CN117100901A - Photoinitiated biological tissue adhesive, gel sheet, preparation method and application - Google Patents

Abstract

The invention discloses a light-initiated biological tissue adhesive, a gel sheet, a preparation method and an application. The biological tissue adhesive is cured by a bifunctional polymer and a photoinitiator under light initiation; it is produced in natural materials through amidation reaction. Two active units, catechol group and olefin group, are simultaneously introduced into the polymer to produce a bifunctional polymer. The mixed solution of the bifunctional polymer and photoinitiator can achieve gelation and moderate mechanical strength under light triggering. strength. By adjusting the grafting ratio of the two active units on the natural polymer, the tissue bonding strength and the mechanical strength of the adhesive itself are respectively realized and regulated, so that the adhesive of the present invention can be used not only for dura (spinal) membrane repair. , and can also be applied to repair other tissue wounds.

Description

Photoinitiated biological tissue adhesive, gel sheet, preparation method and application

Technical field

The invention belongs to the technical field of biotechnology, and in particular relates to a photoinitiated biological tissue adhesive, gel sheet, preparation method and application.

Background technique

The dura (spinal) membrane is the outermost layer of the surface membrane of the brain and spinal cord. It is a connective tissue fiber membrane. The dura (spinal) membrane is a layer of protective structure. When it is damaged, it is often accompanied by cerebrospinal fluid leakage. That is, the cerebrospinal fluid leaks from the dura (spinal) membrane gap and the covered bone under the action of the pressure gradient inside and outside the skull and spinal canal. The phenomenon of leakage from quality defects. Continuous cerebrospinal fluid leakage will not only cause disorder of cerebrospinal fluid circulation dynamics, postural headache and dizziness, but also further increase the risk of complications such as nervous system infection, poor incision healing, and even intracranial and intraspinal hemorrhage. These complications will consume more medical resources and significantly increase the financial burden on patients; even patients may receive repeated invasive treatments, which not only have poor effects, but also lead to severe disability or death as the condition worsens. According to the cause, the clinical classification of cerebrospinal fluid leakage can be divided into: traumatic cerebrospinal fluid leakage, postoperative cerebrospinal fluid leakage and idiopathic cerebrospinal fluid leakage. Postoperative cerebrospinal fluid leakage is one of the common complications of neurosurgery and spine surgery. Although with the advancement of surgical technology and the application of new skull base repair materials, the incidence of postoperative cerebrospinal fluid leakage has gradually decreased, the effect is still not ideal. Studies have shown that complications related to cerebrospinal fluid leakage after infratentorial craniotomy are as high as 32%, and the incidence of cerebrospinal fluid leakage after spinal surgery is 5% to 13%. The prevention of cerebrospinal fluid leakage is higher than the treatment. In addition to various therapeutic measures to prevent cerebrospinal fluid leakage during surgery, timely repair of dural rupture during surgery is a key step to prevent postoperative cerebrospinal fluid leakage. Therefore, various clinical reasons can be solved to prevent cerebrospinal fluid leakage. Damage to the (spinal) membrane or loose sutures can lead to cerebrospinal fluid leakage, and this technology can repair the dura (spinal) membrane with controllable strong adhesion in biologically humid environments and irregular shapes.

Existing technology: A method of repairing the dura (spinal) membrane with tight suturing is to use autologous tissue (fat, muscle, fat, bone fragments, etc.) or artificial materials to perform enlarged and molded sutures. The main focus is on surgical strategies and techniques, but sutures are not excluded. The effect may be poor or the leak may be caused by another tear, so how to repair and close the first barrier of the dura (spinal) membrane is particularly important.

Another way is to use fibrin adhesives, that is, human-derived medical biological protein adhesives (Gulax) and porcine-derived medical biological protein adhesives (Anchor, Beixiu glue, etc.), sealing agents (Hydrogel, composite adhesive, etc.). However, this method is only a simple adhesive filling, and the viscosity is not high; and it is easy to be washed and displaced, and the sealing effect is not good; especially for high-flow cerebrospinal fluid leaks, involving the skull base or irregular areas, it has little effect in sealing cerebrospinal fluid leaks; therefore, this method is also Controlled and effective adhesion cannot be performed on the boundary of the defect, especially in high-flow areas, which is uncontrollable and cannot guarantee effective adhesion.

Contents of the invention

In response to the above problems, the present invention provides a controllable light-initiated biological tissue adhesive, gel sheet, preparation method and application, which simultaneously introduces catechol groups on carboxylic acid-containing natural polymers through amidation reaction. and olefin groups to produce a bifunctional polymer. The grafting ratio of the two active units on the natural polymer can be adjusted as needed to achieve and regulate the tissue bonding strength and the mechanical strength of the adhesive itself. , the mixed solution of bifunctional polymer and photoinitiator can achieve gelation and moderate mechanical strength under light triggering.

In order to achieve the above objects, the technical solution of the present invention is:

A photoinitiated biological tissue adhesive, which is cured by a bifunctional polymer and a photoinitiator under light initiation; the bifunctional polymer is a natural polymer containing carboxylic acid through an amidation reaction. Catechol groups and double bond groups are introduced.

Natural polymers are polymers from nature, such as starch, cellulose, lignin, etc., which are all natural polymers. The natural polymers in the photoinitiated biological tissue adhesive contained in this application are natural polymers containing carboxylic acid. , with a molecular weight of 1000-100000 Da, specifically sodium carboxymethyl cellulose, sodium carboxymethyl cellulose, hyaluronic acid, sodium hyaluronate, sodium alginate, carboxymethyl chitin or carboxymethyl chitosan, etc. Natural polymers.

Preferably, the bifunctional polymer is obtained through a condensation reaction between a natural polymer and amino-containing catechol, amino-containing olefin and amidation condensation agent.

Preferably, the mass ratio of the natural polymer, amino-containing catechol, amino-containing olefin and amidation condensation agent is 1:0.005-20:0.005-20:0.1-1000. The biological tissue within this range After the adhesive is cured, a hydrogel system with good adhesion effect and mechanical strength can be obtained.

Preferably, the amino-containing catechol is selected from the group consisting of dopamine, 6-hydroxydopamine, 5-hydroxydopamine, 3,4-dihydroxybenzylamine, norepinephrine, norepinephrine hydrochloride, 3,4 -Dihydroxynorephedrine or discoidein.

Preferably, the amino-containing olefin is selected from 2-amino methacrylate, 3-butene-1-amine, 2-methylallylamine, methacrylamide, pent-4-en-1-amine or 4 -Vinylbenzylamine.

Preferably, the amidation condensation agent is selected from: 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride, active esters (such as N,N' -carbonyldiimidazole, etc.), carbodiimides (such as dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-(3-dimethylaminopropyl)- 3-Ethylcarbodiimide (EDCl, etc.), onium salts (HATU-PF6, HCTU-PF6, BOP, etc.), organophosphorus (DPPCl, MPTA, BOP-Cl, etc.) and other types (such as triphenyl Phosphate-polyhalomethanes, triphenylphosphorus-hexachloroacetone, triphenylphosphorus-NBS, 3-acyl-2-thiazoline, etc.).

Preferably, the photoinitiator is selected from: phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, aromatic ketone photoinitiator, condensed ring aromatic hydrocarbon photoinitiator, polysilane photoinitiator Any one or more of acylphosphonate photoinitiators, azo photoinitiators or metal-organic complexes.

Preferably, the mass ratio of the bifunctional polymer to the photoinitiator is 1:0.01-10.

Based on the same inventive concept, the present invention also provides a preparation method for photoinitiated biological tissue adhesive, which includes the following steps:

S1: Dissolve the natural polymer containing carboxylic acid in the solvent to prepare the natural polymer solution;

S2: Add the amidation reagent to the natural polymer solution prepared in step S1, then add amino-containing catechol and amino-containing olefins in the dark, and use NaOH solution to adjust the pH of the above solution to 5.5- 7.5, obtain the reaction solution;

S3: Precipitate the reaction solution obtained in step S2 in ice ethanol, centrifuge at low temperature, and repeat this step 2-3 times to obtain a bifunctional polymer;

S4: After dissolving the bifunctional polymer obtained in step S3, add a photoinitiator to dissolve to obtain the biological tissue adhesive;

The biological tissue adhesive triggers cross-linking and solidification of the bifunctional polymer and the photoinitiator under light exposure.

Preferably, the solvent in step S1 is one of water, N,N-dimethylformamide DMF or dimethyl sulfoxide DMSO, or a mixture thereof.

Preferably, the mass ratio of the carboxylic acid-containing natural polymer to the solvent is 1:0.1-1000.

Preferably, the wavelength of light used in the cross-linking reaction between the bifunctional polymer and the photoinitiator is 100-1000 nm, and the illumination time is 1-600S.

Based on the same inventive concept, the present invention also provides a biological tissue gel sheet, which is the photoinitiated biological tissue adhesive of the above embodiment or the photoinitiated biological tissue adhesive obtained by the preparation method. The gel after the tissue adhesive is cured by light can be tailored according to the needs to obtain corresponding sheet products, such as sheet products with length, width and height of 4cm*5cm*0.5 or 8cm*10cm*0.5cm.

Based on the same inventive concept, the present invention also provides an application of a photoinitiated biological tissue adhesive in the preparation of bioadhesive products. The photoinitiated biological tissue adhesive is the photoinitiated biological tissue adhesive. Or obtained by the preparation method described.

Furthermore, light-initiated biological tissue adhesives are used in dura (spinal) membrane repair, peritoneal and pleural repair, damaged blood vessel repair, and adhesive repair of skin and mucous membranes.

Furthermore, the bioadhesive product may be in the form of an adhesive with certain fluidity directly packaged in an injection gun, or may be a cured gel sheet.

Due to the adoption of the above technical solutions, the present invention has the following advantages and positive effects compared with the prior art:

The biological tissue adhesive provided by the invention is based on a light-responsive bifunctional natural polymer. The bifunctional natural polymer introduces both catechol groups and olefin groups on the natural polymer containing carboxylic acid through amidation reaction. Active units are produced. Then the bifunctional polymer and the photoinitiator can achieve the curing of the adhesive and its adhesion to biological tissue under light triggering. Therefore, this tissue adhesive is only composed of carboxylic acid-modified natural polymers and photoinitiators, without other small molecule reagents, which can greatly reduce the possibility of tissue inflammation, and the biosafety and degradability of natural polymers are also improved. Provides strong safety guarantee for tissue application; at the same time, the bifunctional polymer can be conveniently and efficiently triggered by light to gel, has time and space controllability, and is easy and flexible to use; by regulating both the catechol group and the olefin group The grafting ratio of active units on the natural polymer can achieve and regulate the required adhesive strength of biological tissue and the mechanical strength of the adhesive itself, and is suitable for repairing leaks in the dura (spinal) membrane.

The preparation method of the biological tissue adhesive of the present invention has a simple system and strong adjustability. The bifunctional polymer only requires one-step chemical synthesis, the preparation process and components are simple, and the specific structure and grafting ratio of the two active components are highly adjustable. .

Description of drawings

Figure 1 is a graph showing the toxicity level test on microglia (BV2) of biological tissue adhesives with different volume concentrations in Example 1 of the present invention;

Figure 2 is the rheological behavior of the biological tissue adhesive before photo-crosslinking in Example 1 of the present invention;

Figure 3 is the rheological behavior of the biological tissue adhesive after photo-crosslinking in Example 1 of the present invention;

Figure 4 is a sealed cerebrospinal fluid biological tissue model constructed from the fish bladder of the present invention. The biological tissue adhesive of Example 1 is used to seal the leak (the blade makes an incision of about 1.5cm). After the physiological saline is injected, the liquid oozes out from the leak. Obviously; then, the light-triggered cross-linking of the adhesive for 15 seconds was used to repair the gap, and water was injected into the biological tissue model to simulate the filling effect of cerebrospinal fluid. It was seen that after complete water injection, there was no liquid seepage or leakage from the leak, and the repair effect was good;

Figure 5 is a closed cerebrospinal fluid biological tissue model constructed from the fish bladder of the present invention. 15 seconds after sealing the leak with the biological tissue adhesive of Example 1, it can be seen that no liquid oozes out or leaks out of the leak after complete water injection, and then it is administered by hand. The ultimate burst pressure of 25-35cmH ₂ O simulates the transient increase in intracranial pressure caused by changes in posture, sneezing, coughing and vomiting after surgery. It is seen that the adhesive colloid expands as the tear breaks, and no leakage of normal saline is seen. , showing good adhesion and mechanical elasticity;

Figure 6 is a closed cerebrospinal fluid biological tissue model constructed from porcine intestinal mucosa according to the present invention. The biological tissue adhesive of Example 1 is used to seal the leakage opening (the blade makes an incision of about 1.5cm). After the physiological saline is injected, the liquid leaks out from the leakage opening. , the leakage was obvious; then, the gap was repaired by cross-linking the light-triggered adhesive for 15 seconds, and water was injected into the biological tissue model to simulate the filling effect of cerebrospinal fluid. After complete water injection, there was no leakage or leakage of liquid from the leakage opening, and the repair effect was good. A variety of biological tissues verify the biological effectiveness of the adhesive;

Figure 7 is a rat model of cerebrospinal fluid leakage constructed by the present invention, using the biological tissue adhesive and physiological saline of Example 1 to repair the rat cerebrospinal fluid leakage animal model. A in Figure 7 shows the observation of wound healing and cerebrospinal fluid leakage; Figure 7 C shows the body weight changes of each group after feeding; B in Figure 7 shows the whole HE stained tissue sections of the rat scalp, galea, dura mater, arachnoid mater and pia mater after sudden death;

Figure 8 is a picture of the cell growth morphology under a light microscope when biological tissue adhesive diluted at a volume concentration of 6.3% and a normal saline blank control group were added to the microglial cell line BV2 culture system.

Detailed ways

Current tissue adhesives offer a variety of materials for wound management and are used in a wide variety of medical settings, ranging from minor to life-threatening tissue injuries. Compared to traditional wound closure methods (i.e. suturing and suturing), tissue adhesives are relatively easy to use, enable rapid application, and minimize tissue damage. In addition, tissue adhesives can act as hemostatic agents to control bleeding and provide a tissue healing environment at the wound site. An ideal tissue adhesive should possess many properties, including: (1) biocompatibility and nontoxicity; (2) the ability to form robust interactions with tissue; (3) mechanical similarity to the adhesive tissue; (4) ) mechanical ability to withstand repeated application of stress to the tissue; (5) acceptable swelling properties to minimize tissue compression; and (6) biodegradability compatible with the rate of tissue healing.

However, existing adhesives only meet some of these requirements and still face some limitations and unsolved challenges (e.g., weak adhesive strength and poor mechanical properties, complex systems, low biosafety, causing inflammation, etc.), These limit their use and there is room for further improvement.

Since there is cerebrospinal fluid in the dura (spinal) membrane, the adhesive has greater adhesion in a wet or liquid environment, and needs to maintain a good sealing effect under the pressure inside and outside the skull and spinal canal. The present invention designs and prepares a photoinitiated biological tissue adhesive to solve the existing problems in dura (spinal) membrane repair. The biological tissue adhesive includes a bifunctional polymer, a bifunctional polymer and a photoinitiator. The mixed solution can gel under photoinitiation, and the gel after gelation has excellent mechanical strength. The bifunctional polymer of the present invention simultaneously introduces catechol units and olefin units into a natural polymer containing carboxylic acid through an amidation reaction. The natural polymer has excellent biocompatibility and degradability and is used as a brain Meningeal repair provides a strong safety guarantee, allowing the adhesive to fuse with the tissue and degrade after being applied to the tissue; the catechol unit and the olefin unit provide biological sites for the adhesive to bind to the tissue that needs to be repaired. The adhesive has excellent mechanical properties. In particular, the catechol unit has an efficient chemical interaction with the amino or sulfur groups present in large amounts in biological tissues to obtain stable chemical bonds, making it highly adhesive even in humid environments. Strength, and the repaired breach will not be damaged under greater pressure; the bifunctional polymer can easily and efficiently trigger gelation through light, has time and space controllability, and is easy and flexible to use. The present invention can also adjust the grafting ratio of the two active units on the natural polymer as needed to realize and regulate the subsequent tissue bonding strength and the mechanical strength of the adhesive, so that the adhesive provided by the present invention is not only suitable for hard Brain (spinal) membrane repair, but also used in other tissue trauma, such as peritoneal and pleural repair, blood vessel damage repair, skin and mucous membrane adhesive repair, etc.

The photoinitiated biological tissue adhesive, gel sheet, preparation method and application proposed by the present invention will be further described in detail below with reference to the accompanying drawings and specific examples. The advantages and features of the present invention will become clearer from the following description.

Example 1

S1: Add 2.0g sodium carboxymethylcellulose to 200mL deionized water and stir at room temperature to obtain sodium carboxymethylcellulose solution;

S2: Add 1.38g of amidation reagent 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride to the sodium carboxymethylcellulose solution in step S1, Dissolve to obtain a uniform solution; add 114 mg dopamine hydrochloride and 100 mg methacrylic acid 2-amino hydrochloride to the above mixed solution, and stir in the dark until completely dissolved; use 0.5M NaOH solution to adjust the pH of the above solution to 6.5. Stir at room temperature for 24h to obtain a reaction solution;

S3: Precipitate the reaction solution obtained in step S2 in 800 mL of glacial ethanol, and centrifuge at low temperature to obtain the polymer; dissolve the polymer in 200 mL of deionized water again, precipitate in glacial ethanol again, and obtain the bifunctional polymer through low-temperature centrifugation and freeze-drying. ;

S4: Dissolve 45 mg of bifunctional polymer in 3 mL of deionized water to obtain a bifunctional polymer solution, add 6 mg of photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, shake or Stir and wait until it is completely dissolved to obtain a biological tissue adhesive; use a UV lamp with a wavelength of 365 nm to illuminate the obtained biological tissue adhesive for 2 seconds to complete the photo-crosslinking process and obtain a cured biological tissue adhesive.

1. Cytotoxicity test

The CCK-8 kit was selected to test the cytotoxicity of the obtained tissue adhesive. The CCK-8 kit is a fast, highly sensitive, non-radioactive assay based on WST-8 that is widely used in cell proliferation and cytotoxicity. Color detection kit. CCK-8 solution can be added directly to cell samples without pre-preparing various components. WST-8 can be reduced by some dehydrogenases in mitochondria to produce orange-yellow formazan in the presence of electronic coupling reagents. The more and faster the cells proliferate, the darker the color; the greater the cytotoxicity, the lighter the color. For the same cells, there is a linear relationship between the depth of color (the amount of formazan produced) and the number of cells. WST-8 is an upgraded replacement product of MTT. It has obvious advantages compared with MTT or other MTT similar products, such as XTT, MTS, etc. First, the formazan produced by the reduction of MTT by some dehydrogenases in the mitochondria is not water-soluble and requires a specific solvent to dissolve it; while the formazan produced by WST-8, XTT, and MTS are all water-soluble, which can save the need for subsequent steps. dissolution step. Second, the formazan produced by WST-8 is more soluble than the formazan produced by XTT and MTS. Third, WST-8 is more stable than XTT and MTS, making experimental results more reliable. Fourth, WST-8 has a wider linear range, higher sensitivity, and more stability than MTT, XTT, etc. WST-8 has no obvious toxicity to cells. After adding CCK-8 solution for color development, the plate can be read repeatedly with a microplate reader at different times, making the detection time more flexible and making it easier to determine the optimal detection time.

(1), Make standard curve

1. Inoculate 100 μl of BV2 (microglia) cell suspension (5000 cells/well) in a 96-well plate;

2. Dilute the medium into a cell concentration gradient in equal proportions in sequence. Generally, 5-7 cell concentration gradients are made, with 4-6 duplicate wells in each group;

3. After inoculation, incubate for 2-4 hours to allow the cells to adhere to the wall, then add 10 μL of CK-8 reagent to every 100 μL of culture medium and incubate for a certain period of time. Then measure the OD value and create a standard curve with the number of cells as the abscissa and the OD value as the ordinate. . The number of cells in unknown samples can be determined based on this standard curve. The prerequisite for using this standard curve is that the experimental conditions are completely consistent.

(2) Cell proliferation-toxicity experiment

1. Inoculate 100 μl of cell suspension (5000 cells/well) in a 96-well plate;

2. Place in advance in a 37°C 5% CO ₂ saturated humidity incubator for 24 hours;

3. Use 100 microliters of fresh culture medium to dilute the tissue adhesive to different volume concentrations (50%, 25%, 12.5%, 6.3%, 3.1%, 1.6%, 0.8%, 0.4%, 0.2%) and add In the culture plate containing cells, only microglia culture medium was added to the control group;

4. Cultivate in the incubator for 24 hours;

5. Dilute the CCK-8 reagent with culture medium to a concentration of 10% and add it to the well plate for 30 minutes;

6. Measure the absorbance value at a wavelength of 450nm.

Figure 1 shows the absorbance values of tissue adhesives with different volume concentrations and the blank control group. It can be seen from the figure that except for 50% of the absorbance values of tissue adhesives with different volume concentrations which are slightly smaller than the control group, the others are basically greater than The absorbance value of the control group shows that the synthesized biological tissue adhesive has no cytotoxicity; especially the absorbance values at volume concentrations of 25%, 12.5%, and 6.3% are much greater than the blank control group, indicating that the biological tissue adhesive is also effective against nerve glue. Plasma cells have the function of nourishing and promoting proliferation.

In addition, the BV2 cell line was cultured under a light microscope. The experimental group added diluted biological tissue adhesives with different volume concentrations. The control group did not add any. After culturing in the incubator for 24 hours, it was observed under a light microscope. Figure 8 shows the addition of diluted biological tissue adhesives. Electron microscopy photos of biological tissue adhesive with a volume concentration of 6.3% and the control group. The cell morphology of the experimental group and the control group grew well, and the morphology of the experimental group was more plump. Combined with CCK8 experimental data, it is proven that the hydrogel we designed and synthesized not only has no cytotoxicity, but also has a certain nourishing and proliferative effect on glial cells.

2. Whether it has cross-linking properties after photoinitiation

The rheological test of the biological tissue adhesive in Example 1 before and after photo-crosslinking was performed on a rotational rheometer (ThermoScientific, Mars 60). The rotation time scan mode was selected, the temperature was 37°C, and the rotation rate was set to 0.01/s, 0.1/s, 0.5/s, 1.0/s, 5.0/s and 10/s, scanning for 2 minutes at each rotation rate, and collecting 100 data points at each rotation rate. Apply the bifunctional polymer aqueous solution (1.5wt%) containing the photocrosslinking agent on the rheometer round table with a thickness greater than 1mm, and then the rheological behavior of the adhesive before photocrosslinking can be tested, as shown in Figure 2 shown. The polymer solution applied on the round table is cross-linked by light for about 30 seconds to achieve in-situ cross-linking, and the rheological behavior of the adhesive after cross-linking can be tested, as shown in Figure 3.

As shown in Figures 2 and 3, the loss modulus G" of the sample before cross-linking is greater than its storage modulus G', which means that the system is in a liquid flow state. After cross-linking, its storage modulus G' is greater than its loss modulus G", and can be maintained at a stable value, represents the formation of an elastic three-dimensional network, indicating that the bifunctional polymer and the initiator undergo a cross-linking reaction and solidification after photoinitiation. It also shows from the side that it has bonding strength after curing.

3. Use fish bladder and intestinal mucosa to prepare a simple biological model of cerebrospinal fluid leakage. As shown in Figure 4, the biological tissue adhesive of the embodiment is used to seal the leakage (an incision of about 1.5cm is made with a blade). After injecting normal saline, the liquid leaks from the leakage hole. There was obvious leakage and leakage from the mouth; then, the light-triggered adhesive in Example 1 was cross-linked and bonded for 15 seconds to repair the gap, and water was injected into the biological tissue model to simulate the cerebrospinal fluid filling effect, as shown in Figure 4, after complete water injection There is no liquid leakage or leakage from the leakage opening, and the repair effect is good. . Then, 15 seconds after the biological tissue adhesive seals the leak, no liquid seeps out or leaks out of the leak after complete water injection. Then, the ultimate burst pressure of 25-35cmH ₂ O is given by hand to simulate postoperative body changes and injections. In the case of a transient increase in intracranial pressure caused by sneezing, coughing, and vomiting, the adhesive expands as the tear breaks, and no saline leaks out, demonstrating good adhesion and mechanical elasticity, as shown in Figure 5 Show. Similarly, we chose a sealed cerebrospinal fluid biological tissue model constructed from pig intestinal mucosa to conduct the same experiment. The light-triggered adhesive was cross-linked and bonded for 15 seconds to repair the gap. Water was injected into the biological tissue model to simulate the cerebrospinal fluid filling effect. See Complete Water Injection There was no liquid leakage or leakage from the rear leak, and the repair effect was good. The biological effectiveness of the adhesive was verified from a variety of biological tissues, as shown in Figure 6. As further explained above, the adhesive strength and mechanical strength of the adhesive in this embodiment after light curing make it applicable to dura (spinal) membrane repair.

4. Animal experiments to evaluate the effectiveness and biocompatibility of biological tissue adhesives

A: Select a male SD rat weighing about 160g, take the right forehead (5mm to the right of the sagittal suture, 5mm in front of the coronal suture) to expose the skull, cut the dura mater and subarachnoid space, and simulate a craniotomy cerebrospinal fluid leakage model. An experimental group and a control group were established, with 3 rats in each group. After the cerebrospinal fluid leakage rat model was successfully constructed in the experimental group, 1 ml of the biological tissue adhesive of Example 1 was used to cover the wound. After the sealing was confirmed to be satisfactory, a light source was used to stimulate the adhesive fixation. Suture the wound; similarly to the control group, after the rat model of cerebrospinal fluid leakage is successfully constructed, 1 ml of physiological saline is used to cover the wound. After confirmation that the sealing is satisfactory, a light source is used to stimulate adhesion and fixation, and the wound is sutured.

B: After the experiment is successful, the rats in the experimental group and the control group will be raised for a total of 15 days, and then

(1) Material collection: Take the heads of the rats from the experimental group and the control group in the embedding frame.

(2) Dehydration and wax dipping: Take out the extracted tissue together with the embedding frame, put it into a dehydration basket, and dehydrate it in a dehydration machine using gradient alcohol: 75% alcohol 2h-85% alcohol 2h-90% alcohol 1.5h- 95% alcohol 2h - absolute ethanol I 2h - absolute ethanol II 2h - alcohol benzene 40min - xylene I 40min - xylene II 40min-65°; then dip into wax: melt paraffin I 0.5h-65° melt paraffin II 1h Melt paraffin III at -65° for 2h45min.

(3) Embedding: Embedding the wax-soaked tissue in an embedding machine. First put the melted wax into the embedding frame. Before the wax solidifies, take the tissue out of the dehydration box and put it into the embedding frame according to the requirements of the embedding surface and attach the corresponding label. Cool it on the -20° freezing table until the wax solidifies. Finally, remove the wax block from the embedding frame and trim the wax block.

(4) Slice: Take full-thickness vertical slices of skin, muscle, dura mater, arachnoid mater and cortical brain tissue along the exposed part of the experimental skull. Place the trimmed wax block into a paraffin microtome and slice it into 4 μm thick slices. Flatten the tissue in warm water at 40°C in a spreading machine, pick up the tissue with a glass slide, bake the slices in a 60°C oven, bake the dry wax in the water, take it out and store it at room temperature for later use.

(5) Staining: The classic hematoxylin-eosin staining method (HE) is used for staining, as shown in B in Figure 7. The control group and experimental group respectively show the complete structure of skin, muscle, dura mater, arachnoid membrane and cortical brain tissue. , focusing on observing cortical brain tissue and skin and muscle tissue under 10x and 20x microscopes. The results showed that there were no signs of inflammation, necrosis, hemorrhage, or abnormal proliferation in the skin, muscles, dura mater, arachnoid mater, and cortical brain tissue of the experimental group and the control group, and the biological adhesive was basically absorbed, and no residual or foreign matter was found. etc., suggesting that this bioadhesive has good biocompatibility and degradability.

C: After the experiment was successful, the rats in the experimental group and the control group were raised for a total of 15 days. The wounds of the two groups of rats were observed at the time points of 1, 3, 5, 7, 9, 11, 13, and 15 days. The experimental group and the control group were both No abnormality such as wound redness, swelling, or infection was found. A small amount of cerebrospinal fluid leakage occurred in the control group, but no cerebrospinal fluid leakage was seen in the experimental group. The weight changes of the two groups of rats were detected, as shown in C in Figure 7. There was no statistical difference in the weight curves of the experimental group and the control group, proving that the rats in the experimental group had good weight development and further proved that the biocompatibility was good.

Example 2

S1: Add 3.0g sodium carboxymethylcellulose to 200mL deionized water and stir at room temperature to obtain sodium carboxymethylcellulose solution;

S2: Add 2.76g of the amidation reagent bis(2-oxo-3-oxazolidinyl)phosphoryl chloride to the sodium carboxymethylcellulose solution in step S1, and dissolve to obtain a uniform mixed solution; add 200mg to the above mixed solution. Merepinephrine and 150 mg 3-buten-1-amine were stirred in the dark until completely dissolved; adjust the pH of the above solution to 7.0 with 0.5M NaOH solution and stir at room temperature for 24 hours to obtain a reaction solution;

S3: Precipitate the reaction solution obtained in step S2 in 800 mL of glacial ethanol, and centrifuge at low temperature to obtain the polymer; dissolve the polymer in 200 mL of deionized water again, precipitate in glacial ethanol again, and obtain the bifunctional polymer through low-temperature centrifugation and freeze-drying. ;

S4: Dissolve 45 mg of bifunctional polymer in 3 mL of deionized water to obtain a bifunctional polymer solution, and add 6 mg of photoinitiator 2,5-bis-[4-(diethylamino)-benzylidene]-cyclo pentanone, shake or stir to completely dissolve, and obtain a biological tissue adhesive; use a wavelength 800nm laser to illuminate the obtained biological tissue adhesive for 10 seconds to complete the photo-crosslinking process, and obtain a cured biological tissue adhesive.

Example 3

S1: Add 2.0g sodium hyaluronate to a mixed solvent of 120mL deionized water and 80mL N,N-dimethylformamide, and stir at room temperature to obtain a sodium hyaluronate solution;

S2: Add 0.69g of the amidation reagent N,N'-carbonyldiimidazole to the sodium hyaluronate solution in step S1, and dissolve to obtain a uniform mixed solution; add 57mg of 5-hydroxydopamine hydrochloride and 50mg to the above mixed solution. 4-Vinylbenzylamine, stir in the dark until completely dissolved; use 0.5M NaOH solution to adjust the pH of the above solution to 6.0, and stir at room temperature for 24 hours to obtain a reaction solution;

S3: Precipitate the reaction solution obtained in step S2 in 800 mL of glacial ethanol, and centrifuge at low temperature to obtain the polymer; dissolve the polymer in 200 mL of deionized water again, precipitate in glacial ethanol again, and obtain the bifunctional polymer through low-temperature centrifugation and freeze-drying. ;

S4: Dissolve 30 mg of bifunctional polymer in 3 mL of deionized water to obtain a bifunctional polymer solution, add 3 mg of photocross initiator phenyleosin Y, shake or stir to completely dissolve, and obtain a biological tissue adhesive; The obtained biological tissue adhesive is illuminated with a 530nm LED for 20 seconds to complete the photo-cross-linking process, and the cured biological tissue adhesive is obtained.

The embodiments of the present invention have been described in detail above with reference to the accompanying drawings, but the present invention is not limited to the above-mentioned embodiments. Even if various changes are made to the present invention, if these changes fall within the scope of the claims of the present invention and equivalent technologies, they will still fall within the protection scope of the present invention.

Claims (17)

1. A photoinitiated biological tissue adhesive, wherein the biological tissue adhesive is cured by irradiation of light from a bifunctional polymer and a photoinitiator;

the difunctional polymer is prepared by introducing catechol groups and double bond groups into a natural polymer containing carboxylic acid through amidation reaction.

2. The photoinitiated biological tissue adhesive according to claim 1, wherein the carboxylic acid-containing natural polymer is carboxymethyl cellulose, sodium carboxymethyl cellulose, hyaluronic acid, sodium hyaluronate, sodium alginate, carboxymethyl chitin, or carboxymethyl chitosan.

3. The photoinitiated biological tissue adhesive according to claim 1, wherein the bifunctional polymer is specifically obtained by condensation reaction of the carboxylic acid-containing natural polymer with an amino-containing catechol, an amino-containing alkene and an amidation condensing agent.

4. The photoinitiated biological tissue adhesive according to claim 3, wherein the mass ratio of the carboxylic acid-containing natural polymer, the amino-containing catechol, the amino-containing alkene, and the amidation condensing agent is 1:0.005-20:0.005-20:0.1-1000.

5. The method of preparing a photoinitiated biological tissue adhesive according to claim 3 or 4, wherein the amino group-containing catechol is selected from the group consisting of dopamine, 6-hydroxydopamine, 5-hydroxydopamine, 3, 4-dihydroxybenzylamine, norepinephrine hydrochloride, 3, 4-dihydroxynorephedrine, and cortex lycii radicis.

6. The method of preparing a photoinitiated biological tissue adhesive according to claim 3 or 4, wherein the amino group-containing alkene is selected from the group consisting of 2-amino methacrylate, 3-butene-1-amine, 2-methallylamine, methacrylamide, pent-4-en-1-amine, and 4-vinylbenzylamine.

7. The method of preparing a photoinitiated biological tissue adhesive according to claim 3 or 4, wherein the amidation condensing agent is selected from the group consisting of: 4- (4, 6-dimethoxy-triazin-2-yl) -4-methylmorpholine hydrochloride, active esters, carbodiimides, diisopropylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, etc.), onium salts, organic phosphorus, triphenylphosphine-polyhalomethane, triphenylphosphine-hexachloroacetone, triphenylphosphine-NBS, 3-acyl-2-thiothiazoline, or nitrotetrafluoroborate ion.

8. The photoinitiated biological tissue adhesive according to claim 1, wherein the photoinitiator is selected from the group consisting of: any one or more of phenyl (2, 4, 6-trimethyl benzoyl) lithium phosphate, aryl ketone photoinitiator, polycyclic aromatic hydrocarbon photoinitiator, polysilane photoinitiator, acyl phosphonate photoinitiator, azo photoinitiator or metal organic complex.

9. The photoinitiated biological tissue adhesive according to claim 1 or 8, wherein the mass ratio of the bifunctional polymer to the photoinitiator is 1:0.01-10.

10. A method of preparing a photoinitiated biological tissue adhesive according to any of claims 1 to 9, comprising the steps of:

s1: dissolving a natural polymer containing carboxylic acid in a solvent to prepare a natural polymer solution;

s2: adding an amidation reagent into the natural polymer solution prepared in the step S1, then adding catechol containing amino and olefin containing amino under the condition of avoiding light, and adopting NaOH solution to adjust the pH value of the solution to 5.5-7.5 to obtain a reaction solution;

s3: precipitating the reaction solution obtained in the step S2 in glacial ethanol, centrifuging at low temperature, and repeating the step for 2-3 times to obtain a difunctional polymer;

s4: dissolving the difunctional polymer obtained in the step S3, and adding a photoinitiator for dissolving to obtain the biological tissue adhesive;

under the irradiation of light, the biological tissue adhesive initiates the crosslinking reaction and solidification of the difunctional polymer and the photoinitiator.

11. The method of preparing a photoinitiated biological tissue adhesive according to claim 10, wherein the solvent in step S1 is one or a mixture of water, N-dimethylformamide or dimethyl sulfoxide.

12. The method of preparing a photoinitiated biological tissue adhesive according to claim 10 or 11, wherein the mass ratio of the carboxylic acid-containing natural polymer to the solvent is 1:0.1-1000.

13. The method for preparing a photoinitiated biological tissue adhesive according to claim 10, wherein the wavelength of light is 100-1000nm and the irradiation time is 1-600S when the crosslinking reaction is induced by the difunctional polymer and the photoinitiator.

14. A biological tissue gel sheet, characterized in that the biological tissue gel sheet is a photoinitiated biological tissue adhesive according to any one of claims 1 to 9 or a sheet-like product obtained after photoinitiated curing of the photoinitiated biological tissue adhesive obtained by the production method according to any one of claims 10 to 13.

15. Use of a photoinitiated biological tissue adhesive according to any of claims 1 to 9 or obtainable by a method according to any of claims 10 to 13 for the preparation of a bioadhesive product.

16. The use of a photoinitiated biological tissue adhesive according to claim 15 for the preparation of a bioadhesive product, wherein the photoinitiated biological tissue adhesive is applied for repair of dura mater (spinal) membranes, repair filling of peritoneum and pleura, repair of vascular damage, and adhesive repair of skin mucosa.

17. Use of a photoinitiated biological tissue adhesive according to claim 15 in the preparation of a bioadhesive product, wherein the bioadhesive product is a sheet-like product.