Double-crosslinked fibrin gel, raw material composition thereof, kit, and use thereof

Abstract

The present invention provides a double cross-linked fibrin gel. The double cross-linked fibrin gel of the present invention is a solid hydrogel composed of a mesh structure having a closing function and a mesh structure having an adhesive function, the mesh structure having a closing function is a three-dimensional fibrin mesh structure, and the mesh structure having an adhesive function is a three-dimensional photosensitive gel mesh structure, in which there is a set of fibrin mesh structures inside each photosensitive gel mesh channel, each set of fibrin mesh structures is continuous as a whole, and the three-dimensional fibrin mesh structure exists in a disordered manner on the surface and inside of the solid hydrogel as a whole. The present invention also provides a raw material composition and a kit for preparing the double cross-linked fibrin gel, and the use of the kit in preparing an in situ rapid-clotting hemostatic material. The double cross-linked fibrin gel prepared using the kit has a rapid and efficient hemostatic effect and can be widely used for hemostasis of accidental wounds and surgical wounds.

Description

[Technical Field]

[0001] The present invention relates to the field of biomedical materials, and in particular to a double-crosslinked fibrin gel for hemostasis of bleeding due to accidental trauma or surgery, and a method for using the same. [Background technology]

[0002] Uncontrolled bleeding after trauma or during surgery is a leading cause of death worldwide, causing more than 2 million deaths each year. Uncontrolled bleeding during surgery or trauma often leads to complications and adverse outcomes. Therefore, controlling bleeding is an important measure to reduce complications and mortality and improve patient outcomes.

[0003] Currently, several topical hemostatic materials have been developed to help control bleeding during surgery. Commonly used surgical sealants on the market include fibrin glue and synthetic tissue adhesives. Fibrin glue is the most widely used hemostatic agent with excellent biocompatibility. It supports hemostasis in various surgical procedures by simulating the coagulation cascade reaction and forming fibrin clots at the bleeding site, blocking bleeding. However, fibrin glue loses its adhesive strength due to continuous tissue tension and blood flow, making it susceptible to being washed away by blood flow. This is detrimental to its hemostatic function. Furthermore, its hemostatic effect is limited by its poor adhesive strength to wet tissue. On the other hand, synthetic tissue adhesives, such as cyanoacrylate glue, have limited use due to their high cytotoxicity and difficulty in removal, despite their excellent adhesive ability.

[0004] To overcome the current bottleneck of bioadhesives, namely the low adhesive strength of bioadhesives on wet tissue surfaces, prior art has focused on the use of methacryloylated gelatin as a hemostatic gel material. This double-bond modified gelatin is obtained by functionalizing the free amino groups of gelatin with methacrylic anhydride to form methacrylamide groups. Under irradiation with specific wavelengths, a photoinitiator within the material absorbs light energy to generate free radicals, which then form bonds between methacryloylated gelatin molecules, resulting in the formation of a solid gel. Methacryloylated gelatin possesses excellent biocompatibility, as well as excellent mechanical properties and adhesive strength. However, its lack of procoagulant properties limits its hemostatic ability. Methacryloylated gelatin has a long photocuring time of 5–10 seconds, making it susceptible to being washed away by blood flow during the photocuring process. Therefore, in severe bleeding, a large amount of blood weakens its adhesive ability. To address this drawback, Luo et al. introduced hemocoagulase, which has clotting properties, into methacryloylated gelatin. The resulting hemostatic gel exhibited improved hemostatic efficacy. (Guo Y, Wang Y, Zhao X, et al. Snake extract-laden hemostatic bioadhesive gel cross-linked by visible light. Sci Adv. 2021. 7(29).) However, the hemocoagulase-loaded methacryloylated gel constructed in this study had limited hemocoagulase activity. Only a small amount of hemocoagulase on the gel surface was able to come into contact with blood. Furthermore, the fibrinogen concentration in blood was low (2–4 g / L), and the fibrin cross-links formed were insufficient to close the wound. Furthermore, methacryloylated gelatin's ability to close the wound was weak before light curing was complete. These factors significantly limited its hemostatic efficacy.In 2020, Wang et al. incorporated free thrombin and thrombin-loaded liposomes into methacryloylated gelatin to construct a thrombin-methacryloylated gelatin hydrogel. This hydrogel promoted hemostasis through early thrombin release and subsequently regulated multistage healing in diabetic wounds through sustained thrombin release (Chongyang W, Tianyi W, Guangwang L, et al. Promoting coagulation and activating SMAD3 phosphorylation in wound healing via a dual-release thrombin-hydrogel. Chemical Engineering Journal. 2020. 397(C).). The drawbacks of the gel constructed in this study were similar to those of Luo et al.'s study. The amount of thrombin released from the gel was low, resulting in limited contact with fibrinogen in the blood, which prevented the formation of fibrin bridges, resulting in weak wound closure and reduced hemostatic efficacy.

[0005] Furthermore, the prior art has also reported the use of other photocurable materials to produce hemostatic materials. For example, Chinese Patent Publication CN111116973A discloses a polyvinyl alcohol hemostatic porous material with active hemostatic properties. The sponge is obtained by photocatalytic crosslinking of modified polyvinyl alcohol, and then polymers with active hemostatic properties, such as chitosan and / or thrombin, are added to impart active hemostatic properties to the sponge. However, the hemostatic time reported in this paper is only 90 to 100 seconds, which inevitably results in a reduced hemostatic effect. This is due to the following reasons: 1) preformed sponges cannot fully contact wet tissue, resulting in a lower closure effect than gels formed in situ; 2) thrombin in the sponge is not easily released in a dry state, limiting its procoagulant function; and 3) the fibrinogen concentration in blood is low (2 to 4 g / L), resulting in insufficient fibrin crosslinks to close the wound. Therefore, it is difficult to meet the demand for rapid hemostasis when massive bleeding occurs during surgery. Furthermore, if there is a bleeding wound on an organ or the body surface, the expansion of the sponge limits its ability to compress the wound, weakening the hemostatic effect to some extent. Furthermore, when the sponge is removed, it may adhere to the hemostatic area, causing secondary damage to the wound.

[0006] A suitable hemostatic material should be independent of the in vivo clotting mechanism, be able to exert hemostatic effects even when the in vivo clotting mechanism fails, have high adhesive properties to wet tissue, and have a suitable rate of coagulation and hemostatic action. Therefore, it is particularly important to invent a new hemostatic material that can solve the problems of existing hemostatic materials, such as poor adhesive properties to wet tissue and limited hemostatic efficacy. Summary of the Invention [Problem to be solved by the invention]

[0007] In order to solve the above-mentioned drawbacks of the prior art, the main object of the present invention is to provide an adhesive that can achieve both the effects of accelerating coagulation and improving adhesiveness, thereby enabling rapid hemostasis and rapid gelation, and having high adhesiveness.

[0008] Another object of the present invention is to provide a raw material composition capable of preparing said adhesive, and a kit for clinical dissemination and use of said adhesive.

[0009] A further object of the present invention is to provide a method for stopping bleeding using the kit. [Means for solving the problem]

[0010] To achieve the above objectives, the present invention adopts the following technical solutions.

[0011] In a first aspect, the present invention provides a double-crosslinked fibrin gel, which is a solid hydrogel composed of a network structure having a closing function and a network structure having an adhesive function, wherein the network structure having a closing function is formed before the network structure having an adhesive function. The network structure having a closing function is a three-dimensional fibrin network, and the network structure having an adhesive function is a three-dimensional photosensitive gel network, wherein each photosensitive gel network channel contains a set of fibrin networks, each set of fibrin networks is generally continuous, and the three-dimensional fibrin network is generally present in a disordered manner across the surface and interior of the solid hydrogel.

[0012] In the double-crosslinked fibrin gel of the present invention, the 3D fibrin network functions as a scaffold and increases the strength of the gel. Its formation process converts fibrinogen in the blood into fibrin, thereby pre-clotting the wound. As the fibrin content increases, the gelation time of the solid hydrogel decreases, its adhesive strength to tissue decreases, but the gel pores become larger, improving its procoagulant function. The 3D photosensitive gel network contributes to the gel's strength and adhesive strength to tissue. As the photosensitive gel network content increases in the solid hydrogel, its adhesive strength to tissue also increases, but the gelation time also increases, the gel pores become smaller, and its procoagulant function decreases. Considering the different effects of the above two networks on the overall hemostatic performance of the gel, the present invention further optimized the proportions of the two networks in the gel through experiments. In a preferred double-crosslinked fibrin gel, the volume ratio of the three-dimensional fibrin network to the three-dimensional photosensitive gel network is 0.5 to 3, preferably 0.5 to 2, and most preferably 1. At these optimal volume ratios, the two networks provide excellent hemostatic performance to the entire gel; in particular, when the volume ratio of the fibrin network to the photosensitive gel network reaches 1:1, the hemostatic performance of the gel is optimized, and coagulation can be rapidly promoted while improving the strength and adhesive force of the gel.

[0013] In the double-crosslinked fibrin gel of the present invention, the photosensitive gel can be formed by photocrosslinking of various existing photocurable polymeric materials (i.e., photosensitive materials). The photosensitive material may be a methacryloylated polymer or its derivative, a polyacrylate polymer or its derivative, or a polymer composite material containing them.

[0014] Furthermore, the methacryloylated high molecular weight polymer or derivative thereof may be any one or a mixture of two or more selected from the group consisting of methacryloylated gelatin or derivatives thereof, methacryloylated hyaluronic acid or derivatives thereof, methacryloylated sodium alginate or derivatives thereof, methacryloylated silk fibroin or derivatives thereof, methacryloylated chitosan or derivatives thereof, and methacryloylated carboxymethyl chitosan or derivatives thereof. The high molecular weight polyacrylate polymer or derivative thereof may be selected from polyether diacrylate or derivatives thereof, or polyethylene glycol diacrylate or derivatives thereof. The most preferred photosensitive material of the present invention is methacryloylated gelatin or derivatives thereof, or methacryloylated silk fibroin or derivatives thereof.

[0015] Furthermore, the derivatives of the methacryloylated high molecular weight polymer include polymers in which one or more of its functional groups have been modified. The modifiable functional groups of the methacryloylated gelatin include one or more of amino, carboxy, mercapto, hydroxy, or guanidino. The derivatives of the methacryloylated hyaluronic acid include polymers in which one or more of its functional groups have been modified, and the modifiable functional groups include one or more of hydroxy, carboxy, acetamide, or hydroxymethyl. The derivatives of the methacryloylated sodium alginate include polymers in which one or more of its functional groups have been modified, and the modifiable functional groups include one or more of carboxy and hydroxy. The derivatives of the methacryloylated silk fibroin include polymers in which one or more of its functional groups have been modified, and the modifiable functional groups include one or more of amino, carboxy, mercapto, hydroxy, or guanidino. The derivatives of methacryloylated chitosan include polymers in which one or more of the functional groups have been modified or which have undergone multiple chemical reactions, where the functional groups that can be modified include one or two of amino or hydroxy, and the multiple chemical reactions that can occur include one or more of alkylation, acylation, carboxymethylation, hydrolysis, oxidation, and reduction chemical reactions.

[0016] The molecular weight of the methacryloylated high molecular weight polymer or its derivative is in the range of 5 to 400 kDa, and the molecular weight of the polyacrylate high molecular weight polymer or its derivative is in the range of 700 to 1000 kDa.

[0017] Furthermore, polymer composite material systems containing the methacryloylated polymer or its derivatives include methacryloylated gelatin-polyvinyl alcohol systems, methacryloylated gelatin-polyurethane systems, methacryloylated gelatin-polylactic acid systems, methacryloylated gelatin-cellulose systems, methacryloylated hyaluronic acid-polyvinyl alcohol systems, methacryloylated hyaluronic acid-polyurethane systems, methacryloylated hyaluronic acid-polylactic acid systems, methacryloylated hyaluronic acid-cellulose systems, methacryloylated sodium alginate-polyvinyl alcohol systems, methacryloylated sodium alginate-polyurethane systems, methacryloylated sodium alginate-polylactic acid systems, and methacryloylated sodium alginate-cellulose systems. The polymer may include one or more of methacryloylated silk fibroin-polyvinyl alcohol systems, methacryloylated silk fibroin-polyurethane systems, methacryloylated silk fibroin-polylactic acid systems, methacryloylated silk fibroin-cellulose systems, methacryloylated chitosan-polyvinyl alcohol systems, methacryloylated chitosan-polyurethane systems, methacryloylated chitosan-polylactic acid systems, methacryloylated chitosan-cellulose systems, methacryloylated carboxymethyl chitosan-polyvinyl alcohol systems, methacryloylated carboxymethyl chitosan-polyurethane systems, methacryloylated carboxymethyl chitosan-polylactic acid systems, and methacryloylated carboxymethyl chitosan-cellulose systems.

[0018] In the double-crosslinked fibrin gel of the present invention, the fibrin network may be formed by enzymatic crosslinking of fibrinogen, which may be any one of human fibrinogen, bovine fibrinogen, and porcine fibrinogen.

[0019] In a second aspect, the present invention provides a raw material composition for preparing the double cross-linked fibrin adhesive according to the first aspect of the present invention, comprising Composition A and Composition B, wherein Composition A comprises 10 to 200 parts by weight of a photosensitive material, 1 to 3 parts by weight of a photoinitiator, 0.14 to 0.28 parts by weight of an enzyme, and 1.11 to 8.88 parts by weight of a water-soluble inorganic calcium salt, and Composition B comprises 5 to 100 parts by weight of the photosensitive material, 1 to 2 parts by weight of a photoinitiator, and 30 to 50 parts by weight of fibrinogen, and the mass ratio of Composition A to Composition B is 1.4:10 to 14:1, preferably 1.4:1 to 1.4:10, more preferably 1.4:1 to 1.4:5, and most preferably 1.4:1.

[0020] In a preferred embodiment of the present invention, the weight part of the photosensitive material in Composition A is greater than the weight part of the photosensitive material in Composition B. The above weight parts of the photosensitive materials in Composition A and Composition B are advantageous for the enzyme in Composition A and the fibrinogen in Composition B to undergo an enzymatic reaction to form a stabilized fibrin network.

[0021] In the raw material composition of the present invention, the fibrinogen can form a fibrin network through an enzymatic cross-linking reaction under the action of the enzyme, and the photosensitive material can form a photosensitive gel through a photo-cross-linking reaction under the action of the photoinitiator. Therefore, a dual-crosslinking fibrin adhesive can be prepared by mixing Composition A and Composition B in the raw material composition of the present invention in the above mass ratio and then irradiating them with light. This adhesive is a solid hydrogel, whose structure contains both a 3D fibrin network and a 3D photosensitive gel network. Each photosensitive gel network channel contains a set of fibrin networks, and each set of fibrin networks is connected as a whole. The 3D fibrin network is distributed randomly throughout the surface and interior of the solid hydrogel. When this solid hydrogel is formed on a bleeding wound, it instantly (approximately 1 s) forms a fibrin clot on the surface of the wound, pre-clotting the wound and preventing blood loss. Furthermore, the enzymes in the fibrin clot convert fibrinogen in the blood into clots, thereby efficiently promoting coagulation. Furthermore, under photoexcitation, the photosensitive material can form a photocurable gel within 5 to 10 seconds. The photocurable gel has high adhesive strength, withstands the impact of blood flow, and prevents fibrin crosslinks from being washed away by blood. In short, in the raw material composition of the present invention, after mixing Composition A and Composition B, a fibrin network serving as a scaffold is immediately formed, and then a photosensitive gel is also rapidly formed. The fibrin network is covered by the photosensitive gel that is formed later.

[0022] In the present invention, we have experimentally found that the mass ratio of Composition A to Composition B in the raw material composition is related to the wound pre-closure effect and adhesive strength of the double cross-linked fibrin adhesive. When the mass ratio of Composition A to Composition B is within the range of 1.4:10 to 1.4:1, as the proportion of Composition A increases, the procoagulation function, pre-closure effect, and adhesive strength of the double cross-linked fibrin adhesive increase. When the mass ratio of Composition A to Composition B is within the range of 1.4:1 to 14:1, as the proportion of Composition A increases, the procoagulation function, pre-closure effect, and adhesive strength of the double cross-linked fibrin adhesive do not increase further. This indicates that a mass ratio of Composition A to Composition B of 1.4:1 achieves the best hemostatic effect and the best material utilization. At this ratio, the volume ratio of the two networks generated by cross-linking reaches approximately 1:1, resulting in optimal procoagulation function and adhesive strength for the prepared gel.

[0023] In a preferred raw material composition of the present invention, composition A contains 80 to 200 parts by weight of a photosensitive material, 1 to 3 parts by weight of a photoinitiator, 0.14 to 0.28 parts by weight of an enzyme, and 1.11 to 8.88 parts by weight of a water-soluble inorganic calcium salt, and composition B contains 30 to 100 parts by weight of a photosensitive material, 1 to 2 parts by weight of a photoinitiator, and 30 to 50 parts by weight of fibrinogen.

[0024] In a more preferred raw material composition of the present invention, composition A contains 100 to 200 parts by weight of a photosensitive material, 1 to 3 parts by weight of a photoinitiator, 0.14 to 0.28 parts by weight of an enzyme, and 1.11 to 8.88 parts by weight of a water-soluble inorganic calcium salt, and composition B contains 30 to 50 parts by weight of a photosensitive material, 1 to 2 parts by weight of a photoinitiator, and 30 to 50 parts by weight of fibrinogen.

[0025] In the most preferred raw material composition of the present invention, composition A contains 100 to 150 parts by weight of a photosensitive material, 1 to 3 parts by weight of a photoinitiator, 0.14 to 0.28 parts by weight of an enzyme, and 1.11 to 8.88 parts by weight of a water-soluble inorganic calcium salt, and composition B contains 30 to 50 parts by weight of a photosensitive material, 1 to 2 parts by weight of a photoinitiator, and 30 to 50 parts by weight of fibrinogen.

[0026] In a third aspect, the present invention also provides a method for preparing a liquid precursor composition, the method comprising the steps of: preparing a mixed solution of a photosensitive material and a photoinitiator in a solvent; mixing the mixed solution with a solution containing hemopexin and calcium ions to obtain a first precursor solution, controlling the concentration ratio of the photosensitive material, photoinitiator, enzyme, and calcium ions in the first precursor solution to 10-200:1-3:0.14-0.28:1.11-8.88; and mixing the mixed solution with a solution containing fibrinogen to obtain a second precursor solution, controlling the concentration ratio of the photosensitive material, photoinitiator, and fibrinogen in the second precursor solution to 5-100:1-2:30-50, thereby obtaining a liquid precursor composition containing the first precursor solution and the second precursor solution. The liquid precursor composition may be further processed by conventional methods to obtain a solid precursor composition such as a freeze-dried powder, sponge, or granules.

[0027] In the preparation method of the present invention, the time for leaving the first precursor solution in a room temperature environment is controlled to be less than 30 minutes in order to maintain the activity of the photosensitive material in the first precursor solution.

[0028] In the preparation method of the present invention, taking into consideration the uniform distribution of fibrinogen and the photocrosslinking rate of the photosensitive material, it is preferable to control the concentration of the photosensitive material in the first precursor solution to be higher than 0.5% (w / v) and the concentration of the photosensitive material in the second precursor solution to be lower than the concentration of the photosensitive material in the first precursor solution. This allows the fibrinogen solution to be more easily dispersed uniformly in the second precursor solution, which contains a relatively low concentration of photosensitive material. When the two precursor solutions are thoroughly mixed, they come into rapid and complete contact with the enzyme, which immediately causes complete enzymatic crosslinking and forms a uniformly distributed fibrin network. Furthermore, a high concentration of the photosensitive material in the first precursor solution increases the overall concentration of the photosensitive material in the mixed two precursor solutions, achieving a more ideal concentration required for gelation. This is advantageous for shortening the photocrosslinking time and improving the adhesion and strength of the gel.

[0029] In the preparation method of the present invention, when the first precursor solution and the second precursor solution are prepared, the temperature of the mixed solution is preferably controlled to 37° C. or less.

[0030] A more preferred method for preparing the raw material composition injection of the present invention is specifically as follows: Step 1) of preparing a first mixed solution by dissolving a photosensitive material and a photoinitiator in a solvent, and controlling the concentration ratio of the photosensitive material to the photoinitiator to be 10 to 200:1 to 3 and the concentration of the photosensitive material to be 0.5% to 30% (w / v); Step 2) of preparing a second mixed solution by dissolving a photosensitive material and a photoinitiator in a solvent, and controlling the concentration ratio of the photosensitive material to the photoinitiator to be 5 to 100:1 to 2 and the concentration of the photosensitive material to be lower than that of the first mixed solution described in step 1); Step 3) mixing the first mixed solution prepared in step 1) with a solution containing an enzyme and calcium ions to obtain a first precursor solution, and controlling the concentration ratio of the photosensitive material, the photoinitiator, the enzyme, and the calcium ions to 10-200:1-3:0.14-0.28:1.11-8.88; and step 4) mixing the second mixed solution prepared in step 2) with a solution containing fibrinogen to obtain a second precursor solution, and controlling the concentration ratio of the photosensitive material, photoinitiator, and fibrinogen to 5-100:1-2:30-50.

[0031] In the preparation method of the present invention, the solution containing the enzyme and calcium ions is prepared by adding a solvent and a water-soluble inorganic calcium salt solution to the enzyme, dissolving the enzyme completely, and forming a Ca 2+ The enzyme activity in the resulting solution is 500 IU to 2000 IU / ml, Ca 2+ The concentration is preferably controlled to 60 to 100 mmol / L.

[0032] In the preparation method of the present invention, the concentration of fibrinogen in the fibrinogen-containing solution is preferably 5% to 10% (w / v).

[0033] In a preferred preparation method of the present invention, the concentration of the photosensitive material in the first precursor solution is controlled to 1% to 30% (w / v), more preferably 8% to 30% (w / v), and even more preferably 10% to 20%.

[0034] In a preferred preparation method of the present invention, the enzyme activity in the first precursor solution is controlled to 200 IU / ml or more, preferably 500 IU / ml or more, more preferably 1000 IU / ml or more.

[0035] In a preferred preparation method of the present invention, the concentration of calcium ions in the first precursor solution is controlled to 20 mmol / L or more, preferably 30 mmol / L or more, more preferably 40 mmol / L or more.

[0036] In a preferred preparation method of the present invention, the concentration of the photosensitive material in the second precursor solution is controlled to be 0.5% (w / v) or more and not more than the concentration of the photosensitive material in the first precursor solution, more preferably 1% (w / v) or more and not more than the concentration of the photosensitive material in the first precursor solution, and even more preferably 1% to 10% (w / v) and not more than the concentration of the photosensitive material in the first precursor solution.

[0037] In a preferred preparation method of the present invention, the concentration of fibrinogen in the second precursor solution is controlled to 3% (w / v) or more, more preferably 3% to 5% (w / v).

[0038] In a fourth aspect, the present invention also provides a kit for preparing a double crosslinked fibrin gel according to the first aspect of the present invention, comprising a first precursor reagent and a second precursor reagent packaged individually, the first precursor reagent containing 10 to 200 parts by weight of a photosensitive material, 1 to 3 parts by weight of a photoinitiator, 0.14 to 0.28 parts by weight of an enzyme, and 3.33 to 5.55 parts by weight of a water-soluble inorganic calcium salt, and the second precursor reagent containing 5 to 100 parts by weight of a photosensitive material, 1 to 2 parts by weight of a photoinitiator, and 30 to 50 parts by weight of fibrinogen, and the mass ratio of the first precursor reagent to the second precursor reagent is 1.4:10 to 14:1, preferably 1.4:1 to 1.4:10, more preferably 1.4:1 to 1.4:5, and most preferably 1.4:1.

[0039] In a preferred kit of the present invention, the first precursor reagent contains 80 to 200 parts by weight of a photosensitive material, 1 to 3 parts by weight of a photoinitiator, 0.14 to 0.28 parts by weight of an enzyme, and 3.33 to 5.55 parts by weight of a water-soluble inorganic calcium salt, and the second precursor reagent contains 30 to 100 parts by weight of a photosensitive material, 1 to 2 parts by weight of a photoinitiator, and 30 to 50 parts by weight of fibrinogen.

[0040] In a more preferred kit of the present invention, the first precursor reagent contains 100 to 200 parts by weight of a photosensitive material, 1 to 3 parts by weight of a photoinitiator, 0.14 to 0.28 parts by weight of an enzyme, and 3.33 to 5.55 parts by weight of a water-soluble inorganic calcium salt, and the second precursor reagent contains 30 to 50 parts by weight of a photosensitive material, 1 to 2 parts by weight of a photoinitiator, and 30 to 50 parts by weight of fibrinogen.

[0041] In the most preferred kit of the present invention, the first precursor reagent contains 100 to 150 parts by weight of a photosensitive material, 1 to 3 parts by weight of a photoinitiator, 0.14 to 0.28 parts by weight of an enzyme, and 3.33 to 5.55 parts by weight of a water-soluble inorganic calcium salt, and the second precursor reagent contains 30 to 50 parts by weight of a photosensitive material, 1 to 2 parts by weight of a photoinitiator, and 30 to 50 parts by weight of fibrinogen.

[0042] In the kit of the present invention, the photosensitive material contained in the first precursor reagent and the second precursor reagent is a photosensitive biohydrogel material, which may be any of various existing photocurable polymer materials. The photoinitiator contained in the first precursor reagent and the second precursor reagent is a substance that absorbs light energy and generates free radicals when irradiated with light of a specific wavelength. The photoinitiator can absorb light energy and generate free radicals, which then form bonds between molecules of the photosensitive material, thereby rapidly forming a solid gel. A suitable photosensitive material should have good biocompatibility and degradability, as well as good mechanical and adhesive properties.

[0043] In the raw material composition or kit of the present invention, the photosensitive material may be a methacryloylated high molecular weight polymer or a derivative thereof, a polyacrylate high molecular weight polymer or a derivative thereof, or a polymer composite material system containing these.

[0044] Furthermore, the methacryloylated high molecular weight polymer or derivative thereof may be any one or a mixture of two or more selected from the group consisting of methacryloylated gelatin or derivatives thereof, methacryloylated hyaluronic acid or derivatives thereof, methacryloylated sodium alginate or derivatives thereof, methacryloylated silk fibroin or derivatives thereof, methacryloylated chitosan or derivatives thereof, and methacryloylated carboxymethyl chitosan or derivatives thereof. The high molecular weight polyacrylate polymer or derivative thereof may be selected from polyether diacrylate or derivatives thereof, or polyethylene glycol diacrylate or derivatives thereof. The most preferred photosensitive material of the present invention is methacryloylated gelatin or derivatives thereof, or methacryloylated silk fibroin or derivatives thereof.

[0045] Furthermore, the derivatives of the methacryloylated high molecular weight polymer include polymers in which one or more of its functional groups have been modified. The modifiable functional groups of the methacryloylated gelatin include one or more of amino, carboxy, mercapto, hydroxy, or guanidino. The derivatives of the methacryloylated hyaluronic acid include polymers in which one or more of its functional groups have been modified, and the modifiable functional groups include one or more of hydroxy, carboxy, acetamide, or hydroxymethyl. The derivatives of the methacryloylated sodium alginate include polymers in which one or more of its functional groups have been modified, and the modifiable functional groups include one or more of carboxy and hydroxy. The derivatives of the methacryloylated silk fibroin include polymers in which one or more of its functional groups have been modified, and the modifiable functional groups include one or more of amino, carboxy, mercapto, hydroxy, or guanidino. The derivatives of methacryloylated chitosan include polymers in which one or more of its functional groups have been modified or subjected to multiple chemical reactions, where the functional groups that can be modified include one or two of amino or hydroxy, and the multiple chemical reactions that can occur include one or more of alkylation, acylation, carboxymethylation, hydrolysis, oxidation, and reduction chemical reactions.

[0046] The molecular weight of the methacryloylated high molecular weight polymer or its derivative is in the range of 5 to 400 kDa, and the molecular weight of the polyacrylate high molecular weight polymer or its derivative is in the range of 700 to 1000 kDa.

[0047] Furthermore, polymer composite material systems containing the methacryloylated polymer or its derivatives include methacryloylated gelatin-polyvinyl alcohol systems, methacryloylated gelatin-polyurethane systems, methacryloylated gelatin-polylactic acid systems, methacryloylated gelatin-cellulose systems, methacryloylated hyaluronic acid-polyvinyl alcohol systems, methacryloylated hyaluronic acid-polyurethane systems, methacryloylated hyaluronic acid-polylactic acid systems, methacryloylated hyaluronic acid-cellulose systems, methacryloylated sodium alginate-polyvinyl alcohol systems, methacryloylated sodium alginate-polyurethane systems, methacryloylated sodium alginate-polylactic acid systems, and methacryloylated sodium alginate-cellulose systems. The polymer may include one or more of methacryloylated silk fibroin-polyvinyl alcohol systems, methacryloylated silk fibroin-polyurethane systems, methacryloylated silk fibroin-polylactic acid systems, methacryloylated silk fibroin-cellulose systems, methacryloylated chitosan-polyvinyl alcohol systems, methacryloylated chitosan-polyurethane systems, methacryloylated chitosan-polylactic acid systems, methacryloylated chitosan-cellulose systems, methacryloylated carboxymethyl chitosan-polyvinyl alcohol systems, methacryloylated carboxymethyl chitosan-polyurethane systems, methacryloylated carboxymethyl chitosan-polylactic acid systems, and methacryloylated carboxymethyl chitosan-cellulose systems.

[0048] In the preferred raw material composition or kit of the present invention, the photoinitiator may be any one or more compositions selected from lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, ethyl 2,4,6-trimethylbenzoylphenylphosphinate, 2-methyl-1-(4-methylthiophenyl)-2-morpholinopropan-1-one, methyl 2-benzoylbenzoate, 2-benzyl-2-dimethylamino-1-(4-morpholinophenyl)butanone, and 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], and is most preferably lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate.

[0049] In the preferred raw material composition or kit of the present invention, the enzyme may be any one selected from human thrombin, recombinant human thrombin, bovine thrombin, porcine thrombin, and hemocoagulase.

[0050] In the preferred raw material composition or kit of the present invention, the fibrinogen may be any one selected from human fibrinogen, bovine fibrinogen, and porcine fibrinogen.

[0051] In a preferred raw material composition or kit of the present invention, the water-soluble inorganic calcium salt may be selected from calcium chloride, calcium nitrate, or calcium sulfate, but is most preferably calcium chloride.

[0052] In a preferred kit of the present invention, the first precursor reagent and / or the second precursor reagent further include auxiliary materials and / or additives. The auxiliary materials are one or more selected from glycine, arginine hydrochloride, sodium citrate, sucrose, and sodium chloride. The additives are one or more selected from growth factors, interleukins, vitamins, and silver ions. The growth factors may further be one or more selected from platelet growth factors, epidermal growth factors, and fibroblast growth factors. The interleukins may further be one or more selected from interleukin 2, interleukin 6, and interleukin 8. The vitamins may further be one or more selected from vitamin B, vitamin C, vitamin E, and vitamin K.

[0053] In the kit of the present invention, the first precursor reagent and / or the second precursor reagent may be in a number of specific pharmaceutical or clinically acceptable dosage forms, such as a lyophilized powder, a sponge, or a granule.

[0054] The kit of the present invention may further include a separately packaged preparation solvent, which may be any one or a mixture of a plurality of phosphate buffer solution, HEPES biological buffer solution, 0.9% sodium chloride solution, calcium chloride solution, and deionized water. The preparation solvent is preferably in the form of an injection.

[0055] The kit of the present invention may further include an instruction manual that describes how to use the kit.

[0056] In a fifth aspect, the present invention also provides a method for performing on-site rapid hemostasis on a bleeding wound using the kit according to the fourth aspect of the present invention, the method comprising: preparing the first precursor reagent and the second precursor reagent into injectable solutions using a preparation solvent, respectively; uniformly injecting or spraying the solutions onto the bleeding wound; and then irradiating the wound with light in a wavelength range of 290 to 480 nm for 10 to 60 seconds to rapidly form a solid hydrogel in situ at the bleeding wound.

[0057] The bleeding wound includes organ bleeding caused by accidental trauma or during surgery, and the organ may be the liver, spleen, kidney, gastrointestinal tract, heart, or skin.

[0058] In the use of the present invention, when the kit is injected into a bleeding wound, (1) it instantly (approximately 1 second) forms a fibrin clot on the surface of the wound, pre-clotting the wound and preventing blood outflow, thereby strengthening the weak closure effect of the photosensitive material until photocuring is complete. (2) Furthermore, the enzymes in the fibrin clot convert fibrinogen in the blood into clots, effectively promoting coagulation. (3) Furthermore, under photoexcitation, the photosensitive material can form a photocurable gel within 5 to 10 seconds. The photocurable gel has high adhesive strength, can withstand impact from blood flow, and prevents fibrin crosslinks from being washed away by blood. Therefore, by using the kit or kit of the present invention to prepare a double-crosslinked fibrin gel in situ, it is possible to combine the rapid generation of fibrin crosslinks and the high adhesive strength due to photocrosslinking to obtain a double-crosslinked fibrin gel with a fibrin crosslinked network and a photocrosslinked network structure. [Effects of the Invention]

[0059] Compared with the prior art, the advantages of the present invention are as follows: short gelation time, fast setting speed, strong adhesive strength to wet tissue, and good hemostatic effect. (1) The double cross-linked fibrin gel kit of the present invention generates fibrin cross-links immediately (about 1 s) after mixing, which acts as a preliminary closure and blocks the impact of blood flow. (2) The enzyme in the double cross-linked fibrin gel kit of the present invention can convert fibrinogen in blood into fibrin cross-links, and has efficient coagulation promoting ability. (3) The double-crosslinked fibrin gel kit of the present invention can undergo a photocrosslinking reaction within 5 to 10 seconds under the excitation of ultraviolet or visible light to form a photocurable gel, which imparts high adhesive strength to wet tissue and prevents the fibrin crosslinks from being washed away by blood. The double cross-linked fibrin gel according to the present invention has an excellent coagulation promoting function, a fast setting rate, high adhesive strength to wet tissues, and a rapid hemostatic effect, and can therefore be used for hemostasis of bleeding from the liver, spleen, kidneys, heart, gastrointestinal tract, and skin following accidental trauma or surgery. [Brief explanation of the drawings]

[0060] [Figure 1] 1 is an SEM image of fibrin crosslinks in Comparative Example 1. [Figure 2] 1 is an SEM image of photocrosslinking of methacryloylated gelatin in the precursor solution of Comparative Example 2. [Figure 3] 1 is an SEM image of the double-crosslinked fibrin gel of Example 1. [Figure 4] The hemostasis time of Examples 1, 9, 14, 19, 25, and 31 and Comparative Examples 1 to 6 was compared. [Figure 5] The amount of blood loss in Examples 1, 9, 14, 19, 25, and 31 and Comparative Examples 1 to 6 was compared. DETAILED DESCRIPTION OF THE INVENTION

[0061] The technical problems to be solved, technical solutions, and beneficial effects of the present invention will be described in detail below with reference to specific examples. The following examples are intended to help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any way. However, those skilled in the art may make minor modifications and improvements without departing from the concept of the present invention, which fall within the scope of protection of the present invention.

[0062] The present invention provides a double-crosslinked fibrin gel, which is a solid hydrogel composed of both a three-dimensional photosensitive gel network and a three-dimensional fibrin network. The microstructure of this double-crosslinked fibrin gel is shown in Figure 3, and it has both a fibrin-crosslinked network structure and a photosensitive material-crosslinked porous structure. Each channel of the porous structure contains a set of fibrin networks, and each set of fibrin networks is generally continuous. The three-dimensional fibrin network is generally present in a disordered manner across the surface and interior of the solid hydrogel. The fibrin network structure functions as a scaffold for the photosensitive gel network channels, and the pore walls of the photosensitive gel porous structure surround the fibrin network structure.

[0063] The double-crosslinked fibrin gel is prepared by the following method. (1) Preparation of Composition A solution: A calcium ion-containing enzyme solution is dissolved in a mixed solution containing a photosensitive material and a photoinitiator, and the mixture is mixed uniformly to obtain Composition A solution containing a photosensitive material, a photoinitiator, and an enzyme. The concentration of the photosensitive material in the resulting Composition A solution is controlled to 1% (w / v) or more, preferably 3% (w / v) or more, and more preferably 3% to 20% (w / v), and the enzyme activity is controlled to 200 IU / mL or more, preferably 500 IU / mL or more, and more preferably 1000 IU / mL or more. (2) Preparation of Composition B Solution: The fibrinogen solution is added to the mixed solution containing the photosensitive material and photoinitiator, and the mixture is mixed uniformly to obtain Composition B solution containing the photosensitive material, photoinitiator, and fibrinogen. The concentration of the photosensitive material in the resulting Composition B solution is controlled to 0.5% (w / v) or more, preferably 1% to 10% (w / v), and the concentration of fibrinogen is controlled to 3% (w / v) or more, preferably 3% to 5% (w / v). (3) Storage method: The obtained composition A solution and composition B solution are freeze-dried in a volume ratio of 1:10 to 10:1, and stored in the form of a sponge. (4) Preparation of double-crosslinked fibrin gel using the above freeze-dried sponge: Sponge-like component A and sponge-like component B were dissolved in a solvent, respectively, to obtain injectable solutions of component A and component B. Equal volumes of component A solution and component B solution were uniformly injected / sprayed onto the bleeding site, and then irradiated with blue light or ultraviolet light for 10 to 60 seconds to rapidly form a solid hydrogel in situ. Preferred injection devices for use with the injectable solution include a double syringe, syringe, or Pasteur pipette. In the above preparation form, the solvent may be any one or a combination of a phosphate buffer solution, a HEPES biological buffer solution, a 0.9% sodium chloride solution, a calcium chloride solution, and deionized water, and the amount used is not particularly limited and can be prepared according to the actually required concentration. Based on the above embodiments, the present invention will be further illustrated by the following examples. [Example]

[0064] The method for preparing the double-crosslinked fibrin gel in this example includes the following steps: (1) Preparation of methacryloylated gelatin-lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate precursor solution: The desired volume of 0.9% sodium chloride solution was added to powdered lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate and dissolved by heating in a water bath to obtain two concentrations of lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate solution (0.25% (w / v) and 0.5% (w / v)). Desired concentrations of phenyl(2,4,6-trimethylbenzoyl)phosphinate lithium solution were added to desired weights of solid methacryloylated gelatin and dissolved by heating in a water bath to obtain two mass / volume percentage (w / v) methacryloylated gelatin-lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate precursor solutions: 26% (w / v) methacryloylated gelatin-0.5% (w / v) phenyl(2,4,6-trimethylbenzoyl)phosphinate lithium, and 10% (w / v) methacryloylated gelatin-0.25% (w / v) phenyl(2,4,6-trimethylbenzoyl)phosphinate lithium. (2) Preparation of hemopexin solution: A calcium chloride solution of the desired volume and concentration was injected into hemopexin and completely dissolved to obtain a hemopexin solution with a hemopexin activity of 2000 IU / mL. 2+ The concentration is 80mmol / L. (3) Preparation of fibrinogen solution: The desired weight of fibrinogen was slowly added to preheated 0.9% sodium chloride solution until completely dissolved, yielding a 10% (w / v) fibrinogen solution. (4) Preparation of component A solution: The hemopexin solution obtained in step (2) was added to the 26% (w / v) methacryloylated gelatin-0.5% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate precursor solution obtained in step (1) and mixed uniformly to obtain component A solution: 13% (w / v) methacryloylated gelatin-0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate-1000 IU / mL hemopexin. (5) Preparation of component B solution: The fibrinogen solution obtained in step (3) was added to the precursor solution of 10% (w / v) methacryloylated gelatin-0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate obtained in step (1) and mixed uniformly to obtain component B solution: 5% (w / v) methacryloylated gelatin-0.125% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate-5% (w / v) fibrinogen. (6) Storage: The obtained component A solution and component B solution were freeze-dried in a volume ratio of 1:1 and stored as sponges. (7) Method of Use: Sponge-like components A and B were dissolved in a 0.9% sodium chloride solution at a 1:1 volume ratio to obtain injectable solutions of components A and B. Equal volumes of component A and B solutions were placed in a double syringe, and the components A and B solutions were injected / sprayed into the bleeding site using a nozzle. After irradiating with blue light for 10-60 seconds, a solid hydrogel was formed in situ. The resulting gel had a 1:1 volume ratio of fibrin crosslinks to photocrosslinks. (8) The structure of the solid gel is shown in Figure 3. This is a solid hydrogel composed of both a three-dimensional fibrin network and a three-dimensional photosensitive gel network, with one set of fibrin networks present inside the porous structure of the cross-linked methacryloylated gelatin formed, each set of fibrin networks being continuous as a whole, and the three-dimensional fibrin network being present in a disordered manner throughout the surface and interior of the solid hydrogel. [Example]

[0065] A mixed solution of 10% (w / v) methacryloylated gelatin, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 1000 IU / mL hemopexin was prepared as Component A solution, and a mixed solution of 5% (w / v) methacryloylated gelatin, 0.125% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 5% (w / v) fibrinogen was prepared as Component B solution. The composition, preparation method, and usage method were the same as those in Example 1, except that the concentration of methacryloylated gelatin in Component A solution prepared in step (4) was adjusted to 10% (w / v). [Example]

[0066] A mixed solution of 8% (w / v) methacryloylated gelatin, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 1000 IU / mL hemopexin was prepared as Component A solution, and a mixed solution of 5% (w / v) methacryloylated gelatin, 0.125% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 5% (w / v) fibrinogen was prepared as Component B solution. The composition, preparation method, and usage method were the same as those in Example 1, except that the concentration of methacryloylated gelatin in Component A solution prepared in step (4) was adjusted to 8% (w / v). [Example]

[0067] A mixed solution of 5% (w / v) methacryloylated gelatin, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 1000 IU / mL hemopexin was prepared as Component A solution, and a mixed solution of 5% (w / v) methacryloylated gelatin, 0.125% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 5% (w / v) fibrinogen was prepared as Component B solution. The composition, preparation method, and usage method of Component A solution prepared in step (4) were the same as those in Example 1, except that the concentration of methacryloylated gelatin was 5% (w / v). In the gel obtained in this manner, the volume ratio of fibrin crosslinks to photocrosslinks was 2:1. [Example]

[0068] A mixed solution of 13% (w / v) methacryloylated gelatin, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 1000 IU / mL hemopexin was prepared as Component A solution, and a mixed solution of 3% (w / v) methacryloylated gelatin, 0.125% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 5% (w / v) fibrinogen was prepared as Component B solution. The composition, preparation method, and usage method were the same as those in Example 1, except that the concentration of methacryloylated gelatin in Component B solution prepared in step (5) was adjusted to 3% (w / v). [Example]

[0069] A mixed solution of 13% (w / v) methacryloylated gelatin, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 500 IU / mL hemopexin was prepared as Component A, and a mixed solution of 5% (w / v) methacryloylated gelatin, 0.125% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 5% (w / v) fibrinogen was prepared as Component B. The composition, preparation method, and usage method were the same as those in Example 1, except that the hemopexin activity of Component A prepared in step (4) was adjusted to 500 IU / mL. [Example]

[0070] A mixed solution of 13% (w / v) methacryloylated gelatin, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 250 IU / mL hemopexin was prepared as Component Solution A, and a mixed solution of 5% (w / v) methacryloylated gelatin, 0.125% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 5% (w / v) fibrinogen was prepared as Component Solution B. The composition, preparation method, and usage method were the same as those in Example 1, except that the hemopexin activity of Component Solution A prepared in step (4) was adjusted to 250 IU / mL. [Example]

[0071] A mixed solution of 13% (w / v) methacryloylated gelatin, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 1000 IU / mL hemopexin was prepared as Component A solution, and a mixed solution of 5% (w / v) methacryloylated gelatin, 0.125% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 3% (w / v) fibrinogen was prepared as Component B solution. The composition, preparation method, and usage method of Component B solution prepared in step (5) were the same as those in Example 1, except that the fibrinogen concentration was 3% (w / v). In the gel obtained in this manner, the volume ratio of fibrin crosslinks to photocrosslinks was 1:2. [Example]

[0072] A mixed solution of 8% (w / v) methacryloylated hyaluronic acid, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 1000 IU / mL hemopexin was prepared as component A, and a mixed solution of 5% (w / v) methacryloylated hyaluronic acid, 0.125% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 5% (w / v) fibrinogen was prepared as component B. The preparation and use methods were the same as in Example 1, except that: 1. In this example, the photosensitive material in Components A and B was methacryloylated hyaluronic acid, and the concentration of methacryloylated hyaluronic acid in the Component A solution was 8% (w / v), and 2. The preparation of the precursor solution of methacryloylated hyaluronic acid-lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate did not require heating and could be carried out at room temperature. In the gel thus obtained, the volume ratio of fibrin crosslinks to photocrosslinks was 1:1. [Example]

[0073] A mixed solution of 5% (w / v) methacryloyl hyaluronic acid, 0.25% (w / v) phenyl (2,4,6-trimethylbenzoyl) phosphinate lithium, and 1000 IU / mL hemopexin was prepared as component A solution, and a mixed solution of 5% (w / v) methacryloyl hyaluronic acid, 0.125% (w / v) phenyl (2,4,6-trimethylbenzoyl) phosphinate lithium, and 5% (w / v) fibrinogen was prepared as component B solution. The composition, preparation method, and usage method were the same as in Example 9, except that the concentration of methacryloyl hyaluronic acid in component A solution was adjusted to 5% (w / v). In the gel obtained in this way, the volume ratio of fibrin crosslinking to photocrosslinking was 2:1. [Example]

[0074] The mixed solution of 8% (w / v) methacryloyl hyaluronic acid-0.25% (w / v) phenyl (2,4,6-trimethylbenzoyl) phosphinic acid lithium-1000IU / mL hemopexin is prepared as component A solution, and the mixed solution of 3% (w / v) methacryloyl hyaluronic acid-0.125% (w / v) phenyl (2,4,6-trimethylbenzoyl) phosphinic acid lithium-5% (w / v) fibrinogen is prepared as component B solution.Other than adjusting the concentration of methacryloyl hyaluronic acid in component B solution to 3% (w / v), its composition, preparation method and usage method are the same as those in Example 9. [Example]

[0075] A mixed solution of 8% (w / v) methacryloyl hyaluronic acid, 0.25% (w / v) lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate, and 500 IU / ml hemopexin was prepared as component A solution, and a mixed solution of 5% (w / v) methacryloyl hyaluronic acid, 0.125% (w / v) lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate, and 5% (w / v) fibrinogen was prepared as component B solution. Except for adjusting the hemopexin activity of component A solution to 500 IU / ml, its composition, preparation method, and usage method were the same as those in Example 9. [Example]

[0076] A mixed solution of 8% (w / v) methacryloylated hyaluronic acid, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 1000 IU / mL hemopexin was prepared as component A solution, and a mixed solution of 5% (w / v) methacryloylated hyaluronic acid, 0.125% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 3% (w / v) fibrinogen was prepared as component B solution. The composition, preparation method, and usage method of component B solution were the same as those of Example 9, except that the fibrinogen concentration was adjusted to 3% (w / v). In the gel obtained in this way, the volume ratio of fibrin crosslinking to photocrosslinking was 1:2. [Example]

[0077] A mixed solution of 8% (w / v) methacryloylated sodium alginate, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 1000 IU / mL hemopexin was prepared as component A, and a mixed solution of 5% (w / v) methacryloylated sodium alginate, 0.125% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 5% (w / v) fibrinogen was prepared as component B. In this example, the photosensitive material in components A and B was methacryloylated sodium alginate, but the preparation and use methods were the same as in Example 9. In the gel obtained in this way, the volume ratio of fibrin crosslinks to photocrosslinks was 1:1. [Example]

[0078] A mixed solution of 5% (w / v) methacryloylated sodium alginate, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 1000 IU / mL hemopexin was prepared as Component A, and a mixed solution of 5% (w / v) methacryloylated sodium alginate, 0.125% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 5% (w / v) fibrinogen was prepared as Component B. The composition, preparation, and usage of Component A were the same as those of Example 14, except that the concentration of methacryloylated sodium alginate in Component A was adjusted to 5% (w / v). In the gel obtained in this manner, the volume ratio of fibrin crosslinks to photocrosslinks was 2:1. [Example]

[0079] A mixed solution of 8% (w / v) methacryloylated sodium alginate, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 1000 IU / mL hemopexin was prepared as Component A, and a mixed solution of 3% (w / v) methacryloylated sodium alginate, 0.125% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 5% (w / v) fibrinogen was prepared as Component B. The composition, preparation, and usage of Component B were the same as those of Example 14, except that the concentration of methacryloylated sodium alginate in Component B was adjusted to 3% (w / v). [Example]

[0080] A mixed solution of 8% (w / v) methacryloylated sodium alginate, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 500 IU / mL hemopexin was prepared as Component A, and a mixed solution of 5% (w / v) methacryloylated sodium alginate, 0.125% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 5% (w / v) fibrinogen was prepared as Component B. The hemopexin activity in Component A was adjusted to 500 IU / mL, but the composition, preparation method, and usage method were the same as in Example 14. [Example]

[0081] A mixed solution of 8% (w / v) methacryloylated sodium alginate, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 1000 IU / mL hemopexin was prepared as Component A solution, and a mixed solution of 5% (w / v) methacryloylated sodium alginate, 0.125% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 3% (w / v) fibrinogen was prepared as Component B solution. The composition, preparation method, and usage method of Component B solution were the same as those of Example 14, except that the fibrinogen concentration in Component B solution was adjusted to 3% (w / v). In the gel obtained in this manner, the volume ratio of fibrin crosslinks to photocrosslinks was 1:2. [Example]

[0082] A mixed solution of 10% (w / v) methacryloylated silk fibroin, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 1000 IU / mL hemopexin was prepared as component A solution, and a mixed solution of 5% (w / v) methacryloylated silk fibroin, 0.125% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 5% (w / v) fibrinogen was prepared as component B solution. In this example, the photosensitive material in components A and B was methacryloylated silk fibroin, and the preparation and use methods were the same as in Example 9, except that the concentration of methacryloylated silk fibroin in component A solution was 10% (w / v). In the resulting gel, the volume ratio of fibrin crosslinks to photocrosslinks was 1:1. [Example]

[0083] A mixed solution of 8% (w / v) methacryloylated silk fibroin, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 1000 IU / mL hemopexin was prepared as Component A, and a mixed solution of 5% (w / v) methacryloylated silk fibroin, 0.125% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 5% (w / v) fibrinogen was prepared as Component B. The composition, preparation method, and usage method of Component A were the same as those of Example 19, except that the concentration of methacryloylated silk fibroin in Component A was adjusted to 8% (w / v). [Example]

[0084] A mixed solution of 5% (w / v) methacryloylated silk fibroin, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 1000 IU / mL hemopexin was prepared as Component A, and a mixed solution of 5% (w / v) methacryloylated silk fibroin, 0.125% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 5% (w / v) fibrinogen was prepared as Component B. The composition, preparation, and usage of Component A were the same as those of Example 19, except that the concentration of methacryloylated silk fibroin in Component A was adjusted to 5% (w / v). In the gel obtained in this manner, the volume ratio of fibrin crosslinks to photocrosslinks was 2:1. [Example]

[0085] A mixed solution of 10% (w / v) methacryloylated silk fibroin, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 1000 IU / mL hemopexin was prepared as component solution A, and a mixed solution of 3% (w / v) methacryloylated silk fibroin, 0.125% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 5% (w / v) fibrinogen was prepared as component solution B. The composition, preparation method, and usage method of component solution B were the same as those of Example 19, except that the concentration of methacryloylated silk fibroin in component solution B was adjusted to 3% (w / v). [Example]

[0086] A mixture of 10% (w / v) methacryloylated silk fibroin, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 500 IU / mL hemopexin was prepared as Component Solution A, and a mixture of 5% (w / v) methacryloylated silk fibroin, 0.125% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 5% (w / v) fibrinogen was prepared as Component Solution B. The hemopexin activity in Component Solution A was adjusted to 500 IU / mL, but the composition, preparation method, and usage method were the same as in Example 19. [Example]

[0087] A mixed solution of 10% (w / v) methacryloylated silk fibroin, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 1000 IU / mL hemopexin was prepared as component A solution, and a mixed solution of 5% (w / v) methacryloylated silk fibroin, 0.125% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 3% (w / v) fibrinogen was prepared as component B solution. The composition, preparation method, and usage method of component B solution were the same as those of Example 19, except that the fibrinogen concentration in component B solution was adjusted to 3% (w / v). In the gel obtained in this way, the volume ratio of fibrin crosslinks to photocrosslinks was 1:2. [Example]

[0088] A mixed solution of 3% (w / v) methacryloylated chitosan, 0.1% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 1000 IU / mL hemopexin was prepared as Component A solution, and a mixed solution of 1% (w / v) methacryloylated chitosan, 0.1% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 5% (w / v) fibrinogen was prepared as Component B solution. In this example, the photosensitive material in Components A and B was methacryloylated chitosan. The preparation and use methods were the same as in Example 9, except that the concentration of methacryloylated chitosan in Component A solution was 3% (w / v), and the concentration of methacryloylated chitosan in Component B solution was 1% (w / v). In the gel obtained in this way, the volume ratio of fibrin crosslinks to photocrosslinks was 1:1. [Example]

[0089] A mixture of 2% (w / v) methacryloylated chitosan, 0.1% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 1000 IU / mL hemopexin was prepared as Component A, and a mixture of 1% (w / v) methacryloylated chitosan, 0.1% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 5% (w / v) fibrinogen was prepared as Component B. The composition, preparation, and usage of Component A were the same as those of Example 25, except that the concentration of methacryloylated chitosan in Component A was adjusted to 2% (w / v). [Example]

[0090] A mixed solution of 1% (w / v) methacryloylated chitosan, 0.1% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 1000 IU / mL hemopexin was prepared as Component A, and a mixed solution of 1% (w / v) methacryloylated chitosan, 0.1% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 5% (w / v) fibrinogen was prepared as Component B. The composition, preparation, and use of Component A were the same as those of Example 25, except that the concentration of methacryloylated chitosan in Component A was adjusted to 1% (w / v). In the gel obtained in this manner, the volume ratio of fibrin crosslinks to photocrosslinks was 2:1. [Example]

[0091] A mixture of 3% (w / v) methacryloylated chitosan, 0.1% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 1000 IU / mL hemopexin was prepared as Component A, and a mixture of 0.5% (w / v) methacryloylated chitosan, 0.1% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 5% (w / v) fibrinogen was prepared as Component B. The composition, preparation, and use of Component B were the same as those of Example 25, except that the concentration of methacryloylated chitosan in Component B was adjusted to 0.5% (w / v). [Example]

[0092] A mixture of 3% (w / v) methacryloylated chitosan, 0.1% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 500 IU / mL hemopexin was prepared as Component Solution A, and a mixture of 1% (w / v) methacryloylated chitosan, 0.1% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 5% (w / v) fibrinogen was prepared as Component Solution B. The hemopexin activity in Component Solution A was adjusted to 500 IU / mL, but the composition, preparation method, and usage method were the same as in Example 25. [Example]

[0093] A mixed solution of 3% (w / v) methacryloylated chitosan, 0.1% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 1000 IU / mL hemopexin was prepared as Component A solution, and a mixed solution of 1% (w / v) methacryloylated chitosan, 0.1% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 3% (w / v) fibrinogen was prepared as Component B solution. The fibrinogen concentration in Component B solution was adjusted to 3% (w / v), but its composition, preparation method, and usage method were the same as those in Example 25. In the gel obtained in this manner, the volume ratio of fibrin crosslinks to photocrosslinks was 1:2. [Example]

[0094] A mixed solution of 20% (w / v) polyether F127 diacrylate, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 1000 IU / mL hemopexin was prepared as Component A solution, and a mixed solution of 10% (w / v) polyether F127 diacrylate, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 5% (w / v) fibrinogen was prepared as Component B solution. In this example, the photosensitive material in Components A and B was polyether F127 diacrylate, and the preparation and use methods were the same as in Example 9, except that the concentration of polyether F127 diacrylate in Component A solution was 20% (w / v) and the concentration of polyether F127 diacrylate in Component B solution was 10% (w / v). In the gel thus obtained, the volume ratio of fibrin crosslinks to photocrosslinks was 1:1. [Example]

[0095] A mixed solution of 15% (w / v) polyether F127 diacrylate, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 1000 IU / mL hemopexin was prepared as Component A, and a mixed solution of 10% (w / v) polyether F127 diacrylate, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 5% (w / v) fibrinogen was prepared as Component B. The composition, preparation, and usage of Component A were the same as those of Example 31, except that the concentration of polyether F127 diacrylate in Component A was adjusted to 15% (w / v). [Example]

[0096] A mixed solution of 10% (w / v) polyether F127 diacrylate, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 1000 IU / mL hemopexin was prepared as Component A, and a mixed solution of 10% (w / v) polyether F127 diacrylate, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 5% (w / v) fibrinogen was prepared as Component B. The composition, preparation, and use of Component A were the same as those of Example 31, except that the concentration of polyether F127 diacrylate in Component A was adjusted to 10% (w / v). In the resulting gel, the volume ratio of fibrin crosslinks to photocrosslinks was 2:1. [Example]

[0097] A mixed solution of 20% (w / v) polyether F127 diacrylate, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 1000 IU / mL hemopexin was prepared as Component A, and a mixed solution of 5% (w / v) polyether F127 diacrylate, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 5% (w / v) fibrinogen was prepared as Component B. The composition, preparation, and usage of Component B were the same as those of Example 31, except that the concentration of polyether F127 diacrylate in Component B was adjusted to 5% (w / v). [Example]

[0098] A mixture of 20% (w / v) polyether F127 diacrylate, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 500 IU / mL hemopexin was prepared as Component A, and a mixture of 10% (w / v) polyether F127 diacrylate, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 5% (w / v) fibrinogen was prepared as Component B. The hemopexin activity of Component A was adjusted to 500 IU / mL, but the composition, preparation method, and usage method were the same as in Example 31. [Example]

[0099] A mixed solution of 20% (w / v) polyether F127 diacrylate, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 1000 IU / mL hemopexin was prepared as Component A solution, and a mixed solution of 10% (w / v) polyether F127 diacrylate, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 3% (w / v) fibrinogen was prepared as Component B solution. The composition, preparation method, and usage method of Component B solution were the same as those of Example 31, except that the fibrinogen concentration in Component B solution was adjusted to 3% (w / v). In the gel obtained in this manner, the volume ratio of fibrin crosslinks to photocrosslinks was 1:2.

[0100] <Comparative Example 1> A lyophilized fibrin glue containing an enzyme reagent and a fibrinogen reagent (Wugu Laishi, purchased from Shanghai Laishi Co., Ltd.) was used externally. The enzyme reagent and fibrinogen reagent were prepared in separate solutions according to the manufacturer's instructions, mixed, and enzymatic cross-linking was carried out for approximately 1 second to obtain the fibrin glue with the microstructure shown in Figure 1.

[0101] <Comparative Example 2> The precursor solution of 9% (w / v) methacryloylated gelatin-0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate was prepared in the same manner as the component A solution in Example 2, except that no hemopexin was added to the solution.

[0102] <Comparative Example 3> The mixed solution of 13% (w / v) methacryloylated gelatin-0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate-1000 IU / mL hemopexin had the same ingredients and preparation method as the component A solution in Example 2.

[0103] <Comparative Example 4> The 5% (w / v) methacryloylated gelatin-0.125% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate-5% (w / v) fibrinogen mixed solution had the same ingredients and was prepared in the same manner as in step (5) of Example 1.

[0104] <Comparative Example 5> A mixed solution of 30% (w / v) methacryloylated sericin, 0.5% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 1000 IU / mL hemopexin was prepared as component A, and a mixed solution of 20% (w / v) methacryloylated sericin, 0.5% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 5% (w / v) fibrinogen was prepared as component B. In this comparative example, the photosensitive material in components A and B was methacryloylated sericin, the concentration of methacryloylated sericin in the component A solution was 30% (w / v), the concentration of lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate was 0.5% (w / v), and the concentration of methacryloylated sericin in the component B solution was 20% (w / v), and the concentration of lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate was 0.5% (w / v), except that the preparation method was the same as in Example 9. In the gel thus obtained, the volume ratio of fibrin crosslinks to photocrosslinks was 1:1.

[0105] <Comparative Example 6> A mixture of 10% (w / v) methacryloylated dextran, 0.25% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 1000 IU / mL hemopexin was prepared as Component A solution, and a mixture of 10% (w / v) methacryloylated dextran, 0.125% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, and 5% (w / v) fibrinogen was prepared as Component B solution. In this comparative example, the photosensitive material in Components A and B was methacryloylated dextran, and the concentration of methacryloylated dextran in Component A solution was 10% (w / v), and the concentration of methacryloylated dextran in Component B solution was 5% (w / v), except that the preparation method was the same as in Example 9. In the gel thus obtained, the volume ratio of fibrin crosslinks to photocrosslinks was 1:1.

[0106] Performance Test In order to confirm the performance of the double-crosslinked fibrin gels obtained in Examples 1 to 36 and the hydrogels of Comparative Examples 1 to 6, a performance test of gelation time, an adhesive strength test, and an animal hemostatic experiment were carried out on each of them as follows.

[0107] Gel time test Detects: The above Examples 1 to 36 and Comparative Examples 1 to 6 Detection method: Rheological analysis was performed on Examples 1-36 and Comparative Examples 1-6 to compare their gelation times, and the results are shown in Table 1. Specific operating procedure: Dynamic rheological experiments were performed at 37°C using a HAAKE RS6000 opto-rheometer with a parallel plate (P20 TiL, 20 mm diameter) geometry. Time-sweep oscillatory tests of the hydrogels of Examples 1-36 and Comparative Examples 1-6 were performed at 5% strain and a frequency of 1 Hz for 300 seconds. Strain sweeps were performed on the pregelling solutions to verify linear response. The gel point was determined when the torsional modulus (G') exceeded the loss modulus (G'').

[0108] Adhesion Strength Test The above Examples 1 to 36 and Comparative Examples 1 to 6 Detection method: Specific procedure: Pigskin was cut into a 40 mm x 20 mm rectangle, and two pieces of pigskin were bonded together using 500 μl of Examples 1 to 36 and Comparative Examples 1 to 6. The mixed solutions of components A and B in Examples 1 to 36 and the precursor solutions in Comparative Examples 2 to 6 were irradiated with blue light of the same wavelength band for 60 seconds. Next, the adhesive strength was tested at a strain rate of 1 mm / min. Figure 2 shows the microstructure of the gel formed after photocrosslinking of the precursor solution in Comparative Example 2. Figure 3 shows the microstructure of the gel formed after photocrosslinking of the mixed solution in Example 1. The reading when the gel peeled off from the pigskin was recorded and used as the adhesive strength (kPa). The detection results are shown in Table 1.

[0109] Hemostatic effect test Detects: Examples 1, 9, 14, 19, 25, and 31 of the present invention, and Comparative Examples 1 to 6 Detection method: Rabbit liver surface 1cm incision bleeding model: After anesthetizing, New Zealand white rabbits were secured to a surgical table with their abdomens exposed. A midline abdominal incision was made to expose the liver, and a 1cm x 0.5cm bleeding model was created on the liver. Using weighed filter paper and components A and B from Examples 1, 9, 14, 19, 25, and 31, the resulting mixed solutions and the precursor solutions from Comparative Examples 1 to 6 were injected using the injection method described in step (7) of Example 1. The bleeding site was covered with a hemostatic agent until bleeding stopped (in the present Examples and Comparative Examples 2 to 6, the bleeding site was covered and treated with blue light in the same wavelength range). The bleeding time and blood loss were recorded. The results are shown in Table 1, Figures 4 and 5.

[0110] [Table 1]

[0111] The values ​​for hemostasis time and blood loss are expressed as (mean ± standard deviation). Result analysis:

[0112] As can be seen from Figure 1, in the fibrin adhesive of Comparative Example 1, only fibrin crosslinks form a network structure. As can be seen from Figure 2, after the precursor solution of Comparative Example 2 is irradiated, only the methacryloylated gelatin is photocrosslinked to form a porous structure. As can be seen from Figure 3, the mixed solution of Components A and B in Example 1 of the present invention can have both a fibrin crosslinked network structure and a methacryloylated gelatin crosslinked porous structure after irradiation, and a continuous three-dimensional fibrin network structure is distributed throughout the channels of the formed methacryloylated gelatin crosslinked porous structure. As can be seen from Table 1, the gelation times of Examples 1 to 36 ranged from 1 to 3 s, and for the same photosensitive material, the gelation time increased as the photocrosslinking ratio increased. Meanwhile, for all types of photosensitive materials selected in Examples 1 to 36, the gelation times at a specific double crosslinking ratio were significantly shorter than those of Comparative Examples 2 to 4 (the gelation time of Comparative Example 2 was 8 s, the gelation time of Comparative Example 3 was 9 s, and the gelation time of Comparative Example 4 was 14 s).

[0113] As can be seen from Table 1, the adhesive strength of Examples 1 to 36 ranged from 82 to 132 kPa. When the photosensitive material was the same, the adhesive strength of the gel decreased as the concentration of the photosensitive material decreased. On the other hand, all types of photosensitive materials selected in Examples 1 to 36 had higher adhesive strengths at specific double crosslinking ratios than those of each comparative example (the adhesive strength of Comparative Example 1 was 6 kPa, the adhesive strength of Comparative Example 2 was 80 kPa, the adhesive strength of Comparative Example 3 was 76 kPa, the adhesive strength of Comparative Example 4 was 70 kPa, the adhesive strength of Comparative Example 5 was 29 kPa, and the adhesive strength of Comparative Example 6 was 45 kPa).

[0114] As can be seen from Table 1 and Figures 4 and 5, the hemostasis times in Examples 1, 9, 14, 19, 25, and 31 were 6 seconds to 24 seconds, all of which were significantly shorter than the 40 seconds or more in Comparative Examples 1 to 6. The average blood loss in Examples 1, 9, 14, 19, 25, and 31 was 12 mg to 37 mg, which was significantly lower than the blood loss of 90 mg or more in Comparative Examples 1 to 6.

[0115] In summary, when the double-crosslinked fibrin gel of the present invention is applied to a bleeding wound, it immediately (approximately 1 s) forms a fibrin clot, which "preliminarily" closes the wound and blocks blood outflow. Furthermore, enzymes within the fibrin clot convert fibrinogen in the blood into clots, providing an efficient procoagulation effect. Furthermore, the photosensitive material forms a photosensitive gel under light excitation, which has strong adhesive properties to wet tissue, providing a "strong" wound closure effect. The interaction between the fibrin crosslinked structure and the photocrosslinked structure provides both preliminarily wound closure and strong tissue adhesion, thereby achieving excellent hemostatic effects.

[0116] Although the specific embodiments of the present invention have been described in detail above, the present invention is not limited to the specific embodiments, and any variations, modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention do not affect the essence of the present invention and are all intended to be included in the scope of protection of the claims of the present invention.

Claims

1. 1. A raw material composition for preparing a rapid hemostatic double cross-linked fibrin adhesive in situ by mixing at a bleeding wound, comprising: The composition A includes composition A and composition B, wherein composition A includes 10 to 200 parts by weight of a photosensitive material, 1 to 3 parts by weight of a photoinitiator, 0.14 to 0.28 parts by weight of an enzyme, and 1.11 to 8.88 parts by weight of a water-soluble inorganic calcium salt; composition B includes 5 to 100 parts by weight of a photosensitive material, 1 to 2 parts by weight of a photoinitiator, and 30 to 50 parts by weight of fibrinogen; the mass ratio of composition A to composition B is 1.4:10 to 14:1; the photosensitive material is a polymer composite material system including a methacryloylated high-molecular polymer, a high-molecular polymer of polyacrylate, or a high-molecular polymer of polyacrylate; the methacryloylated high-molecular polymer is methacryloylated gelatin, methacryloylated hyaluronic acid, methacryloylated alginic acid sodium, or the like. the enzyme is any one selected from human thrombin, recombinant human thrombin, bovine thrombin, porcine thrombin, and hemocoagulase; the double-crosslinked fibrin adhesive is a solid hydrogel composed of both a three-dimensional fibrin meshwork and a three-dimensional photosensitive gel meshwork, the three-dimensional fibrin meshwork being continuous throughout the entire inner wall of each of the photosensitive gel meshworks, the three-dimensional fibrin meshworks of each set being continuous as a whole, and the three-dimensional fibrin meshwork being distributed in a disordered manner throughout the surface and interior of the solid hydrogel.

2. 2. The raw material composition according to claim 1, wherein the weight parts of the photosensitive material in composition A are greater than the weight parts of the photosensitive material in composition B.

3. 2. The raw material composition according to claim 1, wherein the mass ratio of said composition A to said composition B is 1.4:1 to 1.4:

10.

4. 2. The raw material composition according to claim 1, wherein composition A comprises 80 to 200 parts by weight of a photosensitive material, 1 to 3 parts by weight of a photoinitiator, 0.14 to 0.28 parts by weight of an enzyme, and 1.11 to 8.88 parts by weight of a water-soluble inorganic calcium salt, and composition B comprises 30 to 100 parts by weight of a photosensitive material, 1 to 2 parts by weight of a photoinitiator, and 30 to 50 parts by weight of fibrinogen.

5. 2. The raw material composition according to claim 1, wherein the high molecular weight polyacrylate is selected from the group consisting of polyether diacrylate and polyethylene glycol diacrylate.

6. The polymer composite material systems containing the methacryloylated polymer include methacryloylated gelatin-polyvinyl alcohol systems, methacryloylated gelatin-polyurethane systems, methacryloylated gelatin-polylactic acid systems, methacryloylated gelatin-cellulose systems, methacryloylated hyaluronic acid-polyvinyl alcohol systems, methacryloylated hyaluronic acid-polyurethane systems, methacryloylated hyaluronic acid-polylactic acid systems, methacryloylated hyaluronic acid-cellulose systems, methacryloylated sodium alginate-polyvinyl alcohol systems, methacryloylated sodium alginate-polyurethane systems, methacryloylated sodium alginate-polylactic acid systems, methacryloylated sodium alginate-cellulose systems, methacryloylated silk fibroin- 2. The raw material composition according to claim 1, which is any one or more selected from polyvinyl alcohol-based, methacryloylated silk fibroin-polyurethane-based, methacryloylated silk fibroin-polylactic acid-based, methacryloylated silk fibroin-cellulose-based, methacryloylated chitosan-polyvinyl alcohol-based, methacryloylated chitosan-polyurethane-based, methacryloylated chitosan-polylactic acid-based, methacryloylated chitosan-cellulose-based, methacryloylated carboxymethyl chitosan-polyvinyl alcohol-based, methacryloylated carboxymethyl chitosan-polyurethane-based, methacryloylated carboxymethyl chitosan-polylactic acid-based, and methacryloylated carboxymethyl chitosan-cellulose-based materials.

7. 2. The raw material composition according to claim 1, wherein the photosensitive material is methacryloylated gelatin or methacryloylated silk fibroin.

8. 2. The raw material composition according to claim 1, wherein the photoinitiator is one or more compounds selected from the group consisting of lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, ethyl 2,4,6-trimethylbenzoylphenylphosphinate, 2-methyl-1-(4-methylthiophenyl)-2-morpholinopropan-1-one, methyl 2-benzoylbenzoate, 2-benzyl-2-dimethylamino-1-(4-morpholinophenyl)butanone, and 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide].

9. 2. The raw material composition according to claim 1, wherein the fibrinogen is any one selected from the group consisting of human fibrinogen, bovine fibrinogen, and porcine fibrinogen.

10. 7. The raw material composition according to claim 1, wherein the water-soluble inorganic calcium salt is calcium chloride, calcium nitrate, or calcium sulfate.

11. 7. The raw material composition according to claim 1, wherein the raw material composition is in the form of a freeze-dried powder, an injection, a sponge, or granules.

12. the method includes the steps of preparing a mixed solution by dissolving a photosensitive material and a photoinitiator in a solvent, mixing the mixed solution with a solution containing hemopexin and calcium ions to obtain a first precursor solution, controlling the concentration ratio of the photosensitive material, the photoinitiator, the enzyme, and the calcium ions in the first precursor solution to 10-200:1-3:0.14-0.28:1.11-8.88, and controlling the storage time of the first precursor solution in a room temperature environment to less than 30 minutes; and mixing the mixed solution with a solution containing fibrinogen to obtain a second precursor solution, controlling the concentration ratio of the photosensitive material, the photoinitiator, and the fibrinogen in the second precursor solution to 5-100:1-2:30-50, and obtaining a liquid precursor composition containing the first precursor solution and the second precursor solution, The method for preparing a raw material composition according to any one of claims 1 to 6, wherein the photosensitive material is a methacryloylated high molecular weight polymer, a high molecular weight polymer of polyacrylate, or a polymer composite material system containing a methacryloylated high molecular weight polymer or a high molecular weight polymer of polyacrylate, and the methacryloylated high molecular weight polymer is any one or a mixture of two or more selected from the group consisting of methacryloylated gelatin, methacryloylated hyaluronic acid, methacryloylated sodium alginate, methacryloylated silk fibroin, methacryloylated chitosan, and methacryloylated carboxymethyl chitosan.

13. The liquid raw material composition is further treated by a freeze-drying method to obtain a solid raw material composition, 13. The method of claim 12, wherein the solid source composition is a freeze-dried powder, a sponge, or a granule.

14. 13. The method of claim 12, wherein the concentration of the photosensitive material in the first precursor solution is controlled to be higher than 0.5% (w / v), and the concentration of the photosensitive material in the second precursor solution is controlled to be lower than the concentration of the photosensitive material in the first precursor solution.

15. in particular, Step 1) preparing a first mixed solution by dissolving a photosensitive material and a photoinitiator in a solvent, and controlling the concentration ratio of the photosensitive material to the photoinitiator to be 10 to 200:1 to 3 and the concentration of the photosensitive material to be 0.5% to 30% (w / v); Step 2) preparing a second mixed solution by dissolving a photosensitive material and a photoinitiator in a solvent, and controlling the concentration ratio of the photosensitive material to the photoinitiator to be 5 to 100:1 to 2, and the concentration of the photosensitive material to be lower than that of the first mixed solution described in step 1); 3) mixing the first mixed solution prepared in 1) with a solution containing an enzyme and calcium ions to obtain a first precursor solution, and controlling the concentration ratio of the photosensitive material, the photoinitiator, the enzyme, and the calcium ions to 10-200:1-3:0.14-0.28:1.11-8.88; and step 4) mixing the second mixed solution prepared in step 2) with a solution containing fibrinogen to obtain a second precursor solution, and controlling the concentration ratio of the photosensitive material, the photoinitiator, and the fibrinogen to be 5-100:1-2:30-50.

16. The solution containing the enzyme and calcium ions is prepared by adding a solvent and a water-soluble inorganic calcium salt solution to the enzyme, dissolving it completely, and then adding Ca ２＋ The enzyme activity in the resulting solution is 500 IU to 2000 IU / ml, Ca ２＋ The method according to claim 12, wherein the concentration is controlled to be 60 to 100 mmol / L.

17. 13. The method according to claim 12, wherein the concentration of fibrinogen in the fibrinogen-containing solution is 5% to 10% (w / v).

18. 13. The method of claim 12, wherein the concentration of the photosensitive material in the first precursor solution is controlled to be 1% to 30% (w / v).

19. 13. The method of claim 12, wherein the enzyme activity in the first precursor solution is controlled to be 200 IU / ml or more.

20. 13. The method of claim 12, wherein the concentration of calcium ions in the first precursor solution is controlled to be 20 mmol / L or more.

21. 13. The method of claim 12, wherein the concentration of the photosensitive material in the second precursor solution is controlled to be 1% to 10% (w / v) and equal to or less than the concentration of the photosensitive material in the first precursor solution.

22. 13. The method of claim 12, wherein the concentration of fibrinogen in the second precursor solution is controlled to be 3% (w / v) or higher.

23. A kit for preparing a double-crosslinked fibrin gel capable of rapid on-site hemostasis of a bleeding wound, comprising a first precursor reagent and a second precursor reagent packaged separately from each other; The double-crosslinked fibrin gel prepared by the kit is a solid hydrogel composed of a three-dimensional fibrin meshwork and a three-dimensional photosensitive gel meshwork, the three-dimensional fibrin meshwork is continuous across the entire inner wall of each photosensitive gel meshwork, the three-dimensional fibrin meshwork of each set is continuous as a whole, the three-dimensional fibrin meshwork is generally present in a disordered manner on the surface and inside of the solid hydrogel, the volume ratio of the three-dimensional fibrin meshwork to the three-dimensional photosensitive gel meshwork is 0.5 to 3, and the three-dimensional fibrin meshwork is formed in advance by enzymatic crosslinking of fibrinogen, the first precursor reagent contains 10 to 200 parts by weight of a photosensitive material, 1 to 3 parts by weight of a photoinitiator, 0.14 to 0.28 parts by weight of an enzyme, and 3.33 to 5.55 parts by weight of a water-soluble inorganic calcium salt; the second precursor reagent contains 5 to 100 parts by weight of a photosensitive material, 1 to 2 parts by weight of a photoinitiator, and 30 to 50 parts by weight of fibrinogen, the mass ratio of the first precursor reagent to the second precursor reagent being 1.4:10 to 14:1; the photosensitive material is a methacryloylated high-molecular-weight polymer, a polyacrylate high-molecular-weight polymer, or a polymer composite material system containing a methacryloylated high-molecular-weight polymer or a polyacrylate high-molecular-weight polymer; and the enzyme is any one selected from human thrombin, recombinant human thrombin, bovine thrombin, porcine thrombin, and hemocoagulase.

24. 24. The kit of claim 23, wherein the mass ratio of the first precursor reagent to the second precursor reagent is 1.4:1 to 1.4:

10.

25. 24. The kit of claim 23, wherein the first precursor reagent contains 80 to 200 parts by weight of a photosensitive material, 1 to 3 parts by weight of a photoinitiator, 0.14 to 0.28 parts by weight of an enzyme, and 3.33 to 5.55 parts by weight of a water-soluble inorganic calcium salt, and the second precursor reagent contains 30 to 100 parts by weight of a photosensitive material, 1 to 2 parts by weight of a photoinitiator, and 30 to 50 parts by weight of fibrinogen.

26. The kit according to claim 23, characterized in that the methacryloylated high molecular weight polymer is any one or a mixture of two or more selected from methacryloylated gelatin, methacryloylated hyaluronic acid, methacryloylated sodium alginate, methacryloylated silk fibroin, methacryloylated chitosan, and methacryloylated carboxymethyl chitosan.

27. 24. The kit of claim 23, wherein the photosensitive material is methacryloylated gelatin or methacryloylated silk fibroin.

28. 24. The kit of claim 23, wherein the high molecular weight polyacrylate polymer is selected from polyether diacrylate or polyethylene glycol diacrylate.

29. The polymer composite material systems containing the methacryloylated polymer include methacryloylated gelatin-polyvinyl alcohol systems, methacryloylated gelatin-polyurethane systems, methacryloylated gelatin-polylactic acid systems, methacryloylated gelatin-cellulose systems, methacryloylated hyaluronic acid-polyvinyl alcohol systems, methacryloylated hyaluronic acid-polyurethane systems, methacryloylated hyaluronic acid-polylactic acid systems, methacryloylated hyaluronic acid-cellulose systems, methacryloylated sodium alginate-polyvinyl alcohol systems, methacryloylated sodium alginate-polyurethane systems, methacryloylated sodium alginate-polylactic acid systems, methacryloylated sodium alginate-cellulose systems, methacryloylated silk fibroin- The kit according to claim 23, wherein the kit is any one or more selected from the group consisting of polyvinyl alcohol-based, methacryloylated silk fibroin-polyurethane-based, methacryloylated silk fibroin-polylactic acid-based, methacryloylated silk fibroin-cellulose-based, methacryloylated chitosan-polyvinyl alcohol-based, methacryloylated chitosan-polyurethane-based, methacryloylated chitosan-polylactic acid-based, methacryloylated chitosan-cellulose-based, methacryloylated carboxymethyl chitosan-polyvinyl alcohol-based, methacryloylated carboxymethyl chitosan-polyurethane-based, methacryloylated carboxymethyl chitosan-polylactic acid-based, and methacryloylated carboxymethyl chitosan-cellulose-based.

30. The kit according to claim 23, wherein the photoinitiator is any one or more compositions selected from lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, ethyl 2,4,6-trimethylbenzoylphenylphosphinate, 2-methyl-1-(4-methylthiophenyl)-2-morpholinopropan-1-one, methyl 2-benzoylbenzoate, 2-benzyl-2-dimethylamino-1-(4-morpholinophenyl)butanone, and 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide].

31. 24. The kit according to claim 23, wherein the fibrinogen is any one selected from human fibrinogen, bovine fibrinogen, and porcine fibrinogen.

32. 24. The kit of claim 23, wherein the water-soluble inorganic calcium salt is selected from calcium chloride, calcium nitrate, or calcium sulfate.

33. 24. The kit according to claim 23, wherein the first precursor reagent and / or the second precursor reagent further comprise auxiliary materials and / or additives, the auxiliary materials being one or more selected from glycine, arginine hydrochloride, sodium citrate, sucrose, and sodium chloride, and the additives being one or more selected from growth factors, interleukins, vitamins, and silver ions.

34. 34. The kit of claim 33, wherein the growth factor is one or more selected from the group consisting of platelet growth factor, epidermal growth factor, and fibroblast growth factor.

35. 34. The kit of claim 33, wherein the interleukin is one or more selected from interleukin 2, interleukin 6, and interleukin 8.

36. 34. The kit of claim 33, wherein the vitamin is one or more selected from vitamin B, vitamin C, vitamin E, or vitamin K.

37. 24. The kit of claim 23, further comprising an individually packaged formulation solvent, wherein the formulation solvent is a mixture of any one or more of phosphate buffer solution, HEPES biological buffer, 0.9% sodium chloride solution, calcium chloride solution, or deionized water.

38. 24. The kit of claim 23, further comprising instructions for use with the kit.

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