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WO2024078129A1 - 双交联纤维蛋白凝胶、其原料组合物和试剂盒及其应用 - Google Patents

双交联纤维蛋白凝胶、其原料组合物和试剂盒及其应用 Download PDF

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Publication number
WO2024078129A1
WO2024078129A1 PCT/CN2023/113503 CN2023113503W WO2024078129A1 WO 2024078129 A1 WO2024078129 A1 WO 2024078129A1 CN 2023113503 W CN2023113503 W CN 2023113503W WO 2024078129 A1 WO2024078129 A1 WO 2024078129A1
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Prior art keywords
methacrylated
derivative
solution
photosensitive material
fibrinogen
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PCT/CN2023/113503
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English (en)
French (fr)
Inventor
毛峥伟
余丽莎
王伟林
丁元
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Zhejiang University ZJU
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Zhejiang University ZJU
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Priority claimed from CN202211249717.0A external-priority patent/CN115920118B/zh
Priority claimed from CN202211244546.2A external-priority patent/CN115671372B/zh
Application filed by Zhejiang University ZJU filed Critical Zhejiang University ZJU
Priority to JP2024538770A priority Critical patent/JP7772430B2/ja
Priority to US18/841,788 priority patent/US12377188B2/en
Priority to KR1020247021479A priority patent/KR20240110979A/ko
Publication of WO2024078129A1 publication Critical patent/WO2024078129A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/02—Surgical adhesives or cements; Adhesives for colostomy devices containing inorganic materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/001—Use of materials characterised by their function or physical properties
    • A61L24/0031—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/046—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/08—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/10—Polypeptides; Proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/10—Polypeptides; Proteins
    • A61L24/106—Fibrin; Fibrinogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
    • A61L26/0028—Polypeptides; Proteins; Degradation products thereof
    • A61L26/0042—Fibrin; Fibrinogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/04—Materials for stopping bleeding

Definitions

  • 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.
  • 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.
  • 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.
  • fibrin glue will decrease under 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.
  • 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.
  • 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.
  • 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.
  • Luo et al. introduced snake venom hemocoagulase with coagulation function into methacryloyl gelatin, and the constructed hemostatic gel 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.
  • 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 4 g/L), and the fibrin crosslinks formed are insufficient to block the wound.
  • 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.
  • 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 raw material composition and 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.
  • 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.
  • 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.
  • 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.
  • the present invention further optimizes the ratio of the two networks in the gel through experiments.
  • 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.
  • 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.
  • 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.
  • photocurable polymer materials i.e., photosensitive materials
  • 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.
  • 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.
  • 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
  • the molecular weight of the above-mentioned methacrylylated 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.
  • 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
  • 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.
  • the present invention provides a raw material composition for preparing the double cross-linked fibrin adhesive described in the first aspect of the present invention, comprising composition A and composition B; in parts by weight, the composition A comprises 10 to 200 parts of photosensitive material, 1 to 3 parts of photoinitiator, 0.14 to 0.28 parts of enzyme and 1.11 to 8.88 parts of water-soluble inorganic calcium salt, and the composition B 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 composition A to the 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; most preferably 1.4:1.
  • the weight of the photosensitive material in the composition A is greater than the weight of the photosensitive material in the composition B.
  • the weight of the photosensitive materials in the composition A and the composition B is conducive to the enzyme reaction between the enzyme in the composition A and the fibrinogen in the composition B to form a stable fibrin network structure.
  • the fibrinogen can form a fibrin network through an enzyme cross-linking reaction under the action of the enzyme, and the photosensitive material can form a photosensitive gel through a photocross-linking reaction under the action of the photoinitiator. Therefore, after the composition A and the composition B in the raw material composition described in the present invention are mixed according to the mass ratio, a double cross-linked fibrin adhesive can be prepared by irradiation.
  • the adhesive is a solid hydrogel, and a three-dimensional fibrin network and a three-dimensional photosensitive gel network exist in its structure at the same time; each of the photosensitive gel network channels 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 network is disorderly distributed on the surface and inside of the solid hydrogel.
  • this solid hydrogel When this solid hydrogel is formed at a bleeding wound site, it can instantly (about 1 second) form a fibrin clot on the wound surface, which plays a role in preliminary 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 a highly efficient blood coagulation function; further, under light excitation, the photosensitive material can form a photocurable gel within 5 to 10 seconds; 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.
  • the composition A and the composition B can instantly form a fibrin network to play a scaffolding role after mixing, and a photosensitive gel will also be formed rapidly later, and the photosensitive gel formed later is wrapped around the fibers in the fibrin network.
  • the mass ratio of the composition A to the composition B is related to the preliminary wound sealing effect and adhesion strength of the double cross-linked fibrin adhesive: when the mass ratio of the composition A to the composition B is in the range of 1.4:10 to 1.4:1, as the proportion of the composition A increases, the procoagulant function of the double cross-linked fibrin adhesive is improved, the preliminary wound sealing effect is increased, and the adhesion strength increases; when the mass ratio of the composition A to the composition B is in the range of 1.4:1 to 14:1, as the proportion of the composition A increases, the procoagulant function, the preliminary wound sealing effect and the adhesion strength of the double cross-linked fibrin adhesive do not further increase.
  • the mass ratio of the composition A to the composition B is 1.4:1, the best hemostatic effect can be achieved and the best material utilization rate can be achieved.
  • the volume ratio of the two networks produced by cross-linking can reach about 1:1, which can bring the best procoagulant function and adhesion strength to the prepared gel.
  • the composition A comprises 80 to 200 parts of photosensitive material, 1 to 3 parts of photosensitive material, Initiator, 0.14-0.28 parts of enzyme and 1.11-8.88 parts of water-soluble inorganic calcium salt
  • the composition B comprises 30-100 parts of photosensitive material, 1-2 parts of photoinitiator and 30-50 parts of fibrinogen.
  • the composition A comprises 100 to 200 parts of photosensitive material, 1 to 3 parts of photoinitiator, 0.14 to 0.28 parts of enzyme and 1.11 to 8.88 parts of water-soluble inorganic calcium salt
  • the composition B comprises 30 to 50 parts of photosensitive material, 1 to 2 parts of photoinitiator and 30 to 50 parts of fibrinogen, by weight.
  • the composition A comprises, by weight, 100 to 150 parts of photosensitive material, 1 to 3 parts of photoinitiator, 0.14 to 0.28 parts of enzyme and 1.11 to 8.88 parts of water-soluble inorganic calcium salt
  • the composition B comprises 30 to 50 parts of photosensitive material, 1 to 2 parts of photoinitiator and 30 to 50 parts of fibrinogen.
  • the present invention also provides a method for preparing the raw material composition, comprising: preparing a mixed solution in which a photosensitive material and a photoinitiator are dissolved in a solvent, mixing the mixed solution with a solution containing thrombin and calcium ions to obtain a first precursor solution, and controlling the concentration ratio of the photosensitive material, photoinitiator, enzyme and calcium ions in the first precursor solution to be 10-200:1-3:0.14-0.28:1.11-8.88; mixing the mixed solution with a solution containing fibrinogen to obtain a second precursor solution, and controlling the concentration ratio of the photosensitive material, photoinitiator and fibrinogen in the second precursor solution to be 5-100:1-2:30-50; thereby, a liquid raw material composition comprising the first precursor solution and the second precursor solution can be obtained; and the liquid raw material composition can also be further processed according to conventional methods to obtain a solid raw material composition, such as a lyophilized powder, sponge or granules.
  • a solid raw material composition such as a
  • the first precursor solution in order to maintain the activity of the photosensitive material in the first precursor solution, the first precursor solution is kept at room temperature for less than 30 minutes.
  • the concentration of the photosensitive material in the first precursor solution in order to take into account both the uniformity of fibrinogen dispersion and the speed at which the photosensitive material completes photocrosslinking, it is preferred to control the concentration of the photosensitive material in the first precursor solution to be greater than 0.5% (w/v), and the concentration of the photosensitive material in the second precursor solution to be lower than that in the first precursor solution.
  • the fibrinogen solution is more easily uniformly dispersed in the second precursor solution with a relatively low concentration of the photosensitive material, and can quickly and fully contact the enzyme after the two precursor solutions are mixed, and completely enzyme crosslinking occurs instantly to form a uniformly distributed fibrin network; at the same time, the higher concentration of the photosensitive material in the first precursor solution can increase the overall concentration of the photosensitive material after the two precursor solutions are mixed, so that it reaches a more ideal concentration required for gelation, which is beneficial to shorten the photocrosslinking time and increase the adhesion and strength of the gel.
  • the temperature of the mixed solution when the first precursor solution and the second precursor solution are prepared, it is preferred to control the temperature of the mixed solution to be no higher than 37°C.
  • a further preferred method of preparing the raw material composition injection of the present invention specifically comprises the following steps:
  • the second mixed solution prepared in 2) is mixed with a solution containing fibrinogen to obtain a second precursor solution, wherein the concentration ratio of the photosensitive material, the photoinitiator and the fibrinogen is controlled to be 5-100:1-2:30-50.
  • the solution containing enzyme and calcium ions is preferably prepared according to the following method: adding a solvent and a water-soluble inorganic calcium salt solution to the enzyme, and obtaining an enzyme solution containing Ca2 + after complete dissolution, and controlling the enzyme activity in the obtained solution to be 500IU-2000IU/ml and the Ca2 + concentration to be 60-100mmol/L.
  • the concentration of fibrinogen in the fibrinogen-containing solution is preferably 5% to 10% (w/v).
  • the concentration of the photosensitive material in the first precursor solution is controlled to be 1% to 30% (w/v), further preferably 8% to 30% (w/v), and more preferably 10%-20%.
  • the enzyme activity in the first precursor solution is controlled to be not less than 200 IU/ml, preferably not less than 500 IU/ml; more preferably not less than 1000 IU/ml.
  • the calcium ion concentration in the first precursor solution is controlled to be not less than 20 mmol/L, preferably not less than 30mmol/L, more preferably not less than 40mmol/L.
  • the concentration of the photosensitive material in the second precursor solution is controlled to be not less than 0.5% (w/v) and not higher than the concentration of the photosensitive material in the first precursor solution, further preferably not less than 1% (w/v) and not higher than the concentration of the photosensitive material in the first precursor solution, more preferably 1%-10% (w/v) and not higher than the concentration of the photosensitive material in the first precursor solution.
  • the fibrinogen concentration in the second precursor solution is controlled to be no less than 3% (w/v), more preferably 3%-5% (w/v).
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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 illumination conditions 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.
  • 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.
  • 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.
  • 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 methacrylylated gelatin include any one or more of amino, carboxyl, thiol, hydroxyl or guanidine;
  • the derivatives of the methacrylylated 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 methacrylylated 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 methacrylylated 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
  • the molecular weight of the above-mentioned methacrylylated 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.
  • 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
  • the photoinitiator can be selected from any one of the following or a combination of two or more: phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt, 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) phosphate lithium salt.
  • the enzyme can be selected from any one of human thrombin, recombinant human thrombin, bovine thrombin, porcine thrombin or snake venom thrombin.
  • the fibrinogen can be selected from any one of human fibrinogen, bovine fibrinogen or porcine fibrinogen.
  • the water-soluble inorganic calcium salt can be selected from calcium chloride, calcium nitrate or calcium sulfate; calcium chloride is most preferred.
  • 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.
  • 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.
  • 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.
  • the present invention also provides a method for rapidly stopping bleeding in situ at a bleeding wound using the kit described in the fourth aspect of the present invention, comprising: preparing the first precursor reagent and the second precursor reagent into injectable solutions respectively by configuring a solvent, and then simultaneously and uniformly injecting or spraying them on the bleeding wound site, and then irradiating them with light in the 290-480nm band for 10-60s, so that a solid hydrogel can be rapidly 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.
  • a fibrin clot when the kit is injected into a bleeding wound, (1) a fibrin clot can be formed on the wound surface in an instant (about 1 second), which plays a role in initially sealing the wound and blocking blood from flowing out, thereby compensating for the weak sealing 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 strong adhesion, can withstand the impact of blood flow and protect the fibrin cross-links from being washed away by blood.
  • the use of the kit or the kit in situ preparation of double cross-linked fibrin gel described in the present invention can combine the immediate occurrence of fibrin cross-linking and the strong adhesion of photocross-linking to obtain a double cross-linked fibrin gel having a fibrin cross-linked network and a photocross-linked network structure.
  • the advantages of the present invention are: rapid gelation, fast curing speed, strong wet tissue adhesion, and good hemostatic effect:
  • 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 blood flow impact.
  • 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.
  • 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;
  • the double cross-linked fibrin gel provided by the present invention has excellent coagulation promoting 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.
  • 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 between 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.
  • 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:
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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;
  • 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;
  • 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;
  • 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 is continuous as a whole; overall, the three-dimensional fibrin network is randomly distributed on the surface and inside of the solid hydrogel.
  • 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).
  • 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-1000 IU/mL thrombin was prepared as the component A solution.
  • a mixed solution of trimethylbenzoyl) lithium phosphate salt-5% (w/v) fibrinogen is used as the component B solution. Its 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 adjusted to 8% (w/v).
  • 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).
  • the volume ratio of fibrin cross-linking to photocross-linking is 2:1.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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 substantially 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.
  • 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.
  • 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 substantially 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.
  • 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).
  • 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.
  • Its composition, preparation method The method and use are substantially the same as those of Example 14, except that the thrombin activity in the component A solution is adjusted to 500 IU/mL.
  • 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.
  • 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 the gel obtained at this time, the volume ratio of fibrin cross-linking and photo-cross-linking is 1:1.
  • 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).
  • 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).
  • the volume ratio of fibrin cross-linking and photo-cross-linking is 2:1.
  • 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).
  • 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.
  • 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.
  • 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 photo-cross-linking is 1:1.
  • 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).
  • 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.
  • Its 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).
  • the volume ratio of fibrin cross-linking and photo-cross-linking is 2:1.
  • 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).
  • 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.
  • 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.
  • 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).
  • the volume ratio of fibrin cross-linking and photocross-linking is 1:1.
  • 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).
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • the volume ratio of fibrin cross-linking and photo-cross-linking is 1:1.
  • 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.
  • Embodiment 1 Embodiment 1, embodiment 9, embodiment 14, embodiment 19, embodiment 25, embodiment 31 of the present invention, and comparative examples 1 to 6;
  • the gelation time range of Examples 1 to 36 is 1 to 3 s.
  • the gelation time will be extended with the increase of the photocrosslinking ratio.
  • 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).
  • the adhesion strength of Examples 1 to 36 ranges from 82 to 132 kPa.
  • the adhesion strength of the gel decreases with the decrease in the concentration of the photosensitive material.
  • 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).
  • 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.
  • 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.

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Abstract

本发明提供一种双交联纤维蛋白凝胶，它是一种由具有封闭功能的网络结构和具有粘附功能的网络结构组成的固态水凝胶；具有封闭功能的网络结构是三维立体的纤维蛋白网络，具有粘附功能的网络结构是三维立体的光敏凝胶网络；每个光敏凝胶网络孔道内部都有一组纤维蛋白网络，且每一组纤维蛋白网络整体具有连续性；整体上，三维立体的纤维蛋白网络无序地遍布固态水凝胶表面和内部。本发明还提供制备所述双交联纤维蛋白凝胶的原料组合物及试剂盒，以及所述的试剂盒在制备原位快凝止血材料中的应用。所述的试剂盒制备得到的双交联纤维蛋白凝胶具有快速、高效的止血效果，可广泛应用于意外创伤或手术伤口止血。

Description

双交联纤维蛋白凝胶、其原料组合物和试剂盒及其应用 技术领域
本发明属于生物医药材料领域，尤其涉及用于意外创伤或手术出血止血的双交联纤维蛋白凝胶，及其应用方法。
背景技术
创伤后或手术期间发生的无法控制的出血是全球死亡的主要原因，每年造成超过200万人死亡。手术和创伤环境中不受控制的出血通常会致并发症和不良的结果。因此，控制出血量是降低并发症和死亡率、改善患者预后的重要措施。
目前已经开发了一些局部止血材料来辅助控制手术过程中的出血。市面上常用的外科手术密封剂包括纤维蛋白胶和合成组织粘合剂。纤维蛋白胶是最广泛使用的止血剂，具有较好的生物相容性，可辅助各种外科手术中的止血，模拟凝血级联反应，在出血部位原位形成纤维蛋白凝块，封堵出血。然而纤维蛋白胶的黏附强度会受到持续的组织张力和血液的影响而下降，易被血流冲走，不利于发挥其止血性能，且因在湿态组织上的粘附差而限制了其止血效果。另一方面，合成的组织粘合剂如氰基丙烯酸酯粘合剂，尽管具有较好的粘合能力，但因其高细胞毒性和难去除性而限制了其应用。
为了突破当前生物黏合剂在潮湿组织表面低粘附的应用瓶颈。现有技术中，有研究采用甲基丙烯酰化明胶作为止血凝胶材料，这类双键改性的明胶通过甲基丙烯酸酐将明胶的游离氨基官能化为甲基丙烯酰胺基团而获得，在特定波长的光照条件下，材料中的光引发剂吸收光能产生自由基，进而使甲基丙烯酰化明胶分子间成键形成固相凝胶。甲基丙烯酰化明胶具有良好的生物相容性，同时兼具较好的机械性能和粘附力。但甲基丙烯酰化明胶不具有促凝血功能，一定程度上限制了其止血能力；甲基丙烯酰化明胶的光固化时间为5～10秒，光固化时间长，光固化过程中易被血流冲走；在严重出血中，大量的血液削弱了其粘附能力。为了弥补该缺陷，Luo等人的研究将具有凝血功能的蛇毒血凝酶引入甲基丙烯酰化明胶，构建的止血胶提高了止血效果(Guo Y,Wang Y,Zhao X,et al.Snake extract-laden hemostatic bioadhesive gel cross-linked by visible light.Sci Adv.2021.7(29).)。但该研究构建的承载蛇毒血凝酶的甲基丙烯酰化明胶，只有在凝胶表面的极少量的蛇毒血凝酶才能与血液接触，且血液中纤维蛋白原的浓度低(2～4g/L)，形成的纤维蛋白交联不足以封堵伤口；同时在甲基丙烯酰化明胶完成光固化之前，其对伤口的封堵效果较弱，这些均很大程度上限制了其止血效果。Wang等人于2020年构建了针对糖尿病伤口多阶段愈合的凝血酶-甲基丙烯酰化明胶水凝胶，通过将游离凝血酶和负载凝血酶的脂质体掺入到甲基丙烯酰化明胶中，以实现最初的凝血酶释放来促进止血和持续的凝血酶释放来调节糖尿病伤口后期愈合(Chongyang W,Tianyi W,Guangwang L,et al.Promoting coagulation and activating SMAD3phosphorylation in wound healing via a dual-release thrombin-hydrogel.Chemical Engineering Journal.2020.397(C).)。该研究构建凝胶的缺点与Luo等人的研究类似，凝胶释放出的凝血酶量少，且与血液中纤维蛋白原的接触少，无法形成纤维蛋白交联，因对伤口的封堵作用弱而降低了止血效果。
此外，现有技术中还有采用其他光固化材料制备止血材料的报道，例如，中国专利文献CN 111116973 A公开了一种具有主动止血功能的聚乙烯醇止血多孔材料，通过将壳聚糖或(和)凝血酶等这些具有主动止血功能的聚合物添加到改性聚乙烯醇经光催化交联得到的海绵里，赋予海绵主动止血的功效。但是该论文中报道的止血时间为90s～100s，止血时间长，这必然致止血效果差。这是因为，1)预先形成的海绵无法与湿组织充分接触，致封堵效果差于原位形成的凝胶；2)干态状态下海绵中的凝血酶不易游离，从而限制其促凝血功能；3)血液中的纤维蛋白原浓度低(2～4g/L)，形成的纤维蛋白交联不足以封堵伤口。因此难以满足手术中大量出血时的快速止血需求；且在面对脏器或身体表面的伤口出血时，其膨胀压迫伤口的作用效果被局限，一定程度上削弱了止血效果；同时在去除该海绵时，可因其粘连在止血部位而对伤口造成二次损伤。
理想的止血材料应该不依赖于机体凝血机制，甚至当机体凝血障碍时亦可发挥止血作用，同时兼具较好的湿润组织粘附能力和理想的凝血止血速度。因此，发明一种可以解决现有止血材料所存在的湿态组织黏附差和止血效果局限的新型止血材料显得尤为重要。
发明内容
为了克服现有技术中存在的上述缺点，本发明的首要目的在于：提供一种可快速止血、快速凝胶化、具有高粘附性的粘合剂，以期可同时达到促凝血和强粘附效果。
本发明另一个目的在于：提供可制备所述粘合剂的原料组合物及试剂盒，以方便所述粘合剂在临床的推广应用。
本发明再一个目的在于：提供使用所述试剂盒止血的方法。
为了实现上述目的，本发明采取以下技术方案：
第一方面，本发明提供一种双交联纤维蛋白凝胶，它是一种由具有封闭功能的网络结构和具有粘附功能的网络结构组成的固态水凝胶；所述具有封闭功能的网络结构先于具有粘附功能的网络结构形成。所述的具有封闭功能的网络结构是三维立体的纤维蛋白网络，所述的具有粘附功能的网络结构是三维立体的光敏凝胶网络；每个所述的光敏凝胶网络孔道内部都有一组所述的纤维蛋白网络，且每一组所述的纤维蛋白网络整体具有连续性；整体上，所述的三维立体的纤维蛋白网络无序地遍布所述固态水凝胶表面和内部。
本发明所述的双交联纤维蛋白凝胶中，所述的三维立体的纤维蛋白网络作为支架，可起到增强凝胶的强度的作用，并且其形成的过程用于将血液中的纤维蛋白原转化为纤维蛋白，从而起到初步封堵伤口的作用，随着纤维蛋白比例的升高，固态水凝胶的胶凝时间会缩短、组织粘附力会降低，但凝胶孔隙会变大、且促凝血功能会升高。所述三维立体的光敏凝胶网络起到提供凝胶的强度和组织粘附力的作用，随着光敏凝胶网络在所述固态水凝胶中比例的升高，固态水凝胶的组织粘附力也升高，但胶凝时间也会延长、凝胶的孔隙会变小、且促凝血功能会降低。鉴于上述两种网络对凝胶整体止血性能带来的影响不同，本发明通过实验进一步优化了凝胶中两种网络的比例，优选的双交联纤维蛋白凝胶中，所述的三维立体的纤维蛋白网络与所述的三维立体的光敏凝胶网络的体积比为0.5～3；优选0.5～2；最优选1。在这些优选的体积比下，两种网络能够为凝胶整体带来更优的止血性能，尤其是纤维蛋白网络和光敏凝胶网络的体积比到达1:1时，凝胶的止血性能可达到最佳，即可在提高凝胶强度和粘附力的同时实现快速促凝血。
本发明所述的双交联纤维蛋白凝胶中，所述的光敏凝胶可以由现有的多种可发生光固化的高分子材料(即光敏材料)通过光交联形成，所述的光敏材料可以是甲基丙烯酰化的高分子聚合物或其衍生物、聚丙烯酸酯类的高分子聚合物或其衍生物、或包含它们的高分子复合材料体系。
进一步地，以上所述的甲基丙烯酰化的高分子聚合物或其衍生物可以选自以下任意一种或两种以上的混合物：甲基丙烯酰化明胶或其衍生物、甲基丙烯酰化透明质酸或其衍生物、甲基丙烯酰化海藻酸钠或其衍生物、甲基丙烯酰化丝素蛋白或其衍生物、甲基丙烯酰化壳聚糖或其衍生物、甲基丙烯酰化羧甲基壳聚糖或其衍生物。以上所述的聚丙烯酸酯类的高分子聚合物或其衍生物可以选自聚醚二丙烯酸酯或其衍生物、或聚乙二醇二丙烯酸酯或其衍生物。本发明最优选的光敏材料是甲基丙烯酰化明胶或其衍生物、或甲基丙烯酰化丝素蛋白或其衍生物。
进一步地，以上所述的甲基丙烯酰化的高分子聚合物的衍生物包括对其一种或多种官能团修饰后的聚合物。所述的甲基丙烯酰化明胶的可修饰官能团包括氨基、羧基、巯基、羟基或胍基中的任意一种或两种以上；所述的甲基丙烯酰化透明质酸的衍生物包括对其一种或多种官能团修饰后的聚合物，其中可修饰官能团包括羟基、羧基、乙酰氨基或羟甲基中的任意一种或两种以上；所述的甲基丙烯酰化海藻酸钠的衍生物包括对其一种或多种官能团修饰后的聚合物，其中可修饰官能团包括羧基、羟基中的任意一种或两种；所述的甲基丙烯酰化丝素蛋白的衍生物包括对其一种或多种官能团修饰后的聚合物，其中可修饰官能团包括氨基、羧基、巯基、羟基或胍基中的任意一种或两种以上；所述的甲基丙烯酰化壳聚糖的衍生物包括对其一种或多种官能团修饰后的聚合物或发生多种化学反应后的聚合物，其中可修饰官能团包括氨基或羟基中的任意一种或两种，可发生的多种化学反应包括烷基化、酰基化、羧甲基化、水解、氧化、还原化学反 应中的任意一种或两种以上。
以上所述的甲基丙烯酰化的高分子聚合物或其衍生物的分子量范围为5～400kDa，以上所述的聚丙烯酸酯类的高分子聚合物或其衍生物的分子量范围为700～1000kDa。
进一步地，以上所述的包含甲基丙烯酰化的高分子聚合物或其衍生物的高分子复合材料体系包括：甲基丙烯酰化明胶-聚乙烯醇体系、甲基丙烯酰化明胶-聚氨酯体系、甲基丙烯酰化明胶-聚乳酸体系、甲基丙烯酰化明胶-纤维素体系、甲基丙烯酰化透明质酸-聚乙烯醇体系、甲基丙烯酰化透明质酸-聚氨酯体系、甲基丙烯酰化透明质酸-聚乳酸体系、甲基丙烯酰化透明质酸-纤维素体系、甲基丙烯酰化海藻酸钠-聚乙烯醇体系、甲基丙烯酰化海藻酸钠-聚氨酯体系、甲基丙烯酰化海藻酸钠-聚乳酸体系、甲基丙烯酰化海藻酸钠-纤维素体系、甲基丙烯酰化丝素蛋白-聚乙烯醇体系、甲基丙烯酰化丝素蛋白-聚氨酯体系、甲基丙烯酰化丝素蛋白-聚乳酸体系、甲基丙烯酰化丝素蛋白-纤维素体系、甲基丙烯酰化壳聚糖-聚乙烯醇体系、甲基丙烯酰化壳聚糖-聚氨酯体系、甲基丙烯酰化壳聚糖-聚乳酸体系、甲基丙烯酰化壳聚糖-纤维素体系、甲基丙烯酰化羧甲基壳聚糖-聚乙烯醇体系、甲基丙烯酰化羧甲基壳聚糖-聚氨酯体系、甲基丙烯酰化羧甲基壳聚糖-聚乳酸体系、甲基丙烯酰化羧甲基壳聚糖-纤维素体系中的任意一种或两种以上。
本发明所述的双交联纤维蛋白凝胶中，所述的纤维蛋白网络可以由纤维蛋白原通过酶交联形成。所述的纤维蛋白原可以是人纤维蛋白原、牛纤维蛋白原或猪纤维蛋白原中的任意一种。
第二方面，本发明提供一种用于制备本发明第一方面所述的双交联纤维蛋白粘合剂的原料组合物，包括组合物A和组合物B；按重量份计，所述的组合物A包括10～200份光敏材料、1～3份光引发剂、0.14～0.28份酶和1.11～8.88份水溶性无机钙盐，所述的组合物B包括5～100份光敏材料、1～2份光引发剂和30～50份纤维蛋白原；所述的组合物A与所述的组合物B质量比为1.4:10～14:1；优选1.4:1～1.4:10；更优选1.4:1～1.4:5；最优选1.4:1。
本发明优选的方案中，所述的组合物A中光敏材料重量份大于组合物B中光敏材料重量份。所述的组合物A与组合物B光敏材料的重量份，有利于组合物A中酶与组合物B中纤维蛋白原进行酶反应，形成稳定的纤维蛋白网络结构。
本发明所述的原料组合物中，所述的纤维蛋白原可以在所述酶的作用下通过酶交联反应形成纤维蛋白网络，所述的光敏材料可以在所述光引发剂作用下通过光交联反应形成光敏凝胶。因此，将本发明所述原料组合物中的组合物A和组合物B按所述质量比混合后，通过光照，即可以制备得到一种双交联纤维蛋白粘合剂。该粘合剂是一种固态水凝胶，其结构中会同时存在三维立体的纤维蛋白网络和三维立体的光敏凝胶网络；每个所述的光敏凝胶网络孔道内部都有一组所述的纤维蛋白网络，且每一组所述的纤维蛋白网络整体具有连续性；整体上，所述的三维立体的纤维蛋白网络无序地遍布所述固态水凝胶表面和内部。这种固态水凝胶形成于正在出血的伤口部位时，可瞬间(1s左右)在伤口表面形成纤维蛋白凝块，起到初步封堵伤口的作用，阻挡血液流出；同时，纤维蛋白凝块中的酶将血液中的纤维蛋白原转化为凝块，起到高效促凝血功能；进一步的，光激发下，光敏材料在5～10s内能够形成光固化凝胶；光固化凝胶具有较强的粘附力，可以抵挡血流的冲击并保护纤维蛋白交联免被血液冲走。总之，本发明所述的原料组合物中，所述的组合物A和组合物B混合后可瞬间先形成纤维蛋白网络以起到支架作用，稍后光敏凝胶也会迅速形成，后面形成的光敏凝胶包裹在纤维蛋白网络中的纤维上。
本发明通过实验发现，所述的原料组合物中，所述的组合物A与组合物B的质量比与双交联纤维蛋白粘合剂的初步封堵伤口效果和粘附强度有关：当组合物A与组合物B质量比在1.4:10～1.4:1的范围内时，随着组合物A的比例升高，双交联纤维蛋白粘合剂的促凝血功能提高，初步封堵伤口效果升高，且粘附强度增大；当组合物A与组合物B质量比在1.4:1～14:1的范围内时，随着组合物A的比例升高，双交联纤维蛋白粘合剂的促凝血功能、初步封堵伤口效果及粘附强度并未进一步升高。这意味着当组合物A与组合物B质量比为1.4:1时，可取得最佳止血效果，并达到材料最佳利用率。该比例下，交联产生的两种网络的体积比可以达到1:1左右，能够为制得的凝胶带来最优的促凝血功能和粘附强度。
本发明优选的所述原料组合物中，按重量份计，所述的组合物A包括80～200份光敏材料、1～3份光 引发剂、0.14～0.28份酶和1.11～8.88份水溶性无机钙盐，所述的组合物B包括30～100份光敏材料、1～2份光引发剂和30～50份纤维蛋白原。
本发明更优选的所述原料组合物中，按重量份计，所述的组合物A包括100～200份光敏材料、1～3份光引发剂、0.14～0.28份酶和1.11～8.88份水溶性无机钙盐，所述的组合物B包括30～50份光敏材料、1～2份光引发剂和30～50份纤维蛋白原。
本发明最优选的所述原料组合物中，按重量份计，所述的组合物A包括100～150份光敏材料、1～3份光引发剂、0.14～0.28份酶和1.11～8.88份水溶性无机钙盐，所述的组合物B包括30～50份光敏材料、1～2份光引发剂和30～50份纤维蛋白原。
第三方面，本发明还提供制备所述的原料组合物的方法，包括：制备溶剂中溶解有光敏材料和光引发剂的混合溶液，将所述的混合溶液与含有凝血酶和钙离子的溶液混合得到第一前体溶液，控制所述第一前体溶液中光敏材料、光引发剂、酶和钙离子的浓度比为10～200:1～3:0.14～0.28:1.11～8.88；将所述的混合溶液与含有纤维蛋白原的溶液混合得到第二前体溶液，控制所述第二前体溶液中光敏材料、光引发剂和纤维蛋白原的浓度比为5～100:1～2:30～50；由此可得到一种包含第一前体溶液和第二前体溶液的液体原料组合物；还可以将所述的液体原料组合物进一步按照常规方法处理得到固态的原料组合物，例如冻干粉剂、海绵或颗粒。
本发明所述的制备方法中，为了保持第一前体溶液中光敏材料的活性，控制第一前体溶液在室温环境中放置的时间少于30分钟。
本发明所述的制备方法中，为了兼顾纤维蛋白原分散的均匀性和光敏材料完成光交联的速度，优选控制所述第一前体溶液中的光敏材料浓度大于0.5％(w/v)，且所述第二前体溶液中的光敏材料浓度低于所述第一前体溶液中的光敏材料浓度。由此，纤维蛋白原溶液在光敏材料浓度相对较低的第二前体溶液中更容易实现均匀分散，在两种前体溶液混合后能够迅速充分地接触到酶，瞬间发生完全地酶交联形成均匀分布的纤维蛋白网络；同时第一前体溶液中较高的光敏材料浓度又可以提升两种前体溶液混合后的整体光敏材料浓度，使其达到较理想的胶凝所需浓度，有利于缩短光交联时间、增加凝胶粘附力和强度。
本发明所述的制备方法中，所述第一前体溶液和第二前体溶液制备时，优选控制所述混合溶液的温度不高于37℃。
本发明进一步优选的一种制备所述的原料组合物注射剂的方法，具体包括以下步骤：
1)制备溶剂中溶解有光敏材料和光引发剂的第一混合溶液，控制其中光敏材料和光引发剂的浓度比为10～200:1～3，且光敏材料的浓度在0.5％～30％(w/v)；
2)制备溶剂中溶解有光敏材料和光引发剂的第二混合溶液，控制其中光敏材料和光引发剂的浓度比为5～100:1～2，且光敏材料的浓度低于1)所述的第一混合溶液；
3)将1)制备的第一混合溶液与含有酶和钙离子的溶液混合得到第一前体溶液，控制其中光敏材料、光引发剂、酶和钙离子的浓度比为10～200:1～3:0.14～0.28:1.11～8.88；
4)将2)制备的第二混合溶液与含有纤维蛋白原的溶液混合得到第二前体溶液，控制其中光敏材料、光引发剂和纤维蛋白原的浓度比为5～100:1～2:30～50。
本发明所述的制备方法中，所述的含有酶和钙离子的溶液，优选按照以下方法制备：将溶剂和水溶性无机钙盐溶液加入酶中，完全溶解后得到含Ca2+的酶溶液，控制所得溶液中酶活力为500IU～2000IU/ml、Ca2+浓度为60～100mmol/L。
本发明所述的制备方法中，所述的含有纤维蛋白原的溶液中，纤维蛋白原的浓度优选为5％～10％(w/v)。
本发明优选的制备方法中，控制所述的第一前体溶液中光敏材料浓度在1％～30％(w/v)，进一步优选在8％～30％(w/v)，更优选10％-20％。
本发明优选的制备方法中，控制所述的第一前体溶液中酶活力不低于200IU/ml，优选不低于500IU/ml；更优选不低于1000IU/ml。
本发明优选的制备方法中，控制所述的第一前体溶液中钙离子浓度不低于20mmol/L，优选不低于 30mmol/L，更优选不低于40mmol/L。
本发明优选的制备方法中，控制所述的第二前体溶液中光敏材料浓度不低于0.5％(w/v)且不高于所述的第一前体溶液中光敏材料浓度，进一步优选不低于1％(w/v)且不高于所述的第一前体溶液中光敏材料浓度，更优选1％-10％(w/v)且不高于所述的第一前体溶液中光敏材料浓度。
本发明优选的制备方法中，控制所述的第二前体溶液中纤维蛋白原浓度不低于3％(w/v)，更优选3％-5％(w/v)。
第四方面，本发明还提供一种制备本发明第一方面所述的双交联纤维蛋白凝胶的试剂盒，包括相互独立包装的第一前体试剂、第二前体试剂；按重量份计，所述的第一前体试剂含有10～200份光敏材料、1～3份光引发剂、0.14～0.28份酶和3.33～5.55份水溶性无机钙盐，所述的第二前体试剂包括5～100份光敏材料、1～2份光引发剂和30～50份纤维蛋白原；所述的第一前体试剂与所述的第二前体试剂质量比为1.4:10～14:1；优选1.4:1～1.4:10；更优选1.4:1～1.4:5；最优选1.4:1。
本发明优选的所述试剂盒中，按重量份计，所述的第一前体试剂含有80～200份光敏材料、1～3份光引发剂、0.14～0.28份酶和3.33～5.55份水溶性无机钙盐，所述的第二前体试剂含有30～100份光敏材料、1～2份光引发剂和30～50份纤维蛋白原。
本发明更优选的所述试剂盒中，按重量份计，所述的第一前体试剂含有100～200份光敏材料、1～3份光引发剂、0.14～0.28份酶和3.33～5.55份水溶性无机钙盐，所述的第二前体试剂含有30～50份光敏材料、1～2份光引发剂和30～50份纤维蛋白原。
本发明最优选的所述试剂盒中，按重量份计，所述的第一前体试剂含有100～150份光敏材料、1～3份光引发剂、0.14～0.28份酶和3.33～5.55份水溶性无机钙盐，所述的第二前体试剂含有30～50份光敏材料、1～2份光引发剂和30～50份纤维蛋白原。
本发明所述的试剂盒中，所述第一前体试剂和第二前体试剂含有的光敏材料是光敏性的生物水凝胶材料，可以是现有的多种可发生光固化的高分子材料；所述第一前体试剂和第二前体试剂含有的光引发剂是在特定波长的光照条件下，吸收光能后可产生自由基的物质。所述的光引发剂吸收光能后可产生自由基，可使所述的光敏材料分子间成键，因此快速形成固相凝胶。理想的光敏材料应具有良好的生物相容性和可降解性，同时兼具较好的机械性能和粘附性能。
本发明所述原料组合物或所述试剂盒中，所述的光敏材料可以是甲基丙烯酰化的高分子聚合物或其衍生物、聚丙烯酸酯类的高分子聚合物或其衍生物、或包含它们的高分子复合材料体系。
进一步地，以上所述的甲基丙烯酰化的高分子聚合物或其衍生物可以选自以下任意一种或两种以上的混合物：甲基丙烯酰化明胶或其衍生物、甲基丙烯酰化透明质酸或其衍生物、甲基丙烯酰化海藻酸钠或其衍生物、甲基丙烯酰化丝素蛋白或其衍生物、甲基丙烯酰化壳聚糖或其衍生物、甲基丙烯酰化羧甲基壳聚糖或其衍生物。以上所述的聚丙烯酸酯类的高分子聚合物或其衍生物可以选自聚醚二丙烯酸酯或其衍生物、或聚乙二醇二丙烯酸酯或其衍生物。本发明最优选的光敏材料是甲基丙烯酰化明胶或其衍生物、或甲基丙烯酰化丝素蛋白或其衍生物。
进一步地，以上所述的甲基丙烯酰化的高分子聚合物的衍生物包括对其一种或多种官能团修饰后的聚合物。所述的甲基丙烯酰化明胶的可修饰官能团包括氨基、羧基、巯基、羟基或胍基中的任意一种或两种以上；所述的甲基丙烯酰化透明质酸的衍生物包括对其一种或多种官能团修饰后的聚合物，其中可修饰官能团包括羟基、羧基、乙酰氨基或羟甲基中的任意一种或两种以上；所述的甲基丙烯酰化海藻酸钠的衍生物包括对其一种或多种官能团修饰后的聚合物，其中可修饰官能团包括羧基、羟基中的任意一种或两种；所述的甲基丙烯酰化丝素蛋白的衍生物包括对其一种或多种官能团修饰后的聚合物，其中可修饰官能团包括氨基、羧基、巯基、羟基或胍基中的任意一种或两种以上；所述的甲基丙烯酰化壳聚糖的衍生物包括对其一种或多种官能团修饰后的聚合物或发生多种化学反应后的聚合物，其中可修饰官能团包括氨基或羟基中的任意一种或两种，可发生的多种化学反应包括烷基化、酰基化、羧甲基化、水解、氧化、还原化学反应中的任意一种或两种以上。
以上所述的甲基丙烯酰化的高分子聚合物或其衍生物的分子量范围为5～400kDa，以上所述的聚丙烯酸酯类的高分子聚合物或其衍生物的分子量范围为700～1000kDa。
进一步地，以上所述的包含甲基丙烯酰化的高分子聚合物或其衍生物的高分子复合材料体系包括：甲基丙烯酰化明胶-聚乙烯醇体系、甲基丙烯酰化明胶-聚氨酯体系、甲基丙烯酰化明胶-聚乳酸体系、甲基丙烯酰化明胶-纤维素体系、甲基丙烯酰化透明质酸-聚乙烯醇体系、甲基丙烯酰化透明质酸-聚氨酯体系、甲基丙烯酰化透明质酸-聚乳酸体系、甲基丙烯酰化透明质酸-纤维素体系、甲基丙烯酰化海藻酸钠-聚乙烯醇体系、甲基丙烯酰化海藻酸钠-聚氨酯体系、甲基丙烯酰化海藻酸钠-聚乳酸体系、甲基丙烯酰化海藻酸钠-纤维素体系、甲基丙烯酰化丝素蛋白-聚乙烯醇体系、甲基丙烯酰化丝素蛋白-聚氨酯体系、甲基丙烯酰化丝素蛋白-聚乳酸体系、甲基丙烯酰化丝素蛋白-纤维素体系、甲基丙烯酰化壳聚糖-聚乙烯醇体系、甲基丙烯酰化壳聚糖-聚氨酯体系、甲基丙烯酰化壳聚糖-聚乳酸体系、甲基丙烯酰化壳聚糖-纤维素体系、甲基丙烯酰化羧甲基壳聚糖-聚乙烯醇体系、甲基丙烯酰化羧甲基壳聚糖-聚氨酯体系、甲基丙烯酰化羧甲基壳聚糖-聚乳酸体系、甲基丙烯酰化羧甲基壳聚糖-纤维素体系中的任意一种或两种以上。
本发明优选的所述原料组合物或所述试剂盒中，所述的光引发剂可选自以下任意一种或两种以上的组合物：苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐、2-羟基-4′-(2-羟乙氧基)-2-甲基苯丙酮、2,4,6-三甲基苯甲酰基膦酸乙酯、2-甲基-1-[4-甲硫基苯基]-2-吗啉基-1-丙酮、邻苯甲酰苯甲酸甲酯、2-苯基苄-2-二甲基胺-1-(4-吗啉苄苯基)丁酮或2,2-偶氮(2-甲基-N-(2-羟基乙基)丙酰胺)；最优选苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐。
本发明优选的所述原料组合物或所述试剂盒中，所述的酶可以选自人凝血酶、重组人凝血酶、牛凝血酶、猪凝血酶或蛇毒血凝酶中的任意一种。
本发明优选的所述原料组合物或所述试剂盒中，所述的纤维蛋白原可选自人纤维蛋白原、牛纤维蛋白原或猪纤维蛋白原中的任意一种。
本发明优选的原料组合物或试剂盒中，所述的水溶性无机钙盐可选自氯化钙、硝酸钙或硫酸钙；最优选氯化钙。
本发明优选的试剂盒中，所述的第一前体试剂和/或第二前体试剂中还包含辅料和/或添加剂。所述的辅料选自甘氨酸、盐酸精氨酸、枸橼酸钠、蔗糖、氯化钠中的一种或两种以上。所述的添加剂选自生长因子、白细胞介素、维生素、银离子中的一种或两种以上。所述的生长因子可进一步选自血小板生长因子、表皮生长因子或成纤维细胞生长因子中的一种或多种；所述的白细胞介素可进一步选自白细胞介素2、白细胞介素6或白细胞介素8中的一种或多种；所述的维生素可进一步选自维生素B、维生素C、维生素E或维生素K中的一种或多种。
本发明所述的试剂盒中，所述的第一前体试剂和/或第二前体试剂可以是制药或临床上可接受的多种具体剂型，可以是冻干粉剂、海绵或颗粒。
本发明所述的试剂盒中，还可以进一步包括独立包装的配置用溶剂，所述的配置用溶剂可选为磷酸缓冲盐溶液、HEPES生物缓冲液、0.9％氯化钠溶液、氯化钙溶液、去离子水中的任意一种或几种的混合物。所述配置用溶剂的剂型优选注射剂。
本发明所述的试剂盒中，还可以进一步包括说明书，用于说明所述试剂盒的使用方法。
第五方面，本发明还提供利用本发明第四方面所述的试剂盒在出血伤口原位快速止血的方法，包括：通过配置用溶剂将所述的第一前体试剂和第二前体试剂分别制备成可注射溶液状，然后同时均匀的注射或喷涂于出血伤口部位，然后用290～480nm波段的光照射10～60s，可在出血伤口部位原位快速形成固态水凝胶。
所述的出血伤口包括由于意外创伤导致或手术中发生的器官出血；所述的器官可以是肝脏、脾脏、肾脏、胃肠、心脏或皮肤。
本发明所述的应用中，在出血伤口注射所述的试剂盒时，(1)可瞬间(1s左右)在伤口表面先形成纤维蛋白凝块，起到初步封堵伤口的作用，阻挡血液流出，从而弥补光敏材料在光固化完成前的弱封堵作用；(2)同时，纤维蛋白凝块中的酶将血液中的纤维蛋白原转化为凝块，起到高效促凝血功能；(3)进一步 的，光敏材料在光激发下，于5～10s内形成光固化凝胶；光固化凝胶具有较强的粘附力，可以抵挡血流的冲击并保护纤维蛋白交联免被血液冲走。因此，使用本发明所述的试剂盒或试剂盒原位制备双交联纤维蛋白凝胶，可以结合纤维蛋白交联的即刻发生和光交联的强粘附力，得到具有纤维蛋白交联网络和光交联网络结构的双交联纤维蛋白凝胶。
相对于现有技术，本发明的优点在于：快速凝胶化、固化速度快、强湿组织粘附力、止血效果佳：
(1)本发明的双交联纤维蛋白凝胶试剂盒混合后可即刻(1s左右)发生纤维蛋白交联，起到初步封堵作用，阻挡血流冲击。
(2)本发明的双交联纤维蛋白凝胶试剂盒中的酶可将血液中的纤维蛋白原转化为纤维蛋白交联，具有高效促凝血能力。
(3)本发明的双交联纤维蛋白凝胶试剂盒在紫外光或可见光激发下，可在5-10秒内发生光交联反应，形成光固化凝胶，提供强湿组织粘附力，可保护纤维蛋白交联免被血流冲走；
正是由于本发明提供的双交联纤维蛋白凝胶具有很好的促凝血功能、固化速度、湿组织粘附力和快速止血效果，因此可用于意外创伤或手术中的肝脏、脾脏、肾脏、心脏、胃肠和皮肤出血的止血应用。
附图说明
图1是对比例1的纤维蛋白交联的SEM图。
图2是对比例2的前体溶液甲基丙烯酰化明胶光交联的SEM图。
图3是实施例1的双交联纤维蛋白凝胶的SEM图。
图4体现了实施例1、9、14、19、25、31及对比例1～6在止血时间上的对比。
图5体现了实施例1、9、14、19、25、31及对比例1～6在失血量上的对比。
具体实施方式
下面结合具体实施例对本发明要解决的技术问题、技术方案和有益效果进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员，在不脱离本发明构思的前体下，还可以做出若干变形和改进。这些都属于本发明的保护范围。
本发明提供一种双交联纤维蛋白凝胶，它是由三维立体的光敏凝胶网络和三维立体的纤维蛋白网络共同构成的固态水凝胶；其微观结构如图3所示，同时具备纤维蛋白交联的网状结构和光敏材料交联的多孔结构；所述多孔结构的每个孔道内都有一组所述的纤维蛋白网络，且每一组所述的纤维蛋白网络整体具有连续性；整体上，所述的三维立体的纤维蛋白网络无序地遍布所述固态水凝胶表面和内部；所述的纤维蛋白网状结构在所述的光敏凝胶网络孔道内起到支架作用，光敏凝胶多孔结构的孔壁围绕在纤维蛋白网络结构周围。
所述的双交联纤维蛋白凝胶按照以下方法制备得到：
(1)组合物A溶液的制备：将含钙离子的酶溶液加入溶解有光敏材料和光引发剂的混合溶液中，均匀混合后，得到组合物A溶液，包含光敏材料、光引发剂和酶；控制所得组合物A溶液中光敏材料浓度不低于1％(w/v)，优选不低于3％(w/v)，更优选3％-20％(w/v)；同时控制酶活力不低于200IU/mL，优选不低于500IU/mL，更优选不低于1000IU/mL。
(2)组合物B溶液的制备：将纤维蛋白原溶液加入溶解有光敏材料和光引发剂的混合溶液中，均匀混合后，得到组合物B溶液：包含光敏材料、光引发剂和纤维蛋白原。控制所得组合物B溶液中光敏材料浓度不低于0.5％(w/v)，优选1％-10％(w/v)；同时控制纤维蛋白原浓度不低于3％(w/v)，优选3％-5％(w/v)。
(3)储存方法：将得到的组合物A溶液和组合物B溶液按体积比为1：10～10：1分别进行冷冻干燥，成为海绵状后进行储存。
(4)使用上述冻干海绵制备双交联纤维蛋白凝胶：将海绵状A组分和海绵状B组分分别溶于溶剂中，得到可注射溶液状A组分和B组分。将等体积的A组分溶液和B组分溶液均匀的注射/喷涂于出血部位，并用蓝光或紫外光照射10～60s，可在原位快速形成固态水凝胶。作为优选方案，所述的可注射溶液在使用 时的注射工具为双联注射器、注射器、巴氏吸管。
上述制备方案中，所述的溶剂可选为磷酸缓冲盐溶液、HEPES生物缓冲液、0.9％氯化钠溶液、氯化钙溶液、去离子水中的任意一种或几种的组合，且其使用量没有特别限制，可以根据实际需要浓度进行配制。
基于上述实施方式，本发明进一步列举以下实施例予以说明。
实施例1
本实施例的双交联纤维蛋白凝胶制备方法，步骤如下：
(1)甲基丙烯酰化明胶-苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐前体溶液的制备：向粉末状苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐中加入所需体积的0.9％氯化钠溶液，水浴中加热溶解，得到两种浓度的苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐溶液：0.25％(w/v)、0.5％(w/v)；取所需重量的固体甲基丙烯酰化明胶，分别向其中加入所需浓度的苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐溶液，水浴加热溶解，得到两种质量体积百分比(w/v)的甲基丙烯酰化明胶-苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐前体溶液：26％(w/v)甲基丙烯酰化明胶-0.5％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐、10％(w/v)甲基丙烯酰化明胶-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐；
(2)凝血酶溶液的制备：将所需体积和所需浓度的氯化钙溶液注射入凝血酶中，完全溶解后，得到凝血酶活力为2000IU/mL的凝血酶溶液，其中Ca2+浓度为80mmol/L；
(3)纤维蛋白原溶液的制备：取所需重量的纤维蛋白原，缓慢置于预热的0.9％氯化钠溶液中，完全溶解后，得到质量体积百分比(w/v)为10％(w/v)的纤维蛋白原溶液。
(4)A组分溶液的制备：将步骤(2)得到的凝血酶溶液加入步骤(1)得到的26％(w/v)甲基丙烯酰化明胶-0.5％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐前体溶液中，均匀混合后，得到A组分溶液：13％(w/v)甲基丙烯酰化明胶-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶；
(5)B组分溶液的制备：将步骤(3)得到的纤维蛋白原溶液加入步骤(1)得到的10％(w/v)甲基丙烯酰化明胶-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐前体溶液中，均匀混合后，得到B组分溶液：5％(w/v)甲基丙烯酰化明胶-0.125％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原；
(6)储存：将得到的A组分溶液和B组分溶液按照体积比为1:1的比例分别进行冷冻干燥，以海绵状进行储存；
(7)使用方法：将海绵状A组分和B组分按照1:1的体积份额比例分别溶于含有0.9％氯化钠溶液中，得到可注射溶液状的A组分和B组分。将A组分溶液和B组分溶液等体积的装入双联注射器，A组分溶液和B组分溶液通过喷头注射/喷涂在出血部位，随后用蓝光照射10～60s，可原位转变为固态水凝胶。且此时得到的凝胶中，纤维蛋白交联与光交联的体积比例为1:1。
(8)所述的固态凝胶结构如图3所示：它是由三维立体的纤维蛋白网络和三维立体的光敏凝胶网络共同构成的固态水凝胶；并且形成的甲基丙烯酰化明胶交联多孔结构内部都有一组所述的纤维蛋白网络，且每一组所述的纤维蛋白网络整体具有连续性；整体上，所述的三维立体的纤维蛋白网络无序地遍布所述固态水凝胶表面和内部。
实施例2
制备浓度为10％(w/v)甲基丙烯酰化明胶-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为5％(w/v)甲基丙烯酰化明胶-0.125％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其组成、制备方法和使用方法与实施例1大体相同，不同之处仅在于：将步骤(4)制备得到的A组分溶液的甲基丙烯酰化明胶浓度调整为10％(w/v)。
实施例3
制备浓度为8％(w/v)甲基丙烯酰化明胶-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为5％(w/v)甲基丙烯酰化明胶-0.125％(w/v)苯基(2,4,6- 三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其组成、制备方法和使用方法与实施例1大体相同，不同之处仅在于：将步骤(4)制备得到的A组分溶液的甲基丙烯酰化明胶浓度调整为8％(w/v)。
实施例4
制备浓度为5％(w/v)甲基丙烯酰化明胶-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为5％(w/v)甲基丙烯酰化明胶-0.125％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其组成、制备方法和使用方法与实施例1大体相同，不同之处仅在于：将步骤(4)制备得到的A组分溶液的甲基丙烯酰化明胶浓度为5％(w/v)。且此时得到的凝胶中，纤维蛋白交联与光交联的体积比例为2:1。
实施例5
制备浓度为13％(w/v)甲基丙烯酰化明胶-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为3％(w/v)甲基丙烯酰化明胶-0.125％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其组成、制备方法和使用方法与实施例1大体相同，不同之处仅在于：将步骤(5)制备得到的B组分溶液的甲基丙烯酰化明胶浓度调整为3％(w/v)。
实施例6
制备浓度为13％(w/v)甲基丙烯酰化明胶-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-500IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为5％(w/v)甲基丙烯酰化明胶-0.125％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其组成、制备方法和使用方法与实施例1大体相同，不同之处仅在于：将步骤(4)制备得到的A组分溶液的凝血酶活力调整为500IU/mL。
实施例7
制备浓度为13％(w/v)甲基丙烯酰化明胶-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-250IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为5％(w/v)甲基丙烯酰化明胶-0.125％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其组成、制备方法和使用方法与实施例1大体相同，不同之处仅在于：将步骤(4)制备得到的A组分溶液的凝血酶活力调整为250IU/mL。
实施例8
制备浓度为13％(w/v)甲基丙烯酰化明胶-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为5％(w/v)甲基丙烯酰化明胶-0.125％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-3％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其组成、制备方法和使用方法与实施例1大体相同，不同之处仅在于：将步骤(5)制备得到的B组分溶液的纤维蛋白原浓度为3％(w/v)。且此时得到的凝胶中，纤维蛋白交联和光交联的体积比为1:2。
实施例9
制备浓度为8％(w/v)甲基丙烯酰化透明质酸-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为5％(w/v)甲基丙烯酰化透明质酸-0.125％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其制备和使用方法与实施例1大体相同，不同之处在于：①本实施例A和B组分中的光敏材料为甲基丙烯酰化透明质酸，且A组分溶液中的甲基丙烯酰化透明质酸浓度为8％(w/v)；②甲基丙烯酰化透明质酸-苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐前体溶液的制备过程不需要加热，可在室温下进行。且此时得到的凝胶中，纤维蛋白交联和光交联的体积比为1:1。
实施例10
制备浓度为5％(w/v)甲基丙烯酰化透明质酸-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐 -1000IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为5％(w/v)甲基丙烯酰化透明质酸-0.125％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其组成、制备方法和使用方法与实施例9大体相同，不同之处仅在于：将A组分溶液的甲基丙烯酰化透明质酸浓度调整为5％(w/v)。且此时得到的凝胶中，纤维蛋白交联和光交联的体积比为2:1。
实施例11
制备浓度为8％(w/v)甲基丙烯酰化透明质酸-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为3％(w/v)甲基丙烯酰化透明质酸-0.125％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其组成、制备方法和使用方法与实施例9大体相同，不同之处仅在于：将B组分溶液的甲基丙烯酰化透明质酸浓度调整为3％(w/v)。
实施例12
制备浓度为8％(w/v)甲基丙烯酰化透明质酸-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-500IU/ml凝血酶的混合溶液作为A组分溶液，制备浓度为5％(w/v)甲基丙烯酰化透明质酸-0.125％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其组成、制备方法和使用方法与实施例9大体相同，不同之处在于：将A组分溶液的凝血酶活力调整为500IU/ml。
实施例13
制备浓度为8％(w/v)甲基丙烯酰化透明质酸-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为5％(w/v)甲基丙烯酰化透明质酸-0.125％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-3％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其组成、制备方法和使用方法与实施例9大体相同，不同之处在于：将B组分溶液的纤维蛋白原浓度调整为3％(w/v)。且此时得到的凝胶中，纤维蛋白交联和光交联的体积比例为1:2。
实施例14
制备浓度为8％(w/v)甲基丙烯酰化海藻酸钠-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为5％(w/v)甲基丙烯酰化海藻酸钠-0.125％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其制备方法和使用方法与实施例9大体相同，不同之处仅在于：本实施例A和B组分中的光敏材料为甲基丙烯酰化海藻酸钠。且此时得到的凝胶中，纤维蛋白交联和光交联的体积比为1:1。
实施例15
制备浓度为5％(w/v)甲基丙烯酰化海藻酸钠-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为5％(w/v)甲基丙烯酰化海藻酸钠-0.125％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其组成、制备方法和使用方法与实施例14大体相同，不同之处仅在于：调整A组分溶液中的甲基丙烯酰化海藻酸钠浓度为5％(w/v)。且此时得到的凝胶中，纤维蛋白交联和光交联的体积比为2:1。
实施例16
制备浓度为8％(w/v)甲基丙烯酰化海藻酸钠-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为3％(w/v)甲基丙烯酰化海藻酸钠-0.125％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其组成、制备方法和使用方法与实施例14大体相同，不同之处仅在于：调整B组分溶液中的甲基丙烯酰化海藻酸钠浓度为3％(w/v)。
实施例17
制备浓度为8％(w/v)甲基丙烯酰化海藻酸钠-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-500IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为5％(w/v)甲基丙烯酰化海藻酸钠-0.125％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其组成、制备方 法和使用方法与实施例14大体相同，不同之处仅在于：调整A组分溶液中的凝血酶活力为500IU/mL。
实施例18
制备浓度为8％(w/v)甲基丙烯酰化海藻酸钠-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为5％(w/v)甲基丙烯酰化海藻酸钠-0.125％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-3％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其组成、制备方法和使用方法与实施例14大体相同，不同之处仅在于：调整B组分溶液中的纤维蛋白原浓度为3％(w/v)。且此时得到的凝胶中，纤维蛋白交联和光交联的体积比为1:2。
实施例19
制备浓度为10％(w/v)甲基丙烯酰化丝素蛋白-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为5％(w/v)甲基丙烯酰化丝素蛋白-0.125％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其制备方法和使用方法与实施例9大体相同，不同之处仅在于：本实施例中A和B组分中的光敏材料为甲基丙烯酰化丝素蛋白，且A组分溶液中的甲基丙烯酰化丝素蛋白浓度为10％(w/v)。且此时得到的凝胶中，纤维蛋白交联和光交联的体积比为1:1。
实施例20
制备浓度为8％(w/v)甲基丙烯酰化丝素蛋白-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为5％(w/v)甲基丙烯酰化丝素蛋白-0.125％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其组成、制备方法和使用方法与实施例19大体相同，不同之处仅在于：调整A组分溶液中的甲基丙烯酰化丝素蛋白浓度为8％(w/v)。
实施例21
制备浓度为5％(w/v)甲基丙烯酰化丝素蛋白-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为5％(w/v)甲基丙烯酰化丝素蛋白-0.125％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其组成、制备方法和使用方法与实施例19大体相同，不同之处仅在于：调整A组分溶液中的甲基丙烯酰化丝素蛋白浓度为5％(w/v)。且此时得到的凝胶中，纤维蛋白交联和光交联的体积比为2:1。
实施例22
制备浓度为10％(w/v)甲基丙烯酰化丝素蛋白-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为3％(w/v)甲基丙烯酰化丝素蛋白-0.125％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其组成、制备方法和使用方法与实施例19大体相同，不同之处仅在于：调整B组分溶液中的甲基丙烯酰化丝素蛋白浓度为3％(w/v)。
实施例23
制备浓度为10％(w/v)甲基丙烯酰化丝素蛋白-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-500IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为5％(w/v)甲基丙烯酰化丝素蛋白-0.125％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其组成、制备方法和使用方法与实施例19大体相同，不同之处仅在于：调整A组分溶液中的凝血酶活力为500IU/mL。
实施例24
制备浓度为10％(w/v)甲基丙烯酰化丝素蛋白-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为5％(w/v)甲基丙烯酰化丝素蛋白-0.125％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-3％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其组成、制备方法和使用方法与实施例19大体相同，不同之处仅在于：调整B组分溶液中的纤维蛋白原浓度为3％(w/v)。且此时得到的凝胶中，纤维蛋白交联和光交联的体积比为1:2。
实施例25
制备浓度为3％(w/v)甲基丙烯酰化壳聚糖-0.1％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为1％(w/v)甲基丙烯酰化壳聚糖-0.1％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其制备方法和使用方法与实施例9大体相同，不同之处在于：本实施例的A和B组分中的光敏材料为甲基丙烯酰化壳聚糖，且A组分溶液中的甲基丙烯酰化壳聚糖浓度为3％(w/v)，B组分溶液中的甲基丙烯酰化壳聚糖浓度为1％(w/v)。且此时得到的凝胶中，纤维蛋白交联和光交联的体积比为1:1。
实施例26
制备浓度为2％(w/v)甲基丙烯酰化壳聚糖-0.1％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为1％(w/v)甲基丙烯酰化壳聚糖-0.1％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其组成、制备方法和使用方法与实施例25大体相同，不同之处仅在于：调整A组分溶液中的甲基丙烯酰化壳聚糖浓度为2％(w/v)。
实施例27
制备浓度为1％(w/v)甲基丙烯酰化壳聚糖-0.1％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为1％(w/v)甲基丙烯酰化壳聚糖-0.1％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其组成、制备方法和使用方法与实施例25大体相同，不同之处仅在于：调整A组分溶液中的甲基丙烯酰化壳聚糖浓度为1％(w/v)。且此时得到的凝胶中，纤维蛋白交联和光交联的体积比2:1。
实施例28
制备浓度为3％(w/v)甲基丙烯酰化壳聚糖-0.1％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为0.5％(w/v)甲基丙烯酰化壳聚糖-0.1％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其组成、制备方法和使用方法与实施例25大体相同，不同之处仅在于：调整B组分溶液中的甲基丙烯酰化壳聚糖浓度为0.5％(w/v)。
实施例29
制备浓度为3％(w/v)甲基丙烯酰化壳聚糖-0.1％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-500IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为1％(w/v)甲基丙烯酰化壳聚糖-0.1％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其组成、制备方法和使用方法与实施例25大体相同，不同之处仅在于：调整A组分溶液中的凝血酶活力为500IU/mL。
实施例30
制备浓度为3％(w/v)甲基丙烯酰化壳聚糖-0.1％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为1％(w/v)甲基丙烯酰化壳聚糖-0.1％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-3％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其组成、制备方法和使用方法与实施例25大体相同，不同之处仅在于：调整B组分溶液中的纤维蛋白原浓度为3％(w/v)。且此时得到的凝胶中，纤维蛋白交联和光交联的体积比为1:2。
实施例31
制备浓度为20％(w/v)聚醚F127二丙烯酸酯-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为10％(w/v)聚醚F127二丙烯酸酯-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其制备方法和使用方法与实施例9大体相同，不同之处在于：本实施例的A和B组分中的光敏材料为聚醚F127二丙烯酸酯，且A组分溶液中的聚醚F127二丙烯酸酯浓度为20％(w/v)，B组分溶液中的聚醚F127二丙烯酸酯浓度为10％(w/v)。且此时得到的凝胶中，纤维蛋白交联和光交联的体积比为1:1。
实施例32
制备浓度为15％(w/v)聚醚F127二丙烯酸酯-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为10％(w/v)聚醚F127二丙烯酸酯-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其组成、制备方法和使用方法与实施例31大体相同，不同之处仅在于：调整A组分溶液中的聚醚F127二丙烯酸酯浓度为15％(w/v)。
实施例33
制备浓度为10％(w/v)聚醚F127二丙烯酸酯-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为10％(w/v)聚醚F127二丙烯酸酯-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐--5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其组成、制备方法和使用方法与实施例31大体相同，不同之处仅在于：调整A组分溶液中的聚醚F127二丙烯酸酯浓度为10％(w/v)。且此时得到的凝胶中，纤维蛋白交联和光交联的体积比2:1。
实施例34
制备浓度为20％(w/v)聚醚F127二丙烯酸酯-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为5％(w/v)聚醚F127二丙烯酸酯-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其组成、制备方法和使用方法与实施例31大体相同，不同之处仅在于：调整B组分溶液中的聚醚F127二丙烯酸酯浓度为5％(w/v)。
实施例35
制备浓度为20％(w/v)聚醚F127二丙烯酸酯-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-500IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为10％(w/v)聚醚F127二丙烯酸酯-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其组成、制备方法和使用方法与实施例31大体相同，不同之处仅在于：调整A组分溶液中的凝血酶活力为500IU/mL。
实施例36
制备浓度为20％(w/v)聚醚F127二丙烯酸酯-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为10％(w/v)聚醚F127二丙烯酸酯-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-3％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其组成、制备方法和使用方法与实施例31大体相同，不同之处仅在于：调整B组分溶液中的纤维蛋白原浓度为3％(w/v)。且此时得到的凝胶中，纤维蛋白交联和光交联的体积比为1:2。
对比例1
外用冻干纤维蛋白粘合剂(护固莱士，购于上海莱士)，包括酶试剂和纤维蛋白原试剂。将酶试剂和纤维蛋白原试剂按其说明书分别配制成溶液，混合后约1s完成酶交联得到纤维蛋白粘合剂，该粘合剂微观结构如图1所示。
对比例2
9％(w/v)甲基丙烯酰化明胶-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐前体溶液，其制备方法与实施例2的A组分溶液大体相同，不同之处仅在于：溶液中不添加凝血酶。
对比例3
13％(w/v)甲基丙烯酰化明胶-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶混合溶液，组分和制备方法与实施例2的A组分溶液相同。
对比例4
5％(w/v)甲基丙烯酰化明胶-0.125％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原混合溶液，组分和制备方法与实施例1的步骤(5)相同。
对比例5
制备浓度为30％(w/v)甲基丙烯酰化丝胶蛋白-0.5％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为20％(w/v)甲基丙烯酰化丝胶蛋白-0.5％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其制备方法与实施例9大体相同，不同之处在于：本对比例中的A和B组分中的光敏材料为甲基丙烯酰化丝胶蛋白，且A组分溶液中的甲基丙烯酰化丝胶蛋白浓度为30％(w/v)，苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐浓度为0.5％(w/v)，B组分溶液中的甲基丙烯酰化丝胶蛋白浓度为20％(w/v)，苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐浓度为0.5％(w/v)。且此时得到的凝胶中，纤维蛋白交联和光交联的体积比为1:1。
对比例6
制备浓度为10％(w/v)甲基丙烯酰化葡聚糖-0.25％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-1000IU/mL凝血酶的混合溶液作为A组分溶液，制备浓度为10％(w/v)甲基丙烯酰化葡聚糖-0.125％(w/v)苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐-5％(w/v)纤维蛋白原的混合溶液作为B组分溶液。其制备方法与实施例9大体相同，不同之处在于：本对比例中的A和B组分中的光敏材料为甲基丙烯酰化葡聚糖，且A组分溶液中的甲基丙烯酰化葡聚糖浓度为10％(w/v)，B组分溶液中的甲基丙烯酰化葡聚糖浓度为5％(w/v)。且此时得到的凝胶中，纤维蛋白交联和光交联的体积比为1:1。
性能测试
为验证实施例1～36得到的双交联纤维蛋白凝胶及对比例1～6水凝胶的性能，下面分别对其进行胶凝时间性能测试、粘附强度测试和动物止血实验。
胶凝时间测试
检测对象：
前述的实施例1～36，以及对比例1-6；
检测方法：
对实施例1～36及对比例1～6进行流变学分析，来比较其胶凝时间，其结果见表1。具体操作方法：使用具有平行板(P20TiL，20-mm直径)几何结构的HAAKE RS6000光流变仪在37℃下进行动态流变实验。实施例1～36和对比例1～6水凝胶的时间扫描振荡测试在5％应变、1Hz频率下进行300秒。对预凝胶溶液进行应变扫描以验证线性响应。凝胶点在扭转模量(G’)超过损耗模量(G”)时确定。
粘附强度测试
前述的实施例1～36，以及对比例1～6；
检测方法：
具体操作：将猪皮切成40毫米×20毫米的长方形，两片猪皮间用500μl的实施例1～36及对比例1～6进行粘合，对实施例1～36的A组分和B组分的混合溶液及对比例2～6的前体溶液进行相同波段蓝光的光照60s。之后以1mm/min的应变速率进行粘合强度的测试。对比例2的前体溶液光照交联后形成的凝胶微观结构如图2所示；实施例1混合溶液光照交联后形成的凝胶微观结构如图3所示。记录凝胶从猪皮脱落时的读数，即为粘附强度(Kpa)。检测结果见表1。
止血效果测试
检测对象：
本发明的实施例1、实施例9、实施例14、实施例19、实施例25、实施例31，以及对比例1～6；
检测方法：
兔肝表面1cm切口出血模型：将新西兰大白兔麻醉后，暴露腹部，固定在手术台，腹部正中切口，暴露肝脏，在肝脏上造1cm*0.5cm的出血模型；分别用已称重的滤纸、实施例1、9、14、19、25、31的A组分和B组分按照实施例1步骤(7)所述的注射方法注射得到的混合溶液、以及对比例1～6的前体溶液作为止血材料覆盖在出血部位(其中本发明各实施例和对比例2-6均在覆盖同时使用相同波段蓝光光照处理)，直至出血停止，记录出血时间和失血量，其结果见表1、图4和图5。
表1
止血时间和失血量的数值用(均值±标准差)来表示。
结果分析：
由图1可知，对比例1的纤维蛋白胶粘合剂中仅有纤维蛋白交联呈网状结构。由图2可知，对比例2前体溶液光照后仅有甲基丙烯酰化明胶光交联呈多孔结构。由图3可知，本发明实施例1的A、B组分混合溶液光照后可同时具有纤维蛋白交联的网状结构和甲基丙烯酰化明胶交联的多孔结构，且形成的甲基丙烯酰化明胶交联多孔结构的孔道内都分布有整体连续的三维立体纤维蛋白网络结构。
由表1可知，实施例1～36的胶凝时间范围为1～3s，在光敏材料相同的情况下，胶凝时间会随着光交联比例的提高而延长，但实施例1～36所选择的所有种类的光敏材料在特定的双交联比例下的胶凝时间均显著低于对比例2-4的胶凝时间(对比例2的胶凝时间为8s，对比例3的胶凝时间为9s，对比例4的胶凝时间为14s)。
由表1可知，实施例1～36的粘附强度范围为82～132kPa，在光敏材料相同的情况下，凝胶的粘附强度随着光敏材料浓度的降低而减小，但实施例1～36所选择的所有种类的光敏材料在特定的双交联比例下的粘附强度均高于各对比例的粘附强度(对比例1的粘附强度为6kPa，对比例2的粘附强度为80kPa，对比例3的粘附强度为76kPa，对比例4的粘附强度为70kPa，对比例5的粘附强度为29kPa，对比例6的粘附强度为45kPa)。
由表1和图4、图5可知，实施例1、实施例9、实施例14、实施例19、实施例25和实施例31的止血时间为6s～24s，均显著低于对比例1～6的40s以上的止血时间。实施例1、实施例9、实施例14、实施例19、实施例25和实施例31的平均失血量为12mg～37mg，均显著低于对比例1～6的90mg以上的失血量。
总之，本发明的双交联纤维蛋白凝胶，应用在出血伤口时，可即刻(1s左右)形成纤维蛋白凝块，起到“初步”封堵伤口作用，阻挡血液流出；同时，纤维蛋白凝块中的酶将血液中的纤维蛋白原转化凝块，起到高效的促凝血效果；进一步的，光敏材料在光激发下，形成光敏凝胶，光敏凝胶具有强湿组织粘附力，起到“强”封闭伤口效果。纤维蛋白交联和光交联结构相互作用，兼具初步封堵伤口和强组织粘附功能，从而达到优异的止血效果。
以上对本发明的具体实施例进行了详细介绍。需要理解的是，本发明并不局限于特定实施方式，凡在本发明的精神和原则之内所作的任何变形或修改、等同替换和改进等，并不影响本发明的实质内容，均应包含在本发明权利要求的保护范围之内。

Claims (46)

  1. 一种可在出血伤口原位快速止血的双交联纤维蛋白凝胶，其特征在于：它是一种由具有封闭功能的网络结构和具有粘附功能的网络结构组成的固态水凝胶；所述的具有封闭功能的网络结构是三维立体的纤维蛋白网络，所述的具有粘附功能的网络结构是三维立体的光敏凝胶网络；每个所述的光敏凝胶网络孔道内部都有一组所述的纤维蛋白网络，且每一组所述的纤维蛋白网络整体具有连续性；整体上，所述的三维立体的纤维蛋白网络无序地遍布所述固态水凝胶表面和内部；所述的三维立体的纤维蛋白网络与所述的三维立体的光敏凝胶网络的体积比为0.5～3；所述的纤维蛋白网络先于光敏凝胶形成；所述的纤维蛋白网络由纤维蛋白原通过酶交联形成；所述的光敏凝胶由光敏材料通过光交联形成，所述的光敏材料是甲基丙烯酰化的高分子聚合物或其衍生物、聚丙烯酸酯类的高分子聚合物或其衍生物、或包含甲基丙烯酰化的高分子聚合物或聚丙烯酸酯类的高分子聚合物的高分子复合材料体系。
  2. 如权利要求1所述的双交联纤维蛋白凝胶，其特征在于：所述的甲基丙烯酰化的高分子聚合物或其衍生物选自以下任意一种或两种以上的混合物：甲基丙烯酰化明胶或其衍生物、甲基丙烯酰化透明质酸或其衍生物、甲基丙烯酰化海藻酸钠或其衍生物、甲基丙烯酰化丝素蛋白或其衍生物、甲基丙烯酰化壳聚糖或其衍生物、甲基丙烯酰化羧甲基壳聚糖或其衍生物。
  3. 如权利要求1所述的双交联纤维蛋白凝胶，其特征在于：所述的光敏材料是甲基丙烯酰化明胶或其衍生物、或甲基丙烯酰化丝素蛋白或其衍生物。
  4. 如权利要求1所述的双交联纤维蛋白凝胶，其特征在于：所述的聚丙烯酸酯类的高分子聚合物或其衍生物选自聚醚二丙烯酸酯或其衍生物、或聚乙二醇二丙烯酸酯或其衍生物。
  5. 如权利要求1所述的双交联纤维蛋白凝胶，其特征在于：所述的包含甲基丙烯酰化的高分子聚合物的高分子复合材料体系选自甲基丙烯酰化明胶-聚乙烯醇体系、甲基丙烯酰化明胶-聚氨酯体系、甲基丙烯酰化明胶-聚乳酸体系、甲基丙烯酰化明胶-纤维素体系、甲基丙烯酰化透明质酸-聚乙烯醇体系、甲基丙烯酰化透明质酸-聚氨酯体系、甲基丙烯酰化透明质酸-聚乳酸体系、甲基丙烯酰化透明质酸-纤维素体系、甲基丙烯酰化海藻酸钠-聚乙烯醇体系、甲基丙烯酰化海藻酸钠-聚氨酯体系、甲基丙烯酰化海藻酸钠-聚乳酸体系、甲基丙烯酰化海藻酸钠-纤维素体系、甲基丙烯酰化丝素蛋白-聚乙烯醇体系、甲基丙烯酰化丝素蛋白-聚氨酯体系、甲基丙烯酰化丝素蛋白-聚乳酸体系、甲基丙烯酰化丝素蛋白-纤维素体系、甲基丙烯酰化壳聚糖-聚乙烯醇体系、甲基丙烯酰化壳聚糖-聚氨酯体系、甲基丙烯酰化壳聚糖-聚乳酸体系、甲基丙烯酰化壳聚糖-纤维素体系、甲基丙烯酰化羧甲基壳聚糖-聚乙烯醇体系、甲基丙烯酰化羧甲基壳聚糖-聚氨酯体系、甲基丙烯酰化羧甲基壳聚糖-聚乳酸体系、甲基丙烯酰化羧甲基壳聚糖-纤维素体系中的任意一种或两种以上。
  6. 如权利要求1所述的双交联纤维蛋白凝胶，其特征在于：所述的纤维蛋白原是人纤维蛋白原、牛纤维蛋白原或猪纤维蛋白原中的任意一种。
  7. 一种用于在出血伤口通过混合原位制备快速止血用双交联纤维蛋白粘合剂的原料组合物，其特征在于：包括组合物A和组合物B；按重量份计，所述的组合物A包括10～200份光敏材料、1～3份光引发剂、0.14～0.28份酶和1.11～8.88份水溶性无机钙盐，所述的组合物B包括5～100份光敏材料、1～2份光引发剂和30～50份纤维蛋白原；所述的组合物A与所述的组合物B质量比为1.4:10～14:1；所述的光敏材料是甲基丙烯酰化的高分子聚合物或其衍生物、聚丙烯酸酯类的高分子聚合物或其衍生物、或包含甲基丙烯酰化的高分子聚合物或聚丙烯酸酯类的高分子聚合物的高分子复合材料体系；所述的甲基丙烯酰化的高分子聚合物或其衍生物选自以下任意一种或两种以上的混合物：甲基丙烯酰化明胶或其衍生物、甲基丙烯酰化透明质酸或其衍生物、甲基丙烯酰化海藻酸钠或其衍生物、甲基丙烯酰化丝素蛋白或其衍生物、甲基丙烯酰化壳聚糖或其衍生物、甲基丙烯酰化羧甲基壳聚糖或其衍生物；所述的酶选自人凝血酶、重组人凝血酶、牛凝血酶、猪凝血酶或蛇毒血凝酶中的任意一种；所述的双交联纤维蛋白粘合剂是由三维立体的纤维 蛋白网络和三维立体的光敏凝胶网络共同构成的固态水凝胶；每个所述的光敏凝胶网络孔道内部都有一组所述的纤维蛋白网络，且每一组所述的纤维蛋白网络整体具有连续性；整体上，所述的三维立体的纤维蛋白网络无序地遍布所述固态水凝胶表面和内部。
  8. 如权利要求7所述的原料组合物，其特征在于：所述的组合物A中光敏材料重量份大于组合物B中光敏材料重量份。
  9. 如权利要求7所述的原料组合物，其特征在于：所述的组合物A与所述的组合物B质量比为1.4:1～1.4:10。
  10. 如权利要求7所述的原料组合物，其特征在于：按重量份计，所述的组合物A包括80～200份光敏材料、1～3份光引发剂、0.14～0.28份酶和1.11～8.88份水溶性无机钙盐，所述的组合物B包括30～100份光敏材料、1～2份光引发剂和30～50份纤维蛋白原。
  11. 如权利要求7所述的原料组合物，其特征在于：所述的聚丙烯酸酯类的高分子聚合物或其衍生物选自聚醚二丙烯酸酯或其衍生物、或聚乙二醇二丙烯酸酯或其衍生物。
  12. 如权利要求7所述的原料组合物，其特征在于：所述的包含甲基丙烯酰化的高分子聚合物的高分子复合材料体系选自甲基丙烯酰化明胶-聚乙烯醇体系、甲基丙烯酰化明胶-聚氨酯体系、甲基丙烯酰化明胶-聚乳酸体系、甲基丙烯酰化明胶-纤维素体系、甲基丙烯酰化透明质酸-聚乙烯醇体系、甲基丙烯酰化透明质酸-聚氨酯体系、甲基丙烯酰化透明质酸-聚乳酸体系、甲基丙烯酰化透明质酸-纤维素体系、甲基丙烯酰化海藻酸钠-聚乙烯醇体系、甲基丙烯酰化海藻酸钠-聚氨酯体系、甲基丙烯酰化海藻酸钠-聚乳酸体系、甲基丙烯酰化海藻酸钠-纤维素体系、甲基丙烯酰化丝素蛋白-聚乙烯醇体系、甲基丙烯酰化丝素蛋白-聚氨酯体系、甲基丙烯酰化丝素蛋白-聚乳酸体系、甲基丙烯酰化丝素蛋白-纤维素体系、甲基丙烯酰化壳聚糖-聚乙烯醇体系、甲基丙烯酰化壳聚糖-聚氨酯体系、甲基丙烯酰化壳聚糖-聚乳酸体系、甲基丙烯酰化壳聚糖-纤维素体系、甲基丙烯酰化羧甲基壳聚糖-聚乙烯醇体系、甲基丙烯酰化羧甲基壳聚糖-聚氨酯体系、甲基丙烯酰化羧甲基壳聚糖-聚乳酸体系、甲基丙烯酰化羧甲基壳聚糖-纤维素体系中的任意一种或两种以上。
  13. 如权利要求7-12任意一项所述的原料组合物，其特征在于：所述的光敏材料是甲基丙烯酰化明胶或其衍生物、或甲基丙烯酰化丝素蛋白或其衍生物。
  14. 如权利要求7-12任意一项所述的原料组合物，其特征在于：所述的光引发剂选自以下任意一种或两种以上的组合物：苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐、2-羟基-4'-(2-羟乙氧基)-2-甲基苯丙酮、2,4,6-三甲基苯甲酰基膦酸乙酯、2-甲基-1-[4-甲硫基苯基]-2-吗啉基-1-丙酮、邻苯甲酰苯甲酸甲酯、2-苯基苄-2-二甲基胺-1-(4-吗啉苄苯基)丁酮或2,2-偶氮(2-甲基-N-(2-羟基乙基)丙酰胺)。
  15. 如权利要求7-12任意一项所述的原料组合物，其特征在于：所述的纤维蛋白原选自人纤维蛋白原、牛纤维蛋白原或猪纤维蛋白原中的任意一种。
  16. 如权利要求7-12任意一项所述的原料组合物，其特征在于：所述的水溶性无机钙盐为氯化钙、硝酸钙或硫酸钙。
  17. 如权利要求7-12任意一项所述的原料组合物，其特征在于：所述的原料组合物是冻干粉剂、注射剂、海绵或颗粒。
  18. 制备权利要求7-12任意一项所述的原料组合物的方法，包括：制备溶剂中溶解有光敏材料和光引发剂的混合溶液，将所述的混合溶液与含有凝血酶和钙离子的溶液混合得到第一前体溶液，控制所述第一前体溶液中光敏材料、光引发剂、酶和钙离子的浓度比为10～200:1～3:0.14～0.28:1.11～8.88；控制所述的第一前体溶液在室温环境下的保存时间小于30分钟；将所述的混合溶液与含有纤维蛋白原的溶液混合得到 第二前体溶液，控制所述第二前体溶液中光敏材料、光引发剂和纤维蛋白原的浓度比为5～100:1～2:30～50；由此得到包含所述第一前体溶液和所述第二前体溶液的液体原料组合物；所述的光敏材料是甲基丙烯酰化的高分子聚合物或其衍生物、聚丙烯酸酯类的高分子聚合物或其衍生物、或包含甲基丙烯酰化的高分子聚合物或聚丙烯酸酯类的高分子聚合物的高分子复合材料体系；所述的甲基丙烯酰化的高分子聚合物或其衍生物选自以下任意一种或两种以上的混合物：甲基丙烯酰化明胶或其衍生物、甲基丙烯酰化透明质酸或其衍生物、甲基丙烯酰化海藻酸钠或其衍生物、甲基丙烯酰化丝素蛋白或其衍生物、甲基丙烯酰化壳聚糖或其衍生物、甲基丙烯酰化羧甲基壳聚糖或其衍生物。
  19. 如权利要求18所述的方法，其特征在于：将所述的液体原料组合物进一步按照冷冻干燥方法处理得到固态的原料组合物，所述的固态的原料组合物为冻干粉剂、海绵或颗粒。
  20. 如权利要求18所述的方法，其特征在于：控制所述第一前体溶液中的光敏材料浓度大于0.5％(w/v)，且所述第二前体溶液中的光敏材料浓度低于所述第一前体溶液中的光敏材料浓度。
  21. 如权利要求18所述的方法，其特征在于，具体包括以下步骤：
    1)制备溶剂中溶解有光敏材料和光引发剂的第一混合溶液，控制其中光敏材料和光引发剂的浓度比为10～200:1～3，且光敏材料的浓度在0.5％～30％(w/v)；
    2)制备溶剂中溶解有光敏材料和光引发剂的第二混合溶液，控制其中光敏材料和光引发剂的浓度比为5～100:1～2，且光敏材料的浓度低于1)所述的第一混合溶液；
    3)将1)制备的第一混合溶液与含有酶和钙离子的溶液混合得到第一前体溶液，控制其中光敏材料、光引发剂、酶和钙离子的浓度比为10～200:1～3:0.14～0.28:1.11～8.88；
    4)将2)制备的第二混合溶液与含有纤维蛋白原的溶液混合得到第二前体溶液，控制其中光敏材料、光引发剂和纤维蛋白原的浓度比为5～100:1～2:30～50。
  22. 如权利要求18或21任意一项所述的方法，其特征在于，所述的含有酶和钙离子的溶液按照以下方法制备：将溶剂和水溶性无机钙盐溶液加入酶中，完全溶解后得到含Ca2+的酶溶液，控制所得溶液中酶活力为500IU～2000IU/ml、Ca2+浓度为60～100mmol/L。
  23. 如权利要求18或21任意一项所述的方法，其特征在于，所述的含有纤维蛋白原的溶液中，纤维蛋白原的浓度为5％～10％(w/v)。
  24. 如权利要求18或21任意一项所述的方法，其特征在于，控制所述的第一前体溶液中光敏材料浓度在1％～30％(w/v)。
  25. 如权利要求18或21任意一项所述的方法，其特征在于，控制所述的第一前体溶液中酶活力不低于200IU/ml。
  26. 如权利要求18或21任意一项所述的方法，其特征在于，控制所述的第一前体溶液中钙离子浓度不低于20mmol/L。
  27. 如权利要求18或21任意一项所述的方法，其特征在于，控制所述的第二前体溶液中光敏材料浓度在1％-10％(w/v)且不高于所述的第一前体溶液中光敏材料浓度。
  28. 如权利要求18或21任意一项所述的方法，其特征在于，控制所述的第二前体溶液中纤维蛋白原浓度不低于3％(w/v)。
  29. 一种用于制备权利要求1-6任意一项所述的双交联纤维蛋白凝胶的试剂盒，包括相互独立包装的第一前体试剂、第二前体试剂；按重量份计，所述的第一前体试剂含有10～200份光敏材料、1～3份光引发剂、0.14～0.28份酶和3.33～5.55份水溶性无机钙盐，所述的第二前体试剂包括5～100份光敏材料、1～2份 光引发剂和30～50份纤维蛋白原；所述的第一前体试剂与所述的第二前体试剂质量比为1.4:10～14:1；所述的光敏材料是甲基丙烯酰化的高分子聚合物或其衍生物、聚丙烯酸酯类的高分子聚合物或其衍生物、或包含甲基丙烯酰化的高分子聚合物或聚丙烯酸酯类的高分子聚合物的高分子复合材料体系；所述的酶选自人凝血酶、重组人凝血酶、牛凝血酶、猪凝血酶或蛇毒血凝酶中的任意一种。
  30. 如权利要求29所述的试剂盒，其特征在于：所述的第一前体试剂与所述的第二前体试剂的质量比为1.4:1～1.4:10。
  31. 如权利要求29所述的试剂盒，其特征在于：按重量份计，所述的第一前体试剂含有80～200份光敏材料、1～3份光引发剂、0.14～0.28份酶和3.33～5.55份水溶性无机钙盐，所述的第二前体试剂含有30～100份光敏材料、1～2份光引发剂和30～50份纤维蛋白原。
  32. 如权利要求29所述的试剂盒，其特征在于：所述的甲基丙烯酰化的高分子聚合物或其衍生物选自以下任意一种或两种以上的混合物：甲基丙烯酰化明胶或其衍生物、甲基丙烯酰化透明质酸或其衍生物、甲基丙烯酰化海藻酸钠或其衍生物、甲基丙烯酰化丝素蛋白或其衍生物、甲基丙烯酰化壳聚糖或其衍生物、甲基丙烯酰化羧甲基壳聚糖或其衍生物。
  33. 如权利要求29所述的试剂盒，其特征在于：所述的光敏材料是甲基丙烯酰化明胶或其衍生物、或甲基丙烯酰化丝素蛋白或其衍生物。
  34. 如权利要求29所述的试剂盒，其特征在于：所述的聚丙烯酸酯类的高分子聚合物或其衍生物选自聚醚二丙烯酸酯或其衍生物、或聚乙二醇二丙烯酸酯或其衍生物。
  35. 如权利要求29所述的试剂盒，其特征在于：所述的包含甲基丙烯酰化的高分子聚合物的高分子复合材料体系选自甲基丙烯酰化明胶-聚乙烯醇体系、甲基丙烯酰化明胶-聚氨酯体系、甲基丙烯酰化明胶-聚乳酸体系、甲基丙烯酰化明胶-纤维素体系、甲基丙烯酰化透明质酸-聚乙烯醇体系、甲基丙烯酰化透明质酸-聚氨酯体系、甲基丙烯酰化透明质酸-聚乳酸体系、甲基丙烯酰化透明质酸-纤维素体系、甲基丙烯酰化海藻酸钠-聚乙烯醇体系、甲基丙烯酰化海藻酸钠-聚氨酯体系、甲基丙烯酰化海藻酸钠-聚乳酸体系、甲基丙烯酰化海藻酸钠-纤维素体系、甲基丙烯酰化丝素蛋白-聚乙烯醇体系、甲基丙烯酰化丝素蛋白-聚氨酯体系、甲基丙烯酰化丝素蛋白-聚乳酸体系、甲基丙烯酰化丝素蛋白-纤维素体系、甲基丙烯酰化壳聚糖-聚乙烯醇体系、甲基丙烯酰化壳聚糖-聚氨酯体系、甲基丙烯酰化壳聚糖-聚乳酸体系、甲基丙烯酰化壳聚糖-纤维素体系、甲基丙烯酰化羧甲基壳聚糖-聚乙烯醇体系、甲基丙烯酰化羧甲基壳聚糖-聚氨酯体系、甲基丙烯酰化羧甲基壳聚糖-聚乳酸体系、甲基丙烯酰化羧甲基壳聚糖-纤维素体系中的任意一种或两种以上。
  36. 如权利要求29-35任意一项所述的试剂盒，其特征在于：所述的光引发剂选自以下任意一种或两种以上的组合物：苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐、2-羟基-4′-(2-羟乙氧基)-2-甲基苯丙酮、2,4,6-三甲基苯甲酰基膦酸乙酯、2-甲基-1-[4-甲硫基苯基]-2-吗啉基-1-丙酮、邻苯甲酰苯甲酸甲酯、2-苯基苄-2-二甲基胺-1-(4-吗啉苄苯基)丁酮或2,2-偶氮(2-甲基-N-(2-羟基乙基)丙酰胺)。
  37. 如权利要求29-35任意一项所述的试剂盒，其特征在于：所述的纤维蛋白原选自人纤维蛋白原、牛纤维蛋白原或猪纤维蛋白原中的任意一种。
  38. 如权利要求29-35任意一项所述的试剂盒，其特征在于：所述的水溶性无机钙盐选自氯化钙、硝酸钙或硫酸钙。
  39. 如权利要求29-35任意一项所述的试剂盒，其特征在于：所述的第一前体试剂和/或第二前体试剂中还包含辅料和/或添加剂；所述的辅料选自甘氨酸、盐酸精氨酸、枸橼酸钠、蔗糖、氯化钠中的一种或两种以上；所述的添加剂选自生长因子、白细胞介素、维生素、银离子中的一种或两种以上。
  40. 如权利要求39所述的试剂盒，其特征在于：所述的生长因子选自血小板生长因子、表皮生长因子或成纤维细胞生长因子中的一种或多种。
  41. 如权利要求39所述的试剂盒，其特征在于：所述的白细胞介素可选自白细胞介素2、白细胞介素6或白细胞介素8中的一种或多种。
  42. 如权利要求39所述的试剂盒，其特征在于：所述的维生素选自维生素B、维生素C、维生素E或维生素K中的一种或多种。
  43. 如权利要求29-35任意一项所述的试剂盒，其特征在于：进一步包括独立包装的配置用溶剂，所述的配置用溶剂为磷酸缓冲盐溶液、HEPES生物缓冲液、0.9％氯化钠溶液、氯化钙溶液或去离子水中的任意一种或几种的混合物。
  44. 如权利要求29-35任意一项所述的试剂盒，其特征在于：进一步包括试剂盒使用说明书。
  45. 使用权利要求29-35任意一项所述的试剂盒在出血伤口原位快速止血的方法，包括：利用配置用溶剂将所述试剂盒中的第一前体试剂和第二前体试剂分别制备成可注射溶液状，然后同时均匀的注射或喷涂于出血伤口，再用290～480nm波段的光照射10～60s，在出血伤口原位快速形成作为凝止血材料的固态水凝胶。
  46. 如权利要求45所述的方法，其特征在于：所述的出血伤口包括由于意外创伤导致或手术中发生的器官出血；所述的器官是肝脏、脾脏、肾脏、胃肠、心脏或皮肤。
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