Two-component in-situ adhesive based on supercharged proteins, method for producing the same, and use

Abstract

This application provides a two-component in-situ adhesive based on supercharged proteins, as well as a method for producing and using the same. The two-component in-situ adhesive of this application can rapidly gel in situ at the site of tissue wound, exhibits excellent inter-tissue adhesion strength, and shows excellent adhesive performance even on wet tissue surfaces. After gelling, its gel surface is flexible and can be used to achieve effects such as hemostasis of wet wounds and wound repair. For example, it can be used for vascular reconstruction, occlusion, and hemostasis assistance, and suturing assistance at craniocerebral suture sites. Furthermore, the adhesive of this application has advantages in terms of being easy and quick to manufacture, not requiring special equipment, and having rapid adhesion and biocompatibility. It can be made into a next-generation portable instantaneous biomedical gel and has extremely large application prospects in situations such as acute bleeding at wound sites and complex wound treatment.

Description

Technical Field

[0001] (Cross - reference to related applications) This application claims the priority of a Chinese patent application with the application number 202310822652.2 and the invention title "Two - component in - situ adhesive based on supercharged proteins and its manufacturing method and use", which was filed on July 5, 2023, and all of its content is incorporated herein by reference.

[0002] This application relates to the field of biomaterial technology, specifically to a two - component in - situ adhesive based on supercharged proteins and its manufacturing method and use.

Background Art

[0003] Tissue adhesives refer to a type of biomaterial that can create an interaction force with internal tissues, thereby causing adhesion between tissues or between tissues and non-tissues (e.g., implants), and that also possesses functions such as controlling bleeding (hemostasis) and preventing the flow of gases and fluids (sealing). With the advancement of modern medicine, surgical procedures are required not only to restore function and appearance to the greatest extent possible, but also to minimize patient suffering as much as possible. Currently, suturing or stapling is commonly used in surgeries to treat large tissue defects or bleeding points. However, while this method physically pulls tissue together for repair, it does not yield satisfactory results. The main reasons for this are that sutures and staples do not provide additional effects such as wound healing or anti-inflammatory action beyond simple physical pulling, sutures and staples can cause damage and bleeding of surrounding tissues during surgery, and after wound healing, they can cause scarring and additional pain from suture removal. These problems can be effectively solved by using medical tissue adhesives. Furthermore, the rapid hemostasis and wound adhesion functions of tissue adhesives are particularly suitable for the immediate treatment of wounds that are difficult to suture in wartime environments. They can also treat some internal wounds that are difficult to treat with hemostatic bandages. Moreover, tissue adhesives rapidly seal wounds and prevent further contact with the outside, thus providing infection prevention. According to reports, during wartime, many casualties occur between the time of injury and arrival at field hospitals. The use of highly efficient tissue adhesives with added hemostatic and analgesic agents could save many lives. Additionally, with the advancement of clinical medical technology, minimally invasive techniques are already widely used in various clinical departments. However, minimally invasive surgery involves small wounds and has limitations in field of view and maneuverability, making it difficult to use the conventional wound tissue mechanical fixation techniques mentioned above during the procedure. Therefore, tissue adhesives have extremely broad application prospects in fields such as surgical procedures, cosmetic surgery, and battlefield medical care.

[0004] However, achieving ideal adhesion at tissue interfaces is an extremely challenging task, primarily for two reasons. First, the presence of a large amount of liquid on the tissue surface prevents a large amount of adhesive from penetrating the hydration layer and generating effective intermolecular forces at the tissue interface, significantly reducing the adhesive's tackiness. Second, due to its flexible nature, tissue has extremely high compliance requirements for adhesives; that is, it requires the adhesive to be able to adaptively deform and adjust in response to tissue movement. At the same time, biocompatibility is also of paramount importance. Consequently, this places extremely high demands on both the adhesive properties and component composition of the adhesive, greatly increasing the difficulty of developing ultra-strong tissue adhesives.

[0005] Based on the current state of research described above, the research and development of instantaneous in-situ adhesives based on bioengineered proteins is of great importance to both basic scientific research and biomedical applications. [Overview of the project] [Problems that the invention aims to solve]

[0006] The present invention aims to provide a two-component instantaneous in-situ adhesive based on bioengineered proteins that exhibits excellent adhesive performance, as well as a method for producing and using the same. [Means for solving the problem]

[0007] To achieve the above objectives, this application provides the following technical solution.

[0008] In a first aspect, the present application provides a two-component situ adhesive based on a supercharged protein, which is, (1) Elastin-like proteins rich in amino functional groups, (2) A compound containing a succinimide group, and

[0009] Preferably, the elastin-like protein rich in the amino functional group comprises n [(VPGKG)9] repeating units, with every two [(VPGKG)9] repeating units separated by spacers (VPGXG), where n is an integer between 3 and 18, and X is any natural amino acid other than proline, and / or the succinimide group-containing compound is any one or more selected from succinimidyl succinate-polyethylene glycol succinimidyl succinate (SS-PEG-SS), succinimidyl acetate (succinimidyl acetate), and dithiobis(succinimidyl propionate) (3,3'-dithiodipropionic acid di(N-succinimidyl)).

[0010] More preferably, in the elastin-like protein rich in amino functional groups, n in the number 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 succinimidylsuccinic acid-polyethylene glycol-succinimidylsuccinic acid (SS-PEG-SS).

[0011] In the specific implementation plan, the elastin-like protein rich in the amino functional group contains or is composed of an amino acid sequence selected from 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 succinimidylsuccinic acid-polyethylene glycol-succinimidylsuccinic acid (SS-PEG-SS), where the average relative molecular weight of polyethylene glycol is 2000 to 10000.

[0012] In a preferred specific embodiment, the elastin-like protein rich in amino functional groups comprises or is composed of an amino acid sequence selected from SEQ ID NOs: 3, SEQ ID NOs: 4, and SEQ ID NOs: 5, and / or the succinimide group-containing compound is succinimidylsuccinic acid-polyethylene glycol-succinimidylsuccinic acid (SS-PEG-SS), where the average relative molecular weight of polyethylene glycol is 5000 to 10000.

[0013] Sequence ID 1 [ka] (Here, the underlined part is the repeating unit [(VPGKG)9], and the bolded part is the spacer (VPGXG)) Sequence ID 2 [ka] (Here, the underlined part is the repeating unit [(VPGKG)9], and the bolded part is the spacer (VPGXG)) Sequence ID 3 [ka] (Here, the underlined part is the repeating unit [(VPGKG)9], and the bolded part is the spacer (VPGXG).) Sequence ID 4 [ka] (Here, the underlined part is the repeating unit [(VPGKG)9], and the bolded part is the spacer (VPGXG).) Sequence ID 5 [ka] (Here, the underlined part is the repeating unit [(VPGKG)9], and the bolded part is the spacer (VPGXG).)

[0014] In a specific embodiment, the elastin-like protein rich in amino functional groups exists alone in the form of an aqueous solution, and the succinimide group-containing compound exists in the form of a solid powder. During use, the succinimide group-containing compound is dissolved in ultrapure water and then mixed with the elastin-like protein aqueous solution, after which gelation occurs to form an in-situ adhesion layer.

[0015] Preferably, the mass-volume concentration of the aqueous solution of the elastin-like protein rich in amino functional groups is 50 - 650 mg / mL, preferably 200 - 400 mg / mL, more preferably 200 mg / mL. Preferably, during use, the succinimide group-containing compound is prepared in an aqueous solution with a mass-volume concentration of 0.5 - 1.5 g / mL, preferably 0.5 - 1.0 g / mL, and most preferably 0.75 g / mL. Preferably, during use, the aqueous solution of the succinimide group-containing compound and the aqueous solution of the elastin-like protein rich in amino functional groups are mixed according to a volume ratio of 1:(0.5 - 2.0), preferably a volume ratio of 1:1. Preferably, after the two solutions are mixed, the gelation time is 0 - 120 s.

[0016] In a second aspect, the present application provides a method for manufacturing the two-component in-situ adhesive described in the first aspect above, and the method includes: Step 1) of mixing the elastin-like protein rich in amino functional groups with water to produce an aqueous solution of the elastin-like protein rich in amino functional groups; Step 2) of dissolving the succinimide group-containing compound in water to produce an aqueous solution of the succinimide group-containing compound; Optionally, Step 3) of respectively packaging (loading) the aqueous solution of the elastin-like protein rich in amino functional groups produced in Step 1) and the aqueous solution of the succinimide group-containing compound produced in Step 2) into two-component syringes.

[0017] Preferably, the mass-volume concentration of the aqueous solution of the amino-functional group-rich elastin-like protein produced in step 1) is 50 to 650 mg / mL, preferably 200 to 400 mg / mL, more preferably 200 mg / mL. Preferably, the mass-volume concentration of the aqueous solution of the succinimide group-containing compound prepared in step 2) is 0.5 to 1.5 g / mL, preferably 0.5 to 1.0 g / mL, and most preferably 0.75 g / mL.

[0018] In a third aspect, the present application provides the use of the two-component in-situ adhesive described in the first aspect above in the manufacture of agents for hemostasis, repair and / or regeneration of wounds of tissue or organs. Preferably, the tissue is at least one of the following: meniscal tissue, muscle tissue, tendon tissue, bone tissue, connective tissue, epidermal tissue, or mucosal tissue. Preferably, the organ is at least one of the skin, heart, liver, kidney, brain, or blood vessels. Preferably, the repair involves promoting at least one of the following: closure of the damaged site, cell adhesion, proliferation, or differentiation. More preferably, the agent is used for vascular reconstruction, occlusion, hemostasis assistance, or suturing assistance at craniocerebral suture sites. More preferably, the drug is used for epidermal adhesion or intracerebral adhesion. [Effects of the Invention]

[0019] The two-component in-situ adhesive based on supercharged proteins provided in this application can be applied in situ to the surface of tissue wounds. It achieves rapid in-situ gelation at the tissue wound site by utilizing a room-temperature EDC-NHS reaction between amino groups on an elastin-like protein rich in amino functional groups and active succinimide in a succinimide group-containing compound, and a rapid EDC-NHS reaction between amino groups on biological tissue proteins and active succinimide (see reaction formula I below). It exhibits excellent inter-tissue adhesion strength and excellent adhesive performance even on wet tissue surfaces. [ka]

[0020] After gelling, the gel surface is flexible and can be used to achieve effects such as hemostasis of wet wounds and wound repair. For example, it can be used for vascular reconstruction, occlusion, and hemostasis assistance, as well as suturing assistance at craniocerebral suture sites. Furthermore, the two-component in-situ adhesive of this application has advantages in terms of a simple and rapid manufacturing process, the absence of special equipment, and rapid adhesion and biocompatibility, making it a next-generation portable instantaneous biomedical gelling agent with extremely large application potential in situations such as acute bleeding from wounds and complex wound treatment.

[0021] One or more embodiments are illustrated by photographs in the corresponding drawings, and these illustrative descriptions are not limiting to the embodiments. Here, the term “exemplary” means “used as an example, embodiment, or explanatory element.” There is no need to interpret any embodiment described “exemplary” as being superior or better than other embodiments. [Brief explanation of the drawing]

[0022] [Figure 1] The mechanical curve of adhesion between the two-component in-situ adhesive prepared in Example 1 and degreased pigskin is shown, where the horizontal axis represents the adhesive tensile length (i.e., displacement, in mm) and the vertical axis represents the overlap shear strength (in kPa). [Figure 2]The cured form of the two-component adhesive formed based on SS-PEG-SS containing PEG of different molecular weights produced in Example 2 and the elastin-like protein shown in SEQ ID NO: 3 is shown, where AB1 is a two-component situ-in-situ adhesive based on the elastin-like protein shown in SEQ ID NO: 3 and SS-PEG-SS (where the average relative molecular weight of PEG is 1000), AB2 is a two-component situ-in-situ adhesive based on the elastin-like protein shown in SEQ ID NO: 3 and SS-PEG-SS (where the average relative molecular weight of PEG is 2000), AB3 is a two-component situ-in-situ adhesive based on the elastin-like protein shown in SEQ ID NO: 3 and SS-PEG-SS (where the average relative molecular weight of PEG is 5000), and AB4 is a two-component situ-in-situ adhesive based on the elastin-like protein shown in SEQ ID NO: 3 and SS-PEG-SS (where the average relative molecular weight of PEG is 10000). [Figure 3] The adhesive strength of the five types of two-component situ-in-situ adhesives produced in Examples 1 and 3 on defatted pigskin is shown, where the x-axis indicates the type of two-component situ-in-situ adhesive, where A1B is a two-component situ-in-situ adhesive based on elastin-like protein and SS-PEG-SS (where the average relative molecular weight of PEG is 5000) as shown in Sequence ID No. 1, A2B is a two-component situ-in-situ adhesive based on elastin-like protein and SS-PEG-SS (where the average relative molecular weight of PEG is 5000) as shown in Sequence ID No. 2, and A3B is shown in Sequence ID No. 3. A4B is a two-component situ-based adhesive based on elastin-like protein and SS-PEG-SS (where the average relative molecular weight of PEG is 5000), A5B is a two-component situ-based adhesive based on elastin-like protein and SS-PEG-SS (where the average relative molecular weight of PEG is 5000), as shown in SEQ ID NO: 4, and A5B is a two-component situ-based adhesive based on elastin-like protein and SS-PEG-SS (where the average relative molecular weight of PEG is 5000), as shown in SEQ ID NO: 5. The vertical axis indicates the overlap shear strength (in kPa). [Figure 4]A two-component protein gel (Figure B) was extruded onto moist skin (Figure A), and after about 20 seconds, the gel solidified with the naked eye (Figure C). Subsequently, the tissue interface to which the gel was attached was washed with running water for 1 minute (Figure D), and it was confirmed with the naked eye that the protein gel did not detach (Figure E). Even after vigorously kneading the gel surface with a finger, some of the protein gel remained attached to the tissue surface (Figure F). [Modes for carrying out the invention]

[0023] To clarify the purpose, technical proposal and merits of this application, the technical proposal in the embodiments of this application is described clearly and completely below, and it is clear that the embodiments described are only a selection of embodiments of this application, not all embodiments. All other embodiments obtained by a person skilled in the art without creative work based on the embodiments of this application are all within the scope of protection of this application.

[0024] Unless otherwise explicitly stated, the term "includes" or its variations "inclusion" or "contains" throughout the specification and claims should be interpreted as including the described element or component, rather than excluding other elements or components.

[0025] Furthermore, in order to better illustrate the present application, many specific details are provided in the following specific embodiments. Those skilled in the art should understand that the present application can be carried out even without some of the specific details. In some embodiments, in order to clarify the purpose of the present application, details of raw materials, elements, methods, means, etc., which are familiar to those skilled in the art, are not described.

[0026] The present application will be described in detail below.

[0027] On the other hand, the present application provides a two-component in-situ adhesive based on a supercharged protein containing an elastin-like protein rich in amino functional groups and a succinimide group-containing compound.

[0028] Preferably, the elastin-like protein rich in amino functional groups is an engineered expression protein with (VPGKG)9 as the basic repeating unit, where every two repeating units are separated by (VPGXG), the number of repeating units n is 3 to 18, preferably 8 to 18, and X in the spacer (VPGXG) is any natural amino acid other than proline.

[0029] In a preferred specific implementation, in the spacer (VPGXG), X is valine, alanine, leucine, isoleucine, or arginine.

[0030] In a preferred specific implementation, the elastin-like protein rich in amino functional groups comprises, but is composed of, an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

[0031] Preferably, the succinimide group-containing compound is any one or more selected from succinimidylsuccinic acid-polyethylene glycol-succinimidylsuccinic acid (SS-PEG-SS), succinimidyl acetate, and dithiobis(succinimidylpropionate), more preferably succinimidylsuccinic acid-polyethylene glycol-succinimidylsuccinic acid (SS-PEG-SS), and even more preferably, in the SS-PEG-SS, the average relative molecular weight of PEG is 2000 to 10000, preferably 5000 to 10000.

[0032] On the other hand, the present application provides a method for producing a two-component in-situ adhesive based on supercharged proteins as described above, and this method is Step 1) involves mixing the elastin-like protein rich in amino functional groups with water to produce an aqueous solution of the elastin-like protein rich in amino functional groups, wherein the mass-volume concentration of the aqueous solution of the elastin-like protein rich in amino functional groups is preferably 50 to 650 mg / mL, preferably 200 to 400 mg / mL, and more preferably 200 mg / mL. Step 2) involves dissolving the succinimide group-containing compound in water to produce an aqueous solution of the succinimide group-containing compound, wherein the mass volume concentration of the aqueous solution of the succinimide group-containing compound is preferably 0.5 to 1.5 g / mL, preferably 0.5 to 1.0 g / mL, and more preferably 0.75 g / mL. The procedure includes step 3) selectively loading an aqueous solution of the elastin-like protein rich in the amino functional group produced in step 1) and an aqueous solution of the succinimide group-containing compound produced in step 2) into two-component syringes.

[0033] Furthermore, this application provides for the use of the above-described two-component in-situ adhesive in the manufacture of agents for hemostasis, repair, and / or regeneration of tissue wounds. For example, the agent may be used for vascular reconstruction, occlusion, hemostasis assistance, or suturing assistance at craniocerebral suture sites.

[0034] The present invention will be further illustrated below with examples. In the following examples, unless otherwise specified, all biological and chemical materials used are commercially available, and the elastin-like proteins used can be produced by any suitable technique known to those skilled in the art.

[0035] In the following examples, the amino-functional group-rich elastin-like proteins used were all synthesized by Beijing Lanthanide Biotechnology Co., Ltd., the SS-PEG-SS containing PEG of different molecular weights used were all purchased from Xi'an Kaixin Biotechnology Co., Ltd., the biological tissues used were all isolated by the inventor himself from experimental animals purchased on the market using conventional isolation methods, and the pig intestine casings used (product name: "Pixiaoqian Natural Pig Intestine Casings," purchased from Shenzhen Pin Yuepin Food Co., Ltd.) were purchased on the market by the inventor.

[0036] (Example 1) 1) 200 mg of elastin-like protein rich in amino functional groups is added to 1 mL of ultrapure water and mixed uniformly to obtain a homogeneous 200 mg / mL protein solution, which is denoted as component A solution (i.e., elastin-like protein solution), and the elastin-like protein has the amino acid sequence shown in Sequence ID No. 3. 2) Weigh out 1 g of succinimidylsuccinic acid-polyethylene glycol-succinimidylsuccinic acid (SS-PEG-SS) powder (where the average relative molecular weight of PEG is 5000), add it to 1 mL of ultrapure water, and mix uniformly to obtain the B component solution (i.e., the SS-PEG-SS solution). 3) The two-component in-situ adhesive A3B of the present invention was obtained by loading the A component solution and the B component solution into two-component syringes, respectively.

[0037] During use, the A and B component solutions were sequentially applied to the surface of soft tissue samples of pig skin, pig meat, pig liver, and pig heart, and after uniform mixing, the samples were quickly wrapped over the gel surface with a substrate sheet of the same type to induce 2s gelation and form an in-situ adhesion layer. At this point, the volume ratio of the elastin-like protein solution to the SS-PEG-SS solution was 1:1.

[0038] Using a SHIMADZU SLBL-500N tensile testing machine, the adhesion performance of adhesives was evaluated to characterize the adhesive strength of adhesives on soft tissue samples of defatted pigskin, pork, pig liver, and pig heart, using shear adhesion strength.

[0039] Figure 1 shows the mechanical adhesion curves of the adhesive manufactured in this embodiment on soft tissue samples of defatted pigskin. Here, the three curves are from three parallel experiments, and the manufacturing and testing processes are consistent. Figure 1 shows that the adhesive strength obtained from testing the two-component in-situ adhesive of the present invention, based on the elastin-like protein and SS-PEG-SS indicated by Sequence ID No. 3, is 53.54 ± 7.47 kPa. As can be seen from these results, the two-component in-situ adhesive of the present invention, based on the elastin-like protein and S-PEG-SS indicated by Sequence ID No. 3, exhibits good adhesion performance on biological epidermal tissue.

[0040] (Example 2) In this embodiment, three types of two-component in-situ adhesives of the present application (AB1, AB2, AB3, and AB4) are manufactured by the following steps, where, AB1 is a two-component in-situ adhesive based on the elastin-like protein shown in Sequence ID No. 3 and an SS-PEG-SS solution (where 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 Sequence ID No. 3 and an SS-PEG-SS solution (where 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 Sequence ID No. 3 and an SS-PEG-SS solution (where the average relative molecular weight of PEG is 5000). AB4 is a two-component in-situ adhesive based on the elastin-like protein shown in Sequence ID No. 3 and an SS-PEG-SS solution (where the average relative molecular weight of PEG is 10,000). The manufacturing method is as follows: 200 mg of elastin-like protein having the amino acid sequence shown in Sequence ID No. 3 was added to 1 mL of ultrapure water and mixed uniformly to obtain a homogeneous 200 mg / mL protein solution (component A solution). An SS-PEG-SS solution containing PEG of different molecular weights was prepared as a 750 mg / mL aqueous solution to produce component B solution. Components A and B solutions were loaded into the two plungers of a two-component syringe to form a pre-filled two-component gel.

[0041] During use, the A and B component solutions were pressed into place in a 1:1 volume ratio to form a two-component adhesive.

[0042] During the gelation process of AB1, AB2, AB3, and AB4, the curing time and morphology (see Figure 2) were observed, and the bursting strength (see Table 1) was detected.

[0043] Method for detecting burst strength: After properly preparing the pig intestinal casing (including washing the casing to remove excess salt and immersing it in water to maintain elasticity), it was mounted on a burst test fixture. After confirming that the seal was good, a 0.5 mm diameter hole was made in the center of the test fixture to simulate a tissue leakage point. Two-component adhesives AB1, AB2, AB3, and AB4 were pressed into the intestinal casing membrane on the burst test fixture to seal the burst site. The total volume pressed in was 1 mL. After the pressing was completed, timing was started and stopped when the two-component instant adhesive had completely hardened. The time taken was defined as the hardening time of the two-component instant adhesive. Air was forced into a sealed test fixture, and gas was introduced into the fixture via a pressure gauge. When the adhesive that had adhered to and hardened on the intestinal casing could no longer withstand the increased pressure, gas leaked out from the perforations on the intestinal casing, causing the pressure gauge reading to drop instantaneously. The highest reading on the pressure gauge was recorded and represented as the burst strength of the two-component instantaneous in-situ adhesive with that particular formulation ratio.

[0044] [Table 1]

[0045] Figure 2 shows the SS-PEG-SS adhesive containing PEG of different molecular weights, manufactured in this embodiment, after complete gelation and removal from the burst test fixture. The results in Figure 2 show that SS-PEG-SS containing PEG of different molecular weights has a different effect on the burst strength of the adhesive, with higher PEG molecular weights resulting in faster curing speed, higher burst strength, and harder gel. When two-component gels are applied to the body, higher gel hardness increases discomfort, so a higher burst strength and harder gel are not necessarily better. Furthermore, when two-component gels are applied to the body, an appropriate curing time (e.g., 5s to 2min) is essential, as this not only facilitates the physician's operation but also avoids situations where the gel cannot be quickly removed after accidental application.

[0046] Considering all of the above and taking these results into account, the adhesives of this application prepared using SS-PEG-SS (especially the latter two) with average relative molecular weights of PEG of 2000, 5000, and 10000 all exhibit excellent overall performance in moist biological tissues, and different combinations can be selected depending on the route of use and site of action.

[0047] (Example 3) In this embodiment, four types of two-component situ-in-situ adhesives of the present invention (A1B, A2B, A4B, and A5B) can be produced by the following steps, where A1B is a two-component situ-in-situ adhesive based on the elastin-like protein indicated by SEQ ID NO: 1 and SS-PEG-SS (where the average relative molecular weight of PEG is 5000); A2B is a two-component situ-in-situ adhesive based on the elastin-like protein indicated by SEQ ID NO: 2 and SS-PEG-SS (where the average relative molecular weight of PEG is 5000); A4B is a two-component situ-in-situ adhesive based on the elastin-like protein indicated by SEQ ID NO: 4 and SS-PEG-SS (where the average relative molecular weight of PEG is 5000); and A5B is a two-component situ-in-situ adhesive based on the elastin-like protein indicated by SEQ ID NO: 5 and SS-PEG-SS (where the average relative molecular weight of PEG is 5000). 1) Add 200 mg each of elastin-like proteins rich in the four amino functional groups indicated by SEQ ID NOs: 1, 2, 4, and 5 to 1 mL of ultrapure water, mix uniformly to obtain homogeneous 200 mg / mL protein solutions, and label them A1, A2, A4, and A5 component solutions, respectively. 2) Weigh out 1 g of succinimidylsuccinate-polyethylene glycol-succinimidylsuccinate powder, add it to 1 mL of ultrapure water, and mix uniformly to obtain the B component solution. 3) The two-component instantaneous in-situ adhesives A1B, A2B, A4B, and A5B of this application were obtained by loading the A1, A2, A4, or A5 solution and the B solution into two-component syringes.

[0048] During use, the A1, B or A2, B or A4, B or A5, and B component solutions were sequentially applied to the surface of a defatted pig skin soft tissue sample, mixed uniformly, and then quickly wrapped over the gel surface with a substrate sheet of the same type to induce 2s gelation and form an in-situ adhesion layer. Here, the volume ratio of the elastin-like protein solution rich in amino functional groups to the SS-PEG-SS solution was 1:1.

[0049] The four types of two-component instant situ adhesives A1B, A2B, A4B, and A5B manufactured in this embodiment were each applied to defatted pigskin tissue using the wrap-over method, and tensile tests were performed (the method was the same as in Example 1). The results and the test results for the two-component instant situ adhesive A3B manufactured in Example 1 are shown in Figure 3. Figure 3 shows that the measured adhesive strength of the two-component situ adhesive A1B, based on the elastin-like protein indicated by Sequence ID No. 1 and SS-PEG-SS (where the average relative molecular weight of PEG is 5000), was 21.24 ± 2.97 kPa, and the measured adhesive strength of the two-component situ adhesive A2B, based on the elastin-like protein indicated by Sequence ID No. 2 and SS-PEG-SS (where the average relative molecular weight of PEG is 5000), was 31.91 ± 3.05 kPa. The measured adhesive strength of the two-component in-situ adhesive A3B of this application, based on the elastin-like protein and SS-PEG-SS (where the average relative molecular weight of PEG is 5000) shown in SEQ ID NO: 3, is 53.54 ± 7.47 kPa (i.e., the adhesive strength tested in Example 1), the measured adhesive strength of the two-component in-situ adhesive A4B of this application, based on the elastin-like protein and SS-PEG-SS (where the average relative molecular weight of PEG is 5000) shown in SEQ ID NO: 4, is 51.49 ± 3.86 kPa, and the measured adhesive strength of the two-component in-situ adhesive A5B of this application, based on the elastin-like protein and SS-PEG-SS (where the average relative molecular weight of PEG is 5000) shown in SEQ ID NO: 5, is 50.22 ± 1.81 kPa.

[0050] As can be seen from the results above, the two-component situ-in-situ adhesives of this application, based on the elastin-like protein represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 and SS-PEG-SS, all exhibit good adhesion performance on moist biological tissues, can meet clinical tissue adhesion requirements, and can provide diverse solutions for different tissue adhesion requirements. Here, the adhesion performance of the two-component situ-in-situ adhesives of this application, based on the elastin-like protein represented by SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 and SS-PEG-SS, is high, and in particular, the adhesion performance of the two-component situ-in-situ adhesive A3B, based on the elastin-like protein represented by SEQ ID NO: 3 and SS-PEG-SS, is the best. When the sequence length is larger than the sequence represented by SEQ ID NO: 3, the adhesion strength does not increase rapidly (A3B, A4B, and A5B have almost the same adhesion strength), but the protein expression efficiency decreases, so SEQ ID NO: 3 is an excellent elastin-like protein sequence.

[0051] (Example 4) In the example, the adhesion effect of the two-component in-situ adhesive AB4 of the present invention, manufactured in Example 2, to moist skin was tested by the following steps: A two-component protein gel (Figure 4, Figure B) was extruded onto moist skin (Figure 4, Figure A), and after about 20 seconds, the gel solidified (Figure 4, Figure C) and the tissue interface to which the gel was attached was washed with running water for 1 minute (Figure 4, Figure D), and it was confirmed with the naked eye that the protein gel had not come off (Figure 4, Figure E). Even after vigorously kneading the gel surface with a finger, some of the protein gel remained attached to the tissue surface (Figure 4, Figure F).

[0052] The above examples illustrate that the two-component instantaneous in-situ adhesive of the present invention can rapidly gel in situ on the surface of moist biological tissue, exhibits excellent adhesive performance, and can be used to achieve effects such as hemostasis of moist wounds and wound repair. For example, it can be used for vascular reconstruction, occlusion, and hemostasis assistance, as well as suturing assistance at craniocerebral suture sites.

[0053] Finally, the above embodiments are merely for illustrative purposes and not to limit the technical proposal of the present application. Although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that it is still possible to modify the technical proposal described in each of the above embodiments or to make equivalent substitutions to some of the technical features, and that such modifications or substitutions will not cause the essence of the corresponding technical proposal to deviate from the scope of the technical proposal of each embodiment of the present application. [Industrial applicability]

[0054] The two-component in-situ adhesive based on supercharged proteins provided in this application can rapidly gel in situ at the site of tissue wound, exhibits excellent inter-tissue adhesion strength, and demonstrates excellent adhesive performance even on wet tissue surfaces. After gelation, the gel surface is flexible and can be used to achieve effects such as hemostasis of wet wounds and wound repair. Furthermore, the adhesive of this application has advantages in terms of ease and speed of manufacture, the absence of the need for special equipment, rapid adhesion, and biocompatibility, making it a next-generation portable instantaneous biomedical gelling agent with extremely large application potential in situations such as acute bleeding from wounds and complex wound treatment.

Claims

1. (1) Elastin-like proteins rich in amino functional groups, (2) A succinimide group-containing compound, A two-component situ adhesive based on supercharged proteins.

2. The aforementioned elastin-like protein rich in amino functional groups contains n [(VPGKG) ９ ] Includes repeating units [(VPGKG) ９ The repeating units are separated every two by a spacer (VPGXG), where n is an integer between 3 and 18, and X is any natural amino acid other than proline. and / or the succinimide group-containing compound is any one or more selected from succinimidylsuccinate-polyethylene glycol-succinimidylsuccinate, succinimidyl acetate, and dithiobis(succinimidylpropionate). The two-component in-situ adhesive according to feature 1.

3. In the elastin-like protein rich in the aforementioned amino functional group, the repeating unit [(VPGKG) ９ The number n in ] 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 succinimidylsuccinic acid-polyethylene glycol-succinimidylsuccinic acid. The two-component in-situ adhesive according to feature 2.

4. The elastin-like protein rich in the aforementioned amino functional groups contains or is composed of an amino acid sequence selected from 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 succinimidylsuccinic acid-polyethylene glycol-succinimidylsuccinic acid, where the average relative molecular weight of polyethylene glycol is 2000 to 10000. The two-component in-situ adhesive according to feature 3.

5. The elastin-like protein rich in the aforementioned amino functional groups contains or is composed of an amino acid sequence selected from SEQ ID NOs: 3, SEQ ID NOs: 4, and SEQ ID NOs:

5. and / or the succinimide group-containing compound is succinimidylsuccinic acid-polyethylene glycol-succinimidylsuccinic acid, where the average relative molecular weight of polyethylene glycol is 5,000 to 10,000. The two-component in-situ adhesive according to feature 4.

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

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

8. The mass-volume concentration of the aqueous solution of the elastin-like protein rich in the aforementioned amino functional group is 200 to 400 mg / mL. and / or, the mass-volume concentration of the aqueous solution of the succinimide group-containing compound is 0.5 to 1.0 g / mL. and / or, at the time of use, an aqueous solution of the succinimide group-containing compound and an aqueous solution of the elastin-like protein rich in amino functional groups are mixed in a 1:1 volume ratio. The two-component in-situ adhesive according to feature 7.

9. A method for producing a two-component in-situ adhesive according to any one of claims 1 to 8, Step 1) involves mixing the elastin-like protein rich in amino functional groups with water to produce an aqueous solution of the elastin-like protein rich in amino functional groups, Step 2) to dissolve the succinimide group-containing compound in water to produce an aqueous solution of the succinimide group-containing compound, Step 3) comprises selectively loading an aqueous solution of the elastin-like protein rich in the amino functional group produced in Step 1) and an aqueous solution of the succinimide group-containing compound produced in Step 2) into two-component syringes. A method for manufacturing a two-component in-situ adhesive.

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

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

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

13. The aforementioned tissue is at least one of the following: meniscal tissue, muscle tissue, tendon tissue, bone tissue, connective tissue, epidermal tissue, or mucosal tissue. and / or the organ is at least one of the skin, heart, liver, kidney, brain, or blood vessels, and / or, the repair involves promoting at least one of the following: closure of the damaged site, cell adhesion, proliferation, or differentiation. The use described in feature 12.

Images