Two-component in-situ adhesive based on supercharged protein, and preparation method therefor and use thereof

Abstract

Provided in the present application are a two-component in-situ adhesive based on a supercharged protein, and a preparation method therefor and the use thereof. The two-component in-situ adhesive of the present application can be gelated in situ and rapidly at a tissue wound, exhibiting excellent intensity of adhesion between tissues, and can also exhibit an excellent adhesion performance even on a wet tissue surface; and after gelation, the gelated surface thereof is soft, and can be used for achieving the effects of hemostasis, wound repair, etc. of wet wounds, such as revascularization occlusion-assisted hemostasis and craniocerebral suture site-assisted suturing. In addition, the adhesive of the present application also has the advantages of simple and rapid preparation, no need for special devices, and fast adhesion and biocompatibility, etc., can be used as part of a new generation of portable instant biomedical adhesives, and has great application prospects in cases such as acute wound bleeding and complex wound surface treatment.

Description

A two-component in-situ adhesive based on supercharged protein and its preparation method and use

[0001] Cross-references

[0002] This application claims priority to the Chinese patent application filed on July 5, 2023, with application number 202310822652.2, and invention name “A two-component in-situ adhesive based on supercharged protein, its preparation method and use”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present application relates to the field of biomaterial technology, and in particular to a two-component in-situ adhesive based on supercharged proteins, and a preparation method and use thereof. Background Art

[0004] Tissue adhesives are a class of biomaterials that can interact with tissues in the body, thereby inducing adhesion between tissues or between tissues and non-tissues (such as implants), and have functions such as controlling bleeding (hemostasis) and preventing the flow of gases and liquids (sealing). With the advancement of modern medicine, surgical procedures must not only maximize the restoration of function and appearance, but also minimize patient pain. Currently, sutures or rivets are often used to physically close and repair tissues in surgeries involving larger defects or bleeding points. However, these methods have not been very effective for the following reasons: Sutures and rivets, beyond simply physically closing the wound, do not provide additional benefits such as wound healing and anti-inflammatory properties; sutures and rivets may cause damage to surrounding tissues and bleeding during surgery; and scarring may occur after wound healing, leading to the additional pain of suture removal. Medical tissue adhesives can effectively address these issues. Furthermore, the rapid hemostasis and wound-sealing capabilities of tissue adhesives are particularly suitable for immediate treatment of difficult-to-suture wounds in wartime environments, even for internal wounds that are difficult to treat with hemostatic bandages. Furthermore, while quickly sealing wounds, tissue adhesives also prevent further contact with the outside world, thereby preventing infection. It is reported that a large proportion of battlefield deaths in war occur between injury and arrival at a field hospital. The use of highly effective tissue adhesives incorporating hemostatic and analgesic agents could save many more lives. Furthermore, with the advancement of clinical medicine, minimally invasive techniques have become widely used across clinical disciplines. However, due to the small incisions, limited field of view, and operational flexibility of minimally invasive surgery, traditional mechanical tissue fixation techniques are difficult to implement. Therefore, tissue adhesives hold great promise for broad application in surgery, plastic surgery, and battlefield rescue.

[0005] However, achieving ideal tissue interface adhesion is very challenging for two main reasons: on the one hand, the presence of a large amount of liquid on the tissue surface makes it impossible for a large amount of adhesive to break through the hydration layer and generate effective intermolecular forces at the tissue interface, thereby greatly reducing the adhesion performance of the adhesive; on the other hand, the soft nature of the tissue leads to extremely high compliance requirements for the adhesive, that is, the adhesive is required to adaptively deform and adjust with tissue activities. Biocompatibility is also a point that needs to be paid great attention. Therefore, this places extremely high demands on the adhesion properties and composition of the adhesive, greatly increasing the difficulty of developing ultra-strong tissue adhesives.

[0006] Based on the above research status, the development of transient in situ adhesives based on bioengineered proteins is of great significance for basic scientific research and biomedical applications.

[0007] Summary of the Invention

[0008] Purpose of the Invention

[0009] The purpose of the present application is to provide a two-component instant in-situ adhesive based on bioengineering protein with excellent bonding performance, as well as a preparation method and use thereof.

[0010] Solution

[0011] To achieve the above objectives, this application provides the following technical solutions:

[0012] In a first aspect, the present application provides a two-component in-situ adhesive based on a supercharged protein, comprising:

[0013] (1) Elastin-like proteins rich in amino functional groups, and

[0014] (2) Compounds containing a succinimide group.

[0015] Preferably, the amino-functional group-rich elastin-like protein comprises n [(VPGKG)9] repeating units, and every two [(VPGKG)9] repeating units are separated by a spacer (VPGXG); wherein n is an integer between 3 and 18, and X is any natural amino acid except proline; and / or, the succinimide-containing compound is any one or more selected from the following: disuccinimidyl succinate polyethylene glycol (SS-PEG-SS), succinimidyl acetate, and disulfide disuccinimidyl propionate.

[0016] Further preferably, in the amino-functional elastin-like protein, the number n of repeating units [(VPGKG)9] is an integer between 8 and 18, and / or in the spacer (VPGXG), X is valine, arginine, leucine, isoleucine or alanine; and / or the succinimide-containing compound is disuccinimidyl succinate polyethylene glycol (SS-PEG-SS).

[0017] In a specific embodiment, the amino-functional elastin-like protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5; and / or the succinimide-containing compound is disuccinimidyl succinate polyethylene glycol (SS-PEG-SS), wherein the average relative molecular weight of the polyethylene glycol is between 2000 and 10000.

[0018] In a preferred embodiment, the amino-functional elastin-like protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5; and / or the succinimide-containing compound is disuccinimidyl succinate polyethylene glycol (SS-PEG-SS), wherein the average relative molecular weight of the polyethylene glycol is between 5,000 and 10,000.

[0019] Among them, the underlined part is the repeating unit [(VPGKG)9], and the bold part is the spacer (VPGXG);

[0020] Among them, the underlined part is the repeating unit [(VPGKG)9], and the bold part is the spacer (VPGXG);

[0021] Among them, the underlined part is the repeating unit [(VPGKG)9], and the bold part is the spacer (VPGXG);

[0022] The underlined part is the repeating unit [(VPGKG)9], and the bold part is the spacer (VPGXG).

[0023] The underlined part is the repeating unit [(VPGKG)9], and the bold part is the spacer (VPGXG).

[0024] In a specific embodiment, the amino-functional group-rich elastin-like protein exists alone in the form of an aqueous solution, and the succinimide-containing compound exists in the form of a solid powder. When used, the succinimide-containing compound is dissolved in ultrapure water and then mixed with the elastin-like protein aqueous solution, and then gelled to form an in situ adhesion layer.

[0025] Preferably, the mass volume concentration of the aqueous solution of the amino functional group-rich elastin-like protein is 50 to 650 mg / mL, preferably between 200 and 400 mg / mL, more preferably 200 mg / mL;

[0026] Preferably, when used, the succinimide-containing compound is prepared into an aqueous solution with a mass volume concentration of 0.5 to 1.5 g / mL, preferably 0.5 to 1.0 g / mL, and most preferably 0.75 g / mL;

[0027] Preferably, when used, the aqueous solution of the succinimide-containing compound and the aqueous solution of the amino-functional group-rich elastin-like protein are mixed in a volume ratio of 1: (0.5-2.0), preferably 1:1;

[0028] Preferably, after the two solutions are mixed, the gelation time is 0 to 120 seconds.

[0029] In a second aspect, the present application provides a method for preparing the two-component in-situ adhesive as described in the first aspect, comprising the following steps:

[0030] 1) mixing the amino functional group-rich elastin-like protein with water to prepare an aqueous solution of the amino functional group-rich elastin-like protein;

[0031] 2) dissolving the succinimide-containing compound in water to prepare an aqueous solution of the succinimide-containing compound;

[0032] And optionally, 3) the aqueous solution of the amino-functional group-rich elastin-like protein prepared in step 1) and the aqueous solution of the succinimide-containing compound prepared in step 2) are respectively encapsulated into a two-component syringe.

[0033] Preferably, the mass volume concentration of the aqueous solution of the amino functional group-rich elastin-like protein prepared in step 1) is 50 to 650 mg / mL, preferably between 200 and 400 mg / mL, more preferably 200 mg / mL;

[0034] Preferably, the mass volume concentration of the aqueous solution of the succinimide-containing compound prepared in step 2) is 0.5-1.5 g / mL, preferably 0.5-1.0 g / mL, and most preferably 0.75 g / mL.

[0035] In a third aspect, the present application provides use of the two-component in-situ adhesive as described in the first aspect above in the preparation of a medicament for hemostasis, repair and / or regeneration of tissue or organ wounds;

[0036] Preferably, the tissue is at least one of meniscus tissue, muscle tissue, tendon tissue, bone tissue, connective tissue, epidermal tissue or mucosal tissue;

[0037] Preferably, the organ is at least one of skin, heart, liver, kidney, brain or blood vessel;

[0038] Preferably, the repair is to promote at least one of closure of the damaged site, cell adhesion, proliferation or differentiation;

[0039] More preferably, the drug is used for assisting hemostasis in vascular reconstruction and occlusion or assisting suturing of cranial suture sites;

[0040] More preferably, the drug is used for epidermal adhesion or intracerebral adhesion. Beneficial effects

[0041] The supercharged protein-based two-component in-situ adhesive provided herein can be applied in situ to tissue wound surfaces. It utilizes the room-temperature EDC-NHS reaction between the amino groups on elastin-like proteins rich in amino functional groups and the active succinimide of a succinimide-containing compound. Simultaneously, the amino groups on biological tissue proteins also rapidly undergo an EDC-NHS reaction with the active succinimide (see Reaction Formula I below). This achieves in-situ, rapid gelation at the tissue wound site, exhibiting excellent intertissue adhesion strength, and exhibiting excellent adhesive properties even on moist tissue surfaces.

[0042] After gelation, the adhesive surface is soft and can be used to achieve moist wound hemostasis, wound repair and other effects, such as vascular reconstruction and occlusion to assist hemostasis, and cranial suture to assist suturing. At the same time, the two-component in-situ adhesive of the present application also has the advantages of simple and fast preparation process, no need for special equipment, as well as rapid adhesion and biocompatibility. It can be used as a new generation of portable instant biomedical glue, and has great application prospects in situations such as acute wound bleeding and complex wound treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] One or more embodiments are exemplarily illustrated by the accompanying figures, and these exemplary illustrations do not limit the embodiments. The term "exemplary" is used herein to mean "serving as an example, example, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

[0044] FIG1 shows the bonding mechanics curve of the two-component in-situ adhesive prepared in Example 1 on defatted pigskin; wherein the abscissa shows the adhesive tensile length (i.e., displacement, in mm), and the ordinate shows the lap shear strength (in kPa);

[0045] Figure 2 shows the morphology of the two-component adhesive formed by SS-PEG-SS containing PEG of different molecular weights and the elastin-like protein shown in SEQ ID NO: 3 after curing prepared in Example 2; wherein, AB1 is a two-component in situ adhesive based on the elastin-like protein shown in SEQ ID NO: 3 and SS-PEG-SS (wherein the average relative molecular weight of PEG is 1000), AB2 is a two-component in situ adhesive based on the elastin-like protein shown in SEQ ID NO: 3 and SS-PEG-SS (wherein the average relative molecular weight of PEG is 2000), AB3 is a two-component in situ adhesive based on the elastin-like protein shown in SEQ ID NO: 3 and SS-PEG-SS (wherein the average relative molecular weight of PEG is 5000), and AB4 is a two-component in situ adhesive based on the elastin-like protein shown in SEQ ID NO: 3 and SS-PEG-SS (wherein the average relative molecular weight of PEG is 10000).

[0046] Figure 3 shows the adhesive strength of the five two-component in-situ adhesives prepared in Examples 1 and 3 on defatted pig skin; wherein the abscissa shows the type of the two-component in-situ adhesive, wherein A1B is a two-component in-situ adhesive based on the elastin-like protein as shown in SEQ ID NO: 1 and SS-PEG-SS (wherein the average relative molecular weight of PEG is 5000), A2B is a two-component in-situ adhesive based on the elastin-like protein as shown in SEQ ID NO: 2 and SS-PEG-SS (wherein the average relative molecular weight of PEG is 5000), A3B is a two-component in-situ adhesive based on the elastin-like protein as shown in SEQ ID NO: 3 and SS-PEG-SS (wherein the average relative molecular weight of PEG is 5000), A4B is a two-component in-situ adhesive based on the elastin-like protein as shown in SEQ ID NO: 4 and SS-PEG-SS (wherein the average relative molecular weight of PEG is 5000); A5B is a two-component in-situ adhesive based on the elastin-like protein as shown in SEQ ID NO: 5 NO: 5 shows a two-component in-situ adhesive of elastin-like protein and SS-PEG-SS (wherein the average relative molecular weight of PEG is 5000); the ordinate shows the lap shear strength (unit: kPa).

[0047] Figure 4 shows the extrusion of a two-component protein glue (Figure B) onto wet skin (Figure A). After about 20 seconds, the colloid coagulation was visible to the naked eye (Figure C). Subsequently, the tissue interface with the glue was rinsed with running water for 1 minute (Figure D), and it was visible to the naked eye that the protein glue did not fall off (Figure E). Then, the glue surface was vigorously rubbed with the fingers, and some protein glue was still attached to the tissue surface (Figure F). DETAILED DESCRIPTION

[0048] To make the objectives, technical solutions, and advantages of this application more clear, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of this application, not all of them. Based on the embodiments of this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0049] Unless expressly stated otherwise, throughout the specification and claims, the term "comprise" or variations such as "include" or "comprising", etc., will be understood to include the stated elements or components but not to exclude other elements or other components.

[0050] In addition, in order to better illustrate the present application, numerous specific details are provided in the following detailed description. It should be understood by those skilled in the art that the present application can be implemented without certain specific details. In some embodiments, raw materials, components, methods, means, etc. well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present application.

[0051] Hereinafter, this application will be described in detail.

[0052] In one aspect, the present application provides a two-component in-situ adhesive based on a supercharged protein, which comprises an elastin-like protein rich in amino functional groups and a compound containing a succinimide group.

[0053] Preferably, the amino-functional group-rich elastin-like protein is an engineered expression protein with (VPGKG)9 as the basic repeating unit, with (VPGXG) spaced between every two repeating units, the number of repeating units n is between 3 and 18, preferably between 8 and 18, and X in the spacer (VPGXG) is any natural amino acid except proline.

[0054] In a preferred embodiment, in the spacer (VPGXG), X is valine, alanine, leucine, isoleucine or arginine.

[0055] In a preferred embodiment, the amino-rich elastin-like protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

[0056] Preferably, the succinimide-containing compound is any one or more selected from the following: disuccinimidyl succinate polyethylene glycol (SS-PEG-SS), succinimidyl acetate, disulfide disuccinimidyl propionate, more preferably disuccinimidyl succinate polyethylene glycol (SS-PEG-SS), and further preferably, in the SS-PEG-SS, the average relative molecular weight of PEG is between 2000-10000, preferably between 5000-10000.

[0057] On the other hand, the present application provides a method for preparing the two-component in-situ adhesive based on supercharged protein as described above, which comprises the following steps:

[0058] 1) mixing the amino-functional group-rich elastin-like protein with water to prepare an aqueous solution of the amino-functional group-rich elastin-like protein; preferably, the mass volume concentration of the amino-functional group-rich elastin-like protein aqueous solution is 50 to 650 mg / mL, preferably between 200 and 400 mg / mL, and more preferably 200 mg / mL;

[0059] 2) dissolving the succinimide-containing compound in water to prepare an aqueous solution of the succinimide-containing compound; preferably, the mass volume concentration of the aqueous solution of the succinimide-containing compound is 0.5 to 1.5 g / mL, preferably 0.5 to 1.0 g / mL, and more preferably 0.75 g / mL;

[0060] And optionally, 3) the aqueous solution of the amino-functional group-rich elastin-like protein prepared in step 1) and the aqueous solution of the succinimide-containing compound prepared in step 2) are respectively encapsulated into a two-component syringe.

[0061] In addition, the present application provides the use of the above-mentioned two-component in-situ adhesive in the preparation of a drug for hemostasis, repair and / or regeneration of tissue wounds. For example, the drug is used for assisting hemostasis in vascular reconstruction and occlusion or assisting suturing of cranial suture sites.

[0062] The present application is further illustrated below by way of examples. In the following examples, unless otherwise specified, the biological and chemical materials used are commercially available products. The elastin-like proteins used can be prepared by any suitable technique known to those skilled in the art.

[0063] In the following examples, the amino-functional group-rich elastin used was synthesized by Beijing Lanthanide Biotechnology Co., Ltd.; the SS-PEG-SS containing PEG of different molecular weights used was purchased from Xi'an Kaixin Biological Co., Ltd.; the biological tissues used were obtained by the inventors from commercially available experimental animals through conventional separation methods; and the pig casings used (trade name: "Pi Xiaojian Natural Pig Casings", purchased from Shenzhen Pinyuepin Food Co., Ltd.) were obtained by the inventors through commercial purchase.

[0064] Example 1

[0065] 1) adding 200 mg of amino-rich elastin-like protein to 1 mL of ultrapure water and mixing thoroughly to obtain a homogeneous 200 mg / mL protein solution, designated as component A solution (i.e., elastin-like protein solution); the elastin-like protein has the amino acid sequence shown in SEQ ID NO: 3;

[0066] 2) Weigh 1 g of disuccinimidyl succinate polyethylene glycol (SS-PEG-SS) powder (wherein the average relative molecular weight of PEG is 5000) and add it to 1 mL of ultrapure water. Mix well to obtain a component B solution (i.e., SS-PEG-SS solution).

[0067] 3) The component A solution and the component B solution are respectively encapsulated into a two-component syringe to obtain the two-component in-situ adhesive A3B described in the present application.

[0068] During use, the A and B component solutions are sequentially applied to the surface of soft tissue samples of pig skin, pork, pig liver, and pig heart. After mixing, they are quickly overlapped to the adhesive surface with a film of the same type and gelled for 2 seconds to form an in situ adhesion layer. The volume ratio of the elastin-like solution to the SS-PEG-SS solution is 1:1.

[0069] The adhesion performance of the adhesives was evaluated using shear bond strength using a SHIMADZU SLBL-500N tensile testing machine to characterize the adhesive bond strength on defatted pig skin, pork, pig liver, and pig heart soft tissue samples.

[0070] Figure 1 shows the adhesion mechanics of the adhesive prepared in this example on a defatted porcine soft tissue sample. The three curves are from three parallel experiments, all prepared and tested using the same procedures. Figure 1 shows that the bond strength of the two-component in-situ adhesive based on the elastin-like protein represented by SEQ ID NO: 3 and SS-PEG-SS was 53.54 ± 7.47 kPa. This result demonstrates that the two-component in-situ adhesive based on the elastin-like protein represented by SEQ ID NO: 3 and SS-PEG-SS exhibits excellent adhesion to biological epidermal tissue.

[0071] Example 2

[0072] In this embodiment, three two-component in-situ adhesives of the present application were prepared by the following steps: AB1, AB2, AB3, and AB4, wherein:

[0073] AB1 is a two-component in-situ adhesive based on the elastin-like protein shown in SEQ ID NO: 3 and a SS-PEG-SS solution (wherein the average relative molecular weight of PEG is 1000);

[0074] AB2 is a two-component in-situ adhesive based on the elastin-like protein shown in SEQ ID NO: 3 and a SS-PEG-SS solution (wherein the average relative molecular weight of PEG is 2000);

[0075] AB3 is a two-component in-situ adhesive based on the elastin-like protein shown in SEQ ID NO: 3 and a SS-PEG-SS solution (wherein the average relative molecular weight of PEG is 5000);

[0076] AB4 is a two-component in-situ adhesive based on the elastin-like protein shown in SEQ ID NO: 3 and a SS-PEG-SS solution (wherein the average relative molecular weight of PEG is 10,000);

[0077] The preparation method is as follows: 200 mg of elastin-like protein having the amino acid sequence shown in SEQ ID NO: 3 is added to 1 mL of ultrapure water and mixed thoroughly to obtain a homogeneous 200 mg / mL protein solution (Component A). SS-PEG-SS solutions containing PEGs of varying molecular weights are then prepared as a 750 mg / mL aqueous solution to obtain Component B. Components A and B are then dispensed into the two injectors of a two-component syringe to form a prefilled two-component glue.

[0078] When in use, the A and B component solutions are injected into the original position in a volume ratio of 1:1 to form a two-component glue.

[0079] The curing time and morphology of AB1, AB2, AB3, and AB4 during the gelling process were observed (see Figure 2), and the burst strength was tested (see Table 1).

[0080] Bursting Strength Test Method: After properly treating the hog casing (including cleaning to remove excess salt and soaking it in water to maintain its elasticity), install it on the bursting fixture. After verifying the seal, pierce a 0.5mm diameter hole in the center of the fixture to simulate a tissue leak. Two-component adhesives AB1, AB2, AB3, and AB4 are injected into the casing membrane on the bursting fixture to seal the rupture site, with a total injection volume of 1mL. After the injection is completed, the timer is counted until the two-component instant adhesive is completely cured. The time taken is calculated as the cure time of the two-component instant adhesive. Air is injected into the sealed fixture, and the gas is introduced into the fixture via a pressure gauge. When the cured adhesive attached to the casing cannot withstand the increased air pressure, gas leaks through the perforation in the casing, causing the pressure gauge reading to drop momentarily. The highest pressure gauge reading is recorded as the bursting strength of the two-component instant adhesive with that ratio.

[0081] Table 1: Morphology, curing time and burst strength of glue formed by SS-PEG-SS solutions containing PEG of different molecular weights and elastin-like proteins:

[0082] Figure 2 shows the morphology of the SS-PEG-SS adhesive containing PEG of different molecular weights prepared in this example after being completely gelled and peeled off from the rupture tool. The results in Figure 2 show that the SS-PEG-SS containing PEG of different molecular weights has different effects on the rupture strength of the adhesive: the higher the PEG molecular weight, the faster the curing speed, the stronger the rupture strength, and the harder the colloid. Considering that when a two-component glue is applied in the body, a higher colloid hardness will increase discomfort, so it is not necessarily the case that the stronger the rupture strength and the harder the colloid, the better the two-component glue; at the same time, when applying a two-component glue in the body, a suitable curing time (for example, 5s to 2min) is necessary, which makes it easier for doctors to operate and avoids the inability to quickly remove the colloid after incorrect application.

[0083] Balancing the above considerations and results: The adhesive of the present application prepared with SS-PEG-SS in which the average relative molecular weight of PEG is 2000, 5000, and 10000 (especially the latter two) has good comprehensive performance on wet biological tissues. Different combinations can be selected according to the route of use and site of action.

[0084] Example 3

[0085] In this example, four two-component in-situ adhesives of the present application were prepared by the following steps: A1B, A2B, A4B, and A5B. A1B is a two-component in-situ adhesive based on the elastin-like protein shown in SEQ ID NO: 1 and SS-PEG-SS (wherein the average relative molecular weight of PEG is 5000), A2B is a two-component in-situ adhesive based on the elastin-like protein shown in SEQ ID NO: 2 and SS-PEG-SS (wherein the average relative molecular weight of PEG is 5000), A4B is a two-component in-situ adhesive based on the elastin-like protein shown in SEQ ID NO: 4 and SS-PEG-SS (wherein the average relative molecular weight of PEG is 5000), and A5B is a two-component in-situ adhesive based on the elastin-like protein shown in SEQ ID NO: 5 and SS-PEG-SS (wherein the average relative molecular weight of PEG is 5000):

[0086] 1) adding 200 mg of each of the four amino-group-rich elastin-like proteins represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5 to 1 mL of ultrapure water and mixing them uniformly to obtain homogeneous 200 mg / mL protein solutions, which were designated as component solutions A1, A2, A4, and A5, respectively;

[0087] 2) Weigh 1 g of disuccinimidyl succinate polyethylene glycol powder and add it to 1 mL of ultrapure water. Mix well to obtain a component B solution.

[0088] 3) The component A1 solution, or the component A2 solution, or the component A4 solution, or the component A5 solution and the component B solution are respectively encapsulated into a two-component syringe to obtain the two-component instant in-situ adhesives A1B, A2B, A4B, and A5B described in the present application.

[0089] During use, the A1, B or A2, B or A4, B or A5, and B component solutions are sequentially applied to the surface of the defatted porcine soft tissue sample. After mixing, they are quickly overlapped with the adhesive surface using a film of the same type and gelled for 2 seconds to form an in situ adhesion layer. The volume ratio of the elastin-like solution rich in amino functional groups to the SS-PEG-SS solution is 1:1.

[0090] The four two-component instant in-situ adhesives A1B, A2B, A4B, and A5B prepared in this example were overlap-bonded on defatted porcine tissue and subjected to tensile testing (the method was the same as in Example 1). The test results of the four two-component instant in-situ adhesives A1B, A2B, A4B, and A5B prepared in this example are shown in FIG3 , as well as the test results of the two-component instant in-situ adhesive A3B prepared in Example 1. FIG3 shows that the two-component in-situ adhesive A1B of the present application based on the elastin-like protein and SS-PEG-SS (wherein the average relative molecular weight of PEG is 5000) and the two-component in-situ adhesive A2B of the present application based on the elastin-like protein and SS-PEG-SS (wherein the average relative molecular weight of PEG is 5000) have a measured bonding strength of 21.24±2.97 kPa, and the two-component in-situ adhesive A2B of the present application based on the elastin-like protein and SS-PEG-SS (wherein the average relative molecular weight of PEG is 5000) have a measured bonding strength of 31.91±3.05 kPa. The measured bonding strength of the two-component in-situ adhesive A3B of the present application based on the elastin-like protein and SS-PEG-SS (wherein the average relative molecular weight of PEG is 5000) as shown in SEQ ID NO: 3 is 53.54±7.47 kPa (i.e., the bonding strength tested in Example 1); the measured bonding strength of the two-component in-situ adhesive A4B of the present application based on the elastin-like protein and SS-PEG-SS (wherein the average relative molecular weight of PEG is 5000) as shown in SEQ ID NO: 4 is 51.49±3.86 kPa; and the measured bonding strength of the two-component in-situ adhesive A5B of the present application based on the elastin-like protein and SS-PEG-SS (wherein the average relative molecular weight of PEG is 5000) as shown in SEQ ID NO: 5 is 50.22±1.81 kPa.

[0091] The above results indicate that the two-component in-situ adhesives of the present invention based on the elastin-like protein and SS-PEG-SS as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 all exhibit excellent adhesion properties on moist biological tissues, meet clinical tissue bonding requirements, and provide diverse solutions for different tissue bonding requirements. Among them, the two-component in-situ adhesives of the present invention based on the elastin-like protein and SS-PEG-SS as shown in SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 exhibited high adhesion properties, with the two-component in-situ adhesive A3B based on the elastin-like protein and SS-PEG-SS as shown in SEQ ID NO: 3 exhibiting the best adhesion properties. When the sequence length is longer than that of SEQ ID NO: 3, the adhesion strength does not increase rapidly (the adhesion strengths of A3B, A4B, and A5B are similar), but the protein expression efficiency is reduced, thus SEQ ID NO: 3 is the preferred elastin-like sequence.

[0092] Example 4

[0093] In this example, the adhesion effect of the two-component in-situ adhesive AB4 of the present application prepared in Example 2 on wet skin was tested by the following steps:

[0094] Two-component protein glue (Figure 4B) was squeezed out on wet skin (Figure 4A), and after about 20 seconds, the colloid coagulation was visible to the naked eye (Figure 4C); then, the tissue interface with the glue was rinsed with running water for 1 minute (Figure 4D), and it was visible to the naked eye that the protein glue did not fall off (Figure 4E); then, the glue surface was vigorously rubbed with fingers, and some protein glue was still attached to the tissue surface (Figure 4F).

[0095] The above embodiments illustrate that the two-component instant in-situ adhesive of the present application can achieve in-situ and rapid gelation on the surface of moist biological tissue, exhibiting excellent adhesion properties, and can be used to achieve moist wound hemostasis, wound repair and other effects, such as vascular reconstruction and occlusion to assist hemostasis, and assist suturing of cranial suture sites.

[0096] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application. Industrial Applicability

[0097] The two-component in-situ adhesive based on supercharged proteins provided in this application can be rapidly gelled in situ at tissue wounds, exhibiting excellent intertissue adhesion strength, and can exhibit excellent adhesion properties even on moist tissue surfaces; after gelation, the adhesive surface is soft and can be used to achieve moist wound hemostasis, wound repair and other effects; at the same time, the adhesive of this application also has the advantages of simple and rapid preparation, no need for special equipment, as well as rapid adhesion and biocompatibility. It can be used as a new generation of portable instantaneous biomedical glue and has great application prospects in situations such as acute wound bleeding and complex wound treatment.

Claims

1. A two-component in-situ adhesive based on supercharged protein, comprising: (1) Elastin-like proteins rich in amino functional groups, and (2) Compounds containing a succinimide group.

2. The two-component in-situ adhesive according to claim 1, characterized in that The amino functional group-rich elastin-like protein comprises n [(VPGKG)9] repeating units, and every two [(VPGKG)9] repeating units are separated by a spacer (VPGXG); wherein n is an integer between 3 and 18, and X is any natural amino acid except proline; And / or, the succinimide-containing compound is any one or more selected from the following: disuccinimidyl succinate polyethylene glycol, succinimidyl acetate, dithiodisuccinimidyl propionate.

3. The two-component in-situ adhesive according to claim 2, characterized in that: In the amino functional group-rich elastin-like protein, the number n of repeating units [(VPGKG)9] is an integer between 8 and 18, and / or, in the spacer (VPGXG), X is valine, arginine, leucine, isoleucine or alanine; And / or, the succinimide group-containing compound is disuccinimidyl succinate polyethylene glycol.

4. The two-component in-situ adhesive according to claim 3, characterized in that: The amino functional group-rich elastin-like protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5; And / or, the succinimide group-containing compound is disuccinimidyl succinate polyethylene glycol, wherein the average relative molecular weight of the polyethylene glycol is 2000-10000.

5. The two-component in-situ adhesive according to claim 4, characterized in that: The amino functional group-rich elastin-like protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5; And / or, the succinimide group-containing compound is disuccinimidyl succinate polyethylene glycol, wherein the average relative molecular weight of the polyethylene glycol is 5000-10000.

6. The two-component in-situ adhesive according to any one of claims 1 to 5, characterized in that: The amino-functional group-rich elastin-like protein exists alone in the form of an aqueous solution, and the succinimide-containing compound exists in the form of a solid powder. When used, the succinimide-containing compound is dissolved in ultrapure water, then mixed with the elastin-like protein aqueous solution, and then gelled to form an in-situ adhesion layer.

7. The two-component in-situ adhesive according to claim 6, characterized in that The mass volume concentration of the aqueous solution of elastin-like protein rich in amino functional groups is 50 to 650 mg / mL; and / or, when used, the succinimide group-containing compound is formulated into an aqueous solution with a mass volume concentration of 0.5 to 1.5 g / mL; and / or, when used, the aqueous solution of the succinimide-containing compound and the aqueous solution of the elastin-like protein rich in amino functional groups are mixed in a volume ratio of 1:(0.5-2.0); And / or, the gelation time is 0 to 120 s.

8. The two-component in-situ adhesive according to claim 7, characterized in that: The mass volume concentration of the aqueous solution of elastin-like protein rich in amino functional groups is between 200 and 400 mg / mL; and / or, the mass volume concentration of the aqueous solution of the succinimide-containing compound is 0.5 to 1.0 g / mL; And / or, when used, the aqueous solution of the succinimide-containing compound and the aqueous solution of the elastin-like protein rich in amino functional groups are mixed in a volume ratio of 1:

1.

9. The method for preparing the two-component in-situ adhesive according to any one of claims 1 to 8, comprising the following steps: 1) mixing the amino functional group-rich elastin-like protein with water to prepare an aqueous solution of the amino functional group-rich elastin-like protein; 2) dissolving the succinimide-containing compound in water to prepare an aqueous solution of the succinimide-containing compound; And optionally, 3) the aqueous solution of the amino-functional group-rich elastin-like protein prepared in step 1) and the aqueous solution of the succinimide-containing compound prepared in step 2) are respectively packaged into a two-component syringe.

10. The preparation method according to claim 9, characterized in that: The mass volume concentration of the aqueous solution of the amino functional group-rich elastin-like protein prepared in step 1) is 50 to 650 mg / mL; And / or, the mass volume concentration of the aqueous solution of the succinimide-containing compound prepared in step 2) is 0.5 to 1.5 g / mL.

11. The preparation method according to claim 10, characterized in that: The mass volume concentration of the aqueous solution of the amino functional group-rich elastin-like protein prepared in step 1) is 200-400 mg / mL; And / or, the mass volume concentration of the aqueous solution of the succinimide-containing compound prepared in step 2) is 0.5-1.0 g / mL.

12. Use of the two-component in-situ adhesive according to any one of claims 1 to 8 in the preparation of a medicament for hemostasis, repair and / or regeneration of tissue or organ wounds.

13. The use according to claim 12, characterized in that: The tissue is at least one of meniscus tissue, muscle tissue, tendon tissue, bone tissue, connective tissue, epidermal tissue or mucosal tissue; and / or, the organ is at least one of skin, heart, liver, kidney, brain or blood vessel; And / or, the repair is to promote at least one of closure of the damaged site, cell adhesion, proliferation or differentiation.

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