong adhesive interface on the dynamic, moist, and highly acidic ulcer surface. Summary of the Invention

[0005] This application discloses an acid-triggered stepwise adhesion polymeric tissue adhesive, its preparation method, and its application, which solves the technical problem that traditional polymeric hydrogel adhesives are difficult to deliver endoscopically and provide long-term sealing of gastric ulcer wounds.

[0006] To achieve the above objectives, the technical solution provided in this application is as follows:

[0007] The first aspect of this application provides an acid-triggered stepwise bonding polymeric tissue adhesive, which has an ionized polymer.

[0008] The ionized polymer is prepared by free radical polymerization of monomer components in an alkaline aqueous phase, wherein the monomer components include:

[0009] (a) Anhydride-terminated cationic π-type amino acid vinyl monomers;

[0010] (b) Hydroxyalkyl acrylamide monomers.

[0011] According to the disclosure of the first aspect, the molar ratio of the anhydride-terminated cationic-π-type amino acid vinyl monomer to the hydroxyalkyl acrylamide monomer is (0.5~5):1.

[0012] According to the disclosure of the first aspect, the amino acid unit contained in the anhydride-terminated cationic-π-type amino acid vinyl monomer is selected from one of phenylalanine, tyrosine, and tryptophan.

[0013] According to the disclosure of the first aspect, the anhydride unit contained in the anhydride-terminated cationic-π-type amino acid vinyl monomer is selected from one of maleic anhydride, phthalic anhydride, methylcis-butenidine, and 2,3-dimethylmaleic anhydride.

[0014] According to the disclosure of the first aspect, the anhydride-terminated cationic-π-type amino acid vinyl monomer is selected as anhydride-terminated N-(3-aminopropyl)methacrylamide phenylalanine.

[0015] According to the disclosure of the first aspect, the hydroxyalkyl acrylamide monomers include N,N-bis(2-hydroxyethyl)-2-acrylamide, N-hydroxyethylacrylamide, and N-[tris(hydroxymethyl)methyl]acrylamide.

[0016] The second aspect of this application also discloses a method for preparing the acid-triggered stepwise bonding polymeric tissue adhesive described above, which includes the following steps:

[0017] Synthesize anhydride-terminated cationic π-type amino acid vinyl monomers;

[0018] The anhydride-terminated cationic π-type amino acid vinyl monomer, hydroxyalkyl acrylamide monomer, basic reagent, peroxide initiator and accelerator are dissolved in an aqueous medium and then subjected to free radical polymerization at a temperature of 50~70℃ to obtain the product.

[0019] According to the disclosure of the second aspect, the aqueous medium is selected from one of deionized water, physiological saline, and biological buffer solution;

[0020] And / or, the alkaline reagent is selected from one of sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, and sodium acetate;

[0021] And / or, the peroxide initiator is selected as ammonium persulfate;

[0022] And / or, the promoter is selected as tetramethylethylenediamine.

[0023] According to the disclosure of the second aspect, the total concentration of the two monomers contained in the aqueous medium is 0.2~0.6 mol / mL.

[0024] According to the disclosure of the second aspect, the molar ratio of the basic reagent to the anhydride-terminated cationic-π-type amino acid vinyl monomer is 1:(1~1.2).

[0025] According to the disclosure of the second aspect, the molar ratio of the initiator to the sum of the molar amounts of the two monomers is (0.005~0.02):1.

[0026] According to the disclosure of the second aspect, the molar ratio of the promoter to the sum of the molar amounts of the two monomers is (0.002~0.01):1.

[0027] According to the disclosure of the second aspect, the method for synthesizing the anhydride-terminated cationic-π-type amino acid vinyl monomer comprises:

[0028] After amino-protected amino acid derivatives undergo amidation condensation with amino-substituted acrylamide monomers, deprotection treatment is performed to prepare cationic-π-type amino acid vinyl monomers.

[0029] The cationic π-type amino acid vinyl monomer is obtained by reacting it with an acid anhydride through a nucleophilic acyl substitution reaction.

[0030] The third aspect of this application also discloses the application of the acid-triggered stepwise bonding polymeric tissue adhesive of the present invention in the preparation of acid-sensitive wound repair materials.

[0031] Compared with the prior art, the advantages or beneficial effects of this application include at least the following: This application utilizes the above two monomers to create a polymeric ionized polymer. Firstly, it dissolves in an aqueous medium to form a highly fluid precursor solution, which can be delivered endoscopically in situ, effectively enhancing the control over the treatment process and minimizing operational interference. Secondly, upon contact with gastric acid, the precursor solution undergoes carboxylate protonation and hydrophobic synergistic hydrogen bonding, driving the solution-gel transition and providing interfacial drainage and rapid adhesion, effectively achieving rapid gelation for quick sealing of ulcer wounds. Thirdly, gastric acid causes the acid-sensitive groups of the ionized polymer to gradually detach, exposing amino groups. This enhances the adhesion between the hydrogel and the interface through π-assisted electrostatic interaction, effectively increasing the adhesion strength to the gastric ulcer wound. Furthermore, after the acid-sensitive groups are completely detached, the protonated amino groups and benzene ring structure can form adjacent cation-π adhesion units, achieving long-term stable sealing of the ulcer area, extending the time of tissue adhesive sealing of the gastric ulcer wound to more than 96 hours. Attached Figure Description

[0032] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 The NMR 1H spectrum of the NMA-APA provided in this application;

[0034] Figure 2 The 1H NMR spectrum of NA-Mal provided for this application;

[0035] Figure 3 The NA-Mal provided for this application 0.125 HEAA 0.175 The hydrogen NMR spectrum;

[0036] Figure 4 The NA-Mal provided for this application 0.125 HEAA 0.175 Viscosity test graph of polymer solution;

[0037] Figure 5 The NA-Pht provided for this application 0.2 HEAA 0.3 Gelation test diagram of polymer solution;

[0038] Figure 6 The NA-Mal provided for this application 0.15 HEAA 0.15 The stress-strain curve;

[0039] Figure 7 The NA-Mal provided for this application 0.125 THMA 0.175 Wet bonding kinetics test diagram;

[0040] Figure 8 The NA-Mal provided for this application 0.125 PPA 0.175 Graph of cytotoxicity test;

[0041] Figure 9 NA-Cit provided for this application 0.125 THMA 0.175 Blood compatibility test results;

[0042] Figure 10 The NA-Mal provided for this application 0.2 HEAA 0.1 The in vivo residence time test chart. Detailed Implementation

[0043] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments described in this application without creative effort are within the scope of protection of this application.

[0044] In the following description of this specification, the term "and / or" is used to describe the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, B existing alone, and both A and B existing simultaneously. A and B can be singular or plural; the symbol " / " means "or".

[0045] In the following description of this specification, the term "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions mean any combination of such items, including any combination of single or plural items. For example, "at least one of A, B or C", or "at least one of A, B and C", can mean any one of A, B, C, or A+B, or A+C, or B+C, or A+B+C, where A, B, and C can be single or multiple.

[0046] In the following description of this specification, the sequence of numbers does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and does not constitute any limitation on the execution process of this embodiment.

[0047] In the following description of this specification, numerical ranges should be understood to specifically disclose each intermediate value between the upper and lower limits of the range. Any intermediate value within a stated range, as well as any other stated value or each smaller range between intermediate values ​​within a stated range, are also included in this embodiment, and the upper and lower limits of the smaller ranges may be independently included or excluded from the range.

[0048] Unless otherwise stated, the technical / scientific terms used in this specification have the meanings commonly understood by one of ordinary skill in the art. While this specification describes only preferred materials and methods, any similar or equivalent methods and materials may be used in specific embodiments or test examples. All references to this specification are incorporated by way of citation to disclose and describe the 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.

[0049] To address the challenges of efficient endoscopic delivery and rapid in-situ gelation of existing polymeric tissue adhesives, as well as their insufficient long-term sealing performance on gastric ulcer wounds, a first aspect of this application provides an acid-triggered stepwise adhesive polymeric tissue adhesive comprising an ionized polymer. The ionized polymer is prepared by free radical polymerization of monomer components in an alkaline aqueous phase, the monomer components comprising: (a) an anhydride-terminated cationic-π-type amino acid vinyl monomers; and (b) hydroxyalkyl acrylamide monomers.

[0050] This application embodiment utilizes the above two structural monomers to polymerize ionized polymers. Firstly, this gives the ionized polymers an electrostatic repulsion effect that allows molecular chains to extend, facilitating dissolution in the aqueous medium and forming a highly fluid precursor solution. This allows for in-situ delivery via endoscopy, effectively enhancing the control over the treatment process and minimizing operational interference. Secondly, upon contact with gastric acid, the precursor solution undergoes carboxylate protonation and generates hydrophobic synergistic hydrogen bonds. This drives the solution-gel transition of the precursor solution and also provides interfacial drainage and rapid adhesion, effectively achieving rapid... The gelation process provides rapid sealing of ulcer wounds (primary adhesion effect); thirdly, gastric acid causes some acid-sensitive groups of the ionized polymer to detach and expose amino groups, which can enhance the adhesion between the hydrogel and the interface through π-assisted electrostatic action, effectively improving the adhesion strength to the gastric ulcer wound (secondary adhesion effect); fourthly, after the acid-sensitive groups of the ionized polymer are completely detached, the protonated amino groups and benzene ring structure can form adjacent cation-π adhesion units (tertiary adhesion effect), effectively achieving long-term stable sealing of the ulcer area, increasing the time for tissue adhesive to seal the gastric ulcer wound to more than 96 hours.

[0051] In possible disclosed examples, the molar ratio of the anhydride-terminated cationic-π-type amino acid vinyl monomer to the hydroxyalkyl acrylamide monomer is preferably (0.5~5):1, and can be 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, and any one within the range.

[0052] In possible disclosed examples, the amino acid units contained in the anhydride-terminated cationic-π-type amino acid vinyl monomer are selected from one of phenylalanine, tyrosine, and tryptophan. These amino acids possess adjacent cationic-π structures, which can activate the tertiary adhesion mechanism of adjacent cationic-π adhesion units, effectively achieving long-term stable sealing of the ulcer region.

[0053] In possible disclosed examples, the anhydride unit contained in the anhydride-terminated cationic-π-type amino acid vinyl monomer is selected from one of maleic anhydride, phthalic anhydride, methylcis-butenidine, and 2,3-dimethylmaleic anhydride. These anhydrides can mask the primary amine group of the amino acid structure through acid-sensitive bonds, ensuring both good injectability of the ionized polymer aqueous solution and rapid gelation triggered by acid to seal the wound.

[0054] In possible disclosed examples, the anhydride-terminated cationic-π-type amino acid vinyl monomer is preferably anhydride-terminated N-(3-aminopropyl)methacrylamide phenylalanine.

[0055] In possible public examples, the hydroxyalkylacrylamide monomers comprise N,N-bis(2-hydroxyethyl)-2-acrylamide, N-hydroxyethylacrylamide, and N-[tris(hydroxymethyl)methyl]acrylamide.

[0056] In a second aspect, embodiments of this application also provide a method for preparing the acid-triggered stepwise bonding polymeric tissue adhesive, comprising the steps of:

[0057] Synthesize anhydride-terminated cationic π-type amino acid vinyl monomers;

[0058] The anhydride-terminated cationic π-type amino acid vinyl monomer, hydroxyalkyl acrylamide monomer, basic reagent, peroxide initiator and accelerator are dissolved in an aqueous medium and then subjected to free radical polymerization at a temperature of 50~70℃ to obtain the product.

[0059] In possible public examples, the aqueous medium is selected from deionized water, physiological saline, and biological buffer solution; the alkaline reagent is selected from sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, and sodium acetate; the peroxide initiator is selected as ammonium persulfate; and the accelerator is selected as tetramethylethylenediamine.

[0060] In possible disclosed examples, the total concentration of the two monomers contained in the aqueous medium is 0.2~0.6 mol / mL, which can be 0.2 mol / mL, 0.3 mol / mL, 0.4 mol / mL, 0.5 mol / mL, 0.6 mol / mL, and any one within the range.

[0061] In possible disclosed examples, the molar ratio of the basic reagent to the anhydride-terminated cationic-π-type amino acid vinyl monomer is 1:(1~1.2), which can be 1:1, 1:1.1, 1:1.2, and any one within the range.

[0062] In possible disclosed examples, the molar ratio of the initiator to the sum of the molar amounts of the two monomers is (0.005~0.02):1, which can be 0.005:1, 0.01:1, 0.015:1, 0.02:1, and any one within the range.

[0063] In possible public examples, the molar ratio of the promoter to the sum of the molar amounts of the two monomers is (0.002~0.01):1, which can be 0.002:1, 0.005:1, 0.01:1, or any one within the range.

[0064] This application also provides a method for synthesizing the anhydride-terminated cationic-π-type amino acid vinyl monomer, wherein the method preferably comprises:

[0065] After amino-protected amino acid derivatives undergo amidation condensation with amino-substituted acrylamide monomers, deprotection treatment is performed to prepare cationic-π-type amino acid vinyl monomers.

[0066] The cationic π-type amino acid vinyl monomer is obtained by reacting it with an acid anhydride through a nucleophilic acyl substitution reaction.

[0067] It should be noted that this application provides a method for synthesizing a cationic-π-type amino acid vinyl monomer, taking an anhydride-terminated N-(3-aminopropyl)methacrylamide phenylalanine as an example, which includes:

[0068] 3.18 g of Boc-L-phenylalanine (Boc-L-APA, 0.012 mol), 2.7 g of N-(3-aminopropyl)methacrylamide (NMA, 0.015 mol), and 1.8 g of 1-hydroxybenzotriazole (1-HOBT, 0.013 mol) were dissolved in a mixed organic solvent consisting of 135 mL of dichloromethane and 18 mL of dimethylformamide. Then, 9 mL of triethylamine (0.064 mol) and 4.5 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 0.024 mol) were added to the mixture, and the mixture was stirred at room temperature for 24 hours. The mixture was then washed three times with 50 mL of saturated sodium chloride solution and 50 mL of saturated sodium bicarbonate solution, respectively. The resulting organic phase was dried overnight with anhydrous magnesium sulfate, filtered, and the organic solvent was removed by rotary evaporation to obtain NMA-Boc-APA. NMA-Boc-APA was dissolved in a mixed solution of dichloromethane and trifluoroacetic acid in a volume ratio of 3:1 in 24 mL. After stirring and reacting for 6 hours under ice bath conditions, the mixture was poured into diethyl ether to precipitate the product. The precipitate was then dried under vacuum for 24 hours to obtain the yellow solid product N-(3-aminopropyl)methacrylamide phenylalanine (NMA-APA).

[0069] (1) A method for synthesizing maleic anhydride-terminated N-(3-aminopropyl)methacrylamide phenylalanine (NA-Mal), preferably comprising:

[0070] NMA-APA (5 g, 0.017 mol) was dissolved in 50 mL of deionized water. Sodium bicarbonate (2.9 g, 0.034 mol) was added to the aqueous phase. Then, 3 mL of dimethyl sulfoxide (DMSO) solution containing maleic anhydride (Mal, 1.52 g, 0.016 mol) was added dropwise to the aqueous phase. The mixture was stirred at room temperature for 24 hours. The pH of the aqueous phase was adjusted to 1 using 6 M hydrochloric acid, and the aqueous phase was immediately subjected to four rapid extractions with ethyl acetate. The combined organic phases were washed five times with saturated sodium chloride solution and dried over anhydrous magnesium sulfate. After filtration, ethyl acetate was removed by rotary evaporation, followed by vacuum drying for 24 hours to obtain the white solid product NA-Mal.

[0071] (2) A method for synthesizing methylcis-butenyl dianhydride-terminated N-(3-aminopropyl)methacrylamide phenylalanine (NA-Cit), preferably comprising:

[0072] NMA-APA (5 g, 0.017 mol) was dissolved in 50 mL of deionized water. Sodium bicarbonate (2.9 g, 0.034 mol) was added to the aqueous phase. Then, 3 mL of DMSO solution containing methylcis-butenediantane (Cit, 1.79 g, 0.016 mol) was added dropwise to the aqueous phase. The mixture was stirred at room temperature for 24 hours. The pH of the aqueous phase was adjusted to 1 using 6 M hydrochloric acid, and the aqueous phase was immediately subjected to four rapid extractions with ethyl acetate. The combined organic phases were washed five times with saturated sodium chloride solution and dried over anhydrous magnesium sulfate. After filtration, ethyl acetate was removed by rotary evaporation, followed by vacuum drying for 24 hours to obtain the white solid product NA-Cit.

[0073] (3) A method for synthesizing 2,3-dimethylmaleic anhydride-terminated N-(3-aminopropyl)methacrylamide phenylalanine (NA-Di-Mal), preferably comprising:

[0074] NMA-APA (5 g, 0.017 mol) was dissolved in 50 mL of deionized water. Sodium bicarbonate (2.9 g, 0.034 mol) was added to the aqueous phase. Then, 3 mL of DMSO solution containing 2,3-dimethylmaleic anhydride (Di-Mal, 1.79 g, 0.016 mol) was added dropwise to the aqueous phase. The mixture was stirred at room temperature for 24 hours. The pH of the aqueous phase was adjusted to 1 using 6 M hydrochloric acid, and the aqueous phase was immediately subjected to four rapid extractions with ethyl acetate. The combined organic phases were washed five times with saturated sodium chloride solution and dried over anhydrous magnesium sulfate. After filtration, ethyl acetate was removed by rotary evaporation, followed by vacuum drying for 24 hours to obtain the white solid product NA-Di-Mal.

[0075] (4) A method for synthesizing phthalic anhydride-terminated N-(3-aminopropyl)methacrylamide phenylalanine (NA-Pht), preferably comprising:

[0076] NMA-APA (5 g, 0.017 mol) was dissolved in 50 mL of deionized water. Sodium bicarbonate (2.9 g, 0.034 mol) was added to the aqueous phase. Then, 3 mL of DMSO solution containing phthalic anhydride (Pht, 1.79 g, 0.016 mol) was added dropwise to the aqueous phase. The mixture was stirred at room temperature for 24 hours. The pH of the aqueous phase was adjusted to 1 using 6 M hydrochloric acid, and the aqueous phase was immediately subjected to four rapid extractions with ethyl acetate. The combined organic phases were washed five times with saturated sodium chloride solution and dried over anhydrous magnesium sulfate. After filtration, ethyl acetate was removed by rotary evaporation, followed by vacuum drying for 24 hours to obtain the white solid product NA-Pht.

[0077] In a third aspect, embodiments of this application also provide the application of the acid-triggered stepwise bonding polymeric tissue adhesive described above, specifically the application of the acid-triggered stepwise bonding polymeric tissue adhesive in the preparation of acid-related wound repair materials such as gastric ulcer wounds.

[0078] The technical solution of the present invention will be further described below with reference to specific embodiments. In the following embodiments, the acid-responsive stepwise bonding polymer tissue adhesive prepared is denoted as A. x B y A includes, but is not limited to, NA-Mal, NA-Cit, NA-Di-Mal, NA-Pht, etc.; B monomers include, but are not limited to, N,N-bis(2-hydroxyethyl)-2-acrylamide (PPA), N-hydroxyethylacrylamide (HEAA), N-[tris(hydroxymethyl)methyl]acrylamide (THMA), etc.; x and y represent the molar concentration of the monomers.

[0079] Example 1

[0080] This example demonstrates NA-Mal, a polymeric tissue adhesive for acid-triggered stepwise bonding. 0.125 PPA 0.175 The specific steps for preparing [the substance] are as follows:

[0081] NA-Mal (0.125 mmol) and sodium hydroxide (0.125 mmol) were dissolved in 1 mL of deionized water. The solution was filtered through a sterile membrane with an average pore size of 0.4 μm, and PPA (0.175 mmol) was added. The solution was then deoxygenated by bubbling with nitrogen three times, and ammonium persulfate (0.003 mmol) and tetramethylethylenediamine (0.002 mmol) were immediately added. The polymerization reaction was then carried out at 60 °C for 1 hour to obtain NA-Mal. 0.125 PPA 0.175 Polymer solution.

[0082] Example 2

[0083] This example demonstrates NA-Cit, a polymeric tissue adhesive for acid-triggered stepwise bonding. 0.125 PPA 0.175 The specific steps for preparing [the substance] are as follows:

[0084] NA-Cit (0.125 mmol) and sodium hydroxide (0.125 mmol) were dissolved in 1 mL of deionized water. The solution was filtered through a sterile membrane with an average pore size of 0.4 μm, and PPA (0.175 mmol) was added. After deoxygenation by bubbling with nitrogen three times, ammonium persulfate (0.003 mmol) and tetramethylethylenediamine (0.002 mmol) were immediately added. The polymerization reaction was then carried out at 60 °C for 1 hour to obtain NA-Cit. 0.125 PPA 0.175 Polymer solution.

[0085] Example 3

[0086] This example demonstrates NA-Cit, a polymeric tissue adhesive for acid-triggered stepwise bonding. 0.2 HEAA 0.1 The specific steps for preparing [the substance] are as follows:

[0087] NA-Cit (0.2 mmol) and sodium hydroxide (0.2 mmol) were dissolved in 1 mL of deionized water. The solution was filtered through a sterile membrane with an average pore size of 0.4 μm, and N-hydroxyethyl acrylamide (HEAA, 0.1 mmol) was added. After deoxygenation by bubbling nitrogen three times, ammonium persulfate (0.003 mmol) and tetramethylethylenediamine (0.002 mmol) were immediately added. The polymerization reaction was then carried out at 60 °C for 1 hour to obtain NA-Cit. 0.2 HEAA0.1 Polymer solution.

[0088] Example 4

[0089] This example demonstrates an acid-triggered stepwise bonding polymeric tissue adhesive, NA-Di-Mal. 0.1 PPA 0.175 The specific steps for preparing [the substance] are as follows:

[0090] NA-Di-Mal (0.1 mmol) and sodium hydroxide (0.1 mmol) were dissolved in 1 mL of deionized water. The solution was filtered through a sterile membrane with an average pore size of 0.4 μm and PPA (0.175 mmol) was added. After deoxygenation by bubbling nitrogen three times, ammonium persulfate (0.003 mmol) and tetramethylethylenediamine (0.002 mmol) were immediately added. The polymerization reaction was then carried out at 60 °C for 1 hour to obtain NA-Di-Mal. 0.1 PPA 0.175 Polymer solution.

[0091] Example 5

[0092] This example demonstrates NA-Pht, a polymeric tissue adhesive for acid-triggered stepwise bonding. 0.1 PPA 0.175 The specific steps for preparing [the substance] are as follows:

[0093] NA-Pht (0.1 mmol) and sodium hydroxide (0.1 mmol) were dissolved in 1 mL of deionized water. The solution was filtered through a sterile membrane with an average pore size of 0.4 μm and PPA (0.175 mmol) was added. After deoxygenation by bubbling nitrogen three times, ammonium persulfate (0.003 mmol) and tetramethylethylenediamine (0.002 mmol) were immediately added. The polymerization reaction was then carried out at 60 °C for 1 hour to obtain NA-Pht. 0.1 PPA 0.175 Polymer solution.

[0094] Example 6

[0095] This example demonstrates NA-Mal, a polymeric tissue adhesive for acid-triggered stepwise bonding. 0.125 HEAA 0.175 The specific steps for preparing [the substance] are as follows:

[0096] NA-Mal (0.125 mmol) and sodium hydroxide (0.125 mmol) were dissolved in 1 mL of deionized water. The solution was filtered through a sterile membrane with an average pore size of 0.4 μm and HEAA (0.175 mmol) was added. After deoxygenation by bubbling with nitrogen three times, ammonium persulfate (0.003 mmol) and tetramethylethylenediamine (0.002 mmol) were immediately added. The polymerization reaction was then carried out at 60 °C for 1 hour to obtain NA-Mal. 0.125 HEAA 0.175 Polymer solution.

[0097] Example 7

[0098] This example demonstrates NA-Pht, a polymeric tissue adhesive for acid-triggered stepwise bonding. 0.2 HEAA 0.3 The specific steps for preparing [the substance] are as follows:

[0099] NA-Pht (0.2 mmol) and sodium hydroxide (0.2 mmol) were dissolved in 1 mL of deionized water. The solution was filtered through a sterile membrane with an average pore size of 0.4 μm and HEAA (0.3 mmol) was added. After deoxygenation by bubbling with nitrogen three times, ammonium persulfate (0.005 mmol) and tetramethylethylenediamine (0.003 mmol) were immediately added. The polymerization reaction was then carried out at 60 °C for 1 hour to finally obtain NA-Pht. 0.2 HEAA 0.3 Polymer solution.

[0100] Example 8

[0101] This example demonstrates NA-Mal, a polymeric tissue adhesive for acid-triggered stepwise bonding. 0.15 HEAA 0.15 The specific steps for preparing [the substance] are as follows:

[0102] NA-Mal (0.15 mmol) and sodium hydroxide (0.15 mmol) were dissolved in 1 mL of deionized water. The solution was filtered through a sterile membrane with an average pore size of 0.4 μm and HEAA (0.15 mmol) was added. After deoxygenation by bubbling nitrogen three times, ammonium persulfate (APS, 0.003 mmol) and tetramethylethylenediamine (TEMED, 0.002 mmol) were immediately added. The polymerization reaction was carried out at 60 °C for 1 hour to obtain NA-Mal. 0.15 HEAA 0.15 Polymer solution.

[0103] Example 9

[0104] This example demonstrates NA-Pht, a polymeric tissue adhesive for acid-triggered stepwise bonding. 0.1 THMA 0.2 The specific steps for preparing [the substance] are as follows:

[0105] NA-Pht (0.1 mmol) and sodium hydroxide (0.1 mmol) were dissolved in 1 mL of deionized water. The solution was filtered through a sterile membrane with an average pore size of 0.4 μm and THMA (0.2 mmol) was added. After deoxygenation by bubbling nitrogen three times, ammonium persulfate (0.003 mmol) and tetramethylethylenediamine (0.002 mmol) were immediately added. The polymerization reaction was then carried out at 60 °C for 1 hour to obtain NA-Pht. 0.1 THMA 0.2 Polymer solution.

[0106] Example 10

[0107] This example demonstrates NA-Mal, a polymeric tissue adhesive for acid-triggered stepwise bonding. 0.125 THMA 0.175 The specific steps for preparing [the substance] are as follows:

[0108] NA-Mal (0.125 mmol) and sodium hydroxide (0.125 mmol) were dissolved in 1 mL of deionized water. The solution was filtered through a sterile membrane with an average pore size of 0.4 μm, and THMA (0.175 mmol) was added. After three nitrogen bubbling processes to remove oxygen, ammonium persulfate (0.003 mmol) and tetramethylethylenediamine (0.002 mmol) were immediately added. The polymerization reaction was then carried out at 60 °C for 1 hour to obtain NA-Mal. 0.125 THMA 0.175 Polymer solution.

[0109] Example 11

[0110] This example demonstrates an acid-triggered stepwise bonding polymeric tissue adhesive, NA-Di-Mal. 0.125 THMA 0.175 The specific steps for preparing [the substance] are as follows:

[0111] NA-Di-Mal (0.125 mmol) and sodium hydroxide (0.125 mmol) were dissolved in 1 mL of deionized water. The solution was filtered through a sterile membrane with an average pore size of 0.4 μm and THMA (0.175 mmol) was added. After three nitrogen bubbling processes to remove oxygen, ammonium persulfate (0.003 mmol) and tetramethylethylenediamine (0.002 mmol) were immediately added. The polymerization reaction was then carried out at 60 °C for 1 hour to obtain NA-Di-Mal. 0.125 THMA0.175 Polymer solution.

[0112] Example 12

[0113] This example demonstrates an acid-triggered stepwise bonding polymeric tissue adhesive, NA-Di-Mal. 0.125 THMA 0.175 The specific steps for preparing [the substance] are as follows:

[0114] NA-Cit (0.125 mmol) and sodium hydroxide (0.125 mmol) were dissolved in 1 mL of deionized water. The solution was filtered through a sterile membrane with an average pore size of 0.4 μm and THMA (0.175 mmol) was added. After deoxygenation by bubbling with nitrogen three times, ammonium persulfate (0.003 mmol) and tetramethylethylenediamine (0.002 mmol) were immediately added. The polymerization reaction was then carried out at 60 °C for 1 hour to obtain NA-Di-Mal. 0.125 THMA 0.175 Polymer solution.

[0115] Example 13

[0116] This example demonstrates NA-Mal, a polymeric tissue adhesive for acid-triggered stepwise bonding. 0.125 PPA 0.175 The specific steps for preparing [the substance] are as follows:

[0117] NA-Mal (0.125 mmol) and sodium hydroxide (0.125 mmol) were dissolved in 1 mL of deionized water. The solution was filtered through a sterile membrane with an average pore size of 0.4 μm, and PPA (0.175 mmol) was added. After three nitrogen bubbling processes to remove oxygen, ammonium persulfate (0.003 mmol) and tetramethylethylenediamine (0.002 mmol) were immediately added. The polymerization reaction was then carried out at 60 °C for 1 hour to obtain NA-Mal. 0.125 PPA 0.175 Polymer solution.

[0118] Example 14

[0119] This example demonstrates NA-Cit, a polymeric tissue adhesive for acid-triggered stepwise bonding. 0.125 THMA 0.175 The specific steps for preparing [the substance] are as follows:

[0120] NA-Cit (0.125 mmol) and sodium hydroxide (0.125 mmol) were dissolved in 1 mL of deionized water. The solution was filtered through a sterile membrane with an average pore size of 0.4 μm and THMA (0.175 mmol) was added. After deoxygenation by bubbling nitrogen three times, ammonium persulfate (0.003 mmol) and tetramethylethylenediamine (0.002 mmol) were immediately added. The polymerization reaction was then carried out at 60 °C for 1 hour to obtain NA-Cit. 0.125 THMA 0.175 Polymer solution.

[0121] Example 15

[0122] This example demonstrates an acid-triggered stepwise bonding polymeric tissue adhesive, NA-Di-Mal. 0.15 HEAA 0.2 The specific steps for preparing [the substance] are as follows:

[0123] NA-Di-Mal (0.15 mmol) and sodium hydroxide (0.15 mmol) were dissolved in 1 mL of deionized water. The solution was filtered through a sterile membrane with an average pore size of 0.4 μm and HEAA (0.2 mmol) was added. After deoxygenation by bubbling nitrogen three times, ammonium persulfate (0.003 mmol) and tetramethylethylenediamine (0.002 mmol) were immediately added. The polymerization reaction was then carried out at 60 °C for 1 hour to obtain NA-Di-Mal. 0.15 HEAA 0.2 Polymer solution.

[0124] Example 16

[0125] This example demonstrates NA-Cit, a polymeric tissue adhesive for acid-triggered stepwise bonding. 0.1 PPA 0.2 The specific steps for preparing [the substance] are as follows:

[0126] NA-Cit (0.1 mmol) and sodium hydroxide (0.1 mmol) were dissolved in 1 mL of deionized water. The solution was filtered through a sterile membrane with an average pore size of 0.4 μm, and PPA (0.2 mmol) was added. After deoxygenation by bubbling nitrogen three times, ammonium persulfate (0.003 mmol) and tetramethylethylenediamine (0.002 mmol) were immediately added. The polymerization reaction was then carried out at 60 °C for 1 hour to obtain NA-Cit. 0.1 PPA 0.2 Polymer solution.

[0127] Example 17

[0128] This example demonstrates NA-Mal, a polymeric tissue adhesive for acid-triggered stepwise bonding. 0.175 THMA 0.125 The specific steps for preparing [the substance] are as follows:

[0129] NA-Mal (0.175 mmol) and sodium hydroxide (0.125 mmol) were dissolved in 1 mL of deionized water. The solution was filtered through a sterile membrane with an average pore size of 0.4 μm, and THMA (0.125 mmol) was added. After deoxygenation by bubbling nitrogen three times, ammonium persulfate (0.003 mmol) and tetramethylethylenediamine (0.002 mmol) were immediately added. The polymerization reaction was then carried out at 60 °C for 1 hour to obtain NA-Mal. 0.175 THMA 0.125 Polymer solution.

[0130] Example 18

[0131] This example demonstrates NA-Mal, a polymeric tissue adhesive for acid-triggered stepwise bonding. 0.2 HEAA 0.1 The specific steps for preparing [the substance] are as follows:

[0132] NA-Mal (0.2 mmol) and sodium hydroxide (0.2 mmol) were dissolved in 1 mL of deionized water. The solution was filtered through a sterile membrane with an average pore size of 0.4 μm and HEAA (0.1 mmol) was added. After deoxygenation by bubbling with nitrogen three times, ammonium persulfate (APS, 0.003 mmol) and tetramethylethylenediamine (TEMED, 0.002 mmol) were immediately added. The polymerization reaction was then carried out at 60 °C for 1 hour to obtain NA-Mal. 0.2 HEAA 0.1 Polymer solution.

[0133] Test Example 1: Characterization by Proton NMR Spectroscopy

[0134] 1.1 NMR characterization of NMA-APA (1H NMR spectrum)

[0135] After dissolving 5 mg of NMH-APA in 650 μL of deuterium water, the NMH-APA NMR spectrum was recorded at room temperature using a nuclear magnetic resonance spectrometer. The results were as follows: Figure 1 As shown.

[0136] according to Figure 1 It can be seen that NMA-APA was successfully prepared.

[0137] 1.2 NA-Mal 1H NMR Characterization

[0138] After dissolving 5 mg of NA-Mal in 650 μL of deuterated dimethyl sulfoxide, the NA-Mal NMR spectrum was recorded at room temperature using an NMR spectrometer. The results were as follows: Figure 2 As shown.

[0139] according to Figure 2 It can be seen that NA-Mal was successfully prepared.

[0140] 1.3 NA-Mal 0.125 HEAA 0.175 Nuclear magnetic spectroscopy characterization

[0141] 10 mg NA-Mal 0.125 HEAA 0.175 After adding 650 μL of deuterium water and dissolving completely, the NA-Mal x HEAA y NMR spectrum was recorded at room temperature using a nuclear magnetic resonance spectrometer. The results were as follows: Figure 3 As shown.

[0142] according to Figure 3 It can be seen that NA-Mal 0.125 HEAA 0.175 Successfully prepared.

[0143] Test Example 2: Polymer Solution Viscosity Test

[0144] 100 μL NA-Mal 0.125 HEAA 0.175 The polymer solution was placed in the center of the rheometer disk, and the NA-Mal solution was measured using shear rate scanning mode. 0.125 HEAA 0.175 The shear rate of the polymer solution is 0-100 s. - ¹The viscosity characteristics within a certain range, the results are as follows Figure 4 As shown.

[0145] according to Figure 4 It can be seen that NA-Mal 0.125 HEAA 0.175 The polymer solution has an initial viscosity of less than 10 Pa·s, and its viscosity gradually decreases with increasing shear rate, demonstrating that the polymer tissue binder precursor solution has injectable properties and can be delivered in situ via endoscopy.

[0146] Test Example 3: Polymer Solution Gelation Test

[0147] 100 μL NA-Pht 0.2 HEAA 0.3 The polymer solution was placed in the center of the rheometer disk, and then the disk was filled with simulated gastric fluid. The NA-Pht was immediately measured using a time-scan mode with a frequency of 1 Hz and a strain of 1%.0.2 HEAA 0.3 The gelation behavior of the polymer solution is as follows: Figure 5 As shown.

[0148] according to Figure 5 It can be seen that after 4 seconds, NA-Pht 0.2 HEAA 0.3 The storage modulus of the polymer solution is greater than its loss modulus, demonstrating that the polymer tissue binder precursor solution rapidly gels after 4 seconds of incubation in simulated gastric juice, enabling in-situ gelation via endoscopic delivery.

[0149] Test Example 4: Mechanical Performance Test

[0150] 300 μL NA-Mal 0.15 HEAA 0.15 The polymer solution was injected into 1 mL of simulated gastric fluid and incubated at room temperature for 1 h. Afterward, it was removed and subjected to a universal tensile testing machine using an electromechanical testing instrument with a maximum load of 50 N, at a speed of 5 mm·min. - ¹The rate is used to obtain force-displacement data, and the result is Figure 6 As shown.

[0151] according to Figure 6 It can be seen that NA-Mal 0.15 HEAA 0.15 Its elastic modulus is greater than 30 kPa, making it suitable for sealing gastric wounds.

[0152] Test Example 5: Wet Bonding Kinetics Test

[0153] Fresh stomach tissue was cut into rectangular strips (10 mm × 30 mm) and washed sequentially with PBS and deionized water. Specifically, the surface of the porcine stomach tissue was first lightly dried, and NA-Mal was evenly coated on one side. 0.125 THMA 0.175 A polymer solution was applied, and the other side of the tissue was then overlapped and covered. The bonded double-layered gastric tissue was immersed in simulated gastric juice (SGF) for 10 minutes, 6 hours, and 14 days, followed by a 200 g weight applied for 10 minutes of continuous pressure. Overlap shear tests were performed using a CMT-1503 electromechanical testing instrument equipped with a 50 N load sensor at a tensile rate of 50 mm / min. The results were... Figure 7 As shown.

[0154] according to Figure 7 It can be seen that NA-Mal 0.125 THMA 0.175The instantaneous wet bond strength of the pig stomach was greater than 20 kPa after 10 minutes in a simulated gastric fluid environment, greater than 35 kPa after 6 hours of incubation, and still greater than 10 kPa after 14 days of continuous incubation.

[0155] Test Example 6: Cytotoxicity Test

[0156] First, 10,000 GES-1 cells were seeded in each well of a 96-well plate and cultured at 37°C for 24 hours. Then, the original culture medium was discarded, and cells containing different concentrations of NA-Mal were added. 0.125 PPA 0.175 Cells were cultured in pure cell culture medium for 24 hours. The negative control group used pure cell culture medium, and the positive control group used sterile water. After 24 hours, the culture medium was discarded, and 100 μL of 10% CCK8 was added to each well. After incubation for another 2 hours, the absorption spectrum at 450 nm was measured using a microplate reader. Cell viability was calculated using the following formula. The results were as follows: Figure 8 As shown.

[0157]

[0158] according to Figure 8 It can be seen that different concentrations of NA-Mal 0.125 PPA 0.175 The cell survival rate of the cell culture medium was higher than 90%, indicating that the polymer tissue adhesive of the present invention has excellent biocompatibility.

[0159] Test Example 7: Blood Compatibility Test

[0160] Evaluation of NA-Cit using hemolysis test 0.125 THMA 0.175 Blood compatibility was assessed by taking a 2% fresh rabbit blood cell suspension and mixing it with different concentrations of NA-Citrate. 0.125 THMA 0.175 The samples were co-cultured at 37℃, with PBS as the negative control group and sterile water as the positive control group. After 3 hours, the supernatant was collected, and the absorbance at 545 nm was measured using a UV spectrophotometer. The hemolysis rate was finally calculated using the following formula. Figure 9 As shown.

[0161]

[0162] according to Figure 9 As shown, NA-Cit 0.125 THMA 0.175 The hemolysis rate was less than 5%, indicating that the polymer tissue adhesive of the present invention has excellent blood compatibility.

[0163] Test Example 8: In vivo residence time test

[0164] In vivo imaging was used to observe the in vivo residence time of polymeric tissue adhesives. NA-Mal, used for in vivo monitoring, was prepared by introducing a small amount of N-(3-aminopropyl)methacrylamide hydrochloride during copolymerization and functionalizing it with sulfonated Cyanine 7 NHS ester. 0.2 HEAA 0.1 -Cy7 solution. All rats were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg) and then gastric isolated. 100 μL of NA-Mal was administered using an insulin syringe (20-G needle). 0.2 HEAA 0.1 Cy7 solution was injected into the isolated stomach of rats. In situ monitoring was performed using an in vivo imaging system at 8, 36, 48, 72, and 96 hours post-injection. In the ex vivo imaging study, rats were sacrificed 24 hours after injection, and stomach tissue and major organs were harvested. In vivo imaging was performed at an excitation wavelength of 745 nm and an emission wavelength of 785 nm. The results were... Figure 10 As shown.

[0165] according to Figure 10 It can be seen that NA-Mal 0.2 HEAA 0.1 The fact that the polymeric tissue adhesive of this invention has a residence time of more than 96 hours in the stomach of live rats indicates that it has the ability to effectively seal gastric ulcer wounds.

[0166] The various embodiments in this specification are described in a progressive manner. The same or similar parts between the various embodiments can be referred to each other. Each embodiment focuses on describing the differences from other embodiments.

[0167] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit this application. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of this application.

Claims

1. An acid-triggered stepwise bonding polymeric tissue adhesive, characterized in that, Possesses ionized polymer properties; The ionized polymer is prepared by free radical polymerization of monomer components in an alkaline aqueous phase, wherein the monomer components include: (a) Anhydride-terminated cationic π-type amino acid vinyl monomers; (b) Hydroxyalkyl acrylamide monomers.

2. The polymeric tissue adhesive according to claim 1, characterized in that, The molar ratio of the anhydride-terminated cationic-π-type amino acid vinyl monomer to the hydroxyalkyl acrylamide monomer is (0.5~5):

1.

3. The polymeric tissue adhesive according to claim 1, characterized in that, The amino acid unit contained in the anhydride-terminated cationic-π-type amino acid vinyl monomer is selected from one of phenylalanine, tyrosine, and tryptophan. The anhydride unit contained in the anhydride-terminated cationic-π-type amino acid vinyl monomer is selected from one of maleic anhydride, phthalic anhydride, methylcis-butenidine, and 2,3-dimethylmaleic anhydride.

4. The polymeric tissue adhesive according to claim 3, characterized in that, The anhydride-terminated cationic-π-type amino acid vinyl monomer is selected as anhydride-terminated N-(3-aminopropyl)methacrylamide phenylalanine.

5. The polymeric tissue adhesive according to claim 1, characterized in that, The hydroxyalkyl acrylamide monomers include N,N-bis(2-hydroxyethyl)-2-acrylamide, N-hydroxyethylacrylamide, and N-[tris(hydroxymethyl)methyl]acrylamide.

6. A method for preparing an acid-triggered stepwise bonding polymeric tissue adhesive according to any one of claims 1 to 5, characterized in that it comprises: Synthesize anhydride-terminated cationic π-type amino acid vinyl monomers; The anhydride-terminated cationic π-type amino acid vinyl monomer, hydroxyalkyl acrylamide monomer, basic reagent, peroxide initiator and accelerator are dissolved in an aqueous medium and then subjected to free radical polymerization at a temperature of 50~70℃ to obtain the product.

7. The preparation method according to claim 6, characterized in that, The aqueous medium is selected from one of deionized water, physiological saline, and biological buffer solution; And / or, the alkaline reagent is selected from one of sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, and sodium acetate; And / or, the peroxide initiator is selected as ammonium persulfate; And / or, the promoter is selected as tetramethylethylenediamine.

8. The preparation method according to claim 6, characterized in that, The total concentration of the two monomers contained in the aqueous medium is 0.2~0.6 mol / mL; The molar ratio of the alkaline reagent to the anhydride-terminated cationic-π-type amino acid vinyl monomer is 1:(1~1.2); The molar ratio of the initiator to the sum of the molar amounts of the two monomers is (0.005~0.02):1; The molar ratio of the accelerator to the sum of the molar amounts of the two monomers is (0.002~0.01):

1.

9. The preparation method according to claim 6, characterized in that, The method for synthesizing the anhydride-terminated cationic-π-type amino acid vinyl monomer includes: After amino-protected amino acid derivatives undergo amidation condensation with amino-substituted acrylamide monomers, deprotection treatment is performed to prepare cationic-π-type amino acid vinyl monomers. The cationic π-type amino acid vinyl monomer is obtained by reacting it with an acid anhydride through a nucleophilic acyl substitution reaction.

10. The application of the acid-triggered stepwise bonding polymeric tissue adhesive according to any one of claims 1 to 5 or the acid-triggered stepwise bonding polymeric tissue adhesive prepared by any one of claims 6 to 9 in the preparation of acid-sensitive wound repair materials.

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