An N-hydroxysuccinimide active tissue-adhesive polymer, a tissue sealing film, and a preparation method and application thereof

Abstract

This invention provides an N-hydroxysuccinimide active tissue adhesion polymer, a tissue sealing membrane, its preparation method, and its applications, relating to the field of medical polymer materials technology. The invention involves dissolving acrylic acid, N-vinylpyrrolidone, and azobisisobutyronitrile in dimethyl sulfoxide and performing free radical copolymerization under nitrogen protection and microwave radiation conditions. N-hydroxysuccinimide and dicyclohexylcarbodiimide are added to the resulting carboxyl-containing copolymer system, followed by microwave-assisted activation to convert at least some carboxyl groups into N-hydroxysuccinimide active ester groups. The byproducts are then filtered, precipitated, redissolved, precipitated again, and dried under low-temperature reduced pressure to obtain the tissue adhesion polymer. This invention shortens the preparation cycle of the tissue adhesion polymer through microwave-assisted free radical copolymerization, microwave-assisted activation, and multiple precipitation purification processes, enabling the processing of the tissue adhesion polymer into multilayer tissue sealing membranes, which has promising application prospects.

Description

Technical Field

[0001] This invention relates to the field of medical polymer materials technology, and in particular to an N-hydroxysuccinimide active tissue adhesion polymer, a tissue sealing membrane, its preparation method and application. Background Technology

[0002] Tissue sealing materials form a barrier at tissue wound sites to aid in wound closure and reduce leakage of blood, tissue fluid, or gas, making them valuable in surgical wound closure and tissue repair. In addition to appropriate mechanical strength and flexibility, materials used for tissue sealing should be able to form stable adhesion to moist tissue surfaces and meet the requirements for film-forming processing and subsequent sterilization. Existing tissue sealing materials include bioprotein-based materials, cyanoacrylate-based materials, polyethylene glycol-based materials, and synthetic polymers containing tissue-reactive groups. Different materials still have limitations in terms of wet adhesion, mechanical load-bearing capacity, degradation behavior, and processing performance.

[0003] Polymers containing N-hydroxysuccinimide reactive ester groups can react with primary amine groups in tissue proteins to form amide bonds, thus serving as tissue contact materials for tissue sealing films. Chinese invention patent CN101378791A discloses a tissue adhesive material whose tissue contact layer can contain polymers with structural units of vinylpyrrolidone, acrylic acid, and N-hydroxysuccinimide acrylate. The patent also discloses a preparation route involving the initial preparation of a carboxyl-containing copolymer followed by activation using N-hydroxysuccinimide and dicyclohexylcarbodiimide. This type of post-activation route can introduce tissue-reactive groups, but the polymerization and subsequent activation processes are time-consuming.

[0004] Utility model patent CN214632623U discloses a biodegradable medical film comprising alternating support layers and tissue adhesion layers. It discloses a technical route for preparing a copolymer using acrylic acid, N-vinylpyrrolidone, azobisisobutyronitrile, and dimethyl sulfoxide, followed by activation with N-hydroxysuccinimide and dicyclohexylcarbodiimide. The patent also discloses using a polylactic acid-glycolic acid copolymer to form the support layer and employing an alternating coating method to prepare a multilayer film. In its embodiments, both the copolymerization and activation reactions utilize conventional thermal reaction methods, resulting in a relatively long overall reaction cycle.

[0005] Patent application CN114209873A discloses a bioabsorbable tissue-sealing patch, in which the carrier layer can be formed of polylactic acid-glycolic acid copolymer, and the tissue adhesion layer contains a tissue-reactive polymer with carboxyl, pyrrolidone, and succinimidyl ester groups, and can be formed into a multilayer structure by alternating coatings. This technology further demonstrates that combining polymers containing tissue-reactive groups with film-forming support materials is a feasible way to balance tissue adhesion and mechanical support, but the post-activation stage of the polymer also requires a relatively long reaction time.

[0006] Therefore, existing technologies have disclosed tissue adhesion polymers containing acrylic structural units, N-vinylpyrrolidone (PVP) structural units, and N-hydroxysuccinimide active ester groups, as well as multilayer tissue sealing films formed from such polymers and polylactic acid-glycolic acid copolymers. However, the copolymerization and activation processes in existing preparation routes typically involve long reaction times, affecting preparation efficiency.

[0007] Therefore, it is necessary to provide a preparation method that can shorten the polymerization and activation reaction cycle, and enable the resulting tissue adhesion polymer to still be used to form tissue sealing films with film-forming properties, mechanical properties and tissue adhesion properties. Summary of the Invention

[0008] In view of this, the present invention provides an N-hydroxysuccinimide active tissue adhesion polymer, a tissue sealing membrane, a preparation method thereof, and its application. The present invention shortens the preparation cycle of the tissue adhesion polymer through microwave-assisted free radical copolymerization, microwave-assisted activation, and multiple precipitation purification processes, enabling the processing of the tissue adhesion polymer into a multilayer tissue sealing membrane, which has good application prospects.

[0009] The first aspect of this invention provides a method for preparing an N-hydroxysuccinimide active tissue adhesion polymer, comprising the following steps: S1. Acrylic acid, N-vinylpyrrolidone and azobisisobutyronitrile are dissolved in dimethyl sulfoxide and subjected to free radical copolymerization under nitrogen protection and microwave radiation to obtain a carboxyl-containing copolymer system. S2. Cool the carboxyl-containing copolymer system, add N-hydroxysuccinimide (NHS) and dicyclohexylcarbodiimide, and carry out an activation reaction under microwave radiation to convert at least some of the carboxyl groups in the carboxyl-containing copolymer into N-hydroxysuccinimide active ester groups. S3. Filter the system after the activation reaction to remove dicyclohexylurea, and add the resulting filtrate to a non-solvent to precipitate the target polymer; S4. The precipitated target polymer is redissolved and then subjected to secondary precipitation. The resulting solid is then dried under low temperature and reduced pressure to obtain the N-hydroxysuccinimide active tissue adhesion polymer.

[0010] Preferably, the amounts of acrylic acid, N-vinylpyrrolidone, azobisisobutyronitrile, and dimethyl sulfoxide are 7.2 g, 11.1 g, 0.12 g, and 200 mL, respectively.

[0011] Preferably, the microwave power of the free radical copolymerization reaction is 100~500 W, the reaction temperature is 100~120℃, and the reaction time is 0.5~2 h; more preferably, the microwave power is 300 W, the reaction temperature is 100℃, and the reaction time is 1 h, or the microwave power is 300 W, the reaction temperature is 120℃, and the reaction time is 0.5 h.

[0012] Preferably, 5.74 g of N-hydroxysuccinimide and 12.38 g of dicyclohexylcarbodiimide are added to the carboxyl-containing copolymer system formed by 7.2 g of acrylic acid and 11.1 g of N-vinylpyrrolidone, and the reaction is activated for 2 h under microwave power of 100 W and reaction temperature of 45±2℃.

[0013] Preferably, the activation reaction is carried out in an environment under nitrogen protection and with a relative humidity of less than 30%.

[0014] Preferably, the activation reaction is carried out for 2-3 hours under the conditions of microwave power of 10-100 W and reaction temperature of 45±2℃.

[0015] Preferably, the non-solvent is ethyl acetate or diethyl ether; the precipitated target polymer is redissolved in 100 mL of a mixed solvent of dichloromethane and methanol at a volume ratio of 3:1 or 100 mL of isopropanol, and then ethyl acetate or diethyl ether is added for secondary precipitation. The amount of ethyl acetate or diethyl ether used is 20 to 30 times the mass of the precipitated target polymer, by mass-volume ratio. More preferably, the amount of ethyl acetate or diethyl ether used is 20 times the mass of the precipitated target polymer, by mass-volume ratio.

[0016] Preferably, the precipitation temperature is 0~10℃, the drying temperature is 18~22℃, the absolute pressure is 16 kPa, and the drying time is 24~48 h. More preferably, the low-temperature vacuum drying temperature is 22℃ and the drying time is 36 h.

[0017] A second aspect of the present invention provides an N-hydroxysuccinimide active tissue adhesion polymer prepared according to the above method, the polymer containing an acrylic acid-derived structural unit, an N-vinylpyrrolidone-derived structural unit, and an N-hydroxysuccinimide active ester group formed by the conversion of the carboxyl groups of at least some of the acrylic acid-derived structural units, and the polymer is obtained by microwave-assisted free radical copolymerization and microwave-assisted activation reaction.

[0018] Preferably, the infrared spectrum of the N-hydroxysuccinimide active tissue adhesion polymer is at 1,730 cm⁻¹. -1 Nearby, 1,650~1,670 cm -1 and 1,810 cm -1 Nearby absorption peaks are present, and when deuterated dimethyl sulfoxide is used as the NMR test solvent, its proton NMR spectrum has a signal at a chemical shift of δ2.6~3.0 ppm at which it belongs to the methylene group on the succinimide ring.

[0019] A third aspect of the present invention provides a tissue sealing membrane, comprising a support membrane layer, a tissue adhesion layer, a reinforcing membrane layer and a surface tissue adhesion layer stacked sequentially; wherein the support membrane layer and the reinforcing membrane layer contain a polylactic acid-glycolic acid copolymer; and wherein the tissue adhesion layer and the surface tissue adhesion layer contain the aforementioned N-hydroxysuccinimide active tissue adhesion polymer.

[0020] Preferably, the polylactic acid-glycolic acid copolymer has a lactic acid to glycolic acid molar percentage ratio of 50:50, an intrinsic viscosity of 0.8~1.2 dL / g, and is an ester-terminated polymer with a number-average molecular weight of 7.549 × 10⁻⁶. 4 g / mol, weight-average molecular weight 1.309×10 5 g / mol.

[0021] Preferably, the coating solution forming the supporting film layer and the reinforcing film layer is a 10% by mass polylactic acid-glycolic acid copolymer dichloromethane solution, and the coating solution forming the tissue adhesion layer and the surface tissue adhesion layer is a 10% by mass N-hydroxysuccinimide active tissue adhesion polymer solution, the solvent of which is a mixed solvent of dichloromethane and methanol in a volume ratio of 3:1.

[0022] Preferably, the tissue sealing membrane is sterilized by 25 kGy electron beam irradiation.

[0023] In some specific embodiments of the present invention, at room temperature, the tissue sealing film is packaged in a 15.5 cm × 10 cm paper-plastic bag, the paper-plastic bag is placed in an aluminum foil bag with a size of 21 cm × 13.5 cm, and the aluminum foil bag is sealed.

[0024] A fourth aspect of the present invention provides a method for preparing the tissue sealing membrane, comprising the following steps: A 10% (w / w) polylactic acid-glycolic acid copolymer dichloromethane solution was coated onto a release substrate using a 120 μm wire rod at a coating speed of 30 mm / s to form a support film layer. The coating speeds for the remaining layers were the same. A 10% (w / w) N-hydroxysuccinimide active tissue adhesion polymer solution was coated onto the support film layer using a 50 μm wire rod to form a tissue adhesion layer. The polylactic acid-glycolic acid copolymer dichloromethane solution was then coated onto the tissue adhesion layer using a 50 μm wire rod to form a reinforcing film layer. The N-hydroxysuccinimide active tissue adhesion polymer solution was then coated onto the reinforcing film layer using a 300 μm wire rod to form a surface tissue adhesion layer. The resulting multilayer film was then peeled off from the release substrate and dried under reduced pressure at low temperature for 24 h. In this invention, the wire rod specification is almost equal to the wet film thickness, while the dry film thickness is approximately equal to the wire rod specification multiplied by the solid content.

[0025] The fifth aspect of the present invention provides the use of the N-hydroxysuccinimide active tissue adhesion polymer in the preparation of tissue sealing membranes or medical devices for tissue wound closure or reduction of liquid or gas leakage, and in the preparation of hemostatic materials or tissue repair materials.

[0026] Compared with the prior art, the beneficial technical effects of the present invention are as follows: This invention employs a microwave-assisted method for the free radical copolymerization of acrylic acid and N-vinylpyrrolidone, followed by microwave-assisted activation of the resulting carboxyl-containing copolymer. Compared to conventional thermal polymerization and long-term activation at room temperature, this method shortens the time required for both the copolymerization and activation stages, thereby reducing the overall preparation cycle and improving the efficiency of tissue adhesion polymer preparation.

[0027] This invention involves copolymerization and activation under nitrogen protection and controlled temperature conditions. After the activation reaction, byproduct filtration, first precipitation, redissolution, second precipitation, and low-temperature vacuum drying are performed sequentially. This makes the polymerization reaction, activation reaction, and post-processing steps form a continuous and well-defined preparation process, which is beneficial for subsequent process control and scale-up verification.

[0028] The tissue adhesion polymer prepared by the method of this invention can be alternately coated with polylactic acid-glycolic acid copolymer to form a multilayer tissue sealing film comprising a support film layer, a tissue adhesion layer, a reinforcing film layer, and a surface tissue adhesion layer. This multilayer structure can balance the film-forming and mechanical load-bearing functions of the support material with the tissue contact function of the tissue adhesion polymer. The resulting tissue sealing film possesses film-forming properties, mechanical properties, and in vitro tissue adhesion properties, and can withstand irradiation sterilization treatment. Attached Figure Description

[0029] The present invention will be further described below with reference to the accompanying drawings.

[0030] Figure 1The hydrogen nuclear magnetic resonance spectrum of the N-hydroxysuccinimide active tissue adhesion polymer prepared in Example 1 of this invention; Figure 2 The hydrogen nuclear magnetic resonance spectrum of the N-hydroxysuccinimide active tissue adhesion polymer prepared in Example 2 of this invention; Figure 3 The hydrogen nuclear magnetic resonance spectrum of the N-hydroxysuccinimide active tissue adhesion polymer prepared in Comparative Example 1 of this invention; Figure 4 Fourier transform infrared spectrum of N-hydroxysuccinimide active tissue adhesion polymer prepared in Example 1 of this invention; Figure 5 The image shows the attenuated total reflectance Fourier transform infrared spectrum of the tissue sealing membrane prepared in Example 3 of this invention. Detailed Implementation

[0031] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0032] Unless otherwise specified, all experiments were repeated three times. Analysis of variance (ANOVA) and Duncan's multiple comparison analysis were performed using SPSS 21.0. Results are expressed as mean ± standard deviation, and p < 0.05 was considered statistically significant.

[0033] Example 1 7.2 g of acrylic acid, 11.1 g of N-vinylpyrrolidone and 0.12 g of azobisisobutyronitrile were added to 200 mL of dimethyl sulfoxide and dissolved completely under stirring to form a homogeneous reaction system. The reaction vessel was placed in a microwave reactor and protected with nitrogen gas. During the microwave reaction, nitrogen gas was not continuously introduced. The nitrogen flow rate was 60 mL / min, the gas introduction time was 30 min, the microwave frequency was 2.45 GHz, the stirring speed was 280 rpm, the microwave power was set to 300 W, the reaction temperature was controlled at 120℃, and the radiation reaction was carried out for 0.5 h to carry out the free radical copolymerization reaction. After the reaction was completed, the resulting copolymer system was cooled to room temperature. Under stirring conditions, 5.74 g of N-hydroxysuccinimide and 12.38 g of dicyclohexylcarbodiimide were added. The microwave power was set to 100 W, the reaction system temperature was controlled at 45±2℃, and the reaction was carried out in an environment with a relative humidity of less than 30%. The reaction was carried out under nitrogen protection for 2 h to activate and esterify at least some of the carboxyl groups of the polymer side chains. After the activation reaction was completed, the dicyclohexylurea generated during the reaction was removed by filtration, and the filtrate was retained. The filtrate was added dropwise to 2000 mL of ethyl acetate at a rate of 20 mL / min to precipitate the target polymer. After precipitation for 0.5 h, the filtrate was filtered and the residue was collected. The filter residue was redissolved in 100 mL of a 3:1 mixture of dichloromethane and methanol, and then added dropwise to 2000 mL of ethyl acetate at a rate of 20 mL / min for secondary precipitation. After filtration, the product was dried under reduced pressure at 22 °C and 16 kPa for 48 h to obtain a tissue adhesion polymer with N-hydroxysuccinimide active ester groups. The yield was 15.8 g, with a recovery rate of 68.2%. Its number-average molecular weight was 1.716 × 10⁻⁶. 5 g / mol, weight-average molecular weight is 2.390 × 10⁻⁶ g / mol. 5 g / mol.

[0034] Example 2 The difference from Example 1 is as follows: the free radical copolymerization reaction temperature is 100℃, and the reaction time is 1 h; the activation reaction is carried out at a temperature controlled by circulating cooling of 45±2℃ in an environment with a relative humidity of less than 30%; the filtrate after activation reaction is added dropwise to diethyl ether for the first precipitation; the filter residue obtained from the first precipitation is redissolved in isopropanol and added dropwise to diethyl ether again for a second precipitation. The remaining raw material feed amounts, microwave power, nitrogen protection, filtration, and low-temperature vacuum drying steps are the same as in Example 1. The yield was 13.1 g, with a yield of 56.5%. Its number-average molecular weight was 1.536 × 10⁻⁶. 5 g / mol, weight-average molecular weight 2.723×10 5 g / mol.

[0035] Example 3 A 10% (w / w) polylactic acid-glycolic acid copolymer (PLA-GAC) solution was prepared by dissolving the PLA-GAC copolymer in dichloromethane. The PLA-GAC copolymer had a lactic acid to glycolic acid molar percentage ratio of 50:50, an intrinsic viscosity of 0.8 ± 1.2 dL / g, ester-terminated end groups, and a number-average molecular weight of 7.549 × 10⁻⁶. 4 g / mol, weight-average molecular weight 1.309×10 5 g / mol.

[0036] The tissue adhesion polymer obtained in Example 1 was dissolved in a mixed solvent of dichloromethane and methanol in a volume ratio of 3:1 to prepare a tissue adhesion polymer solution with a mass fraction of 10%.

[0037] Step 1: Using an automated bar coater and a 120 μm bar, the polylactic acid-glycolic acid copolymer solution was coated onto the release substrate surface at a speed of 30 mm / s. After standing at room temperature for 0.5 h, the film thickness was 11.2 μm. Step 2: Using a 50 μm wire bar, the tissue adhesion polymer solution was coated onto the surface of the obtained support membrane at a speed of 30 mm / s. After standing at room temperature for 0.5 h, the membrane thickness was 4.1 μm. Step 3: Using a 50 μm wire bar, the polylactic acid-glycolic acid copolymer solution was coated onto the surface of the obtained tissue adhesion layer at a speed of 30 mm / s. After standing at room temperature for 0.5 h, the film thickness was 3.8 μm. Step 4: Using a 300 μm wire bar, the tissue adhesion polymer solution was coated onto the surface of the resulting reinforced membrane at a speed of 30 mm / s. After standing at room temperature for 8 h, the membrane thickness was 26.1 μm. The film thickness measured in each of the above steps is the thickness of a single layer, that is, the cumulative film thickness minus the thickness of the next layer. The dried multilayer film was peeled off from the release substrate and placed in a vacuum drying oven for low-temperature reduced pressure drying for 24 hours. The vacuum drying temperature was 22℃ and the absolute pressure was 16 kPa.

[0038] The dried tissue sealing membrane was terminally sterilized using an electron beam irradiation source at a dose of 25 kGy.

[0039] Example 4 The difference from Example 3 is that the tissue adhesion layer and the surface tissue adhesion layer use the tissue adhesion polymer prepared in Example 2; the film thickness measured in step 1 is 10.3 μm, the film thickness measured in step 2 is 3.6 μm, the film thickness measured in step 3 is 4.6 μm, and the film thickness measured in step 4 is 25.5 μm.

[0040] Comparative Example 1 The difference from Example 1 is that microwave-assisted free radical copolymerization was not used; instead, the reaction system was heated to 70°C and subjected to free radical copolymerization under nitrogen protection for 12 hours. Microwave-assisted activation was not used; instead, 5.74 g of N-hydroxysuccinimide and 12.38 g of dicyclohexylcarbodiimide were added under stirring at room temperature, and activation was continued for 48 hours. The remaining steps—filtration, first precipitation with ethyl acetate, redissolution in a 3:1 mixture of dichloromethane and methanol, second precipitation with ethyl acetate, and low-temperature vacuum drying—were the same as in Example 1. The yield was 9.2 g, with a recovery rate of 39.7%. Its number-average molecular weight was 1.719 × 10⁻⁶. 5 g / mol, weight-average molecular weight 4.319×10 5 g / mol.

[0041] Comparative Example 2 The difference from Example 3 is that the tissue adhesion layer and the surface tissue adhesion layer use the tissue adhesion polymer prepared in Comparative Example 1. The film thickness was measured to be 8.4 μm in step 1, 3.1 μm in step 2, 3.2 μm in step 3, and 18.9 μm in step 4.

[0042] Test Example 1 1. Film thickness and film thickness of each layer The thickness of the tissue sealing membrane was determined using a membrane thickness measuring instrument with an accuracy of 0.1 μm. Six different locations were randomly selected for each sample, and the measurement was performed three times at each location, with the average value taken; a total of three parallel samples were tested.

[0043] The thickness of each film layer was measured using the same film thickness measuring instrument. Six different locations were randomly selected for each sample, and the measurement was taken three times at each location, with the average value taken; a total of three parallel samples were tested.

[0044] II. Tensile Strength The test was conducted using a tensile testing machine. The sample length was 50 mm and the width was 10 mm. The tensile speed was 30 mm / min. Three parallel samples were tested in each group.

[0045] III. Breaking Through Pressure A rupture machine was used to apply progressively increasing water pressure to the specimen until it ruptured. The specimen size was 50 mm × 50 mm, and three parallel specimens were tested in each group. The water pressure at the time of rupture was recorded.

[0046] IV. Tissue Adhesion Strength The tissue adhesion strength of the tissue sealing films obtained in Example 3, Example 4 and Comparative Example 2 was tested in accordance with YY / T 0729.3-2009 "Test Methods for Adhesive Properties of Tissue Adhesives - Part 3: Tensile Strength".

[0047] Take a tissue sealing membrane sample with an adhesion area of ​​2.5 cm × 5 cm, use glue to fix the surface of the test sample away from the tissue and the surface of the fresh pig liver away from the adhesion interface onto an aluminum mold, and then stretch and separate them at a speed of 10 mm / min, and record the maximum load before adhesion failure.

[0048] Table 1. Physical properties and tissue adhesion properties of tissue sealing films prepared by different processes before irradiation sterilization

[0049] Examples 1 and 2 employed microwave-assisted polymerization and activation reactions, significantly shortening the reaction time compared to traditional thermal reactions. Specifically, microwave-assisted polymerization at 100°C for 1 h and at 120°C for 0.5 h, respectively, yielded carboxyl-containing copolymer intermediates. Subsequent microwave-assisted activation allowed for the effective introduction of NHS active ester groups in a shorter time. In contrast, Comparative Example 1, using traditional thermal reactions, required significantly longer polymerization and activation times. These results demonstrate that microwave-assisted polymerization effectively improves reaction efficiency and shortens the preparation cycle.

[0050] The tissue sealing films prepared in Examples 3, 4, and Comparative Example 2 were further tested for performance. Table 1 shows that the tissue sealing films prepared by microwave-assisted polymer preparation exhibit good film-forming and mechanical properties. Specifically, the tensile strengths of the films obtained in Examples 3 and 4 reached 22.2 ± 2.1 MPa and 18.8 ± 3.4 MPa, respectively, and the burst pressures reached 120.2 ± 2.2 kPa and 22.2 ± 3.8 kPa, respectively, showing no significant decrease compared to the comparative sample prepared using conventional processes. Furthermore, the tissue adhesion strengths of the tissue sealing films obtained in Examples 3 and 4 reached 5.9 ± 0.3 N and 5.8 ± 0.2 N, respectively, higher than the 3.8 ± 0.7 N of Comparative Example 2, indicating that the polymer obtained by the microwave-assisted preparation method can maintain good tissue adhesion properties.

[0051] Table 2. Physical properties and tissue adhesion properties of tissue sealing films prepared by different processes after sterilization by 25 kGy irradiation.

[0052] To evaluate the effect of irradiation sterilization on the performance of tissue sealing membranes, the tissue sealing membranes obtained in Examples 3, 4, and Comparative Example 2 were subjected to 25 kGy irradiation sterilization. The membrane thickness, tensile strength, burst pressure, and tissue adhesion strength were tested before and after sterilization, and the results are shown in Tables 1 and 2. No significant decrease was observed in the membrane thickness, tensile strength, burst pressure, and tissue adhesion strength of any group of tissue sealing membranes. In Examples 3 and 4, the performance indicators remained within similar ranges before and after sterilization, with no significant changes in membrane thickness, tensile strength, burst pressure, and tissue adhesion strength. In Comparative Example 2, after 25 kGy irradiation sterilization, the membrane thickness changed from 30.5 ± 2.3 μm to 29.8 ± 2.6 μm, while the tensile strength, burst pressure, and tissue adhesion strength remained at similar levels, indicating that the irradiation sterilization process did not cause significant damage to the material structure.

[0053] In summary, after 25 kGy irradiation sterilization, the tissue sealing films of each group were still able to maintain good mechanical properties and tissue adhesion properties, indicating that the prepared tissue sealing films can adapt to conventional irradiation sterilization process conditions, and the irradiation process did not have a significant adverse effect on the film-forming structure and functional properties of the materials.

[0054] In vitro degradation experiment: Take 24 tissue sealing films from each of Example 3, Example 4 and Comparative Example 2, each with a size of 50 mm × 50 mm, weigh them, record the weight as m1, and place them in suitable glass bottles. Add 0.1 M NH4OH degradation solution to each glass bottle, place them on a shaker and fix them. Take out the glass bottles daily and shake them vigorously for 10 seconds each to prevent the contents from settling.

[0055] Samples were recovered on days 3, 6, 9, and 12 of degradation, using 6 tablets at each time point. After reaching the corresponding time point, the supernatant was discarded, a small amount of purified water was added, the mixture was vortexed for 30 seconds, and the supernatant was discarded again. Subsequently, the sample was dried under vacuum at 40°C for more than 24 hours, and the mass of the dried residue was weighed to obtain m2.

[0056] Mass loss rate = (m1-m2) / m1×100%.

[0057] Table 3. In vitro mass loss rate of tissue sealing membranes obtained by different preparation processes under 0.1 M ammonium hydroxide conditions.

[0058] To evaluate the in vitro degradation performance of tissue sealing films obtained by different preparation processes, the polymers obtained in Examples 3, 4, and Comparative Example 2 were prepared into films and subjected to in vitro degradation experiments. The changes in the mass loss rate of the samples at different degradation times were recorded, and the results are shown in Table 3. The experimental results show that the tissue sealing films obtained in Examples 3, 4, and Comparative Example 2 all exhibited a degradation trend that gradually increased with the extension of degradation time during the in vitro degradation process. After 3 days of degradation, the mass loss rates of the samples in Examples 3, 4, and Comparative Example 2 were 83.52%, 82.44%, and 86.20%, respectively. With the extension of degradation time, the mass loss rates of the three groups of samples continued to increase, reaching 87.60%, 88.57%, and 93.01% after 6 days of degradation; 90.60%, 91.83%, and 96.99% after 9 days of degradation; and 95.33%, 96.18%, and 98.65% after 12 days of degradation, respectively.

[0059] The results showed that both the tissue sealing membranes prepared by microwave-assisted polymerization and those prepared by conventional thermal reaction processes exhibited good in vitro degradation characteristics, and both showed continuous mass loss during degradation. Although the degradation levels of samples obtained by different preparation processes varied at different time points, the overall degradation trend was basically consistent, indicating that the microwave-assisted reaction process did not change the in vitro degradation characteristics of the polymer materials, and the resulting materials still maintained good absorbability.

[0060] Figures 1-3 The figures show the proton NMR spectra of the polymers obtained in Examples 1, 2, and Comparative Example 1, respectively. The deuterated solvent used in the tests was deuterated dimethyl sulfoxide. The results show that the polymers obtained in Examples 1, 2, and Comparative Example 1 all exhibited the characteristic signal of the methylene group on the succinimide ring in the NHS active ester structure near δ 2.6–3.0 ppm, indicating that after the activation reaction, the carboxyl group in the polymer side chain was successfully converted into the NHS active ester group. Simultaneously, all samples retained the characteristic peaks of the N-vinylpyrrolidone structural unit and the carboxyl copolymer backbone, indicating that the microwave-assisted reaction process did not significantly damage the main polymer structure, and the resulting polymers maintained good structural integrity.

[0061] Figure 4 The Fourier transform infrared spectrum of the N-hydroxysuccinimide active tissue adhesion polymer is shown. Figure 5 The attenuated total reflectance Fourier transform infrared spectrum of the tissue sealing membrane prepared in Example 3.

[0062] Depend on Figure 4 It can be seen that the obtained N-hydroxysuccinimide active tissue adhesion polymer has an effective range of approximately 1810 cm⁻¹. -1 The characteristic absorption peak of the NHS active ester structure appears at approximately 1668 cm⁻¹. -1 The presence of a characteristic absorption peak at the N-vinylpyrrolidone structure, representing the lactam carbonyl group, indicates the successful introduction of NHS active ester groups into the polymer while retaining the PVP structural unit. (Comparison) Figure 4 and Figure 5 It can be seen that the tissue sealing membrane obtained in Example 3 is approximately 1811 cm⁻¹ -1 The characteristic absorption peak of the NHS active ester structure can still be observed at approximately 1650 cm⁻¹. -1The characteristic absorption peak of the lactam carbonyl group in the PVP structure appeared. Although the positions and absorption intensities of some characteristic absorption peaks changed due to the different infrared testing methods used and the differences in sample morphology and film structure, the characteristic absorption peaks of the NHS active ester structure and the PVP structural unit were retained before and after film formation. Therefore, it is shown that the main chemical structure of the tissue adhesion polymer prepared in this invention did not change significantly after coating, drying and film formation, and the NHS active ester groups can be effectively retained in the tissue sealing film.

[0063] In summary, this invention utilizes a microwave-assisted reaction method to prepare NHS active tissue adhesion polymers. While ensuring the effective introduction of NHS active ester groups and the stability of the polymer structure, it effectively shortens the preparation time and improves reaction efficiency in traditional reaction processes. The polymer prepared by this method, after further processing to form a tissue sealing film, still exhibits good film-forming properties, mechanical properties, and tissue adhesion properties. This demonstrates that this method can meet the performance requirements in the preparation of NHS active tissue adhesion materials and has the potential for further large-scale application.

[0064] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for preparing an N-hydroxysuccinimide active tissue adhesion polymer, characterized in that, Includes the following steps: S1. Acrylic acid, N-vinylpyrrolidone and azobisisobutyronitrile are dissolved in dimethyl sulfoxide and subjected to free radical copolymerization under nitrogen protection and microwave radiation to obtain a carboxyl-containing copolymer system. S2. Cool the carboxyl-containing copolymer system, add N-hydroxysuccinimide and dicyclohexylcarbodiimide, and carry out an activation reaction under microwave radiation to convert at least some of the carboxyl groups in the carboxyl-containing copolymer into N-hydroxysuccinimide active ester groups. S3. Filter the system after the activation reaction to remove dicyclohexylurea, and add the resulting filtrate to a non-solvent to precipitate the target polymer; S4. The precipitated target polymer is redissolved and then subjected to secondary precipitation. The resulting solid is then dried under low temperature and reduced pressure to obtain the N-hydroxysuccinimide active tissue adhesion polymer.

2. The preparation method according to claim 1, characterized in that, The microwave power of the free radical copolymerization reaction is 100~500 W, the reaction temperature is 100~120℃, and the reaction time is 0.5~2 h.

3. The preparation method according to claim 1, characterized in that, The activation reaction is carried out in an environment under nitrogen protection and with a relative humidity of less than 30%, with a microwave power of 10~100 W, a reaction temperature of 45±2℃, and a reaction time of 2~3 h.

4. The preparation method according to claim 1, characterized in that, The non-solvent is ethyl acetate or diethyl ether; the precipitated target polymer is redissolved in a mixed solvent of dichloromethane and methanol at a volume ratio of 3:1 or isopropanol, and then ethyl acetate or diethyl ether is added for secondary precipitation. The amount of ethyl acetate or diethyl ether used is 20 to 30 times the mass of the precipitated target polymer by mass-volume ratio.

5. The preparation method according to claim 1, characterized in that, The precipitation temperature is 0~10℃, the low-temperature vacuum drying temperature is 18~22℃, the absolute pressure is 16 kPa, and the drying time is 24~48 h.

6. An N-hydroxysuccinimide active tissue adhesion polymer, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 5.

7. A tissue sealing membrane, characterized in that, It comprises a support film layer, a tissue adhesion layer, a reinforcing film layer, and a surface tissue adhesion layer stacked sequentially; the support film layer and the reinforcing film layer contain polylactic acid-glycolic acid copolymer; the tissue adhesion layer and the surface tissue adhesion layer contain the N-hydroxysuccinimide active tissue adhesion polymer as described in claim 6.

8. A method for preparing the tissue sealing membrane according to claim 7, characterized in that, Includes the following steps: A 10% (w / w) polylactic acid-glycolic acid copolymer dichloromethane solution was coated onto a release substrate using a 120 μm wire rod at a coating speed of 30 mm / s to form a support film layer. A 10% (w / w) N-hydroxysuccinimide active tissue adhesion polymer solution was coated onto the support film layer using a 50 μm wire rod to form a tissue adhesion layer. The polylactic acid-glycolic acid copolymer dichloromethane solution was then coated onto the tissue adhesion layer using a 50 μm wire rod to form a reinforcing film layer. The N-hydroxysuccinimide active tissue adhesion polymer solution was then coated onto the reinforcing film layer using a 300 μm wire rod to form a surface tissue adhesion layer. The resulting multilayer film was then peeled off from the release substrate and dried under reduced pressure at low temperature for 24 h.

9. The use of the N-hydroxysuccinimide active tissue adhesion polymer of claim 6 in the preparation of tissue sealing membranes or medical devices for closure of tissue wounds or reduction of liquid or gas leakage.

10. The use of the N-hydroxysuccinimide active tissue adhesion polymer of claim 6 in the preparation of hemostatic materials or tissue repair materials.

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