CN115920118A - Double-crosslinked fibrin gel, kit and application thereof - Google Patents

Abstract

The invention provides a double-cross-linked fibrin gel, which is a solid hydrogel consisting of a network structure with a sealing function and a network structure with an adhesion function; the network structure with the sealing function is a three-dimensional fibrin network, and the network structure with the adhesion function is a three-dimensional photosensitive gel network; a group of fibrin networks are arranged inside each photosensitive gel network pore channel, and each group of fibrin networks have continuity as a whole; overall, a three-dimensional fibrin network is randomly distributed over the surface and inside of the solid hydrogel. The invention also provides a kit for preparing the double-crosslinked fibrin gel and application of the kit in preparing an in-situ quick-setting hemostatic material. The double-crosslinked fibrin gel prepared by the kit has quick and efficient hemostatic effect, and can be widely applied to hemostasis of accidental wounds or surgical wounds.

Description

Double cross-linked fibrin gel, kit and application thereof

Technical Field

The invention belongs to the field of biomedical materials, and in particular relates to a double-crosslinked fibrin gel used for hemostasis of accidental trauma or surgical bleeding, and an application method thereof.

Background Art

Uncontrolled bleeding after trauma or during surgery is the leading cause of death worldwide, causing more than 2 million deaths each year. Uncontrolled bleeding in surgical and trauma settings often leads to complications and poor outcomes. Therefore, controlling the amount of bleeding is an important measure to reduce complications and mortality and improve patient outcomes.

At present, some local hemostatic materials have been developed to assist in controlling bleeding during surgery. Common surgical sealants on the market include fibrin glue and synthetic tissue adhesives. Fibrin glue is the most widely used hemostatic agent, has good biocompatibility, can assist in hemostasis in various surgical operations, simulate the coagulation cascade reaction, form a fibrin clot in situ at the bleeding site, and block bleeding. However, the adhesion strength of fibrin glue will be reduced by the influence of continuous tissue tension and blood, and it is easily washed away by blood flow, which is not conducive to exerting its hemostatic properties, and its hemostatic effect is limited due to poor adhesion on wet tissue. On the other hand, synthetic tissue adhesives such as cyanoacrylate adhesives, although having good bonding ability, are limited in their application due to their high cytotoxicity and difficulty in removal.

In order to break through the current application bottleneck of low adhesion of bioadhesives on wet tissue surfaces. In the prior art, some studies have used methacrylic gelatin as a hemostatic gel material. This type of double-bond modified gelatin is obtained by functionalizing the free amino groups of gelatin into methacrylamide groups through methacrylic anhydride. Under the illumination of a specific wavelength, the photoinitiator in the material absorbs light energy to produce free radicals, which in turn causes the methacrylic gelatin molecules to form bonds to form a solid phase gel. Methacrylic gelatin has good biocompatibility, as well as good mechanical properties and adhesion. However, methacrylic gelatin does not have a procoagulant function, which limits its hemostatic ability to a certain extent; the photocuring time of methacrylic gelatin is 5 to 10 seconds, the photocuring time is long, and it is easily washed away by blood flow during the photocuring process; in severe bleeding, a large amount of blood weakens its adhesion ability. In order to make up for this defect, Luo et al. introduced snake venom hemocoagulase with coagulation function into methacrylic gelatin, and the hemostatic gel constructed by them improved the hemostatic effect (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 methacrylic gelatin carrying snake venom hemocoagulase constructed in this study can only contact the blood with a very small amount of snake venom hemocoagulase on the surface of the gel, and the concentration of fibrinogen in the blood is low (2-4 g/L), and the fibrin cross-linking formed is not enough to seal the wound; at the same time, before the methacrylic gelatin completes light curing, its sealing effect on the wound is weak, which greatly limits its hemostatic effect. In 2020, Wang et al. constructed a thrombin-methacryloyl gelatin hydrogel for multi-stage healing of diabetic wounds. By incorporating free thrombin and thrombin-loaded liposomes into methacryloyl gelatin, they achieved initial thrombin release to promote hemostasis and sustained thrombin release to regulate the late healing of diabetic wounds (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 shortcomings of the gel constructed in this study are similar to those of Luo et al. The amount of thrombin released by the gel is small, and it has little contact with fibrinogen in the blood, so it cannot form fibrin cross-links, and the hemostatic effect is reduced due to its weak blocking effect on the wound.

In addition, there are reports in the prior art of using other photocurable materials to prepare hemostatic materials. For example, Chinese patent document CN 111116973A discloses a polyvinyl alcohol hemostatic porous material with active hemostatic function. The sponge is endowed with active hemostatic effect by adding polymers with active hemostatic function such as chitosan or (and) thrombin to the sponge obtained by photocatalytic crosslinking of modified polyvinyl alcohol. However, the hemostatic time reported in the paper is 90s to 100s, which is long and inevitably leads to poor hemostatic effect. This is because 1) the pre-formed sponge cannot fully contact the wet tissue, resulting in a poor blocking effect compared to the gel formed in situ; 2) the thrombin in the sponge in the dry state is not easy to be free, thereby limiting its procoagulant function; 3) the fibrinogen concentration in the blood is low (2 to 4g/L), and the fibrin crosslinks formed are not sufficient to block the wound. Therefore, it is difficult to meet the demand for rapid hemostasis when there is massive bleeding during surgery; and when facing bleeding wounds on organs or the surface of the body, the effect of its expansion and compression of the wound is limited, which weakens the hemostasis effect to a certain extent; at the same time, when removing the sponge, it may cause secondary damage to the wound due to its adhesion to the hemostasis site.

An ideal hemostatic material should not rely on the body's coagulation mechanism, and can also play a hemostatic role even when the body has coagulation disorders, while also having good wet tissue adhesion and ideal coagulation and hemostasis speed. Therefore, it is particularly important to invent a new hemostatic material that can solve the poor wet tissue adhesion and limited hemostatic effect of existing hemostatic materials.

Summary of the invention

In order to overcome the above-mentioned shortcomings in the prior art, the primary purpose of the present invention is to provide an adhesive that can quickly stop bleeding, quickly gel, and has high adhesion, so as to achieve both the effects of promoting blood coagulation and strong adhesion.

Another object of the present invention is to provide a kit for preparing the adhesive, so as to facilitate the promotion and application of the adhesive in clinical practice.

Another object of the present invention is to provide a method for hemostasis using the kit.

In order to achieve the above object, the present invention adopts the following technical solutions:

In the first aspect, the present invention provides a double cross-linked fibrin gel, which is a solid hydrogel composed of a network structure with a sealing function and a network structure with an adhesion function; the network structure with a sealing function is formed before the network structure with an adhesion function. The network structure with a sealing function is a three-dimensional fibrin network, and the network structure with an adhesion function is a three-dimensional photosensitive gel network; each of the photosensitive gel network channels has a group of the fibrin networks, and each group of the fibrin networks is continuous as a whole; overall, the three-dimensional fibrin networks are disorderly distributed on the surface and inside of the solid hydrogel.

In the double cross-linked fibrin gel of the present invention, the three-dimensional fibrin network acts as a scaffold to enhance the strength of the gel, and the process of its formation is used to convert fibrinogen in the blood into fibrin, thereby playing a role in preliminary wound occlusion. As the proportion of fibrin increases, the gelation time of the solid hydrogel will be shortened, the tissue adhesion will decrease, but the gel pores will become larger, and the procoagulant function will increase. The three-dimensional photosensitive gel network plays a role in providing the strength and tissue adhesion of the gel. As the proportion of the photosensitive gel network in the solid hydrogel increases, the tissue adhesion of the solid hydrogel also increases, but the gelation time will also be prolonged, the pores of the gel will become smaller, and the procoagulant function will decrease. In view of the different effects of the above two networks on the overall hemostatic performance of the gel, the present invention further optimizes the ratio of the two networks in the gel through experiments. In the preferred double cross-linked fibrin gel, the volume ratio of the three-dimensional fibrin network to the three-dimensional photosensitive gel network is 0.5-3; preferably 0.5-2; most preferably 1. Under these preferred volume ratios, the two networks can bring better hemostatic properties to the gel as a whole, especially when the volume ratio of the fibrin network and the photosensitive gel network reaches 1:1, the hemostatic performance of the gel can reach the best, that is, rapid coagulation can be achieved while improving the strength and adhesion of the gel.

In the double-crosslinked fibrin gel described in the present invention, the photosensitive gel can be formed by photocrosslinking of a variety of existing photocurable polymer materials (i.e., photosensitive materials), and the photosensitive material can be a methacrylated polymer or its derivative, a polyacrylate polymer or its derivative, or a polymer composite material system containing them.

Furthermore, the methacrylylated high molecular polymer or its derivatives described above can be selected from any one of the following or a mixture of two or more thereof: methacrylylated gelatin or its derivatives, methacrylylated hyaluronic acid or its derivatives, methacrylylated sodium alginate or its derivatives, methacrylylated silk fibroin or its derivatives, methacrylylated chitosan or its derivatives, methacrylylated carboxymethyl chitosan or its derivatives. The above-mentioned high molecular polymer of polyacrylate or its derivatives can be selected from polyether diacrylate or its derivatives, or polyethylene glycol diacrylate or its derivatives. The most preferred photosensitive material of the present invention is methacrylylated gelatin or its derivatives, or methacrylylated silk fibroin or its derivatives.

Furthermore, the derivatives of the methacrylylated high molecular polymers mentioned above include polymers with one or more functional groups modified. The modifiable functional groups of the methacryloyl gelatin include any one or more of amino, carboxyl, thiol, hydroxyl or guanidine; the derivatives of the methacryloyl hyaluronic acid include polymers modified with one or more functional groups thereof, wherein the modifiable functional groups include any one or more of hydroxyl, carboxyl, acetylamino or hydroxymethyl; the derivatives of the methacryloyl sodium alginate include polymers modified with one or more functional groups thereof, wherein the modifiable functional groups include any one or two of carboxyl and hydroxyl; the derivatives of the methacryloyl silk fibroin include polymers modified with one or more functional groups thereof, wherein the modifiable functional groups include any one or more of amino, carboxyl, thiol, hydroxyl or guanidine; the derivatives of the methacryloyl chitosan include polymers modified with one or more functional groups thereof or polymers subjected to multiple chemical reactions, wherein the modifiable functional groups include any one or two of amino or hydroxyl, and the multiple chemical reactions that can occur include any one or more of alkylation, acylation, carboxymethylation, hydrolysis, oxidation, and reduction chemical reactions.

The molecular weight of the above-mentioned methacrylated high molecular polymer or its derivative is in the range of 5 to 400 kDa, and the molecular weight of the above-mentioned polyacrylate high molecular polymer or its derivative is in the range of 700 to 1000 kDa.

Furthermore, the polymer composite material system containing methacrylic polymer or its derivatives described above includes: methacrylic gelatin-polyvinyl alcohol system, methacrylic gelatin-polyurethane system, methacrylic gelatin-polylactic acid system, methacrylic gelatin-cellulose system, methacrylic hyaluronic acid-polyvinyl alcohol system, methacrylic hyaluronic acid-polyurethane system, methacrylic hyaluronic acid-polylactic acid system, methacrylic hyaluronic acid-cellulose system, methacrylic sodium alginate-polyvinyl alcohol system, methacrylic sodium alginate-polyurethane system, methacrylic sodium alginate-polylactic acid system, methacrylic sodium alginate-cellulose system, Any one or more of the following: sodium alginate-cellulose system, methacrylated silk fibroin-polyvinyl alcohol system, methacrylated silk fibroin-polyurethane system, methacrylated silk fibroin-polylactic acid system, methacrylated silk fibroin-cellulose system, methacrylated chitosan-polyvinyl alcohol system, methacrylated chitosan-polyurethane system, methacrylated chitosan-polylactic acid system, methacrylated chitosan-cellulose system, methacrylated carboxymethyl chitosan-polyvinyl alcohol system, methacrylated carboxymethyl chitosan-polyurethane system, methacrylated carboxymethyl chitosan-polylactic acid system, and methacrylated carboxymethyl chitosan-cellulose system.

In the double cross-linked fibrin gel of the present invention, the fibrin network can be formed by cross-linking fibrinogen through enzymes. The fibrinogen can be any one of human fibrinogen, bovine fibrinogen or porcine fibrinogen.

In a second aspect, the present invention further provides a kit for preparing the double cross-linked fibrin gel described in the first aspect of the present invention, comprising a first precursor reagent and a second precursor reagent which are independently packaged from each other; in parts by weight, the first precursor reagent contains 10 to 200 parts of photosensitive material, 1 to 3 parts of photoinitiator, 0.14 to 0.28 parts of enzyme and 3.33 to 5.55 parts of water-soluble inorganic calcium salt, and the second precursor reagent comprises 5 to 100 parts of photosensitive material, 1 to 2 parts of photoinitiator and 30 to 50 parts of fibrinogen; 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; most preferably 1.4:1.

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

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

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

In the kit described in the present invention, the photosensitive material contained in the first precursor reagent and the second precursor reagent is a photosensitive biohydrogel material, which can be a variety of existing polymer materials that can undergo photocuring; the photoinitiator contained in the first precursor reagent and the second precursor reagent is a substance that can generate free radicals after absorbing light energy under the condition of light of a specific wavelength. The photoinitiator can generate free radicals after absorbing light energy, which can form bonds between the molecules of the photosensitive material, thereby quickly forming a solid phase gel. An ideal photosensitive material should have good biocompatibility and degradability, and at the same time have good mechanical properties and adhesion properties.

In the kit of the present invention, the photosensitive material may be a methacrylated high molecular polymer or a derivative thereof, a polyacrylate high molecular polymer or a derivative thereof, or a high molecular composite material system containing them.

Furthermore, the methacrylylated high molecular polymer or its derivatives described above can be selected from any one of the following or a mixture of two or more thereof: methacrylylated gelatin or its derivatives, methacrylylated hyaluronic acid or its derivatives, methacrylylated sodium alginate or its derivatives, methacrylylated silk fibroin or its derivatives, methacrylylated chitosan or its derivatives, methacrylylated carboxymethyl chitosan or its derivatives. The above-mentioned high molecular polymer of polyacrylate or its derivatives can be selected from polyether diacrylate or its derivatives, or polyethylene glycol diacrylate or its derivatives. The most preferred photosensitive material of the present invention is methacrylylated gelatin or its derivatives, or methacrylylated silk fibroin or its derivatives.

Furthermore, the derivatives of the methacrylylated high molecular polymers mentioned above include polymers with one or more functional groups modified. The modifiable functional groups of the methacryloyl gelatin include any one or more of amino, carboxyl, thiol, hydroxyl or guanidine; the derivatives of the methacryloyl hyaluronic acid include polymers modified with one or more functional groups thereof, wherein the modifiable functional groups include any one or more of hydroxyl, carboxyl, acetylamino or hydroxymethyl; the derivatives of the methacryloyl sodium alginate include polymers modified with one or more functional groups thereof, wherein the modifiable functional groups include any one or two of carboxyl and hydroxyl; the derivatives of the methacryloyl silk fibroin include polymers modified with one or more functional groups thereof, wherein the modifiable functional groups include any one or more of amino, carboxyl, thiol, hydroxyl or guanidine; the derivatives of the methacryloyl chitosan include polymers modified with one or more functional groups thereof or polymers subjected to multiple chemical reactions, wherein the modifiable functional groups include any one or two of amino or hydroxyl, and the multiple chemical reactions that can occur include any one or more of alkylation, acylation, carboxymethylation, hydrolysis, oxidation, and reduction chemical reactions.

The molecular weight of the above-mentioned methacrylated high molecular polymer or its derivative is in the range of 5 to 400 kDa, and the molecular weight of the above-mentioned polyacrylate high molecular polymer or its derivative is in the range of 700 to 1000 kDa.

Furthermore, the polymer composite material system containing methacrylic polymer or its derivatives described above includes: methacrylic gelatin-polyvinyl alcohol system, methacrylic gelatin-polyurethane system, methacrylic gelatin-polylactic acid system, methacrylic gelatin-cellulose system, methacrylic hyaluronic acid-polyvinyl alcohol system, methacrylic hyaluronic acid-polyurethane system, methacrylic hyaluronic acid-polylactic acid system, methacrylic hyaluronic acid-cellulose system, methacrylic sodium alginate-polyvinyl alcohol system, methacrylic sodium alginate-polyurethane system, methacrylic sodium alginate-polylactic acid system, methacrylic sodium alginate-cellulose system, Any one or more of the following: sodium alginate-cellulose system, methacrylated silk fibroin-polyvinyl alcohol system, methacrylated silk fibroin-polyurethane system, methacrylated silk fibroin-polylactic acid system, methacrylated silk fibroin-cellulose system, methacrylated chitosan-polyvinyl alcohol system, methacrylated chitosan-polyurethane system, methacrylated chitosan-polylactic acid system, methacrylated chitosan-cellulose system, methacrylated carboxymethyl chitosan-polyvinyl alcohol system, methacrylated carboxymethyl chitosan-polyurethane system, methacrylated carboxymethyl chitosan-polylactic acid system, and methacrylated carboxymethyl chitosan-cellulose system.

In the preferred kit of the present invention, the photoinitiator can be selected from any one of the following or a combination of two or more: phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone, 2,4,6-trimethylbenzoylphosphonic acid ethyl ester, 2-methyl-1-[4-methylthiophenyl]-2-morpholinyl-1-propanone, methyl o-benzoylbenzoate, 2-phenylbenzyl-2-dimethylamine-1-(4-morpholinobenzylphenyl)butanone or 2,2-azo (2-methyl-N-(2-hydroxyethyl)propionamide); most preferably phenyl (2,4,6-trimethylbenzoyl) lithium phosphate.

In the preferred kit of the present invention, the enzyme contained in the first precursor reagent can be selected from any one of human thrombin, recombinant human thrombin, bovine thrombin, porcine thrombin or snake venom thrombin.

In the preferred kit of the present invention, the fibrinogen contained in the second precursor reagent can be selected from any one of human fibrinogen, bovine fibrinogen or porcine fibrinogen.

In the preferred kit of the present invention, the water-soluble inorganic calcium salt can be selected from calcium chloride, calcium nitrate or calcium sulfate; calcium chloride is most preferred.

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

In the kit of the present invention, the first precursor reagent and/or the second precursor reagent can be in a variety of specific dosage forms acceptable for pharmaceutical or clinical use, such as lyophilized powder, sponge or granules.

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

The kit of the present invention may further include instructions for describing the method of using the kit.

In a third aspect, the present invention also provides use of the kit of the present invention in preparing an in situ rapid coagulation hemostatic material.

The application of the kit includes: preparing the first precursor reagent and the second precursor reagent into injectable solutions respectively by configuring a solvent, and then injecting or spraying them uniformly on the bleeding wound site at the same time, and then irradiating them with light in the 290-480nm band for 10-60s, so that a solid hydrogel can be quickly formed in situ at the bleeding wound site.

The bleeding wounds include organ bleeding caused by accidental trauma or during surgery; the organs may be the liver, spleen, kidney, gastrointestinal tract, heart or skin.

In the application of the present invention, when the kit is injected into a bleeding wound, (1) a fibrin clot can be formed on the wound surface instantly (about 1 second), which plays a role in preliminary plugging of the wound and blocking blood outflow, thereby compensating for the weak plugging effect of the photosensitive material before the photocuring is completed; (2) at the same time, the enzyme in the fibrin clot converts the fibrinogen in the blood into a clot, which plays a highly efficient procoagulant function; (3) further, the photosensitive material forms a photocurable gel within 5 to 10 seconds under light excitation; the photocurable gel has a strong adhesion force, which can withstand the impact of blood flow and protect the fibrin cross-links from being washed away by blood. Therefore, the use of the kit of the present invention or the in-situ preparation of the double-crosslinked fibrin gel can combine the immediate occurrence of fibrin cross-linking and the strong adhesion of photocrosslinking to obtain a double-crosslinked fibrin gel having a fibrin cross-linked network and a photocrosslinked network structure.

Compared with the prior art, the advantages of the present invention are: rapid gelation, fast curing speed, strong wet tissue adhesion, and good hemostatic effect:

(1) The double-crosslinked fibrin gel kit of the present invention can immediately (about 1 second) cause fibrin crosslinking after mixing, playing a preliminary blocking effect and blocking the impact of blood flow.

(2) The enzyme in the double cross-linked fibrin gel kit of the present invention can convert fibrinogen in the blood into cross-linked fibrin and has a high efficiency in promoting blood coagulation.

(3) The double cross-linked fibrin gel kit of the present invention can undergo a photo-cross-linking reaction within 5-10 seconds under the excitation of ultraviolet light or visible light to form a photocurable gel, which provides strong wet tissue adhesion and can protect the fibrin cross-links from being washed away by the blood flow;

Because the double cross-linked fibrin gel provided by the present invention has excellent coagulation function, curing speed, wet tissue adhesion and rapid hemostasis effect, it can be used for hemostasis of liver, spleen, kidney, heart, gastrointestinal tract and skin bleeding during accidental trauma or surgery.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a SEM image of cross-linked fibrin of Comparative Example 1.

FIG. 2 is a SEM image of the photo-crosslinked methacrylylated gelatin in the precursor solution of Comparative Example 2.

FIG. 3 is a SEM image of the double-crosslinked fibrin gel of Example 1.

FIG4 shows the comparison of hemostasis time of Examples 1, 9, 14, 19, 25, 31 and Comparative Examples 1 to 6.

FIG5 shows the comparison of blood loss between Examples 1, 9, 14, 19, 25, 31 and Comparative Examples 1 to 6.

DETAILED DESCRIPTION

The technical problems, technical solutions and beneficial effects to be solved by the present invention are described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements can also be made without departing from the inventive concept. These all belong to the protection scope of the present invention.

The present invention provides a double cross-linked fibrin gel, which is a solid hydrogel composed of a three-dimensional photosensitive gel network and a three-dimensional fibrin network; its microstructure is shown in Figure 3, and it has both a fibrin cross-linked network structure and a photosensitive material cross-linked porous structure; each pore of the porous structure has a group of the fibrin networks, and each group of the fibrin networks has continuity as a whole; on the whole, the three-dimensional fibrin networks are disorderly distributed on the surface and inside of the solid hydrogel; the fibrin network structure acts as a scaffold in the pores of the photosensitive gel network, and the pore walls of the photosensitive gel porous structure surround the fibrin network structure.

The double cross-linked fibrin gel is prepared according to the following method:

(1) Preparation of Composition A solution: adding an enzyme solution containing calcium ions to a mixed solution containing a photosensitive material and a photoinitiator, and uniformly mixing to obtain a Composition A solution comprising a photosensitive material, a photoinitiator and an enzyme; controlling the concentration of the photosensitive material in the obtained Composition A solution to be not less than 1% (w/v), preferably not less than 3% (w/v), and more preferably 3%-20% (w/v); and controlling the enzyme activity to be not less than 200 IU/mL, preferably not less than 500 IU/mL, and more preferably not less than 1000 IU/mL.

(2) Preparation of Composition B solution: Add the fibrinogen solution to the mixed solution containing the photosensitive material and the photoinitiator, and mix them uniformly to obtain a Composition B solution: comprising the photosensitive material, the photoinitiator and the fibrinogen. The concentration of the photosensitive material in the obtained Composition B solution is controlled to be not less than 0.5% (w/v), preferably 1%-10% (w/v); and the concentration of the fibrinogen is controlled to be not less than 3% (w/v), preferably 3%-5% (w/v).

(3) Storage method: The obtained composition A solution and composition B solution are freeze-dried at a volume ratio of 1:10 to 10:1, respectively, and stored after becoming sponge-like.

(4) Using the above freeze-dried sponge to prepare a double cross-linked fibrin gel: Dissolve the sponge-like component A and the sponge-like component B in a solvent to obtain injectable solution-like components A and B. Inject/spray equal volumes of the component A solution and the component B solution evenly on the bleeding site and irradiate with blue light or ultraviolet light for 10 to 60 seconds to quickly form a solid hydrogel in situ. As a preferred embodiment, the injectable solution is injected with a double syringe, a syringe, or a Pasteur pipette.

In the above preparation scheme, the solvent can be selected from any one or a combination of phosphate buffered saline, HEPES biological buffer, 0.9% sodium chloride solution, calcium chloride solution, and deionized water, and its usage is not particularly limited and can be prepared according to the actual required concentration.

Based on the above-mentioned embodiments, the present invention is further described by the following embodiments.

Example 1

The method for preparing the double cross-linked fibrin gel of this embodiment comprises the following steps:

(1) Preparation of methacrylated gelatin-phenyl (2,4,6-trimethylbenzoyl) lithium phosphate precursor solution: Add the required volume of 0.9% sodium chloride solution to powdered phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, heat and dissolve in a water bath to obtain two concentrations of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate solutions: 0.25% (w/v) and 0.5% (w/v); take the required weight of solid methacrylated gelatin, add the required concentration of phenyl ( The methacryloyl gelatin-phenyl (2,4,6-trimethylbenzoyl) lithium phosphate solution is heated in a water bath to dissolve, and two mass volume percentages (w/v) of methacryloyl gelatin-phenyl (2,4,6-trimethylbenzoyl) lithium phosphate precursor solutions are obtained: 26% (w/v) methacryloyl gelatin-0.5% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, and 10% (w/v) methacryloyl gelatin-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate;

(2) Preparation of thrombin solution: inject a calcium chloride solution of a required volume and concentration into thrombin, and after complete dissolution, obtain a thrombin solution with a thrombin activity of 2000 IU/mL and a Ca ²⁺ concentration of 80 mmol/L;

(3) Preparation of fibrinogen solution: Take the required weight of fibrinogen and slowly place it in a preheated 0.9% sodium chloride solution. After it is completely dissolved, a fibrinogen solution with a mass volume percentage (w/v) of 10% (w/v) is obtained.

(4) Preparation of component A solution: adding the thrombin solution obtained in step (2) to the 26% (w/v) methacryloyl gelatin-0.5% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate precursor solution obtained in step (1), and mixing them evenly to obtain a component A solution: 13% (w/v) methacryloyl gelatin-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin;

(5) Preparation of component B solution: adding the fibrinogen solution obtained in step (3) to the 10% (w/v) methacryloyl gelatin-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate precursor solution obtained in step (1), and uniformly mixing to obtain a component B solution: 5% (w/v) methacryloyl gelatin-0.125% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen;

(6) Storage: freeze-dry the obtained component A solution and component B solution at a volume ratio of 1:1 and store them in a sponge form;

(7) Usage: Dissolve the sponge-like component A and component B in a 0.9% sodium chloride solution in a volume ratio of 1:1 to obtain injectable components A and B. Load equal volumes of the component A solution and the component B solution into a double syringe, inject/spray the component A solution and the component B solution onto the bleeding site through a nozzle, and then irradiate with blue light for 10 to 60 seconds to convert it into a solid hydrogel in situ. In the gel obtained at this time, the volume ratio of fibrin cross-linking to photocross-linking is 1:1.

(8) The structure of the solid gel is shown in FIG3 : it is a solid hydrogel composed of a three-dimensional fibrin network and a three-dimensional photosensitive gel network; and each group of the fibrin networks has continuity as a whole within the formed methacryloyl gelatin cross-linked porous structure; on the whole, the three-dimensional fibrin network is randomly distributed on the surface and inside of the solid hydrogel.

Example 2

A mixed solution of 10% (w/v) methacryloyl gelatin-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 5% (w/v) methacryloyl gelatin-0.125% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen was prepared as the component B solution. The composition, preparation method and use method thereof are substantially the same as those in Example 1, except that the concentration of methacryloyl gelatin in the component A solution prepared in step (4) is adjusted to 10% (w/v).

Example 3

A mixed solution of 8% (w/v) methacryloyl gelatin-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 5% (w/v) methacryloyl gelatin-0.125% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen was prepared as the component B solution. The composition, preparation method and use method thereof are substantially the same as those in Example 1, except that the concentration of methacryloyl gelatin in the component A solution prepared in step (4) is adjusted to 8% (w/v).

Example 4

A mixed solution of 5% (w/v) methacryloyl gelatin-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 5% (w/v) methacryloyl gelatin-0.125% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen was prepared as the component B solution. The composition, preparation method and use method are substantially the same as those in Example 1, except that the concentration of methacryloyl gelatin in the component A solution prepared in step (4) is 5% (w/v). In addition, in the gel obtained at this time, the volume ratio of fibrin cross-linking to photo-cross-linking is 2:1.

Example 5

A mixed solution of 13% (w/v) methacryloyl gelatin-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 3% (w/v) methacryloyl gelatin-0.125% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen was prepared as the component B solution. The composition, preparation method and use method thereof are substantially the same as those in Example 1, except that the concentration of methacryloyl gelatin in the component B solution prepared in step (5) is adjusted to 3% (w/v).

Example 6

A mixed solution of 13% (w/v) methacryloyl gelatin-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-500 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 5% (w/v) methacryloyl gelatin-0.125% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen was prepared as the component B solution. The composition, preparation method and use method are substantially the same as those in Example 1, except that the thrombin activity of the component A solution prepared in step (4) is adjusted to 500 IU/mL.

Example 7

A mixed solution of 13% (w/v) methacryloyl gelatin-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-250 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 5% (w/v) methacryloyl gelatin-0.125% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen was prepared as the component B solution. The composition, preparation method and use method are substantially the same as those in Example 1, except that the thrombin activity of the component A solution prepared in step (4) is adjusted to 250 IU/mL.

Example 8

A mixed solution of 13% (w/v) methacryloyl gelatin-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 5% (w/v) methacryloyl gelatin-0.125% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-3% (w/v) fibrinogen was prepared as the component B solution. The composition, preparation method and use method are substantially the same as those in Example 1, except that the fibrinogen concentration of the component B solution prepared in step (5) is 3% (w/v). In the gel obtained at this time, the volume ratio of fibrin cross-linking and photo-cross-linking is 1:2.

Example 9

A mixed solution of 8% (w/v) methacryloyl hyaluronic acid-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 5% (w/v) methacryloyl hyaluronic acid-0.125% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen was prepared as the component B solution. The preparation and use methods are substantially the same as those in Example 1, except that: ① the photosensitive material in the components A and B of this embodiment is methacryloyl hyaluronic acid, and the concentration of methacryloyl hyaluronic acid in the component A solution is 8% (w/v); ② the preparation process of the methacryloyl hyaluronic acid-phenyl (2,4,6-trimethylbenzoyl) lithium phosphate precursor solution does not require heating and can be carried out at room temperature. In the gel obtained at this time, the volume ratio of fibrin cross-linking and photo-cross-linking is 1:1.

Example 10

A mixed solution of 5% (w/v) methacryloyl hyaluronic acid-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 5% (w/v) methacryloyl hyaluronic acid-0.125% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen was prepared as the component B solution. Its composition, preparation method and use method are substantially the same as those in Example 9, except that the concentration of methacryloyl hyaluronic acid in the component A solution is adjusted to 5% (w/v). In the gel obtained at this time, the volume ratio of fibrin cross-linking and photo-cross-linking is 2:1.

Embodiment 11

A mixed solution of 8% (w/v) methacryloyl hyaluronic acid-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 3% (w/v) methacryloyl hyaluronic acid-0.125% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen was prepared as the component B solution. The composition, preparation method and use method are substantially the same as those in Example 9, except that the concentration of methacryloyl hyaluronic acid in the component B solution is adjusted to 3% (w/v).

Example 12

A mixed solution of 8% (w/v) methacryloyl hyaluronic acid-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-500 IU/ml thrombin was prepared as the component A solution, and a mixed solution of 5% (w/v) methacryloyl hyaluronic acid-0.125% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen was prepared as the component B solution. The composition, preparation method and use method are substantially the same as those in Example 9, except that the thrombin activity of the component A solution is adjusted to 500 IU/ml.

Example 13

A mixed solution of 8% (w/v) methacryloyl hyaluronic acid-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 5% (w/v) methacryloyl hyaluronic acid-0.125% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-3% (w/v) fibrinogen was prepared as the component B solution. Its composition, preparation method and use method are generally the same as those in Example 9, except that the fibrinogen concentration of the component B solution is adjusted to 3% (w/v). And in the gel obtained at this time, the volume ratio of fibrin cross-linking and photo-cross-linking is 1:2.

Embodiment 14

A mixed solution of 8% (w/v) methacryloyl sodium alginate-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 5% (w/v) methacryloyl sodium alginate-0.125% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen was prepared as the component B solution. The preparation method and use method are substantially the same as those in Example 9, except that the photosensitive material in the components A and B of this embodiment is methacryloyl sodium alginate. And in the gel obtained at this time, the volume ratio of fibrin cross-linking and photo-cross-linking is 1:1.

Embodiment 15

A mixed solution of 5% (w/v) methacryloyl sodium alginate-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 5% (w/v) methacryloyl sodium alginate-0.125% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen was prepared as the component B solution. Its composition, preparation method and use method are generally the same as those in Example 14, except that the concentration of methacryloyl sodium alginate in the component A solution is adjusted to 5% (w/v). In the gel obtained at this time, the volume ratio of fibrin cross-linking and photo-cross-linking is 2:1.

Example 16

A mixed solution of 8% (w/v) methacryloyl sodium alginate-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 3% (w/v) methacryloyl sodium alginate-0.125% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen was prepared as the component B solution. The composition, preparation method and use method are substantially the same as those in Example 14, except that the concentration of methacryloyl sodium alginate in the component B solution is adjusted to 3% (w/v).

Embodiment 17

A mixed solution of 8% (w/v) methacryloyl sodium alginate-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-500 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 5% (w/v) methacryloyl sodium alginate-0.125% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen was prepared as the component B solution. The composition, preparation method and use method are substantially the same as those in Example 14, except that the thrombin activity in the component A solution is adjusted to 500 IU/mL.

Embodiment 18

A mixed solution of 8% (w/v) methacryloyl sodium alginate-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 5% (w/v) methacryloyl sodium alginate-0.125% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-3% (w/v) fibrinogen was prepared as the component B solution. Its composition, preparation method and use method are substantially the same as those in Example 14, except that the fibrinogen concentration in the component B solution is adjusted to 3% (w/v). In the gel obtained at this time, the volume ratio of fibrin cross-linking and photo-cross-linking is 1:2.

Embodiment 19

A mixed solution of 10% (w/v) methacrylylated silk fibroin-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 5% (w/v) methacrylylated silk fibroin-0.125% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen was prepared as the component B solution. The preparation method and use method are substantially the same as those in Example 9, except that: the photosensitive material in the A and B components in this embodiment is methacrylylated silk fibroin, and the concentration of methacrylylated silk fibroin in the A component solution is 10% (w/v). In addition, in the gel obtained at this time, the volume ratio of fibrin cross-linking and photo-cross-linking is 1:1.

Embodiment 20

A mixed solution of 8% (w/v) methacryloylated silk fibroin-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 5% (w/v) methacryloylated silk fibroin-0.125% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen was prepared as the component B solution. The composition, preparation method and use method are substantially the same as those in Example 19, except that the concentration of methacryloylated silk fibroin in the component A solution is adjusted to 8% (w/v).

Embodiment 21

A mixed solution of 5% (w/v) methacryloylated silk fibroin-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 5% (w/v) methacryloylated silk fibroin-0.125% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen was prepared as the component B solution. Its composition, preparation method and use method are substantially the same as those in Example 19, except that the concentration of methacryloylated silk fibroin in the component A solution is adjusted to 5% (w/v). In the gel obtained at this time, the volume ratio of fibrin cross-linking and photo-cross-linking is 2:1.

Example 22

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

Embodiment 23

A mixed solution of 10% (w/v) methacryloylated silk fibroin-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-500 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 5% (w/v) methacryloylated silk fibroin-0.125% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen was prepared as the component B solution. The composition, preparation method and use method are substantially the same as those in Example 19, except that the thrombin activity in the component A solution is adjusted to 500 IU/mL.

Embodiment 24

A mixed solution of 10% (w/v) methacryloylated silk fibroin-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 5% (w/v) methacryloylated silk fibroin-0.125% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-3% (w/v) fibrinogen was prepared as the component B solution. Its composition, preparation method and use method are substantially the same as those in Example 19, except that the fibrinogen concentration in the component B solution is adjusted to 3% (w/v). In the gel obtained at this time, the volume ratio of fibrin cross-linking and photo-cross-linking is 1:2.

Embodiment 25

A mixed solution of 3% (w/v) methacryloyl chitosan-0.1% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 1% (w/v) methacryloyl chitosan-0.1% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen was prepared as the component B solution. The preparation method and use method are substantially the same as those in Example 9, except that: the photosensitive material in the A and B components of this embodiment is methacryloyl chitosan, and the concentration of methacryloyl chitosan in the A component solution is 3% (w/v), and the concentration of methacryloyl chitosan in the B component solution is 1% (w/v). And in the gel obtained at this time, the volume ratio of fibrin cross-linking and photocross-linking is 1:1.

Embodiment 26

A mixed solution of 2% (w/v) methacryloyl chitosan-0.1% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 1% (w/v) methacryloyl chitosan-0.1% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen was prepared as the component B solution. The composition, preparation method and use method are substantially the same as those in Example 25, except that the concentration of methacryloyl chitosan in the component A solution is adjusted to 2% (w/v).

Embodiment 27

A mixed solution of 1% (w/v) methacryloyl chitosan-0.1% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 1% (w/v) methacryloyl chitosan-0.1% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen was prepared as the component B solution. The composition, preparation method and use method are substantially the same as those in Example 25, except that the concentration of methacryloyl chitosan in the component A solution is adjusted to 1% (w/v). In the gel obtained at this time, the volume ratio of fibrin cross-linking and photo-cross-linking is 2:1.

Embodiment 28

A mixed solution of 3% (w/v) methacryloyl chitosan-0.1% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 0.5% (w/v) methacryloyl chitosan-0.1% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen was prepared as the component B solution. The composition, preparation method and use method are substantially the same as those in Example 25, except that the concentration of methacryloyl chitosan in the component B solution is adjusted to 0.5% (w/v).

Embodiment 29

A mixed solution of 3% (w/v) methacryloyl chitosan-0.1% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-500 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 1% (w/v) methacryloyl chitosan-0.1% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen was prepared as the component B solution. The composition, preparation method and use method are substantially the same as those in Example 25, except that the thrombin activity in the component A solution is adjusted to 500 IU/mL.

Embodiment 30

A mixed solution of 3% (w/v) methacryloyl chitosan-0.1% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 1% (w/v) methacryloyl chitosan-0.1% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-3% (w/v) fibrinogen was prepared as the component B solution. Its composition, preparation method and use method are substantially the same as those in Example 25, except that the fibrinogen concentration in the component B solution is adjusted to 3% (w/v). In the gel obtained at this time, the volume ratio of fibrin cross-linking and photo-cross-linking is 1:2.

Embodiment 31

A mixed solution of 20% (w/v) polyether F127 diacrylate-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 10% (w/v) polyether F127 diacrylate-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen was prepared as the component B solution. The preparation method and use method are substantially the same as those in Example 9, except that: the photosensitive material in the A and B components of this embodiment is polyether F127 diacrylate, and the concentration of polyether F127 diacrylate in the A component solution is 20% (w/v), and the concentration of polyether F127 diacrylate in the B component solution is 10% (w/v). In the gel obtained at this time, the volume ratio of fibrin cross-linking and photocross-linking is 1:1.

Embodiment 32

A mixed solution of 15% (w/v) polyether F127 diacrylate-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 10% (w/v) polyether F127 diacrylate-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen was prepared as the component B solution. The composition, preparation method and use method are substantially the same as those in Example 31, except that the concentration of polyether F127 diacrylate in the component A solution is adjusted to 15% (w/v).

Embodiment 33

A mixed solution of 10% (w/v) polyether F127 diacrylate-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 10% (w/v) polyether F127 diacrylate-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen was prepared as the component B solution. Its composition, preparation method and use method are generally the same as those in Example 31, except that the concentration of polyether F127 diacrylate in the component A solution is adjusted to 10% (w/v). In the gel obtained at this time, the volume ratio of fibrin cross-linking and photo-cross-linking is 2:1.

Embodiment 34

A mixed solution of 20% (w/v) polyether F127 diacrylate-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 5% (w/v) polyether F127 diacrylate-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen was prepared as the component B solution. The composition, preparation method and use method are substantially the same as those in Example 31, except that the concentration of polyether F127 diacrylate in the component B solution is adjusted to 5% (w/v).

Embodiment 35

A mixed solution of 20% (w/v) polyether F127 diacrylate-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-500 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 10% (w/v) polyether F127 diacrylate-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen was prepared as the component B solution. The composition, preparation method and use method are substantially the same as those in Example 31, except that the thrombin activity in the component A solution is adjusted to 500 IU/mL.

Embodiment 36

A mixed solution of 20% (w/v) polyether F127 diacrylate-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 10% (w/v) polyether F127 diacrylate-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-3% (w/v) fibrinogen was prepared as the component B solution. Its composition, preparation method and use method are substantially the same as those in Example 31, except that the fibrinogen concentration in the component B solution is adjusted to 3% (w/v). In the gel obtained at this time, the volume ratio of fibrin cross-linking and photo-cross-linking is 1:2.

Comparative Example 1

The topical freeze-dried fibrin adhesive (Hugulaishi, purchased from Shanghai Laishi) includes an enzyme reagent and a fibrinogen reagent. The enzyme reagent and the fibrinogen reagent are prepared into solutions according to their instructions, and the enzyme cross-linking is completed in about 1 second after mixing to obtain the fibrin adhesive. The microstructure of the adhesive is shown in Figure 1.

Comparative Example 2

The preparation method of 9% (w/v) methacryloyl gelatin-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate precursor solution is substantially the same as that of the component A solution of Example 2, except that thrombin is not added to the solution.

Comparative Example 3

13% (w/v) methacryloyl gelatin-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin mixed solution, the components and preparation method are the same as those of the component A solution in Example 2.

Comparative Example 4

5% (w/v) methacryloyl gelatin-0.125% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen mixed solution, the components and preparation method are the same as step (5) of Example 1.

Comparative Example 5

A mixed solution of 30% (w/v) methacryloyl sericin-0.5% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 20% (w/v) methacryloyl sericin-0.5% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen was prepared as the component B solution. The preparation method is substantially the same as that of Example 9, except that the photosensitive material in the A and B components in this comparative example is methacryloyl sericin, and the concentration of methacryloyl sericin in the A component solution is 30% (w/v), and the concentration of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate is 0.5% (w/v), and the concentration of methacryloyl sericin in the B component solution is 20% (w/v), and the concentration of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate is 0.5% (w/v). In the gel obtained at this time, the volume ratio of fibrin cross-linking and photo-cross-linking is 1:1.

Comparative Example 6

A mixed solution of 10% (w/v) methacryloyl dextran-0.25% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-1000 IU/mL thrombin was prepared as the component A solution, and a mixed solution of 10% (w/v) methacryloyl dextran-0.125% (w/v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate-5% (w/v) fibrinogen was prepared as the component B solution. The preparation method is substantially the same as that of Example 9, except that: the photosensitive material in the A and B components in this comparative example is methacryloyl dextran, and the concentration of methacryloyl dextran in the A component solution is 10% (w/v), and the concentration of methacryloyl dextran in the B component solution is 5% (w/v). And in the gel obtained at this time, the volume ratio of fibrin cross-linking and photo-cross-linking is 1:1.

Performance Testing

In order to verify the performance of the double cross-linked fibrin gels obtained in Examples 1 to 36 and the hydrogels in Comparative Examples 1 to 6, gelation time performance tests, adhesion strength tests and animal hemostasis experiments were performed on them respectively.

Gel time test

Detection object:

The aforementioned Examples 1 to 36, and Comparative Examples 1 to 6;

Detection method:

Rheological analysis was performed on Examples 1 to 36 and Comparative Examples 1 to 6 to compare their gel time, and the results are shown in Table 1. Specific operating method: Dynamic rheological experiments were performed at 37°C using a HAAKE RS6000 optical rheometer with a parallel plate (P20 TiL, 20-mm diameter) geometry. Time-sweep oscillation tests of the hydrogels of Examples 1 to 36 and Comparative Examples 1 to 6 were performed at 5% strain and 1 Hz frequency for 300 seconds. The pre-gel solution was strain scanned to verify the linear response. The gel point is determined when the torsional modulus (G') exceeds the loss modulus (G").

Adhesion strength test

The aforementioned Examples 1 to 36, and Comparative Examples 1 to 6;

Detection method:

Specific operation: Cut the pigskin into rectangles of 40 mm x 20 mm, and use 500 μl of Examples 1 to 36 and Comparative Examples 1 to 6 to bond two pieces of pigskin. Illuminate the mixed solution of component A and component B of Examples 1 to 36 and the precursor solution of Comparative Examples 2 to 6 with blue light of the same wavelength for 60 seconds. Then test the bonding strength at a strain rate of 1 mm/min. The microstructure of the gel formed after photo-crosslinking of the precursor solution of Comparative Example 2 is shown in Figure 2; the microstructure of the gel formed after photo-crosslinking of the mixed solution of Example 1 is shown in Figure 3. Record the reading when the gel falls off the pigskin, which is the adhesion strength (Kpa). The test results are shown in Table 1.

Hemostatic effect test

Detection object:

Embodiment 1, embodiment 9, embodiment 14, embodiment 19, embodiment 25, embodiment 31 of the present invention, and comparative examples 1 to 6;

Detection method:

1 cm incision bleeding model on rabbit liver surface: After anesthetizing New Zealand white rabbits, the abdomen was exposed and fixed on the operating table. A midline incision was made on the abdomen to expose the liver, and a 1 cm*0.5 cm bleeding model was created on the liver; weighed filter paper, the mixed solution obtained by injecting the A component and the B component of Examples 1, 9, 14, 19, 25, and 31 according to the injection method described in step (7) of Example 1, and the precursor solution of Comparative Examples 1 to 6 were used as hemostatic materials to cover the bleeding site (wherein each of the Examples of the present invention and Comparative Examples 2-6 were covered and irradiated with the same wavelength blue light) until the bleeding stopped, and the bleeding time and blood loss were recorded. The results are shown in Table 1, Figures 4 and 5.

Table 1

The values of hemostasis time and blood loss are expressed as (mean ± standard deviation).

Result analysis:

As shown in Figure 1, only the cross-linked fibrin in the fibrin glue adhesive of Comparative Example 1 presents a mesh structure. As shown in Figure 2, only the methacrylylated gelatin is photo-cross-linked to present a porous structure after the precursor solution of Comparative Example 2 is irradiated. As shown in Figure 3, the mixed solution of components A and B of Example 1 of the present invention can simultaneously have a cross-linked fibrin mesh structure and a cross-linked porous structure of methacrylylated gelatin after irradiation, and the pores of the formed cross-linked porous structure of methacrylylated gelatin are distributed with an overall continuous three-dimensional fibrin network structure.

It can be seen from Table 1 that the gelation time range of Examples 1 to 36 is 1 to 3 s. When the photosensitive materials are the same, the gelation time will be extended with the increase of the photocrosslinking ratio. However, the gelation time of all types of photosensitive materials selected in Examples 1 to 36 at a specific double crosslinking ratio is significantly lower than the gelation time of Comparative Examples 2-4 (the gelation time of Comparative Example 2 is 8 s, the gelation time of Comparative Example 3 is 9 s, and the gelation time of Comparative Example 4 is 14 s).

It can be seen from Table 1 that the adhesion strength of Examples 1 to 36 ranges from 82 to 132 kPa. When the photosensitive material is the same, the adhesion strength of the gel decreases with the decrease in the concentration of the photosensitive material. However, the adhesion strength of all types of photosensitive materials selected in Examples 1 to 36 at a specific double cross-linking ratio is higher than the adhesion strength of each comparative example (the adhesion strength of comparative example 1 is 6 kPa, the adhesion strength of comparative example 2 is 80 kPa, the adhesion strength of comparative example 3 is 76 kPa, the adhesion strength of comparative example 4 is 70 kPa, the adhesion strength of comparative example 5 is 29 kPa, and the adhesion strength of comparative example 6 is 45 kPa).

As shown in Table 1 and Figures 4 and 5, the hemostasis time of Example 1, Example 9, Example 14, Example 19, Example 25 and Example 31 is 6s to 24s, which is significantly lower than the hemostasis time of more than 40s of Comparative Examples 1 to 6. The average blood loss of Example 1, Example 9, Example 14, Example 19, Example 25 and Example 31 is 12mg to 37mg, which is significantly lower than the blood loss of more than 90mg of Comparative Examples 1 to 6.

In summary, the double cross-linked fibrin gel of the present invention, when applied to a bleeding wound, can instantly (about 1 second) form a fibrin clot, which plays a "preliminary" role in plugging the wound and blocking blood from flowing out; at the same time, the enzyme in the fibrin clot converts the fibrinogen in the blood into a clot, which plays an efficient procoagulant effect; further, the photosensitive material forms a photosensitive gel under light excitation, and the photosensitive gel has a strong wet tissue adhesion force, which plays a "strong" wound sealing effect. The fibrin cross-linking and photo-cross-linking structures interact with each other, and have both the initial wound plugging and strong tissue adhesion functions, thereby achieving an excellent hemostatic effect.

The above is a detailed introduction to the specific embodiments of the present invention. It should be understood that the present invention is not limited to a specific implementation mode, and any deformation or modification, equivalent substitution and improvement made within the spirit and principle of the present invention does not affect the essential content of the present invention and should be included in the protection scope of the claims of the present invention.

Claims (19)

1. A double cross-linked fibrin gel, characterized in that: it is a solid hydrogel composed of a network structure with a sealing function and a network structure with an adhesion function; the network structure with a sealing function is a three-dimensional fibrin network, and the network structure with an adhesion function is a three-dimensional photosensitive gel network; each of the photosensitive gel network channels has a group of the fibrin networks inside, and each group of the fibrin networks has continuity as a whole; on the whole, the three-dimensional fibrin networks are disorderly distributed on the surface and inside of the solid hydrogel. 2. The double cross-linked fibrin gel as claimed in claim 1, characterized in that the fibrin network is formed before the photosensitive gel. 3. The double cross-linked fibrin gel as described in claim 1 is characterized in that 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; most preferably 1. 4. The double cross-linked fibrin gel according to claim 1, characterized in that: the photosensitive gel is formed by photosensitive material through photocrosslinking, and the photosensitive material is a methacrylated polymer or its derivative, a polyacrylate polymer or its derivative, or a polymer composite material system containing a methacrylated polymer or a polyacrylate polymer; preferably, the methacrylated polymer or its derivative is selected from any one of the following or a mixture of two or more: methacrylated gelatin or its derivative, methacrylated hyaluronic acid or its derivative, methacrylated sodium alginate or its derivative, methacrylated silk fibroin or its derivative, methacrylated chitosan or its derivative, methacrylated carboxymethyl chitosan or its derivative; preferably, the polyacrylate polymer or its derivative is selected from polyether diacrylate or its derivative, or polyethylene glycol diacrylate or its derivative; the most preferred photosensitive material is methacrylated gelatin or its derivative, or methacrylated silk fibroin or its derivative; preferably, the polymer composite material system containing methacrylated polymer is selected from methacrylated gelatin- Polyvinyl alcohol system, methacrylated gelatin-polyurethane system, methacrylated gelatin-polylactic acid system, methacrylated gelatin-cellulose system, methacrylated hyaluronic acid-polyvinyl alcohol system, methacrylated hyaluronic acid-polyurethane system, methacrylated hyaluronic acid-polylactic acid system, methacrylated hyaluronic acid-cellulose system, methacrylated sodium alginate-polyvinyl alcohol system, methacrylated sodium alginate-polyurethane system, methacrylated sodium alginate-polylactic acid system, methacrylated sodium alginate-cellulose system, methacrylated silk fibroin-polyvinyl alcohol system Any one or more of the following systems: methacrylylated silk fibroin-polyurethane system, methacrylylated silk fibroin-polylactic acid system, methacrylylated silk fibroin-cellulose system, methacrylylated chitosan-polyvinyl alcohol system, methacrylylated chitosan-polyurethane system, methacrylylated chitosan-polylactic acid system, methacrylylated chitosan-cellulose system, methacrylylated carboxymethyl chitosan-polyvinyl alcohol system, methacrylylated carboxymethyl chitosan-polyurethane system, methacrylylated carboxymethyl chitosan-polylactic acid system, and methacrylylated carboxymethyl chitosan-cellulose system. 5. The double cross-linked fibrin gel as claimed in claim 1, characterized in that: the fibrin network is formed by enzymatic cross-linking of fibrinogen; the fibrinogen is any one of human fibrinogen, bovine fibrinogen or porcine fibrinogen. 6. A kit for preparing a double-crosslinked fibrin gel according to any one of claims 1 to 5, comprising a first precursor reagent and a second precursor reagent which are independently packaged from each other; in parts by weight, the first precursor reagent contains 10 to 200 parts of a photosensitive material, 1 to 3 parts of a photoinitiator, 0.14 to 0.28 parts of an enzyme and 3.33 to 5.55 parts of a water-soluble inorganic calcium salt, and the second precursor reagent comprises 5 to 100 parts of a photosensitive material, 1 to 2 parts of a photoinitiator and 30 to 50 parts of fibrinogen; the mass ratio of the first precursor reagent to the second precursor reagent is 1.4:10 to 14:1; the preferred mass ratio is 1.4:1 to 1.4:10; and the most preferred mass ratio is 1.4:1. 7. The kit according to claim 6, characterized in that: by weight, the first precursor reagent contains 80-200 parts of photosensitive material, 1-3 parts of photoinitiator, 0.14-0.28 parts of enzyme and 3.33-5.55 parts of water-soluble inorganic calcium salt, and the second precursor reagent contains 30-100 parts of photosensitive material, 1-2 parts of photoinitiator and 30-50 parts of fibrinogen; preferably, the first precursor reagent contains 100-200 parts of photosensitive material, 1-3 parts of photoinitiator, 0.14-0.28 parts of enzyme and 3.33-5.55 parts of water-soluble inorganic calcium salt. 28 parts of enzyme and 3.33-5.55 parts of water-soluble inorganic calcium salt, the second precursor reagent contains 30-50 parts of photosensitive material, 1-2 parts of photoinitiator and 30-50 parts of fibrinogen; more preferably, the first precursor reagent contains 100-150 parts of photosensitive material, 1-3 parts of photoinitiator, 0.14-0.28 parts of enzyme and 3.33-5.55 parts of water-soluble inorganic calcium salt, and the second precursor reagent contains 30-50 parts of photosensitive material, 1-2 parts of photoinitiator and 30-50 parts of fibrinogen. 8. The kit according to any one of claims 6 to 7, characterized in that: the photosensitive gel is formed by photosensitive material through photocrosslinking, and the photosensitive material is a methacrylated polymer or its derivative, a polyacrylate polymer or its derivative, or a polymer composite material system containing a methacrylated polymer or a polyacrylate polymer; preferably, the methacrylated polymer or its derivative is selected from any one of the following or a mixture of two or more thereof: methacrylated gelatin or its derivative, methacrylated hyaluronic acid or its derivative, methacrylated sodium alginate or its derivative, methacrylated silk fibroin or its derivative, methacrylated chitosan or its derivative, methacrylated carboxymethyl chitosan or its derivative; preferably, the polyacrylate polymer or its derivative is selected from polyether diacrylate or its derivative, or polyethylene glycol diacrylate or its derivative; the most preferred photosensitive material is methacrylated gelatin or its derivative, or methacrylated silk fibroin or its derivative; preferably, the polymer composite material system containing methacrylated polymer is selected from methacrylated gelatin- Polyvinyl alcohol system, methacrylated gelatin-polyurethane system, methacrylated gelatin-polylactic acid system, methacrylated gelatin-cellulose system, methacrylated hyaluronic acid-polyvinyl alcohol system, methacrylated hyaluronic acid-polyurethane system, methacrylated hyaluronic acid-polylactic acid system, methacrylated hyaluronic acid-cellulose system, methacrylated sodium alginate-polyvinyl alcohol system, methacrylated sodium alginate-polyurethane system, methacrylated sodium alginate-polylactic acid system, methacrylated sodium alginate-cellulose system, methacrylated silk fibroin-polyvinyl alcohol system Any one or more of the following systems: methacrylylated silk fibroin-polyurethane system, methacrylylated silk fibroin-polylactic acid system, methacrylylated silk fibroin-cellulose system, methacrylylated chitosan-polyvinyl alcohol system, methacrylylated chitosan-polyurethane system, methacrylylated chitosan-polylactic acid system, methacrylylated chitosan-cellulose system, methacrylylated carboxymethyl chitosan-polyvinyl alcohol system, methacrylylated carboxymethyl chitosan-polyurethane system, methacrylylated carboxymethyl chitosan-polylactic acid system, and methacrylylated carboxymethyl chitosan-cellulose system. 9. The kit according to any one of claims 6 to 7, characterized in that: the photoinitiator is selected from any one of the following or a combination of two or more thereof: phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone, ethyl 2,4,6-trimethylbenzoylphosphonate, 2-methyl-1-[4-methylthiophenyl]-2-morpholinyl-1-propanone, methyl o-benzoylbenzoate, 2-phenylbenzyl-2-dimethylamine-1-(4-morpholinobenzylphenyl)butanone or 2,2-azo (2-methyl-N-(2-hydroxyethyl)propionamide); preferably, the photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate. 10. The kit according to any one of claims 6 to 7, characterized in that the enzyme is selected from any one of human thrombin, recombinant human thrombin, bovine thrombin, porcine thrombin or snake venom thrombin. 11. The kit according to any one of claims 6 to 7, characterized in that the fibrinogen is selected from any one of human fibrinogen, bovine fibrinogen or porcine fibrinogen. 12. The kit according to any one of claims 6 to 7, characterized in that the water-soluble inorganic calcium salt is selected from calcium chloride, calcium nitrate or calcium sulfate; calcium chloride is most preferred. 13. The kit according to any one of claims 6 to 7, characterized in that the kit is a freeze-dried powder, sponge or granules. 14. The kit according to any one of claims 6 to 7, characterized in that: the first precursor reagent and/or the second precursor reagent further contain auxiliary materials and/or additives; the auxiliary materials are selected from one or more of glycine, arginine hydrochloride, sodium citrate, sucrose, and sodium chloride; the additives are selected from one or more of growth factors, interleukins, vitamins, and silver ions; preferably, the growth factors are selected from one or more of platelet growth factor, epidermal growth factor, or fibroblast growth factor; preferably, the interleukins are selected from one or more of interleukin 2, interleukin 6, or interleukin 8; preferably, the vitamins are selected from one or more of vitamin B, vitamin C, vitamin E, or vitamin K. 15. The kit according to any one of claims 6 to 7, characterized in that it further comprises a separately packaged solvent for preparation, wherein the solvent for preparation is any one or a mixture of several of phosphate buffered saline, HEPES biological buffer, 0.9% sodium chloride solution, calcium chloride solution or deionized water. 16. The kit according to any one of claims 6 to 7, further comprising instructions for use. 17. Use of the kit according to any one of claims 6 to 16 in the preparation of in situ rapid coagulation hemostatic materials. 18. The use as claimed in claim 17, characterized in that: the first precursor reagent and the second precursor reagent in the kit are prepared into injectable solutions respectively by configuring a solvent, and then injected or sprayed on the bleeding wound uniformly at the same time, and then irradiated with light in the 290-480nm band for 10-60s, so as to quickly form a solid hydrogel as a coagulation and hemostasis material in situ in the bleeding wound. 19. The use according to claim 18, characterized in that: the bleeding wound includes organ bleeding caused by accidental trauma or during surgery; the organ is liver, spleen, kidney, gastrointestinal tract, heart or skin.

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