A tissue adhesive and a method for preparing the same

Abstract

The present invention belongs to the technical field of medical supplies, and particularly relates to a tissue adhesive and a preparation method thereof. The method uses acrylic acid (AA), N-hydroxysuccinimide acrylate (NAS) and methoxy polyethylene glycol acrylate (mPEGAA) to copolymerize to generate a tissue adhesive. When the NAS chain segment contacts the protein molecule with an amine group, a cross-linking reaction will occur, so that the wound can be tightly bonded. The adhesive has low water solubility and is not easily washed away by body fluids, making the bonding effect more long-lasting. The mPEG chain segment increases the hydrophilicity of the adhesive, making its critical surface tension close to that of human tissue, so that it can be more tightly bonded to the wound.

Description

Technical Field

[0001] The invention belongs to the technical field of medical supplies, and in particular relates to a preparation method and application of a tissue adhesive. Background Art

[0002] For organs that are difficult to suture, such as bones and livers, or tissues that need to be tightly closed, such as meninges and pleura, it is difficult to suture the tissues well, so tissue adhesives can be used to assist. Tissue adhesives need to have good adhesion and tissue compatibility, and can be completely absorbed by the human body. In terms of adhesion performance, adhesives need to solidify quickly to seal wounds. Common theories include adsorption theory, chemical bonding theory, mechanical bonding theory, etc., all of which require breaking through the interface between the two solids to allow interaction between the two solids. The closer the critical surface tension of the two solids, the lower the interfacial tension between the two solids, and the easier it is for the two solids to adhere to each other. The critical surface tension of human tissue is approximately in the range of 36.05 to 72.8 mN / m. After the tissue adhesive solidifies, the critical surface tension must be within this range to be effective.

[0003] Polyacrylic acid is often used as an adhesive due to its good adhesion properties. However, polyacrylic acid is highly water-soluble, easily washed away by body fluids, and difficult to solidify quickly. Therefore, it needs to be modified, but there is still no tissue adhesive that fully meets the application requirements. Summary of the invention

[0004] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a method for preparing and applying a tissue adhesive. The method uses acrylic acid (AA), N-hydroxysuccinimide acrylate (NAS) and methoxy polyethylene glycol acrylate (mPEGAA) to copolymerize to generate a tissue adhesive. When the NAS segment contacts a protein molecule with an amine group, a cross-linking reaction will occur, allowing the wound to be tightly bonded. The adhesive has a low water solubility and is not easily washed away by body fluids, making the bonding effect more long-lasting. The mPEG segment increases the hydrophilicity of the adhesive, making its critical surface tension close to that of human tissue, so that it can be more tightly bonded to the wound.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A tissue adhesive, characterized in that the structure of the tissue adhesive is as follows:

[0007]

[0008] The value range of nmpq is 100-1000.

[0009] A method for preparing a tissue adhesive comprises the following steps:

[0010] (1) Synthesis of N-hydroxysuccinimide acrylate (NAS)

[0011] Dissolve acrylic acid (AA) in tetrahydrofuran to prepare a solution of a certain concentration. Add N-hydroxysuccinimide (NHS) and N,N-dicyclohexylcarbodiimide (DCC) in a certain molar ratio and react for a certain time under certain conditions. After the reaction, remove the solid product and concentrate the liquid to a viscous state to obtain a reaction concentrate. Add a certain amount of ether and filter out the precipitate. Evaporate the ether and collect the solid. Dissolve the solid in an organic solvent for recrystallization and wash to obtain NAS.

[0012] (2) Synthesis of methoxypolyethylene glycol acrylate (mPEGAA)

[0013] Methoxypolyethylene glycol (mPEG) and ethylenediamine (EDA) are dissolved in ether at a certain molar ratio, a certain amount of acryloyl chloride (AC) is added, and the mixture is reacted for a certain time under certain conditions. The mixture is extracted with purified water until neutral, and mPEGAA is obtained after removing the ether and water.

[0014] (3) Tissue adhesive synthesis

[0015] NAS, mPEGAA and AA are mixed and dissolved in ethanol in a certain molar ratio, and a certain amount of initiator is added to react for a certain time under certain conditions. The reactants are placed in a dialysis bag for dialysis, and the tissue adhesive is obtained after drying.

[0016] Preferably, in step (1), the concentration of acrylic acid AA dissolved in tetrahydrofuran is 5-10% w / w.

[0017] Preferably, the certain molar ratio in step (1) is AA:NHS:DCC=1-5:1-5:1-5.

[0018] Preferably, the certain conditions in step (1) are inert gas protection and reaction temperature: 0-20°C.

[0019] Preferably, the certain time in step (1) is 12 to 24 hours.

[0020] Preferably, the certain amount of ether in step (1) is 0.5 to 5 times the volume of the reaction concentrate.

[0021] Preferably, the organic solvent in step (1) is ethanol, isopropanol or chloroform.

[0022] Preferably, the molecular weight of the methoxypolyethylene glycol described in step (2) is 1000 to 10000 Da.

[0023] Preferably, the molar ratio of mPEG:EDA in step (2) is 1:1-5.

[0024] Preferably, the amount of acryloyl chloride in step (2) is 0.5 to 5 times the molar number of mPEG.

[0025] Preferably, the certain condition in step (2) is reflux heating at 25-35°C.

[0026] Preferably, the certain time in step (2) is 3 to 12 hours.

[0027] Preferably, the mixing in a certain molar ratio in step (3) is NAS:mPEGAA:AA=0-5:0-5:0-5.

[0028] Preferably, the certain amount of initiator in step (3) is dibenzoyl peroxide, azobisisobutyronitrile or diisopropylbenzene peroxide in an amount of 1 / 1000 to 1 / 10000 of the total molar number of NAS, mPEGAA and AA.

[0029] Preferably, the certain conditions in step (3) are inert gas protection and reaction temperature: 50-80°C.

[0030] Preferably, the certain time in step (3) is 12 to 36 hours.

[0031] Preferably, the molecular weight cut-off of the dialysis bag in step (3) is 3500 to 14000 Da.

[0032] Preferably, the dialysis condition in step (3) is dialysis in anhydrous ethanol for 12 to 48 hours.

[0033] The present invention further provides a tissue adhesive obtained by the above preparation method.

[0034] The present invention further provides use of the tissue adhesive in tissue bonding.

[0035] Preferably, the application method is: applying the tissue adhesive to the wound or the tissue sealing film to make the wound closely adhered.

[0036] Hydroxysuccinimide ester can react with amine groups to form amides. The present invention grafts hydroxysuccinimide ester onto polyacrylic acid molecular chains, which greatly reduces the water solubility of polyacrylic acid. After contacting with proteins in body fluids, it will react with them to produce crosslinks, and the adhesive will quickly solidify and adhere to the wound. However, hydroxysuccinimide ester is a hydrophobic group, and the critical surface tension will be low after solidification, making it difficult to adhere to human organs. It is necessary to add a hydrophilic group to increase the critical surface tension. Polyethylene glycol is an extremely hydrophilic material. The present invention adjusts the critical surface tension of the adhesive by grafting polyethylene glycol, making it easier to adhere to human organs.

[0037] Compared with the prior art, the present invention has the following advantages and effects:

[0038] (1) The molecular chain of tissue adhesive contains hydroxysuccinimide ester, which will undergo a cross-linking reaction when in contact with protein molecules with amine groups, allowing the wound to be tightly adhered. The adhesive has low water solubility and is not easily washed away by body fluids, making the adhesive effect more long-lasting.

[0039] (2) mPEG is grafted onto the molecular chain of the tissue adhesive to increase the hydrophilicity of the adhesive, making its critical surface tension close to that of human tissue, allowing it to adhere more tightly to the wound. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Infrared spectra of tissue adhesives.

[0041] From the picture, we can see that 815cm -1 The vibration peak of the ester bond appears at 1080cm -1 The carboxyl vibration peak appears at 1210 cm -1 The CN group vibration peak appears at 1700cm -1 The appearance of carbonyl vibration peak indicated that the tissue adhesive was successfully prepared. DETAILED DESCRIPTION

[0042] The present invention is further described in detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. All raw materials and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0043] Example 1

[0044] A method for preparing a tissue adhesive comprises the following steps:

[0045] (1) Synthesis of N-hydroxysuccinimide acrylate (NAS)

[0046] Dissolve acrylic acid (AA) in tetrahydrofuran to prepare a 5% w / w concentration solution. Add N-hydroxysuccinimide (NHS) and N,N-dicyclohexylcarbodiimide (DCC) in a molar ratio of AA:NHS:DCC = 1:1:1, and react at 0°C for 12 hours under nitrogen protection. After the reaction, remove the solid product and concentrate the liquid to a viscous state to obtain a reaction concentrate. Add 0.5 times the volume of ether to the reaction concentrate and filter out the precipitate. Evaporate the ether and collect the solid. Dissolve the solid in ethanol for recrystallization and wash to obtain NAS.

[0047] (2) Synthesis of methoxypolyethylene glycol acrylate (mPEGAA)

[0048] 1000Da methoxypolyethylene glycol (mPEG) and ethylenediamine (EDA) were dissolved in ether at a molar ratio of mPEG:EDA = 1:1, and 0.5 times the molar number of mPEG was added with acryloyl chloride (AC), and the mixture was reacted under reflux heating at 25°C for 3 hours. The mixture was extracted with purified water until neutral, and mPEGAA was obtained after removing the ether and water.

[0049] (3) Tissue adhesive synthesis

[0050] NAS, mPEGAA and AA were mixed and dissolved in ethanol at a molar ratio of NAS:mPEGAA:AA=1:1:1, and dibenzoyl peroxide was added at 1 / 1000 of the total molar number of NAS, mPEGAA and AA, and reacted at 50°C for 12 hours under nitrogen protection. The reactants were placed in a 3500Da dialysis bag, dialyzed in anhydrous ethanol for 12 hours, and dried to obtain a tissue adhesive.

[0051] The application of tissue adhesive in tissue bonding is specifically to apply the tissue adhesive on the sealing film and adhere it to the dura mater wound.

[0052] Example 2

[0053] A method for preparing a tissue adhesive comprises the following steps:

[0054] (1) Synthesis of N-hydroxysuccinimide acrylate (NAS)

[0055] Dissolve acrylic acid (AA) in tetrahydrofuran to prepare a 7.5% w / w concentration solution. Add N-hydroxysuccinimide (NHS) and N,N-dicyclohexylcarbodiimide (DCC) in a molar ratio of AA:NHS:DCC = 5:4:3, and react at 10°C for 16 hours under argon protection. After the reaction, remove the solid product and concentrate the liquid to a viscous state to obtain a reaction concentrate. Add ether 3 times the volume of the reaction concentrate and filter out the precipitate. Evaporate the ether and collect the solid. Dissolve the solid in isopropanol for recrystallization and wash to obtain NAS.

[0056] (2) Synthesis of methoxypolyethylene glycol acrylate (mPEGAA)

[0057] 5000Da methoxypolyethylene glycol (mPEG) and ethylenediamine (EDA) were dissolved in ether at a molar ratio of mPEG:EDA = 1:3, and acryloyl chloride (AC) twice the molar number of mPEG was added, and the mixture was reacted at 30°C under reflux heating for 6 hours. The mixture was extracted with purified water until neutral, and mPEGAA was obtained after removing the ether and water.

[0058] (3) Tissue adhesive synthesis

[0059] NAS, mPEGAA and AA were mixed and dissolved in ethanol at a molar ratio of NAS:mPEGAA:AA=2:0.1:5, and azobisisobutyronitrile was added at 1 / 5000 of the total molar number of NAS, mPEGAA and AA, and reacted at 65°C for 24 hours under argon protection. The reactants were placed in an 8000Da dialysis bag, dialyzed in anhydrous ethanol for 24 hours, and dried to obtain a tissue adhesive.

[0060] The application of tissue adhesive in tissue bonding is specifically to apply the tissue adhesive to the bone fracture for bonding.

[0061] Example 3

[0062] A method for preparing a tissue adhesive comprises the following steps:

[0063] (1) Synthesis of N-hydroxysuccinimide acrylate (NAS)

[0064] Dissolve acrylic acid (AA) in tetrahydrofuran to prepare a 10% w / w concentration solution. Add N-hydroxysuccinimide (NHS) and N,N-dicyclohexylcarbodiimide (DCC) in a molar ratio of AA:NHS:DCC = 3:5:4, and react at 20°C for 24 hours under helium protection. After the reaction, remove the solid product and concentrate the liquid to a viscous state to obtain a reaction concentrate. Add ether 5 times the volume of the reaction concentrate and filter out the precipitate. Evaporate the ether and collect the solid. Dissolve the solid in chloroform for recrystallization and wash to obtain NAS.

[0065] (2) Synthesis of methoxypolyethylene glycol acrylate (mPEGAA)

[0066] 10000Da methoxypolyethylene glycol (mPEG) and ethylenediamine (EDA) were dissolved in ether at a molar ratio of mPEG:EDA = 1:5, and acryloyl chloride (AC) was added at 5 times the molar number of mPEG, and reacted at 35°C under reflux heating for 12 hours. mPEGAA was obtained by extracting with purified water to neutrality and removing ether and water.

[0067] (3) Tissue adhesive synthesis

[0068] NAS, mPEGAA and AA were mixed and dissolved in ethanol at a molar ratio of NAS:mPEGAA:AA=5:4:3, and diisopropylbenzene peroxide was added at 1 / 10,000 of the total molar number of NAS, mPEGAA and AA, and reacted at 80°C for 36 hours under helium protection. The reactants were placed in a 14000Da dialysis bag, dialyzed in anhydrous ethanol for 48 hours, and dried to obtain a tissue adhesive.

[0069] The application of tissue adhesive in tissue bonding is specifically to apply the tissue adhesive to the ruptured part of the liver for bonding.

[0070] Application Examples

[0071] Solidification time test method: flush the inside of the screw bottle with phosphate buffered saline containing calf serum, so that the inner wall of the screw bottle is covered with serum to simulate the wound surface. Dissolve the tissue adhesives of Examples 1-3 in water respectively, add to the screw bottle, put it in a 37°C water bath, and record the solidification time of the adhesive.

[0072] Critical surface tension test method: The tissue adhesives of Examples 1-3 were respectively dissolved in anhydrous ethanol, evenly coated on a plastic wrap from which static electricity had been removed, and dried in an oven at 50°C until completely dry. The mixture was cooled to room temperature, and the contact angles of water, methanol, ethanol, acetic acid and benzene on the surface were measured using a contact angle meter. The cosine of the contact angle was plotted against the surface tension, and the surface tension corresponding to the cosine of the contact angle being 1 was obtained, which was the critical surface tension.

[0073] Peel strength test method: Evenly apply phosphate buffered saline containing calf serum on a glass slide and dry it slightly in the air. Dissolve the tissue adhesives of Examples 1-3 in anhydrous ethanol respectively, evenly apply them on a cling film that has been destaticized, bake in a 50°C oven until completely dry, and stick them on the above glass slide after cooling to room temperature. Put the glass slide with cling film in a 37°C incubator for 30 minutes, take it out, and test the peel strength using a universal material testing machine, with a test speed of 10 mm / min.

[0074] The results are as follows:

[0075]

[0076] From the results, it can be seen that not using NAS will make the adhesive unable to solidify and the peel strength is low; not using mPEGAA will reduce the critical surface tension of the adhesive and make the hydrophilicity poor, which will affect the peel strength.

[0077] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a tissue adhesive, It is characterized in that The structure of the tissue adhesive is as follows: The value ranges of n, m, p and q are all 100-1000; The preparation method of the tissue adhesive comprises the following steps: (1) Synthesis of N-hydroxysuccinimide acrylate (NAS) Dissolve acrylic acid AA in tetrahydrofuran to prepare a solution of a certain concentration, add N-hydroxysuccinimide NHS and N,N-dicyclohexylcarbodiimide DCC, react for a certain time under certain conditions, remove the solid product after the reaction, concentrate the liquid to be viscous, and obtain a reaction concentrated solution; add a certain amount of ether, filter out the precipitate; evaporate the ether, collect the solid, dissolve the solid in an organic solvent for recrystallization, and wash to obtain NAS; (2) Synthesis of methoxypolyethylene glycol acrylate mPEGAA Methoxypolyethylene glycol mPEG and ethylenediamine EDA are dissolved in ether at a certain molar ratio, a certain amount of acryloyl chloride AC is added, reacted for a certain time under certain conditions, extracted with purified water to neutrality, and mPEGAA is obtained after removing ether and water; (3) Synthesis of tissue adhesive NAS, mPEGAA and AA are mixed in a certain molar ratio and dissolved in ethanol, a certain amount of initiator is added, and the mixture is reacted for a certain time under certain conditions, and the tissue adhesive is obtained after dialyzing in a dialysis bag and drying.

2. The preparation method according to claim 1, Features: Step (1) acrylic acid AA is dissolved in tetrahydrofuran at a concentration of 5-10wt%; the molar ratio of AA:NHS:DCC is 1-5:1-5:1-5; the reaction conditions are inert gas protection, reaction temperature: 0-20°C, and reaction time: 12-24 hours; the amount of ether used is 0.5-5 times the volume of the reaction concentrate; and the organic solvent is ethanol, isopropanol or chloroform.

3. The preparation method according to claim 1, Features: The molecular weight of the methoxy polyethylene glycol mPEG described in step (2) is 1000-10000 Da; the molar ratio of mPEG:EDA is 1:1-5; the amount of acryloyl chloride AC used is 0.5-5 times the molar number of mPEG; and the reaction conditions are reflux heating at 25-35°C for 3-12 hours.

4. The preparation method according to claim 1, Features: In step (3), the molar ratio of NAS:mPEGAA:AA is 0.1~5:0.1~5:0.1~5; the amount of initiator used is 1 / 1000 to 1 / 10000 of the total molar number of NAS, mPEGAA and AA; the initiator is dibenzoyl peroxide, azobisisobutyronitrile or diisopropylbenzene peroxide; the reaction conditions are inert gas protection, reaction temperature: 50~80°C, and reaction time: 12~36 hours.

5. The preparation method according to claim 1, Features: In step (3), the molecular weight cutoff of the dialysis bag is 3500-14000 Da; and the dialysis condition is dialysis in anhydrous ethanol for 12-48 hours.

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