Photosensitive tissue sealant as well as preparation method and application thereof

Abstract

The invention relates to a photosensitive tissue sealant and a preparation method thereof. A network of the photosensitive tissue sealant is formed by interweaving CSLA, CSNB and a bridging agent polyethylene glycol derivative; under the irradiation of ultraviolet light, disulfide bonds on CSLA molecules are reversibly broken and reconstructed, so that the gel is endowed with strong adhesive capacity and toughness; under the irradiation of ultraviolet light, an o-nitrobenzene group on the CSNB molecule is converted into o-nitrosobenzaldehyde, the o-nitrosobenzaldehyde reacts with amino to form a C = N bond, and an S-N bond and a C-N bond are formed between the CSLA molecule and the CSNB molecule; the above reactions are the key to realize high adhesion and high crosslinking density of the photosensitive tissue sealing glue. The invention also relates to application of the photosensitive tissue sealing glue in preparation of biological glue, experiments prove that the photosensitive tissue sealing glue has good biocompatibility, short gel forming time, tissue adhesion strength of 86.5 kPa and bursting pressure of 667 mmHg, animal experiments prove that the photosensitive tissue sealing glue has excellent tissue sealing and hemostasis effects in large and small body bleeding scenes, and the photosensitive tissue sealing glue has good application prospects. The method is suitable for high-pressure bleeding on complex sites and has wide application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a photosensitive tissue sealing adhesive, its preparation method, and its application. Background Technology

[0002] In the medical field, especially in surgery and trauma emergency care, hemostasis is a crucial step. Hemostasis of large vessel injuries is particularly challenging because the high pressure and large blood flow in large vessels often render traditional hemostasis methods ineffective. Currently, commonly used clinical hemostasis methods include compression hemostasis, ligation hemostasis, and electrocoagulation hemostasis, but these methods have many limitations when dealing with large vessel bleeding. For example, compression hemostasis requires continuous pressure, which is difficult to maintain for long periods and may affect the blood supply to surrounding tissues; ligation hemostasis may not be secure due to damage to the vessel wall, leading to recurrent bleeding; while electrocoagulation can quickly coagulate blood vessels, it causes significant thermal damage to surrounding tissues and its application is limited in some areas (such as the heart and brain).

[0003] In recent years, tissue sealing adhesives have attracted widespread attention as a novel hemostatic material. Tissue sealing adhesives are materials that can quickly adhere to tissue surfaces and form a sealing layer, preventing blood flow through a physical barrier, thereby achieving hemostasis. However, existing tissue sealing adhesives still have some problems when applied to hemostasis of large blood vessels. First, many tissue sealing adhesives lack sufficient adhesion, making it difficult to firmly adhere to the surface of large blood vessels, especially under the flushing effect of blood, and are prone to detachment. Second, the strength of tissue sealing adhesives is insufficient, unable to withstand the high pressure within large blood vessels, and are easily ruptured by blood flow, leading to hemostasis failure. Furthermore, some tissue sealing adhesives have long curing times, which cannot meet the needs of emergency hemostasis.

[0004] Chitosan, a natural polysaccharide material, possesses excellent biocompatibility, biodegradability, and antibacterial properties, and has been widely used in the biomedical field. However, the relatively low adhesiveness and mechanical strength of pure chitosan limit its application in large vessel hemostasis. To overcome this problem, researchers have attempted to modify chitosan through various methods to improve its adhesiveness and mechanical strength. For example, by using chemical crosslinking and physical mixing methods, chitosan is compounded with other materials to prepare tissue sealing adhesives with better performance. However, these methods often suffer from problems such as complex operation, high cost, and long curing time, making it difficult to meet the needs of practical clinical applications.

[0005] Therefore, developing a highly adhesive, high-burst-pressure chitosan photocurable tissue sealant for hemostasis in large blood vessels has significant clinical and social value. This tissue sealant should be able to cure rapidly within a short time, forming a strong sealing layer to effectively prevent blood leakage from large blood vessels; simultaneously, it should possess sufficient mechanical strength to withstand the high pressure within the large blood vessels, ensuring the durability of the hemostatic effect. Furthermore, the tissue sealant should also have good biocompatibility and antibacterial properties to reduce the risk of postoperative infection and promote wound healing. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention proposes a photosensitive tissue sealing adhesive, its preparation method, and its application.

[0007] The technical solution of this invention is as follows: A method for preparing a photosensitive tissue sealing gel includes the following steps: S1. Chitosan and thioctic acid are coupled by an amide condensation agent to obtain thioctic acid-modified chitosan molecules CSLA, which are then dried and ground to obtain CSLA powder for later use. S2. Chitosan and 4-hydroxymethyl-3-nitrobenzoic acid are coupled by an amide condensation agent to obtain 4-hydroxymethyl-3-nitrobenzoic acid modified chitosan molecules CSNB. After drying, they are ground to obtain CSNB powder for later use. S3. Dissolve and mix CSLA powder, CSNB powder and polyethylene glycol derivative to obtain a mixed solution, and then cure it with ultraviolet light to obtain a photosensitive tissue sealing adhesive.

[0008] Furthermore, the amide condensing agent mentioned in steps S1 and S2 is EDC and NHS.

[0009] Further, the mass ratio of lipoic acid to chitosan in step S1 is (1-5):1.

[0010] Further, in step S2, the mass ratio of 4-hydroxymethyl-3-nitrobenzoic acid to chitosan is (0.1-10):1.

[0011] Further, in step S3, the mass ratio of CSLA powder:CSNB powder:polyethylene glycol derivative is (0.2-20):1:0.15, and the mass concentration of CSLA powder, CSNB powder and polyethylene glycol derivative in the mixed solution is 1-5%.

[0012] Further, the polyethylene glycol derivative mentioned in step S3 is one or more of amino-functionalized polyethylene glycol, amino-functionalized tetra-armed polyethylene glycol, and amino-functionalized octa-armed polyethylene glycol, or a mixture thereof.

[0013] Furthermore, the process parameters for grinding in steps S1 and S2 are as follows: grinding CSLA and CSNB powders to a particle size of 0.2-0.5 mm at a speed of 10000-13000 rpm for a grinding time of 10-15 min.

[0014] Further, the specific process for obtaining the photosensitive tissue sealing gel by dissolving and mixing CSLA powder, CSNB powder and polyethylene glycol derivative in step S3 is as follows: CSLA powder, CSNB powder and polyethylene glycol derivative are mixed at 25±3℃ in the dark, and after mixing and dissolving at 500-1000rpm for 10-16h, the mixture is subjected to ultrasonic degassing in an ice bath for 10-15min. The ultrasonic degassing power is 100-180W and the frequency is 40-50kHz.

[0015] The photosensitive tissue sealing gel was prepared according to the preparation method described above.

[0016] The application of the photosensitive tissue sealing gel in the preparation of bio-adhesives.

[0017] Compared with the prior art, the present invention has at least the following advantages: 1. This application relates to a photosensitive tissue sealing gel and its preparation method, wherein the photosensitive tissue sealing gel network is composed of CSLA, CSNB, and a bridging agent, a polyethylene glycol derivative. During the synthesis of CSLA and CSNB, stable covalent bonds are formed between the photosensitive molecules (LA, NB) and chitosan through EDC / NHS catalysis; under ultraviolet light irradiation, the disulfide bonds on the CSLA molecules undergo reversible breakage and reconstruction, endowing the gel with certain self-healing ability and toughness; secondly, the sulfur free radicals generated during the reaction also form new covalent bonds with chitosan molecules or amino groups on the tissue surface; under ultraviolet light irradiation... The o-nitrophenyl group on the CSNB molecule is converted into o-nitrosobenzaldehyde. The aldehyde group on this intermediate reacts with the amino groups at the ends of chitosan molecules, tissue surfaces, or polyethylene glycol derivatives to form imine bonds (C=N). New covalent bonds (SN and CN bonds) are also formed between CSLA and CSNB molecules. The amino groups at the ends of the polyethylene glycol derivatives can also react with the carboxyl groups on the CSLA and CSNB molecular chains. The polyethylene glycol derivatives and the long chains of CSLA and CSNB molecules intertwine to form physical cross-linking points. These reactions are key to achieving high adhesion and high cross-linking density of the photosensitive tissue sealant.

[0018] 2. This invention also relates to the application of photosensitive tissue sealing adhesive in the preparation of bio-adhesives. Experiments have shown that the photosensitive tissue sealing adhesive has good biocompatibility, a short gelation time (< 3 s), does not cause hemolysis, has a tissue adhesion strength of 86.5 kPa, and a burst pressure of 667 mmHg. Animal experiments have shown that it has excellent tissue sealing and hemostasis effects in cases of rat liver rupture, rabbit femoral artery rupture, and Bama pig femoral artery rupture. This indicates that the photosensitive tissue sealing adhesive prepared by the method of this invention is suitable for complex high-pressure bleeding scenarios and has broad application prospects. Attached Figure Description

[0019] To more clearly illustrate the specific embodiments of the present invention, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0020] Figure 1 This is a flowchart illustrating the preparation process of the photosensitive tissue sealing adhesive described in this invention. Figure 2 These are images showing the state of photosensitive tissue sealing gels of different concentrations before and after UV irradiation in Example 4 of the present invention. Figure 3 This is a scanning electron microscope image of a 1-5% concentration photosensitive tissue sealing gel from Example 4 of the present invention; Figure 4 The gelation time of photosensitive tissue blocking gels of different concentrations in Example 5 of the present invention; Figure 5 The rheological properties of photosensitive tissue blocking adhesives of different concentrations in Example 6 of the present invention; Figure 6 This is a diagram showing the adhesion ability of the photosensitive tissue sealing adhesive of Embodiment 7 of the present invention to different biological tissues; Figure 7 The shear adhesion strength of different gels in Example 8 of this invention; Figure 8 The burst pressure of different gels in Example 9 of this invention; Figure 9 This is a graph showing the cytotoxicity test results of the photosensitive tissue blocking gel in Example 10 of the present invention; Figure 10 This is a graph showing the hemolysis rate detection results of Example 11 of the present invention; Figure 11 This is a graph showing the blood compatibility results of Example Twelve of the present invention; Figure 12 This is a photograph of the hemostatic effect of photosensitive tissue sealing gel on rat liver rupture in Example 13 of the present invention; Figure 13 The hemostatic index of rat liver and heart on the photosensitive tissue blocking gel in Example 13 of this invention; Figure 14This is a photograph of the hemostatic effect of photosensitive tissue sealing adhesive on a ruptured femoral artery in rabbits according to Embodiment Thirteen of the present invention; Figure 15 This is a photograph showing the hemostatic effect of the photosensitive tissue sealing adhesive in the ruptured femoral artery of Bama pigs according to Embodiment Thirteen of the present invention. Detailed Implementation

[0021] The present invention will now be described in further detail. It should be noted that the following specific embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above application content.

[0022] This invention provides a general and / or specific description of the materials and experimental methods used in the experiments. Unless otherwise specified, all experimental or testing methods are conventional methods; all reagents or instruments used, unless otherwise specified, are commercially available conventional products prepared or used using conventional methods.

[0023] Source of materials: Amino-functionalized polyethylene glycol, amino-functionalized tetra-armed polyethylene glycol, and amino-functionalized octa-armed polyethylene glycol: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., item numbers N590849, A595024, and A475436.

[0024] The preparation process of the photosensitive tissue sealing gel for hemostasis of large blood vessels described in this invention is as follows: Figure 1 As shown, the photosensitive tissue sealing gel is composed of two photosensitive molecularly functionalized chitosan polymer networks: the first is chitosan molecule modified with lipoic acid, and the second is chitosan molecule modified with 4-hydroxymethyl-3-nitrobenzoic acid. The stability of the gel network structure is further enhanced by introducing stabilizers, ensuring the gel continues to function. Under ultraviolet light irradiation, the disulfide bonds on the CSLA molecules break and reconstruct. Furthermore, the sulfur free radicals generated during the reaction react with the amino groups on the chitosan molecules or tissue surface to form new covalent bonds. The o-nitrobenzene on the CSNB molecules is converted to o-nitrosobenzaldehyde, which reacts with the amino groups on the chitosan molecules or tissue surface to form imine bonds (C=N). Additionally, new covalent bonds (SN and CN bonds) are formed between CSLA and CSNB molecules, further enhancing the tissue sealing performance of the gel. Specific preparation methods are described in Examples 1 to 3.

[0025] Example 1: Preparation method of photosensitive tissue sealing gel S1. Synthesis of lipoic acid-modified chitosan molecules (CSLA) 1.1) Weigh chitosan powder and dissolve it in a 1.5% (v / v) acetic acid solution. Stir at 500 rpm for 1 h to dissolve the chitosan solution with a concentration of 0.1 mg / mL. 1.2) Weigh lipoic acid powder and dissolve it in ethanol. Use sonication to aid dissolution and obtain a lipoic acid solution with a concentration of 1 mg / mL. Then, weigh EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) in a mass ratio of 0.1:1.91:1.15 as coupling agents and add them to the lipoic acid solution. Stir thoroughly to mix well. 1.3) Load the thioctic acid solution into a syringe and use a micro-injection pump to precisely control the flow rate of the solution to 1 mL / min. Slowly add the solution dropwise to the chitosan solution. The reaction volume ratio of thioctic acid solution to chitosan solution is 0.1:1. Control the reaction temperature at about 25℃ and react in the dark for 12 h with stirring at 500 rpm. 1.4) After the reaction is complete, the obtained solution is placed in a dialysis bag and dialyzed in deionized water for 3 days, changing the deionized water every day to remove unreacted reagents and obtain purified CSLA solution; 1.5) After dialysis, the solution was rapidly frozen at -80°C, and then the frozen sample was vacuum dried to obtain a dried CSLA sample. The prepared sample was stored at 5°C in the dark.

[0026] S2. Synthesis of chitosan molecules modified with 4-hydroxymethyl-3-nitrobenzoic acid (CSNB) 2.1) Weigh out chitosan powder and dissolve it in a 1.5% (v / v) acetic acid solution. Stir at 500 rpm for 1 h to dissolve, and a chitosan solution with a concentration of 0.1 mg / mL is obtained. 2.2) Weigh 4-hydroxymethyl-3-nitrobenzoic acid powder and dissolve it in dimethyl sulfoxide. Use sonication to aid dissolution to obtain a 4-hydroxymethyl-3-nitrobenzoic acid solution with a concentration of 1 mg / mL. Then, weigh EDC and NHS as coupling agents according to the mass ratio of 4-hydroxymethyl-3-nitrobenzoic acid, EDC and NHS of 0.1:0.575:0.345, add them to the 4-hydroxymethyl-3-nitrobenzoic acid solution, and stir thoroughly to mix. 2.3) Load the 4-hydroxymethyl-3-nitrobenzoic acid solution into a syringe, and use a micro-injection pump to precisely control the flow rate of the solution to 1 mL / min. Slowly add the solution dropwise to the chitosan solution. The reaction volume ratio of the 4-hydroxymethyl-3-nitrobenzoic acid solution to the chitosan solution is 0.01:1. Control the reaction temperature at about 25℃, and slowly add the solution to the chitosan solution. Stir at 500 rpm at room temperature and react in the dark for 12 h. 2.4) After the reaction is complete, the obtained solution is placed in a dialysis bag and dialyzed in deionized water for 3 days, changing the deionized water every day to remove unreacted reagents and obtain purified CSNB solution. 2.5) After dialysis, the solution was rapidly frozen at -80°C, and then the frozen sample was vacuum dried to obtain the CSNB sample. The prepared sample was stored at 5°C in the dark.

[0027] S3. Preparation of high-adhesion, high-bursting-pressure photosensitive tissue sealing adhesive 3.1) Place the prepared CSLA and CSNB samples into stainless steel grinding jars and grind them into powder with a particle size of less than 0.5 mm using a high-speed grinder at 10,000 rpm for 10 min to ensure the uniformity and fineness of the samples. 3.2) Weigh the sample reagents using a high-precision electronic balance. Mix CSLA, CSNB, and amino-functionalized polyethylene glycol thoroughly at a fixed mass ratio of 0.2:1:0.15. Add deionized water with a conductivity of less than 1 μS / cm to make the solid-liquid ratio 1%. Stir at around 25°C and 500 rpm in the dark for 12 h. Check the homogeneity of the solution every 2 h to ensure that CSLA, CSNB, and amino-functionalized polyethylene glycol are fully dissolved and mixed evenly. 3.3) The obtained gel solution was transferred to a dedicated ultrasonic container made of corrosion-resistant polypropylene. The ultrasonic container was placed in a 120 W ultrasonic instrument with the frequency set to 40 kHz. Ultrasonic degassing was performed in an ice bath environment to prevent the solution from changing its properties due to temperature rise during the ultrasonic process. The ultrasonic time was 15 min, and the process was paused every 3 min to observe the removal of bubbles in the solution and ensure that there were no obvious bubbles remaining in the solution. 3.4) The ultrasonically degassed gel solution is aseptically transferred into a medical syringe. The injection port is sealed with a sterile sealing film, ensuring that the sealing film fits tightly against the injection port to isolate air and prevent microorganisms and impurities from entering. Finally, the encapsulated photosensitive tissue sealing gel is placed in a special sterile packaging bag and sealed to obtain the encapsulated photosensitive tissue sealing gel. 3.5) Inject an appropriate amount of photosensitive tissue sealing gel onto the wound surface, and irradiate the gel with a 320 nm ultraviolet curing lamp to fully cure the gel and achieve strong adhesion and rapid sealing effect.

[0028] Example 2: Preparation method of photosensitive tissue sealing gel S1. Synthesis of lipoic acid-modified chitosan molecules (CSLA) 1.1) Weigh chitosan powder and dissolve it in a 1.5% (v / v) acetic acid solution. Stir at 500 rpm for 1 h to dissolve the chitosan solution with a concentration of 30 mg / mL. 1.2) Weigh lipoic acid powder and dissolve it in ethanol. Use sonication to aid dissolution and obtain a lipoic acid solution with a concentration of 15 mg / mL. Then, weigh EDC and NHS as coupling agents according to the mass ratio of lipoic acid, EDC and NHS of 5:1.91:1.15 and add them to the lipoic acid solution. Stir and mix thoroughly. 1.3) Load the thioctic acid solution into a syringe and use a micro-injection pump to precisely control the solution flow rate to 1 mL / min. Slowly add the solution to the chitosan solution. The reaction volume ratio of thioctic acid solution to chitosan solution is 2:1. Control the reaction temperature at about 25℃ and react in the dark for 12 h with stirring at 500 rpm. 1.4) After the reaction is complete, the obtained solution is placed in a dialysis bag and dialyzed in deionized water for 3 days, changing the deionized water every day to remove unreacted reagents and obtain purified CSLA solution; 1.5) After dialysis, the solution was rapidly frozen at -80°C, and then the frozen sample was vacuum dried to obtain a dried CSLA sample. The prepared sample was stored at 5°C in the dark.

[0029] S2. Synthesis of chitosan molecules modified with 4-hydroxymethyl-3-nitrobenzoic acid (CSNB) 2.1) Weigh out chitosan powder and dissolve it in a 1.5% (v / v) acetic acid solution. Stir at 500 rpm for 1 h to dissolve, and a chitosan solution with a concentration of 30 mg / mL is obtained. 2.2) Weigh 4-hydroxymethyl-3-nitrobenzoic acid powder and dissolve it in dimethyl sulfoxide. Use sonication to aid dissolution to obtain a 4-hydroxymethyl-3-nitrobenzoic acid solution with a concentration of 60 mg / mL. Then, weigh EDC and NHS as coupling agents according to the mass ratio of 4-hydroxymethyl-3-nitrobenzoic acid, EDC and NHS of 7:0.575:0.345, add them to the 4-hydroxymethyl-3-nitrobenzoic acid solution, and stir thoroughly to mix. 2.3) Load the 4-hydroxymethyl-3-nitrobenzoic acid solution into a syringe, and use a micro-injection pump to precisely control the flow rate of the solution to 1 mL / min. Slowly add the solution dropwise to the chitosan solution. The reaction volume ratio of the 4-hydroxymethyl-3-nitrobenzoic acid solution to the chitosan solution is 3:1. Control the reaction temperature at about 25℃ and slowly add the solution to the chitosan solution. Stir at 500 rpm at room temperature and react in the dark for 12 h. 2.4) After the reaction is complete, the obtained solution is placed in a dialysis bag and dialyzed in deionized water for 3 days, changing the deionized water every day to remove unreacted reagents and obtain purified CSNB solution. 2.5) After dialysis, the solution was rapidly frozen at -80°C, and then the frozen sample was vacuum dried to obtain the CSNB sample. The prepared sample was stored at 5°C in the dark.

[0030] S3. Preparation of high-adhesion, high-bursting-pressure photosensitive tissue sealing adhesive 3.1) Place the prepared CSLA and CSNB samples into stainless steel grinding jars and grind them into powder with a particle size of less than 0.5 mm using a high-speed grinder at 10,000 rpm for 10 min to ensure the uniformity and fineness of the samples. 3.2) Weigh the sample reagents using a high-precision electronic balance. Mix CSLA, CSNB, and amino-functionalized octagonal polyethylene glycol thoroughly at a fixed mass ratio of 5:1:0.15. Add deionized water with a conductivity of less than 1 μS / cm to make the solid-liquid ratio 2.5%. Stir at 500 rpm in the dark at around 25°C for 12 h. Check the homogeneity of the solution every 2 h to ensure that CSLA, CSNB, and amino-functionalized octagonal polyethylene glycol are fully dissolved and mixed evenly. 3.3) The obtained gel solution was transferred to a dedicated ultrasonic container made of corrosion-resistant polypropylene. The ultrasonic container was placed in a 120 W ultrasonic instrument with the frequency set to 40 kHz. Ultrasonic degassing was performed in an ice bath environment to prevent the solution from changing its properties due to temperature rise during the ultrasonic process. The ultrasonic time was 15 min, and the process was paused every 3 min to observe the removal of bubbles in the solution and ensure that there were no obvious bubbles remaining in the solution. 3.4) The ultrasonically degassed gel solution is aseptically transferred into a medical syringe. The injection port is sealed with a sterile sealing film, ensuring that the sealing film fits tightly against the injection port to isolate air and prevent microorganisms and impurities from entering. Finally, the encapsulated photosensitive tissue sealing gel is placed in a special sterile packaging bag and sealed to obtain the encapsulated photosensitive tissue sealing gel. 3.5) Inject an appropriate amount of photosensitive tissue sealing gel onto the wound surface, and irradiate the gel with a 365 nm ultraviolet curing lamp to fully cure the gel and achieve strong adhesion and rapid sealing effect.

[0031] Example 3: Preparation method of photosensitive tissue sealing gel S1. Synthesis of lipoic acid-modified chitosan molecules (CSLA) 1.1) Weigh chitosan powder and dissolve it in a 1.5% (v / v) acetic acid solution. Stir at 500 rpm for 1 h to dissolve the chitosan solution with a concentration of 50 mg / mL. 1.2) Weigh lipoic acid powder and dissolve it in ethanol. Use sonication to aid dissolution and obtain a lipoic acid solution with a concentration of 20 mg / mL. Then, weigh EDC and NHS as coupling agents according to the mass ratio of lipoic acid, EDC and NHS of 10:1.91:1.15 and add them to the lipoic acid solution. Stir and mix thoroughly. 1.3) Load the thioctic acid solution into a syringe and use a micro-injection pump to precisely control the solution flow rate to 1 mL / min. Slowly add the solution to the chitosan solution. The reaction volume ratio of thioctic acid solution to chitosan solution is 10:1. Control the reaction temperature at about 25℃ and react in the dark for 12 h with stirring at 500 rpm. 1.4) After the reaction is complete, the obtained solution is placed in a dialysis bag and dialyzed in deionized water for 3 days, changing the deionized water every day to remove unreacted reagents and obtain purified CSLA solution; 1.5) After dialysis, the solution was rapidly frozen at -80°C, and then the frozen sample was vacuum dried to obtain a dried CSLA sample. The prepared sample was stored at 5°C in the dark.

[0032] S2. Synthesis of chitosan molecules modified with 4-hydroxymethyl-3-nitrobenzoic acid (CSNB) 2.1) Weigh out chitosan powder and dissolve it in a 1.5% (v / v) acetic acid solution. Stir at 500 rpm for 1 h to dissolve the chitosan solution with a concentration of 50 mg / mL. 2.2) Weigh 4-hydroxymethyl-3-nitrobenzoic acid powder and dissolve it in dimethyl sulfoxide. Use sonication to aid dissolution to obtain a 4-hydroxymethyl-3-nitrobenzoic acid solution with a concentration of 100 mg / mL. Then, weigh EDC and NHS as coupling agents according to the mass ratio of 4-hydroxymethyl-3-nitrobenzoic acid, EDC and NHS of 10:0.575:0.345, add them to the 4-hydroxymethyl-3-nitrobenzoic acid solution, and stir thoroughly to mix. 2.3) Load the 4-hydroxymethyl-3-nitrobenzoic acid solution into a syringe, use a micro-injection pump to precisely control the flow rate of the solution to 1 mL / min, and slowly add the solution dropwise to the chitosan solution. The reaction volume ratio of the 4-hydroxymethyl-3-nitrobenzoic acid solution to the chitosan solution is 5:1. Control the reaction temperature at about 25℃, slowly add the solution to the chitosan solution, stir at 500 rpm at room temperature, and react in the dark for 12 h. 2.4) After the reaction is complete, the obtained solution is placed in a dialysis bag and dialyzed in deionized water for 3 days, changing the deionized water every day to remove unreacted reagents and obtain purified CSNB solution. 2.5) After dialysis, the solution was rapidly frozen at -80°C, and then the frozen sample was vacuum dried to obtain the CSNB sample. The prepared sample was stored at 5°C in the dark.

[0033] S3. Preparation of high-adhesion, high-bursting-pressure photosensitive tissue sealing adhesive 3.1) Place the prepared CSLA and CSNB samples into stainless steel grinding jars and grind them into powder with a particle size of less than 0.5 mm using a high-speed grinder at 10,000 rpm for 10 min to ensure the uniformity and fineness of the samples. 3.2) Weigh the sample reagents using a high-precision electronic balance. Mix CSLA, CSNB, and amino-functionalized tetra-arm polyethylene glycol at a fixed mass ratio of 20:1:0.15 thoroughly. Add deionized water with a conductivity of less than 1 μS / cm to make the solid-liquid ratio 5%. Stir at 500 rpm in the dark at around 25°C for 12 h. Check the homogeneity of the solution every 2 h to ensure that CSLA, CSNB, and amino-functionalized tetra-arm polyethylene glycol are fully dissolved and mixed evenly. 3.3) The obtained gel solution was transferred to a dedicated ultrasonic container made of corrosion-resistant polypropylene. The ultrasonic container was placed in a 120 W ultrasonic instrument with the frequency set to 40 kHz. Ultrasonic degassing was performed in an ice bath environment to prevent the solution from changing its properties due to temperature rise during the ultrasonic process. The ultrasonic time was 15 min, and the process was paused every 3 min to observe the removal of bubbles in the solution and ensure that there were no obvious bubbles remaining in the solution. 3.4) The ultrasonically degassed gel solution is aseptically transferred into a medical syringe. The injection port is sealed with a sterile sealing film, ensuring that the sealing film fits tightly against the injection port to isolate air and prevent microorganisms and impurities from entering. Finally, the encapsulated photosensitive tissue sealing gel is placed in a special sterile packaging bag and sealed to obtain the encapsulated photosensitive tissue sealing gel. 3.5) Inject an appropriate amount of photosensitive tissue sealing gel onto the wound surface, and irradiate the gel with a 410 nm UV curing lamp to fully cure the gel and achieve strong adhesion and rapid sealing effect.

[0034] Example 4 Morphology Test The photosensitive tissue sealing gels of different concentrations prepared in Examples 1 to 3 of this invention are shown in the following states before and after UV irradiation: Figure 2As shown, the photosensitive tissue blocking gel with a concentration of 1-5% can all change from solution to solid gel after ultraviolet light irradiation.

[0035] Scanning electron microscope images of the photosensitive tissue sealing gels prepared in Examples 1 to 3 are shown below. Figure 3 As shown, the results indicate that the internal pore size of the photosensitive tissue blocking gel is inversely proportional to the concentration in the solution, suggesting that the higher the solution concentration, the denser the gel network formed.

[0036] Example 5: Gel Formation Time The gelation time of photosensitive tissue blocking gels of different concentrations prepared in Examples 1 to 3 of this invention is as follows: Figure 4 As shown, the results indicate that gelation time is inversely proportional to solution concentration, and a 5% concentration of photosensitive tissue blocking gel can achieve rapid gelation within 3 seconds.

[0037] Example 6: Rheological Performance Testing The rheological properties of photosensitive tissue blocking adhesives at concentrations of 1%, 2.5%, and 5% were tested using a rheometer. The results are as follows: Figure 5 As shown, the results are consistent with the trend of the scanning electron microscope images. The higher the solution concentration, the greater the storage modulus of the formed hydrogel, that is, the stronger the ability of the formed gel to resist deformation.

[0038] Example 7 Adhesion Test Taking the 5% photosensitive tissue sealing gel prepared in Example 3 as an example, adhesion ability tests were performed on various tissues and organs of rats, and the results are as follows: Figure 6 As shown in the figure, the developed photosensitive tissue sealing adhesive has broad and stable adhesion properties to heart, liver, spleen, lung, kidney and muscle tissues.

[0039] Example 8 Shear Adhesion Strength Test Taking the 5% photosensitive tissue sealing gel prepared in Example 3 as an example, a shear adhesion strength test was conducted. The test method was as follows: using pig intestine casing as the biological tissue adhesion test substrate, it was fixed to two transparent plastic test pieces with strong adhesive. The gel solution was injected onto the surface of the pig intestine casing, with the adhesion dimensions on both sides of the pig intestine casing being 25 mm × 10 mm. The gel was fully cured using a UV lamp, and the shear adhesion strength was tested using a universal testing machine at a tensile speed of 5 mm / min. Figure 7As shown, the photosensitive tissue sealant exhibits excellent adhesion properties, with a tissue adhesion strength of 86.5 kPa, which is 10.5 times that of medical fibrin glue. This is superior to HAMA and GelMA gels reported in the literature, as well as pure CSLA gel (69.5 kPa) and pure CSNB gel (59.4 kPa). This indicates that the CSLA and CSNB in ​​the prepared photosensitive tissue sealant are not simply a physical mixture, but rather new chemical bonds (SN and CN bonds) are formed between the two molecules, making the gel network structure more robust and thus achieving high adhesion and high burst pressure characteristics.

[0040] Example 9: Burst Pressure Test Figure 8 To illustrate the burst pressure test results of different photosensitive tissue sealants, the burst pressure test method was as follows: Pigskin was used as the biological tissue adhesion substrate. The pigskin was fixed to the burst pressure testing device, and an 18G syringe needle was used to puncture the center of the pigskin to construct a defect. The tubing was filled with phosphate buffer, and the gel solution was injected into the sample chamber. The gel was fully cured using a UV lamp, and the injection pump was turned on at a flow rate of 10 mL / min. The burst pressure of the tissue sealant was then tested. Taking the 5% photosensitive tissue sealant prepared in Example 3 as an example, the burst pressure test results showed a consistent trend with the shear adhesion strength results. The burst pressure of the prepared photosensitive tissue sealant reached 667 mmHg, which is 4.6 times that of medical fibrin glue, making it suitable for complex high-pressure bleeding scenarios.

[0041] Example 10: Cytotoxicity Test Taking the 5% photosensitive tissue blocking gel prepared in Example 3 as an example, after co-culturing it with cells for 24 h and 48 h, cell viability was detected using CCK-8 assay. The results are as follows: Figure 9 As shown, there was no significant difference in cell activity between the gel group and the control group, indicating that it has good cell compatibility.

[0042] Example 11: Hemolysis Rate Detection Taking the 5% photosensitive tissue blocking gel prepared in Example 3 as an example, the hemolysis rate detection method is as follows: Before the test, fresh blood was drawn from healthy mice, and red blood cells were separated by centrifugation. After removing the supernatant, physiological saline was added to obtain a diluted red blood cell suspension. The gel and red blood cell suspension were co-incubated at 37°C for 1 h, and the supernatant was collected by centrifugation at 1500 rpm. The absorbance was measured at 540 nm using a UV-Vis spectrophotometer. Physiological saline was used as a negative control, and deionized water was used as a positive control. The hemolysis rate of the tissue blocking gel was calculated. Figure 10 The image shows the hemolysis rate test results of the tissue blocking gel. The hemolysis rate of the developed photosensitive tissue blocking gel is less than 5%, indicating that it will not cause a significant hemolytic reaction. Figure 11The image shows the blood compatibility of the photosensitive tissue blocking gel. The results show that the prepared tissue blocking gel does not cause the rupture of red blood cells after co-incubation with red blood cells, indicating that it has good blood compatibility.

[0043] Example 12: Hemostatic Effect in Animals Taking the 5% photosensitive tissue blocking gel prepared in Example 3 as an example, Figure 12 The image shows the hemostatic effect of photosensitive tissue sealing adhesive on liver rupture in rats. The results show that the tissue sealing adhesive prepared by the method described in this invention can quickly seal liver wounds and achieve rapid hemostasis in 20 seconds. Figure 13 The study investigated the hemostatic effects of tissue-blocking gel on rat liver and heart, and the results showed that it could be used for hemostasis of liver and heart rupture. The blood loss and hemostasis time in the gel group were significantly lower than those of medical gauze and fibrin glue.

[0044] Figure 14 The image shows the hemostatic effect of photosensitive tissue sealant on a ruptured femoral artery in rabbits. The results indicate that the tissue sealant can stably adhere to the ruptured femoral artery in rabbits, achieving rapid hemostasis.

[0045] Figure 15 The image shows the hemostatic effect of photosensitive tissue sealant on femoral artery rupture in Bama pigs. The results show that the tissue sealant has the same high hemostatic effect on femoral artery rupture in large animals.

[0046] In summary, the method described in this invention has developed a highly adhesive, high-burst-pressure photosensitive tissue sealing adhesive for hemostasis in large blood vessels, which has significant clinical and social value. The invented tissue sealing adhesive can rapidly cure into a gel within a short time (≤3 s), forming a strong sealing layer that effectively prevents blood leakage from large blood vessels. Simultaneously, it possesses sufficient mechanical strength and tissue adhesion properties to withstand the high pressure within large blood vessels, ensuring the durability of the hemostatic effect. Furthermore, the tissue sealing adhesive exhibits good biocompatibility, and is expected to solve the problems existing in the prior art, providing a more effective, safe, and reliable solution for hemostasis in large blood vessels.

[0047] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention 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 the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A method for preparing a photosensitive tissue sealing gel, characterized in that, Includes the following steps: S1. Chitosan and thioctic acid are coupled by an amide condensation agent to obtain thioctic acid-modified chitosan molecules CSLA, which are then dried and ground to obtain CSLA powder for later use. S2. Chitosan and 4-hydroxymethyl-3-nitrobenzoic acid are coupled by an amide condensation agent to obtain 4-hydroxymethyl-3-nitrobenzoic acid modified chitosan molecules CSNB. After drying, they are ground to obtain CSNB powder for later use. S3. Dissolve and mix CSLA powder, CSNB powder and polyethylene glycol derivative to obtain a mixed solution, and then cure it with ultraviolet light to obtain a photosensitive tissue sealing adhesive.

2. The preparation method according to claim 1, characterized in that, The amide condensing agents mentioned in steps S1 and S2 are EDC and NHS.

3. The preparation method according to claim 2, characterized in that, The mass ratio of lipoic acid to chitosan in step S1 is (1-5):

1.

4. The preparation method according to any one of claims 1-3, characterized in that, The mass ratio of 4-hydroxymethyl-3-nitrobenzoic acid to chitosan in step S2 is (0.1-10):

1.

5. The preparation method according to claim 4, characterized in that, In step S3, the mass ratio of CSLA powder:CSNB powder:polyethylene glycol derivative is (0.2-20):1:0.15, and the mass concentration of CSLA powder, CSNB powder and polyethylene glycol derivative in the mixed solution is 1-5%.

6. The preparation method according to claim 5, characterized in that, The polyethylene glycol derivative mentioned in step S3 is one or more of amino-functionalized polyethylene glycol, amino-functionalized tetra-armed polyethylene glycol, and amino-functionalized octa-armed polyethylene glycol, or a mixture thereof.

7. The preparation method according to claim 6, characterized in that, The process parameters for grinding in steps S1 and S2 are as follows: at a speed of 10000-13000 rpm, grind the CSLA and CSNB powders to a particle size of 0.2-0.5 mm, and grind for 10-15 min.

8. The preparation method according to any one of claims 5-7, characterized in that, The specific process for obtaining the photosensitive tissue sealing gel by dissolving and mixing CSLA powder, CSNB powder and polyethylene glycol derivative in step S3 is as follows: CSLA powder, CSNB powder and polyethylene glycol derivative are mixed at 25±3℃ in the dark, and after mixing and dissolving at 500-1000rpm for 10-16h, the mixture is subjected to ultrasonic degassing in an ice bath for 10-15min. The ultrasonic degassing power is 100-180W and the frequency is 40-50kHz.

9. The photosensitive tissue sealing gel prepared by the preparation method according to any one of claims 1-8.

10. The application of the photosensitive tissue sealing gel according to claim 9 in the preparation of bio-adhesives.

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