CN118576750A - A biosynthetic injectable adhesive and its preparation method and application in intestinal fistula repair - Google Patents

Abstract

The invention discloses a biosynthesis injectable adhesive, a preparation method thereof and application thereof in intestinal fistula repair. The biosynthetically injectable adhesive comprises a copolymer of 3-hydroxybutyric acid and medium-long chain unsaturated hydroxy fatty acid monomers, a dimercapto compound, a photoinitiator and a solvent, can be rapidly crosslinked with tissues in situ through ultraviolet irradiation, forms firm and tough liquid seal, combines the biological effect of promoting cell proliferation by the degradation product of the copolymer material of the 3-hydroxybutyric acid and the medium-long chain unsaturated hydroxy fatty acid monomers, and realizes effective plugging and repairing of the intestinal dynamic wet environment wound.

Description

A biosynthetic injectable adhesive and its preparation method and application in intestinal fistula repair

Technical Field

The invention relates to the technical field of biomedical materials, and in particular to a biosynthetic injectable adhesive and a preparation method thereof and application thereof in intestinal fistula repair.

Background Art

Intestinal fistula refers to the pathological passages between the intestines, between the intestines and other organs or outside the body, which causes the intestinal contents to flow out of the intestinal cavity, causing a series of pathological and physiological changes such as infection and organ dysfunction. The causes can be congenital developmental defects, or acquired inflammation, tumors, trauma and surgical complications. Among them, postoperative intestinal fistula is the most common, accounting for about 75% to 85% of all intestinal fistulas, and this type of intestinal fistula is often serious and complex. It is a common emergency in gastrointestinal surgery, with an incidence of 2%-40%. The condition is critical and the mortality rate can be as high as 36%. It is one of the main causes of death in gastrointestinal surgery patients. The early symptoms are hidden, but the extravasation of digestive fluids can cause serious adverse reactions. Once the condition progresses, it deteriorates rapidly. The treatment time window is short and the treatment is difficult, which is a major problem faced by gastrointestinal surgeons.

Fistula is a key factor in the occurrence of intestinal fistula. The focus of intestinal fistula treatment is to try to close the fistula. In clinical practice, interventional therapy is usually used for fistulas that are difficult to heal on their own. In the early stage, surgical suture is generally performed, and when severe edema and abdominal adhesions occur, diversion enterostomy or intestinal resection is required. The treatment of fistulas by surgical suture is not only time-consuming and laborious, but also inevitably causes secondary damage to tissue puncture, increasing the risk of postoperative infection. Compared with traditional sutures, biological adhesives have many advantages such as easy use, time-saving operation, and prevention of postoperative complications. However, due to the presence of highly corrosive digestive fluids in the intestines, as well as a large amount of dynamic fluid exchange and secretion, higher special requirements are placed on the corrosion resistance and adhesion properties of the repair materials, resulting in the application of adhesives being limited. At present, there is no ideal biomaterial that can achieve ideal closure and repair of intestinal fistulas.

An ideal bioadhesive should meet the following requirements: 1) good biocompatibility and non-toxicity; 2) form a strong bond with moist tissue/organs; 3) suitable mechanical properties, adapted to the modulus of the tissue; 4) sufficient mechanical flexibility to withstand repeated dynamic mechanical forces applied by the tissue; 5) acceptable swelling properties to minimize tissue compression; 6) biodegradability, at a rate compatible with tissue healing. According to different sources, the various medical bioadhesives that have been developed can be roughly divided into two categories: 1) Natural sources: Typical adhesives of this type include fibrin sealants and gelatin-based sealants. Due to their natural origin, they have excellent biocompatibility, can support cell growth, and can be completely absorbed by tissues; 2) Artificial chemical synthesis: Typical adhesives of this type include cyanoacrylate glue (CA glue), which is a single-component tissue sealant synthesized from acrylaldehyde-based resin. It adheres and binds to the target surface within 5-6 seconds after contact with alkaline substances (such as water, blood, human tissue or moisture), and undergoes an exothermic polymerization reaction within 60 seconds to form a strong film. It is easy to use and has rapid bonding properties, and is widely used in medicine, industry and household activities. Among them, cyanoacrylate glue is widely used in medicine, industry and household activities because of its convenience and rapid bonding properties. However, due to its chemical synthesis, it has inherent defects such as poor biocompatibility, difficulty in degradation and toxic degradation products, and is generally only used for the closure of dry wounds. Compared with cyanoacrylate glue, fibrin glue has the following advantages: 1) non-toxic and biocompatible; 2) support cell growth; 3) can be completely absorbed by tissues; 4) the coagulation and degradation time of fibrin glue can be controlled. Although fibrin sealants have many advantages mentioned above, their limitations cannot be ignored: 1) some people have allergic reactions to them; 2) antibodies against fibrinogen and thrombin are formed, leading to coagulopathy and bleeding; 3) spread of infectious diseases; 4) cause systemic embolism; 5) poor mechanical strength.

In summary, all the bioadhesives used in clinical practice have more or less defects, such as poor wet adhesion, inability to resist digestive juice corrosion, hard texture, allergic complications, and cannot meet all the requirements of the application process at the same time. Therefore, it is urgent to develop a bioadhesive that is easy to use, has high adhesion in wet state, and is resistant to digestive juice corrosion to achieve the closure and repair of intestinal fistula.

Summary of the invention

In view of the defects in the prior art, the present invention proposes a biosynthetic injectable adhesive and its preparation method and application in intestinal fistula repair. Specifically, the present invention utilizes the biological effects of the copolymer of 3-hydroxybutyric acid and medium-chain unsaturated hydroxy fatty acid monomers (P(3HB-co-3HA)) material for functional construction, and develops a biosynthetic injectable adhesive that is fast-curing, flexible, highly adhesive, resistant to digestive fluid corrosion, and has good biocompatibility and degradation properties. It is also convenient to combine with an endoscope for minimally invasive surgery, providing an innovative and efficient solution for intestinal fistula repair.

The present invention provides a biosynthetic injectable adhesive, which comprises component A, component B, component C, and component D;

The component A is a copolymer of 3-hydroxybutyric acid and medium- and long-chain unsaturated hydroxy fatty acid monomers;

The component B is a cross-linking agent, and the cross-linking agent is a dithiol compound;

The component C is a photoinitiator, and the photoinitiator is any one of 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone, phenyl-2,4,6-trimethylbenzoyl lithium phosphite, benzoin dimethyl ether, and diphenyl ether;

The component D is a solvent, and the solvent is any one of ethyl acetate, tert-butyl alcohol, tetrahydrofuran, N,N-dimethylformamide, and acetone.

Furthermore, the chain length of the medium-chain unsaturated hydroxy fatty acid monomer is any one of eight to thirteen carbons.

Preferably, the chain length is any one of eight carbons, eleven carbons, and thirteen carbons.

Furthermore, the bis-mercapto compound is any one of dithiothreitol and bis-mercapto polyethylene glycol, wherein the molecular weight of the bis-mercapto polyethylene glycol is any one of 200, 400, 1k, and 2k.

Preferably, the dithiol compound is dithiothreitol.

Furthermore, the molar percentage of the unsaturated hydroxy fatty acid monomer in component A is 10% to 99%, which can be 10%, 15%, 27%, 34%, 40%, 48%, 56%, 67%, 75%, 80%, 87%, 94%, 99%, and is preferably 40-70%.

Furthermore, the weight percentage of component A in the biosynthetic injectable adhesive is 10% to 80%, which can be 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and preferably 20%-50%.

Furthermore, the weight percentage of component B in the biosynthetic injectable adhesive is 1% to 20%, which can be 1%, 2%, 3%, 5%, 7%, 10%, 12%, 14%, 16%, 18%, 20%, preferably 1%-5%.

Furthermore, the weight percentage of component C in the biosynthetic injectable adhesive is 0.01% to 2%, which can be 0.01%, 0.03%, 0.05%, 0.1%, 0.2%, 0.3%, 0.5%, 0.7%, 1%, 1.2%, 1.5%, 2%, and preferably 0.05%-0.5%.

The present invention also provides a method for preparing the biosynthetic injectable adhesive, which comprises the following steps:

(1) dissolving the component A in the component D to obtain a PHA gel precursor solution;

(2) Adding the component B and the component C to the PHA gelling precursor solution of step (1) to obtain a biosynthetic injectable adhesive.

The invention also provides application of the biosynthetic injectable adhesive in preparing soft tissue damage repair materials.

Furthermore, the soft tissue injury repair is preferably intestinal fistula repair.

In summary, compared with the prior art, the present invention achieves the following technical effects:

1. Compared with traditional adhesives that have the defect of failing adhesion due to swelling caused by absorption of body fluids, the biosynthetic injectable adhesive of the present application is tightly adhered to the tissue after in-situ curing through photo-crosslinking, and forms a hydrophobic organic gel network, which can effectively resist excessive swelling caused by infiltration of body fluids, reduce the risk of compression of surrounding tissues and nerves, weakening of adhesion and even adhesive shedding, and ensure its long-term stability during application.

2. Compared with the mechanism of chemical covalent adhesion at the interface of traditional adhesives, the biosynthetic injectable adhesive of the present application achieves firm adhesion to substrates such as tissues through diffusion and topological entanglement mechanisms. This adhesion method, which does not rely on specific functional groups, has the potential to be applied on a variety of substrates and in different pH environments.

3. Compared with traditional physical topological adhesives, the biosynthetic injectable adhesive of the present application is in situ cured by ultraviolet light-induced thiol-ene click reaction, achieving short-term rapid and robust adhesion in a wet environment, and introducing a covalent cross-linking network based on physical entanglement, thereby solving the problem of traditional physical topological adhesives taking a long time to reach peak adhesion and insufficient adhesion.

4. Compared with traditional chemically synthesized adhesives, the biosynthetic injectable adhesive of the present application is prepared using unsaturated double bond P(3HB-co-3HA) material fermented by an engineered strain as raw material. Based on the inherent biological effects of the P(3HB-co-3HA) material, the biosynthetic injectable adhesive of the present application has excellent biosafety and degradation characteristics.

5. Compared with traditional biological patch-shaped adhesives, the biosynthetic injectable adhesive of the present application is not only limited to sealing the outermost layer of the intestine, but also plays a stable closing role on the muscular layer and mucosal layer, and can adapt to various irregular shaped wounds. It can be combined with an endoscope for intestinal minimally invasive surgery, providing a new strategy for the occlusion and repair of intestinal fistulas.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

FIG1 is a synthetic route diagram and a schematic diagram of the adhesion mechanism of a biosynthetic injectable adhesive proposed by the present invention;

FIG2 is a physical picture of the biosynthetic injectable adhesive adhering to various tissues in Example 5 of the present invention;

FIG3 is a fluorescent image of the biosynthetic injectable adhesive in Example 6 of the present invention penetrating fresh pig skin;

FIG4 is a graph showing the rheological properties of the biosynthetic injectable adhesive in Example 7 of the present invention;

FIG5 is a graph showing the anti-swelling and digestive fluid corrosion resistance of the biosynthetic injectable adhesive in Example 8 of the present invention;

FIG6 is a graph showing the lap shear strength of the biosynthetic injectable adhesive in Example 9 of the present invention in different pH media and at different times underwater;

FIG7 is a graph showing the interfacial adhesion capability of the biosynthetic injectable adhesive in Example 10 of the present invention;

FIG8 is a graph showing the in vitro sealing of the small intestine and the bursting pressure of the biosynthetic injectable adhesive in Example 11 of the present invention;

FIG9 is a cell compatibility characterization of the biosynthetic injectable adhesive in Example 12 of the present invention;

FIG10 is a graph showing the in vivo compatibility of the biosynthetic injectable adhesive in Example 13 of the present invention;

FIG. 11 is a diagram showing the application of the biosynthetic injectable adhesive in Example 14 of the present invention for repairing colon defects in living bodies.

DETAILED DESCRIPTION

In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

The present invention is a copolymer (P(3HB-co-3HA)) material of 3-hydroxybutyric acid and medium-long chain unsaturated hydroxy fatty acid monomers synthesized by biological fermentation of an engineering strain, and forms good adhesion with a variety of substrates by utilizing long chain diffusion and topological entanglement. The chain segment diffusion ability of the P(3HB-co-3HA) material is further enhanced in a good solvent, has good wetting and penetration ability to the tissue matrix, and can achieve rapid solidification of the precursor solution and wound sealing by generating a thiol-ene click reaction with a dithiol compound under ultraviolet light irradiation conditions.

The thiol-ene click reaction between the active double bonds and the thiol groups initiated by ultraviolet light is a free radical polymerization reaction, and its reaction rate is closely related to the amount of the dithiol compound used. When the cohesive energy of the adhesive is insufficient, it will also lead to final bonding failure. The size of the cohesive energy is closely related to the double bond ratio in the medium- and long-chain unsaturated hydroxy fatty acid monomer (3HA) and the amount of the dithiol compound used. By adjusting the double bond ratio and the dithiol compound feed ratio, precise control of the curing time and the final bonding strength can be achieved.

Since the medium-chain P (3HB-co-3HA) material with unsaturated double bonds synthesized by biological fermentation of engineered strains has good biocompatibility and biodegradability, its degradation product 3-hydroxybutyric acid is a weakly acidic small molecule with little irritation to cells, and can promote cell proliferation and reduce inflammation to a certain extent. After the adhesive effectively seals the wound, the degradation of the material provides space for the growth of new tissue and accelerates tissue healing.

In summary, the synthesis route and adhesion mechanism of the biosynthetic injectable adhesive are shown in Figure 1. The biosynthetic injectable adhesive can still diffuse into the tissue and undergo topological entanglement in the presence of water, and quickly solidify within 10 seconds under ultraviolet light irradiation, forming a firm seal and maintaining a long-term high-strength adhesion effect.

Biosynthetic injectable adhesive and method for preparing the same:

Ingredients include:

(1) Component A is a copolymer of 3-hydroxybutyric acid and medium-chain unsaturated hydroxy fatty acid monomers (P(3HB-co-3HA));

(2) Component B is a crosslinking agent/curing agent;

(3) Component C is a photoinitiator;

(4) Component D is a solvent.

Specifically, the chain length of the medium-chain unsaturated hydroxy fatty acid monomer (3HA) is any one of eight carbons to thirteen carbons. Preferably, the chain length of 3HA is any one of eight carbons (C8 ⁼ ), eleven carbons (C11 ⁼ ) and thirteen carbons (C13 ⁼ ), and the monomer can provide a reactive terminal double bond.

The active terminal double bond ratio of P(3HB-co-3HAs) in the component A can be controllably adjusted by adjusting the ratio of different fermentation substrates, and the molar percentage (3HA mol%) of the medium-chain unsaturated monomer 3HA, i.e., the active terminal double bond ratio, is between 10% and 99%.

Preferably, when the medium-chain unsaturated monomer 3HA is eleven carbon (C11 ⁼ ) and the 3HA mol% is 33%-99%, it is amorphous and sticky at room temperature, can form good adhesion with a variety of substrates through long-chain diffusion and topological entanglement, and provides sufficient active double bond sites for subsequent chemical modification.

Specifically, B in the component is a crosslinker/curing agent in the system, the crosslinker/curing agent is a bisthiol compound, and the bisthiol compound is any one of dithiothreitol (DTT) and bisthiol polyethylene glycol (SH-(PEG) _n -SH). The molecular weight of the bisthiol polyethylene glycol is any one of 200, 400, 1k, and 2k.

Preferably, the component B is DTT.

Specifically, the component C is a photoinitiator, and the photoinitiator is any one of 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone, phenyl-2,4,6-trimethylbenzoyl lithium phosphite, benzoin dimethyl ether, and diphenyl ether.

Specifically, the component D is a solvent, and the solvent is any one of ethyl acetate, tert-butyl alcohol, tetrahydrofuran, N,N-dimethylformamide, and acetone.

Preferably, the component D is ethyl acetate and tert-butyl alcohol, which are green solvents in the pharmaceutical manufacturing industry.

The specific preparation method comprises the following steps:

(1) dissolving component A in component D to obtain a PHA gel precursor solution;

(2) adding a certain mass fraction of component B and component C to the above-mentioned precursor solution to form the biosynthetic injectable adhesive;

Preferably, the method is as follows:

Component A 3HA mol% C11 ⁼ 40% was dissolved in component D ethyl acetate as a PHA gel precursor, and a crosslinker DTT and a photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone were added thereto to form the biosynthetic injectable adhesive. The subsequent examples used the biosynthetic injectable adhesive prepared by the preferred method (Example 1) to carry out performance verification experiments.

In any embodiment of the present application, the molar percentage of the unsaturated hydroxy fatty acid monomer in component A is 10% to 99%, preferably 40% to 70%.

In any embodiment of the present application, the weight percentage of component A in the biosynthetic injectable adhesive is 10% to 80%, preferably 20% to 50%.

In any embodiment of the present application, the weight percentage of component B in the biosynthetic injectable adhesive is 1% to 20%, preferably 1% to 5%.

In any embodiment of the present application, the weight percentage of the component C in the biosynthetic injectable adhesive is 0.01% to 2%, preferably 0.05% to 0.5%.

Example 1

A method for preparing a biosynthetic injectable adhesive comprises the following steps:

Component A 3HA mol% C11 ⁼ 40% (500 mg) was dissolved in component D ethyl acetate (1 mL) as a PHA gel precursor, and crosslinker DTT (10 mg) and photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone (5 mg) were added thereto to form the biosynthetic injectable adhesive.

Among them, the molar percentage of unsaturated hydroxy fatty acid monomer in component A is 40%, the weight percentage of component A in the biosynthetic injectable adhesive is 50%, the weight percentage of component B in the biosynthetic injectable adhesive is 1%, and the weight percentage of component C in the biosynthetic injectable adhesive is 0.5%.

Example 2

A method for preparing a biosynthetic injectable adhesive comprises the following steps:

Component A 3HA mol% C8＝ (800 mg) with 99% mol% was dissolved in component D ethyl acetate (1 mL) as a PHA gel precursor, and 200 mg of a crosslinking agent (bis-mercapto polyethylene glycol with a molecular weight of 2000) and a photoinitiator benzoin dimethyl ether (20 mg) were added thereto to form the biosynthetic injectable adhesive.

Among them, the molar percentage of unsaturated hydroxy fatty acid monomer in component A is 99%, the weight percentage of component A in the biosynthetic injectable adhesive is 80%, the weight percentage of component B in the biosynthetic injectable adhesive is 20%, and the weight percentage of component C in the biosynthetic injectable adhesive is 2%.

Example 3

A method for preparing a biosynthetic injectable adhesive comprises the following steps:

Component A 3HA mol% C13＝ (100 mg) with 10% mol% was dissolved in component D tert-butyl alcohol (1 mL) as a PHA gel precursor, and 50 mg of a cross-linking agent (bis-mercapto polyethylene glycol with a molecular weight of 400) and a photoinitiator phenyl-2,4,6-trimethylbenzoyl lithium phosphite (0.1 mg) were added thereto to form the biosynthetic injectable adhesive.

Among them, the molar percentage of unsaturated hydroxy fatty acid monomer in component A is 10%, the weight percentage of component A in the biosynthetic injectable adhesive is 10%, the weight percentage of component B in the biosynthetic injectable adhesive is 5%, and the weight percentage of component C in the biosynthetic injectable adhesive is 0.01%.

Example 4 Application of biosynthetic injectable adhesive

The biosynthetic injectable adhesive described in the present application can be used for bonding and sealing soft tissues such as skin and intestines. The specific method is: injecting the photoinduced biosynthetic injectable adhesive into the tissue wound site, and irradiating it under 365nm ultraviolet light for 5 seconds to 1 minute to form a gel to achieve wound sealing.

Example 5 Qualitative Characterization of Biosynthetic Injectable Adhesion to Multiple Tissues

The biosynthetic injectable adhesive in the present application can be injected between the tissue surface and a glass slide coated with gelatin (20% (w/w)) (simulating skin and facilitating light transmission), and form a strong bond between the two substrates within 1 minute of ultraviolet light irradiation. As shown in Figure 2, the biosynthetic injectable adhesive exhibits good adhesion properties to heart, liver, spleen, lung, large intestine and small intestine tissues.

Example 6 Characterization of the penetration ability of biosynthetic injectable adhesive into fresh pig skin

To test the penetration ability of the biosynthetic injectable adhesive of the present invention into tissues. Since the mechanical and biological properties of wet pigskin are similar to those of human skin, wet pigskin is selected as a model tissue. The injectable adhesive precursor solution (CLC11 ⁼ ) containing 6-aminofluorescein (6-AF, excitation wavelength 490nm) is placed on the surface of fresh pigskin (2×2cm ² ). At specific time points, the adhesive-tissue assembly is imaged using a confocal laser scanning microscope to measure the penetration depth of the adhesive over time. Commercial cyanoacrylate glue (CA glue) is set as a control group. As shown in Figure 3, the penetration depth of the biosynthetic injectable adhesive in pigskin increases with time, reaching about 800μm in 5 minutes, which far exceeds the penetration depth of commercial CA glue, breaking through the limitations of CA glue in wet bonding (rapid anionic polymerization triggered by water leads to low permeability). The positive correlation between the above penetration depth and penetration time confirms that the biosynthetic injectable adhesive has good penetration ability into tissue substrates and can form a topological entanglement network with tissues.

Example 7 Rheological Properties of Biosynthetic Injectable Adhesives

To test the injectability, gelation time and structural stability of the biosynthetic injectable adhesive in this application. All rheological measurements were performed on a rheometer (MCR 301, Anton Paar) using a plate with a diameter of 8 mm. Viscosity measurements were performed under a fixed constant strain of 1%, with a shear rate range of 0.01-100 s ^-1 ; oscillation time sweep measurements were performed under a fixed strain of 1.0% and a frequency of 1 Hz; frequency sweep measurements were performed under a fixed constant strain of 1%, with a frequency range of 0.1-10 Hz; amplitude sweep measurements were performed under a fixed frequency of 1 Hz, with a strain range of 0.1-1000%; all test temperatures were maintained at 37°C, and the normal stress F _N was 0 N. As shown in FIG4a , the injectable adhesive exhibits a “shear thinning” phenomenon, has good injectability, is convenient for contact with various irregular tissue substrates and quickly wets and penetrates; as shown in FIG4b , the injectable adhesive can undergo photocrosslinking within 5 seconds under ultraviolet light irradiation conditions, has the ability to cure rapidly, and can quickly cure and form a firm seal after contacting tissue under light; as shown in FIG4c , after the injectable adhesive is cured into a gel, its modulus remains fairly constant within the frequency range of 0.1-10 Hz (this interval includes and exceeds the range of activity of the small intestine), demonstrating the viscoelasticity and long-term network stability of the biosynthetic injectable adhesive after gelation; and this structural stability is further demonstrated in FIG4d , where the data show that the biosynthetic injectable adhesive has a wide linear viscoelastic region, and the yield network fracture strain exceeds 700%, all of which indicate that the biosynthetic injectable adhesive can withstand large deformations/strains and is “fracture-resistant” within the desired application range (anastomosis surgery and postoperative joint movement in vivo).

Example 8 Characterization of the anti-swelling and digestive fluid corrosion resistance of biosynthetic injectable adhesive

In this embodiment, the biosynthetic injectable adhesive after gelation is placed in centrifuge tubes containing phosphate buffered saline (PBS) and artificial simulated intestinal fluid (SIF), and placed in a shaker for incubation (37°C, 300 rpm). The sample is taken out at a specific time point, and the excess water is absorbed from the surface and then weighed and photographed for record. The swelling ratio (%) is defined as Swelling Ratio (%) = (m ₁ -m ₀ /m ₀ ) × 100%, m ₁ is the mass of the sample after swelling at a given time point, and m ₀ is the initial mass of the sample. As shown in Figure 5, after the gelled biosynthetic injectable adhesive was soaked in PBS for 72 hours, the surface morphology shrank slightly and turned white, while the mass remained constant during the soaking process, indicating that the biosynthetic injectable adhesive of the present application has excellent anti-swelling properties; the gelled biosynthetic injectable adhesive only swelled slightly when soaked in SIF, and reached swelling equilibrium within 4 hours (swelling rate was 8.31%). Scanning electron microscopy observation of its cross-section showed that the interior of the gelled biosynthetic injectable adhesive was originally a dense gel network, but a small amount of pore structure appeared after soaking in SIF. These experimental phenomena indicate that the biosynthetic injectable adhesive of the present application has good resistance to corrosion by digestive fluid.

Example 9 Characterization of lap shear strength of biosynthetic injectable adhesive in different pH media and under water for different time periods

In order to test the adaptability of different pH medium environments and underwater long-term adhesion stability of the biosynthetic injectable adhesive in this application. In this embodiment, the biosynthetic injectable adhesive was tested according to the revised ASTM F2255-05 tissue adhesive lap shear strength standard. Two slides (25mm×75mm) were used to store the sample, and a gelatin solution (20% w/w) was applied to the top of the slide (10mm×15mm) and dried at room temperature to obtain a slide coated with gelatin (to simulate the skin and facilitate light penetration). The biosynthetic injectable adhesive precursor CLC11 ⁼ (20μL) was added to the gelatin coating area of the two slides and irradiated under ultraviolet light for 5s for photocrosslinking. The two slides that had been photocrosslinked were immersed in water and different pH (pH=3, 7, 9 and 11) medium liquids, taken out at specific time points, and lap shear tests were performed using a universal mechanical testing machine, with a strain rate of 1mm/min. Commercial cyanoacrylate glue (CA glue) was set as a control group. The shear strength of the biosynthetic injectable adhesive measured at the separation point is shown in Figure 6. After immersion in water or in different pH media for 24 hours, the lap shear strength of the biosynthetic injectable adhesive of the present application decreased to a certain extent, but still maintained a relatively high strength (about 400 kPa). This value is significantly higher than that of commercial CA glue (about 5 kPa), demonstrating its advantages over CA glue in various wet environments, indicating that the biosynthetic injectable adhesive of the present application has the potential to be used in wet environments of different pH in vivo.

Example 10 Characterization of 90° Peel Strength of Biosynthetic Injectable Adhesive

In order to test the interfacial adhesion energy of the biosynthetic injectable adhesive on intestinal tissue in this application. In this embodiment, the biosynthetic injectable adhesive uses a texture analyzer to determine its 90° peel strength on pig intestine. The standard size of the pig intestine is 60mm×15mm. The injectable adhesive precursor CLC11 ⁼ (50μL) was injected onto the outer surface of the pig intestine and fixed on a gelatin-coated slide. After incubation in a humid environment for 3 minutes, the sample was irradiated with ultraviolet light for 5s to complete the sample preparation. The peel test was carried out at a speed of 10mm/min. Commercial cyanoacrylate glue (CA glue) was set as the control group. According to the elastic peeling theory, the interfacial adhesion energy is determined by G=F/d, where F is the average value of the peeling force and d is the width of the adhesion area. (n≥30). As shown in FIG7 , the energy of the biosynthetic injectable adhesive when peeled off from the outer surface of pig intestine reaches 406.78 J/m ² , which is higher than the peeling energy of commercial CA glue (about 128.45 J/m ² ), indicating that the biosynthetic injectable adhesive of the present application has excellent interfacial adhesion ability to intestinal tissue.

Example 11 In vitro sealing of the small intestine and characterization of burst pressure of biosynthetic injectable adhesive

In order to test the viscosity and stability of the biosynthetic injectable adhesive in sealing intestinal tissue under pressure in this application. The bursting pressure of the biosynthetic injectable adhesive in this embodiment is tested according to the revised ASTM F2392-04 standard. A section of pig intestine (about 8 cm) and a pressure gauge are connected to a syringe pump through a plastic tube, and a defect with a diameter of 4 mm is made on the surface of the pig intestine using a puncher. 50 μL of the precursor liquid CLC11 ⁼ of the biosynthetic injectable adhesive is injected into the defect site, and the tissue is sealed after ultraviolet light irradiation for 5 seconds. During the test, air is compressed into the pig intestine at a rate of 0.1 mL/s through a syringe, and the peak pressure recorded after rupture is the bursting pressure (n=3). Commercial cyanoacrylate glue (CA glue) was set as the control group. As shown in Figure 8, the burst pressure of the biosynthetic injectable adhesive on the pig intestine is about 158.67 mmHg, while the burst pressure of the commercial CA glue is about 23 mmHg, which is almost consistent with the maximum pressure of the healthy intestine (about 20 mmHg); further, the adhesion stability of the biosynthetic injectable adhesive was tested by simulating the intestinal humid environment in vitro sealed intestinal perforation experiment, a hole punch was used to make a 5 mm diameter defect on the surface of the pig intestine, and 100 μL of the biosynthetic injectable adhesive precursor CLC11 ⁼ was injected into the defect site, and the tissue was sealed by ultraviolet light irradiation for 30 seconds. Then the closed pig intestine was immersed in PBS buffer and filled with artificial simulated intestinal fluid SIF (pH = 6.8, in order to obtain better visual effects, the red dye Alizarin red was used to dye SIF). As shown in Figure 8, the biosynthetic injectable adhesive can achieve a firm seal of intestinal perforation for 48 hours under conditions of simulated intestinal humid environment, while the commercial CA glue leaked after 4 hours. These data indicate that the biosynthetic injectable adhesive of the present application has excellent sealing ability and long-term stability for intestinal tissue under pressure.

Example 12 Characterization of Cytocompatibility of Biosynthetic Injectable Adhesive

In this embodiment, intestinal epithelial cells IECs were used as model cells to evaluate the cytotoxicity of biosynthetic injectable adhesives. The biosynthetic injectable adhesive precursor CLC11 of the present application ^was evenly coated on the bottom of the cell culture plate after sterilization, and cured under ultraviolet light for 30s to obtain a biosynthetic injectable adhesive coating of about 300 μm thick, and the cells were inoculated on the coating for culture (inoculation density was 3000/well), and directly inoculated on a blank plate as a control. After incubation in a carbon dioxide incubator for 72h, cell viability was tested using live/dead cell fluorescence staining. The live/dead cell staining photo shows that as shown in Figure 9, in the presence of biosynthetic injectable adhesives and in the absence of biosynthetic injectable adhesives, the presence of dead cells was almost not observed. Further, the cell proliferation was tested and counted by the CCK-8 cell viability detection kit, and it was found that the survival rate of IECs cells in the presence of biosynthetic injectable adhesives was higher than 100% (as shown in Figure 9), indicating that the biosynthetic injectable adhesive has good biocompatibility and no obvious cytotoxicity.

Example 13 In vivo compatibility characterization of biosynthetic injectable adhesive

In order to demonstrate the in vivo compatibility of the biosynthetic injectable adhesive of the present application, an in vivo implantation experiment of an animal model was performed in this embodiment, and the results are shown in Figure 10. As shown in Figure 10a, rats were used as experimental subjects to conduct an inflammatory response test of subcutaneous biosynthetic injectable adhesive implantation on the back. After 1 week and 4 weeks of implantation, the full-thickness skin tissue and organ tissue around the implanted sample were collected for pathological analysis, and blood analysis was performed by cardiac puncture of rats to evaluate the safety of the in vivo metabolism of the biosynthetic injectable adhesive. As shown in the HE staining results in Figure 10c, the sample did not cause an inflammatory response in the surrounding skin tissue after implantation, and no obvious inflammatory lesions or signs of damage were observed in key organ tissues. As shown in the blood analysis results in Figure 10b, the blood analysis results of animals with subcutaneous implanted samples were comparable to those of healthy animals, indicating that the biosynthetic injectable adhesive of the present application has excellent biocompatibility.

Example 14 Application of biosynthetic injectable adhesive for repairing colon defects in vivo

In order to demonstrate the effect of the biosynthetic injectable adhesive of the present application on intestinal perforation repair in vivo, the experimental results of the animal colon defect model in this embodiment are shown in Figure 11. As shown in Figure 11a, an intestinal perforation model (diameter 5mm) was established in the colon of rats, and sutures, commercial cyanoacrylate glue (CA glue), and biosynthetic injectable adhesive were used to seal and repair the perforation site, and the colon perforation repair was observed fourteen days after the operation. The experimental results are shown in Figure 11a. There was no leakage of intestinal contents and infection in rats in each group. Among them, the commercial CA glue group had severe tissue adhesion and tissue sclerosis at the defect site; in contrast, there was only a small amount of tissue adhesion in the suture group and the biosynthetic injectable adhesive group, but the colon wall edema was obvious and the colon tube was dilated in the suture group.

The repair effect of the biosynthetic injectable adhesive was evaluated by pathological staining of the colon tissue at the experimental site. The experimental results are shown in Figure 11b. The defect site of the commercial CA glue group was structurally disordered and healed slowly; the mucosa and muscular layer at the defect site of the suture group were significantly changed and poorly arranged, the mucosal glandular tissue was missing, and there was a hematoma under the mucosa (related to acupuncture); in contrast, the defect site where the biosynthetic injectable adhesive was applied was basically filled with muscular mucosal tissue, and although there was no surface mucosal glandular tissue, complete closure was achieved. In addition, PAI-1 and PA immunohistochemistry were used to evaluate the degree of colon tissue fibrosis, and the results are shown in Figure 11c. PAI-1 is a serine protease inhibitor induced by TGF-β1, which promotes intestinal fibrosis and myofibroblast gene expression; PA stimulates fibrinolytic intestinal fibrosis by activating plasminogen reduction. According to the staining results, compared with the suture group and the commercial CA glue group, the expression of PAI-1 in the biosynthetic injectable adhesive group of the present application was significantly reduced and the expression of PA was significantly increased. Compared with sutures and commercial CA glue, the biosynthetic injectable adhesive of the present application exhibits good sealing ability, biocompatibility and a certain ability to prevent postoperative adhesion, which indicates that the biosynthetic injectable adhesive of the present invention has the potential to effectively block and repair intestinal fistulas.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A biosynthetic injectable adhesive, characterized in that the biosynthetic injectable adhesive comprises component A, component B, component C, and component D; The component A is a copolymer of 3-hydroxybutyric acid and medium- and long-chain unsaturated hydroxy fatty acid monomers; The component B is a cross-linking agent, and the cross-linking agent is a dithiol compound; The component C is a photoinitiator, and the photoinitiator is any one of 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone, phenyl-2,4,6-trimethylbenzoyl lithium phosphite, benzoin dimethyl ether, and diphenyl ether; The component D is a solvent, and the solvent is any one of ethyl acetate, tert-butyl alcohol, tetrahydrofuran, N,N-dimethylformamide, and acetone. 2. The biosynthetic injectable adhesive according to claim 1, characterized in that the chain length of the medium- and long-chain unsaturated hydroxy fatty acid monomer is any one of eight to thirteen carbons. 3. The biosynthetic injectable adhesive according to claim 1, characterized in that the bis-thiol compound is any one of dithiothreitol and bis-thiol polyethylene glycol, wherein the molecular weight of the bis-thiol polyethylene glycol is any one of 200, 400, 1k, and 2k. 4 . The biosynthetic injectable adhesive according to claim 2 , wherein the molar percentage of the unsaturated hydroxy fatty acid monomer in component A is 10%-99%. 5 . The biosynthetic injectable adhesive according to claim 1 , wherein the weight percentage of component A in the biosynthetic injectable adhesive is 10% to 80%. 6 . The biosynthetic injectable adhesive according to claim 1 , wherein the weight percentage of component B in the biosynthetic injectable adhesive is 1%-20%. 7 . The biosynthetic injectable adhesive according to claim 1 , wherein the weight percentage of component C in the biosynthetic injectable adhesive is 0.01%-2%. 8. The method for preparing the biosynthetic injectable adhesive according to claim 1, characterized in that the method comprises the following steps: (1) dissolving the component A in the component D to obtain a PHA gel precursor solution; (2) Adding the component B and the component C to the PHA gelling precursor solution of step (1) to obtain a biosynthetic injectable adhesive. 9. Use of the biosynthetic injectable adhesive according to any one of claims 1 to 7 in the preparation of soft tissue damage repair materials. 10. The use according to claim 9, characterized in that the soft tissue injury repair is intestinal fistula repair.