WO2020108670A2 - 一种止血组合物及其制备方法 - Google Patents

一种止血组合物及其制备方法 Download PDF

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WO2020108670A2
WO2020108670A2 PCT/CN2020/074061 CN2020074061W WO2020108670A2 WO 2020108670 A2 WO2020108670 A2 WO 2020108670A2 CN 2020074061 W CN2020074061 W CN 2020074061W WO 2020108670 A2 WO2020108670 A2 WO 2020108670A2
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zeolite
trypsin
coagulation
blood
hemostatic
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PCT/CN2020/074061
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English (en)
French (fr)
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WO2020108670A3 (zh
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范杰
余丽莎
肖丽萍
陈昊
尚小强
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浙江大学
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Priority to EP20728890.3A priority Critical patent/EP3991760A4/en
Priority to KR1020217020561A priority patent/KR20220027801A/ko
Priority to US17/298,945 priority patent/US11951228B2/en
Priority to JP2020508048A priority patent/JP7016049B2/ja
Publication of WO2020108670A2 publication Critical patent/WO2020108670A2/zh
Publication of WO2020108670A3 publication Critical patent/WO2020108670A3/zh
Priority to IL283596A priority patent/IL283596A/en

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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
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/18—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons 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
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/38—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing enzymes
    • 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
    • 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
    • A61L24/0015—Medicaments; Biocides
    • 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
    • A61L24/0036—Porous materials, e.g. foams or sponges
    • 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
    • A61L24/0047—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L24/0052—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material with an inorganic matrix
    • A61L24/0068—Inorganic materials not covered by groups A61L24/0057 or A61L24/0063
    • 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
    • A61L24/108—Specific proteins or polypeptides not covered by groups A61L24/102 - A61L24/106
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/10—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing inorganic materials
    • A61L2300/102—Metals or metal compounds, e.g. salts such as bicarbonates, carbonates, oxides, zeolites, silicates
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • A61L2300/254—Enzymes, proenzymes
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/418—Agents promoting blood coagulation, blood-clotting agents, embolising agents
    • 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 technical field of biomedical materials, and particularly relates to a hemostatic composition and a preparation method thereof.
  • Each step of the coagulation channel reaction process refers to the formation of activated coagulation factors from a coagulation factor, such as the formation of XIIa from coagulation factor XII (activated coagulation factor of XII) in Figure 1, and the activation of coagulation factor IX from XIa to IXa ( IX activated coagulation factor). If one of the reactions of the coagulation channel fails, it will inevitably lead to the coagulation channel not being able to proceed normally and failing to achieve the coagulation function.
  • the inorganic hemostatic material represented by zeolite the process of promoting blood coagulation needs to follow the intrinsic coagulation channel (intrinsic) pathway, that is, it must be strictly in accordance with the following reaction process: first, zeolite activates the coagulation factor XII to form XIIa, thereby starting the internal Derived coagulation waterfall, then XIIa activates coagulation factor XI to form XIa, then XIa activates coagulation factor IX to form IXa, and then IXa activates coagulation factor X to form Xa; at coagulation factor Xa, activates coagulation factor II to form thrombin, and then Thrombin cleaves fibrinogen to form fibrin monomers, and finally forms cross-linked fibrin under the action of factor XIIIa.
  • intrinsic coagulation channel intrinsic pathway
  • hemophilia patients with hemophilia can not achieve coagulation through the coagulation channel due to the congenital lack of VIII, IX, XI coagulation factors in the coagulation channel.
  • hemostasis is much more difficult than that of normal people.
  • a biological hemostatic agent can be thrombin, fibrin glue, etc.
  • thrombin directly cuts fibrinogen to form fibrin monomers, and finally forms cross-linked fibrin under the action of coagulation factor XIIIa.
  • the generally required clotting time is 0.5 ⁇ 1min.
  • Fibrin glue can provide thrombin and fibrinogen, so it can achieve the function of blood coagulation.
  • the blood clotting time is generally 0.5 to 1min.
  • thrombin is used to shear fibrinogen and simultaneously form activated coagulation factor XIIIa; the sheared fibrinogen forms fibrin monomers, which are cross-linked to each other under the action of XIIIa to form a stable cross-linked fibrin coagulation Block, thereby acting as a hemostatic.
  • the preparation of thrombin or fibrin glue is difficult, the purity requirements are high, the storage conditions (-20 °C) are harsh, and the cost is very high (the market price of thrombin is generally 2,000 yuan/mg; the market price of fibrin glue is generally 8,000 yuan / Small package, one-time usage), which leads to low practical value and commercial value.
  • Zeolite is a commonly used inorganic material, and enzyme is a biological material that promotes biological reactions.
  • enzyme is a biological material that promotes biological reactions.
  • the main reason is that zeolite is a "rigid” inorganic material and the enzyme is "Flexible” biomacromolecules, when the enzyme contacts the surface of the zeolite, the rigid zeolite surface easily changes the "flexible” characteristics of the enzyme, that is, the conformation changes; and after the enzyme is combined with the zeolite, part of the enzyme is The contact of the surface of the zeolite reduces the possibility of contact with the reactants, that is, the reaction speed becomes slower, and the combination of the enzyme and the zeolite reduces the activity of the enzyme.
  • zeolite is an inorganic hemostatic material
  • thrombin is a highly effective biological hemostatic preparation; but when the two are combined, most of thrombin (94%) is inactivated on the surface of zeolite, and the coagulation effect of thrombin is extended from 0.5min to 2.2min ( Figure 4), the hemostatic effect is not good, far from meeting the requirements of emergency hemostasis.
  • no inorganic biological enzyme binding hemostatic material with rapid hemostasis, low cost, and effective for hemophilia has been found.
  • the technical problem to be solved by the present invention is to provide an inorganic biological enzyme hemostatic composition that is fast in hemostasis, low in cost, and effective for hemophilia.
  • the inventors controlled the interaction between the zeolite and the enzyme and the enzyme on the surface of the zeolite by selecting specific types of zeolite, specific performance enzymes and specific zeolite to enzyme mass ratio, and modifying the surface of the zeolite The conformation has broken the traditional academic thinking, accidentally for the first time produced an inorganic biological enzyme hemostatic material that is fast, low-cost, and effective for hemophilia.
  • the inventors "regulated" the enzyme in the zeolite by controlling the pore size, the polarization of the divalent metal cation of the zeolite, the ratio of trypsin to zeolite according to the mass ratio of 1:200 to 4:10, and the combined action of the above control means
  • the conformation of the surface the inventor unexpectedly found that trypsin does not enter the internal framework of the zeolite, and trypsin is arranged on the surface of the zeolite according to a regular conformation and orientation (Figure 2), especially the active part of the trypsin does not face the zeolite, which is conducive to " "Capture” the reactants.
  • the hemostatic composition of the present invention does not involve expensive biological agents such as thrombin, and only requires inexpensive and easily available trypsin combined with zeolites with micropores and divalent cations in specific ratios to achieve rapid hemostasis, low cost, and The purpose of hemophilia to effectively stop bleeding.
  • the present invention adopts the following technical solution: the present invention obtains a completely new material by a simple method, a hemostatic composition, the hemostatic composition includes at least zeolite and trypsin, wherein the pores of the zeolite are micropores, The zeolite contains divalent metal cations, and the mass ratio of trypsin to zeolite is 1:200-4:10 ( Figure 2).
  • the divalent metal cations described in the present invention belong to metal cations outside the zeolite framework; the metal cations outside the zeolite framework are cations that balance the negative charge of the zeolite framework, and are located in the channels and cages of the zeolite.
  • the micropores are zeolite pore size below 2nm.
  • the divalent metal cation is selected from any one or more of cobalt ion, nickel ion, calcium ion, magnesium ion, and strontium ion.
  • the mass ratio of the trypsin to zeolite is 1:100 to 3:10; preferably, the mass of the trypsin to zeolite is 1:60 to 2.5:10; preferably, the trypsin to zeolite The mass ratio of 1:50 to 2:10; preferably, the mass ratio of trypsin to zeolite is 1:40 to 1.5:10; preferably, the mass ratio of trypsin to zeolite is 1:20 ⁇ 1:10.
  • the divalent metal cations account for 50% to 95% of the metal cations outside the zeolite framework; preferably, the divalent metal cations account for 60% to 90% of the metal cations outside the zeolite framework; preferably, The divalent metal cations account for 65% to 85% of the metal cations outside the zeolite framework; preferably, the divalent metal cations account for 70% to 80% of the metal cations outside the zeolite framework; preferably, the divalent metal cations Metal cations account for 72% to 78% of the metal cations outside the zeolite framework.
  • the silica-alumina ratio of the zeolite is 1-20; preferably, the silica-aluminum ratio is 1.2-15; preferably, the silica-aluminum ratio is 1.5-4; preferably, the The ratio of silicon to aluminum is 2 to 3.
  • the zeolite is selected from any one or more of type A zeolite, chabazite, ⁇ zeolite, mordenite, X type zeolite, Y type zeolite, and ZSM-5 type zeolite.
  • the second object of the present invention is to provide a method for preparing a hemostatic composition, including the following steps:
  • the mass ratio of zeolite to deionized water of the suspension of zeolite is 1:0.5 to 1:20; preferably, the mass ratio of zeolite to deionized water of the suspension of zeolite is 1: 0.8 to 1:10; preferably, the mass ratio of the zeolite suspension to the deionized water is 1:1 to 1:5; preferably, the zeolite suspension to the zeolite and the removal
  • the mass ratio of ionized water is 1:1.5 ⁇ 1:2.5.
  • the zeolite suspension of the step (1) is subjected to ultrasonic treatment.
  • the time of the ultrasonic treatment is 0.5-30 min
  • the ultrasonic frequency is 20-200 kHz
  • the ultrasonic power is 200-5000 W.
  • the contact temperature in the step (2) is 10 to 37°C; preferably, the contact temperature in the step (2) is 15 to 30°C; preferably, the contact in the step (2) The temperature is 20-25°C.
  • the step (3) after the trypsin is adsorbed on the surface of the zeolite further includes a step of freeze-drying the suspension of the zeolite and trypsin, preferably, the temperature of the freeze-drying is 0°C to -80°C;
  • the lyophilization temperature is -10°C to -60°C; preferably, the lyophilization temperature is -20°C to -50°C; preferably, the lyophilization temperature is -30°C to -45°C.
  • the mass ratio of the trypsin and zeolite is 1:200-4:10; the mass ratio of the trypsin and zeolite is 1:100-3:10; preferably, the mass of the trypsin and zeolite 1:60 ⁇ 2.5:10; preferably, the mass ratio of trypsin to zeolite is 1:50 ⁇ 2:10; preferably, the mass ratio of trypsin to zeolite is 1:40 ⁇ 1.5:10 ; Preferably, the mass ratio of trypsin to zeolite is 1:20 ⁇ 1:10.
  • the third object of the present invention is to provide a hemostatic composite material, which includes any form of hemostatic composition and additives as described above.
  • the additive is selected from any one or more of carriers, antibacterial materials, antistatic materials, and high molecular polysaccharides.
  • the carrier is a base for contacting the hemostatic composition with the wound.
  • the antibacterial material is a material that has the function of killing or inhibiting microorganisms.
  • the carrier is selected from cotton, silk, wool, plastic, cellulose, rayon, polyester, polyurethane, polyethylene foam, polyacrylic acid foam, low density polyether, polyvinyl alcohol, polymethacrylate Any one or more of esters.
  • the antibacterial material is selected from any one or more of nano silver particles, vanillin, and ethyl vanillin compounds.
  • polymer polysaccharide is selected from any one or more of cellulose, lignin, starch, chitosan, and agarose.
  • the fourth object of the present invention is to provide the use of any form of hemostatic composition as described above or the use of any form of hemostatic composite material as described above in the field of hemostasis.
  • the hemostatic effect of the hemostatic composition is much better than that of zeolite alone or trypsin alone, and the clotting time is greatly shortened; in the event of sudden and unexpected hemorrhage, it can very effectively stop bleeding, reduce the risk of death from large artery bleeding, and reduce Damage to important organs; hemophilia patients can also stop bleeding in a very short time.
  • the most effective hemostatic material is blood products represented by thrombin for the emergency of large artery bleeding and the special circumstances of bleeding in hemophilia patients; however, blood products represented by thrombin are expensive (coagulated blood)
  • the price of the enzyme is 2000 yuan/mg), its content is scarce (need to be extracted from human or animal blood), and it is difficult to store (need to be stored at low temperature, which is prone to inactivation in vitro).
  • the performance is equal to or even better than that of thrombin, which can greatly reduce the cost of hemostatic materials and has commercial prospects.
  • the hemostatic composition can be used as an emergency hemostatic material that it carries with him, reducing the risk of hemorrhage and reducing the cost of hemostatic medicine for the life of hemophilia patients.
  • Figure 1 is a schematic diagram of the coagulation channel
  • FIG. 2 is a schematic diagram of the structure of the hemostatic composition of the present invention.
  • FIG. 3 is a comparison chart of the normal blood clotting effect of normal blood clotting time, trypsin, type A zeolite, thrombin and the trypsin type A zeolite hemostatic composition of the present invention
  • FIG. 4 is a comparison diagram of the normal blood coagulation effect of natural blood coagulation time, thrombin, thrombin zeolite composite material, trypsin, and the trypsin A zeolite hemostatic composition of the present invention
  • Fig. 5 is a comparison graph of the normal blood coagulation effect of natural blood coagulation time, trypsin type 4A zeolite composition, trypsin type A zeolite (NH 4 + ) composition, and trypsin type A zeolite hemostatic composition of the present invention.
  • the natural clotting time is the time required for normal people to bleed to clot.
  • 1A type zeolite Na type, that is, monovalent sodium ion accounts for 100% of metal cations outside the zeolite framework, micropores
  • a silica-alumina ratio of 2 is immersed in a 5M magnesium chloride solution at room temperature for 12 hours to obtain the type A zeolite It contains divalent magnesium ions and accounts for 95% of the metal cation content outside the zeolite framework, and monovalent sodium ions account for 5% of the metal cations outside the zeolite framework.
  • thrombin (Sigma T4648 thrombin, manufacturer Sigma-Aldrich) to prepare a 1mg/mL solution, add 50uL to a 2mL centrifuge tube, perform in vitro coagulation experiment at 37°C, and normal blood in advance at 37°C water bath thermostat half an hour. Then add 20uL of 0.2M CaCl 2 and finally add 1mL of constant temperature normal blood. The time when normal blood coagulates is recorded as the coagulation time of thrombin. The coagulation time of thrombin is 0.5min, and its coagulation effect is better (see Figure 3) .
  • thrombin (Sigma T4648 thrombin, manufacturer Sigma-Aldrich) to prepare a 1mg/mL solution, take 50uL into a 2mL centrifuge tube, and perform an in vitro coagulation experiment at 37°C.
  • the lack of X, VIII, IX or XI The factor blood was kept constant in a 37°C water bath for half an hour in advance. Then add 20uL of 0.2M CaCl 2 and finally add 1mL of constant temperature factor X, VIII, IX, or factor XI depleted blood.
  • the time when the factor X, VIII, IX, or factor XI depleted blood coagulated is 0.5, 0.52, 0.51, 0.54min, respectively. This shows that the coagulation effect of thrombin is better (Table 1).
  • thrombin zeolite composite material disperse 5g of type A zeolite in 30ml of distilled water; dissolve thrombin in phosphate buffer pH 7.0 to obtain a thrombin with a mass concentration of 0.1% (Sigma T4648 thrombin, manufacturer Sigma -Aldrich Company) solution 10mL; then the two are mixed in a volume ratio of 3:1 to prepare a mixed solution of 40mL with a weight ratio of type A zeolite to thrombin of 10:1.
  • A-type zeolite composite material 2 Preparation of Thrombin A-type zeolite composite material 2: A-type zeolite (Na type, ie monovalent sodium ion accounts for 100% of metal cations outside the zeolite framework, micropores) with a silica-alumina ratio of 2 is used with a 5M concentration of magnesium chloride solution Immerse at room temperature for 12h.
  • A-type zeolite prepared above uniformly in an aqueous solution to form a suspension of A-type zeolite; make A-type zeolite and thrombin fully contacted (according to the mass ratio of thrombin to A-type zeolite 1:10), Mix at 25°C for 30 min to adsorb thrombin on the surface of type A zeolite, and dry the above solution in vacuum at -20°C for 5 hours.
  • Thrombin is dispersed on the surface of zeolite to obtain thrombin A zeolite composite material 2.
  • Coagulation test with thrombin zeolite composite material the coagulation effect test of thrombin A type zeolite composite material 2 normal blood, lack of X, VIII, IX and XI blood, clotting time: 2, 5, 5, 5.5 , 5.2min.
  • 1A type zeolite Na type, that is, monovalent sodium ion accounts for 100% of metal cations outside the zeolite framework, micropores
  • a silica-alumina ratio of 2 is immersed in a 5M magnesium chloride solution at room temperature for 12 hours to obtain the type A zeolite It contains divalent magnesium ions and accounts for 95% of the metal cation content outside the zeolite framework, and monovalent sodium ions account for 5% of the metal cations outside the zeolite framework.
  • the normal blood coagulation time of trypsin A zeolite hemostatic composition is 0.4min, and the blood coagulation time of blood lacking X, VIII, IX, XI coagulation factors is 0.5, 0.4, 0.4, 0.45min respectively, which shows that trypsin A zeolite
  • the hemostatic composition has a good coagulation effect on normal blood; it also has a good coagulation effect on blood lacking X, VIII, IX, XI coagulation factors, the coagulation time is greatly shortened, and it can promote coagulation of hemophilia patients
  • the blood does not coagulate for a long time (Table 1).
  • Example 1 From the comparison between Example 1 and Comparative Examples 2 and 3, it can be obtained that the hemostatic effect of the trypsin A-type zeolite hemostatic composition is far better than that of the A-type zeolite or trypsin (FIG. 3 ), and the clotting time is greatly shortened.
  • the zeolite (pore structure and metal cation) in the hemostatic composition of the present invention positively regulates the spatial orientation of trypsin, so that the trypsin on the surface of the zeolite promotes the conversion of prothrombin into the clotting channel
  • the activity of thrombin is increased, and its procoagulant activity is better than that of trypsin alone; it is better than that of zeolite alone.
  • hemostatic effect of the hemostatic composition is equivalent to that of thrombin, which can greatly reduce the cost of hemostatic materials and has commercial prospects.
  • Table 1 The clotting time of trypsin type A zeolite hemostatic composition, trypsin and type A zeolite
  • a type zeolite (Na type) with pores with a silica-alumina ratio of 2 as micropores, using one or more room temperature solutions of 5M cobalt chloride, nickel chloride, calcium chloride, magnesium chloride or strontium chloride solution After being immersed for 12 hours, the obtained type A zeolites respectively contain different divalent cations.
  • Trypsin Co-A zeolite hemostatic composition means a hemostatic composition in which a type A zeolite containing cobalt ions is combined with trypsin.
  • Trypsin Co-A/Ni-A zeolite hemostatic composition means a hemostatic composition in which a type A zeolite containing cobalt ions and nickel ions is combined with trypsin.
  • the monovalent cation in the table is sodium ion.
  • the monovalent cation of the zeolite described in Example 2 may also be selected from any one or more of sodium ion, potassium ion, lithium ion, ammonium ion, and hydrogen ion.
  • the monovalent cation described in Example 2 is replaced with any one or more of potassium ion, lithium ion, ammonium ion, and hydrogen ion, the corresponding trypsin zeolite hemostatic composition containing a divalent metal cation can also realize the present invention The coagulation effect.
  • the A-type zeolite with pores with a silica-alumina ratio of 2 as micropores was immersed in a 5M magnesium chloride solution at room temperature for 12 hours.
  • the obtained A-type zeolites contained divalent magnesium ions and accounted for 95% of the metal cation content outside the zeolite framework %, monovalent sodium ions account for 5% of the metal cations outside the zeolite framework.
  • the A-type zeolite (Na type) with pores with a silicon-to-alumina ratio of 2 is microporous, and is immersed in a 5M sodium chloride solution at room temperature for 24 hours.
  • the obtained A-type zeolite with a metal cation of Na as the outer zeolite framework that is, 4A Type zeolite
  • monovalent sodium ions account for 100% of the metal cations outside the zeolite framework.
  • the normal blood clotting time of the trypsin 4A zeolite composition is 5 minutes, and the blood clotting times lacking X, VIII, IX, and XI clotting factors are 6.5, 6, 6, and 6.5 minutes, respectively, which shows that the trypsin 4A zeolite composition It is not ideal for normal blood and blood lacking X, VIII, IX, XI coagulation factors.
  • Example 1 From the comparison between Example 1 and Comparative Example 6, it can be obtained that the coagulation effect of the trypsin 4A zeolite composition is far less than that of the trypsin A zeolite hemostatic composition ( Figure 5), and the trypsin 4A zeolite composition is even better than 4A
  • the coagulation effect of zeolite type or trypsin is poor, indicating that the activity of trypsin in zeolite 4A (without divalent cations) is reduced, or even completely inactivated; it also leads to a reduction in the effect of zeolite in promoting coagulation.
  • the composition formed by zeolite and trypsin has a low procoagulant activity, which does not belong to the hemostatic composition of the present invention.
  • the monovalent cation is selected from any one or more of sodium ion, potassium ion, lithium ion, ammonium ion, and hydrogen ion.
  • the A-type zeolite (Na type) with pores with a silica-alumina ratio of 2 as micropores is immersed in a 5M ammonium chloride solution at room temperature for 24 hours.
  • the obtained A-type zeolite contains monovalent ammonium ions and it occupies the outside of the zeolite framework 60% of the metal cation content, monovalent sodium ions account for 40% of the metal cations outside the zeolite framework.
  • the normal blood clotting time of the trypsin type A zeolite (NH 4 + ) composition is 5.2 min, and the blood clotting times lacking X, VIII, IX, and XI clotting factors are 6.1, 6.4, 6.4, and 6.5 min, respectively.
  • the protease A type zeolite (NH 4 + ) composition is not ideal for coagulation of normal blood and blood lacking X, VIII, IX, and XI coagulation factors.
  • trypsin A-type zeolite (NH 4 + ) composition is far less effective in clotting than the trypsin A-type zeolite hemostatic composition ( Figure 5), trypsin A-type zeolite (NH 4 + ) composition is even worse than the coagulation effect of type A zeolite (NH 4 + , 2.8 min) or trypsin, indicating that the activity of trypsin in type A zeolite (NH 4 + , without divalent cations) is reduced, or even All inactivation will also reduce the effect of zeolite in promoting blood coagulation.
  • 1Beta zeolite (Na type) with pores with a silicon-to-alumina ratio of 15 is microporous, and is immersed in a 0.1M ammonia solution at room temperature for 24 hours.
  • the obtained beta zeolite contains monovalent ammonium ions and accounts for the metal cation content outside the zeolite framework
  • the monovalent sodium ion accounts for 60% of the metal cations outside the zeolite framework.
  • the normal blood clotting time of the trypsin beta zeolite (NH 4 + ) composition is 5.6 min, and the blood clotting times lacking X, VIII, IX, and XI clotting factors are 6.1, 6.4, 6.3, and 6.5 min, respectively.
  • the zeolite ⁇ (NH 4 + ) composition is also not ideal for coagulation of normal blood and blood lacking X, VIII, IX, and XI coagulation factors.
  • the coagulation effect of the trypsin beta zeolite (NH 4 + ) composition is far less than that of the trypsin beta zeolite hemostatic composition.
  • the trypsin beta zeolite (NH 4 + , 2.8 min) composition is even better than the beta zeolite (NH 4 + ) or pancreas
  • the protease's coagulation effect is poor, indicating that the activity of trypsin in ⁇ zeolite (NH 4 + , no divalent cations) is reduced, or even completely inactivated, which also leads to a reduction in the effect of zeolite in promoting coagulation.
  • A-type zeolite (Na type) with pores with a silica-alumina ratio of 2 as micropores is immersed in a 5M magnesium chloride solution at room temperature for 12 hours.
  • the obtained A-type zeolite contains divalent magnesium ions and accounts for metal cations outside the zeolite framework At 95% of the content, monovalent sodium ions account for 5% of the metal cations outside the zeolite framework.
  • the normal blood coagulation time of trypsin A-type zeolite composition (1:300) is 2.8 min.
  • trypsin A-type zeolite composition (1:300) to blood lacking X, VIII, IX, and XI clotting factors, the blood Does not solidify for a long time. It can be seen that the ratio of type A zeolite to trypsin needs to be specified in order to play a good role in promoting blood coagulation; when the ratio of trypsin to zeolite is less than 1:200, the effect of promoting blood coagulation is poor.
  • A-type zeolite (Na type) with pores with a silica-alumina ratio of 2 as micropores is immersed in a 5M magnesium chloride solution at room temperature for 12 hours.
  • the obtained A-type zeolite contains divalent magnesium ions and accounts for metal cations outside the zeolite framework At 95% of the content, monovalent sodium ions account for 5% of the metal cations outside the zeolite framework.
  • trypsin type A zeolite composition (5:10) to normal blood and blood lacking X, VIII, IX, and XI clotting factors, the blood does not coagulate for a long time. It can be seen that the ratio of type A zeolite to trypsin needs to be specific in order to play a good role in promoting blood coagulation; when the ratio of trypsin to zeolite is higher than 4:10, it will cause anticoagulation phenomenon in the blood and aggravate the massive loss of blood .
  • Example 1 and Comparative Examples 3, 9, and 10 From the comparison between Example 1 and Comparative Examples 3, 9, and 10, it can be obtained that the mass ratio of zeolite to trypsin in the composition of the present invention needs to meet a specific mass ratio range (1:200 ⁇ 4:10) When the mass ratio of the two is outside the specific mass ratio range (Comparative Example 9 and Comparative Example 10), the coagulation time of the formed zeolite and trypsin complex on blood is longer than that of trypsin alone, That is, the procoagulant activity is lower than that of trypsin alone, and the highly effective hemostatic property of the present invention cannot be achieved.
  • the normal blood clotting time of the trypsin MCM-41 composition is 5 minutes, and the blood clotting times lacking X, VIII, IX, and XI clotting factors are 5.5, 6.3, 6.2, and 6.1 minutes, respectively, which shows that the trypsin MCM-41 composition It is not ideal for normal blood and blood lacking X, VIII, IX, XI coagulation factors.
  • the composition of trypsin MCM-41 is even worse than that of MCM-41 (3 min) or trypsin.
  • A-type zeolite (Na type, pore size is 5nm) with a silica-to-alumina ratio of 2 was immersed in a 5M magnesium chloride solution at room temperature for 12 hours.
  • the obtained A-type zeolite contained divalent magnesium ions and accounted for metal cations outside the zeolite framework At 95% of the content, monovalent sodium ions account for 5% of the metal cations outside the zeolite framework.
  • the normal blood clotting time of the trypsin mesoporous A-type zeolite composition is 5.5 min, and the blood clotting times lacking X, VIII, IX, and XI clotting factors are 5.6, 5.3, 5.2, and 5.1 min, respectively.
  • the A-type zeolite composition is not ideal for coagulation of normal blood and blood lacking X, VIII, IX, and XI coagulation factors.
  • the composition of trypsin mesoporous A zeolite is even worse than that of mesoporous A zeolite or trypsin.
  • Example 1 From the comparison between Example 1 and Comparative Examples 11 and 12, it can be obtained that the procoagulant effect of the trypsin mesoporous zeolite composition is lower than that of the trypsin zeolite hemostatic composition of the present invention, and the trypsin mesoporous zeolite composition is better than The mesoporous zeolite or trypsin alone has poor coagulation effect. Under the same mass ratio of zeolite to trypsin and the same divalent metal cation, the pore size of the zeolite plays a key role in the hemostatic performance of the hemostatic composition composed of zeolite and trypsin.
  • trypsin will enter the mesopores of the mesoporous zeolite, and biological macromolecules cannot enter the mesoporous pores of the zeolite; when in contact with blood, the trypsin in the mesoporous pores cannot contact the coagulation factors of the coagulation channels in the blood , Can not quickly activate the coagulation channel reaction, so that the effect of promoting coagulation is reduced.
  • the mesopores have a zeolite pore size of 50 nm or less and are larger than micropores.
  • the chabazite (Na type) with pores with a silica-alumina ratio of 1.5 as micropores is immersed in a 5M magnesium chloride solution at room temperature for 12 hours.
  • the resulting chabazite contains divalent magnesium ions and accounts for the content of metal cations outside the zeolite framework 95%, monovalent sodium ion accounts for 5% of the metal cations outside the zeolite framework.
  • the 25mg chabazite prepared above was added to a 2mL centrifuge tube, and an in vitro coagulation experiment was performed at 37°C.
  • the blood lacking factor X, VIII, IX, or XI was preliminarily thermostated in a 37°C water bath for half an hour. Then add 20uL of 0.2M CaCl 2 and finally add 1mL of constant temperature factor X, VIII, IX, or factor XI depleted blood.
  • the chabazite (Na type) with pores with a silica-alumina ratio of 1.5 as micropores is immersed in a 5M magnesium chloride solution at room temperature for 12 hours.
  • the resulting chabazite contains divalent magnesium ions and accounts for the content of metal cations outside the zeolite framework 95%, monovalent sodium ion accounts for 5% of the metal cations outside the zeolite framework.
  • the normal blood clotting time of the trypsin chabazite hemostatic composition is 0.35 min, and the blood clotting times lacking X, VIII, IX, and XI clotting factors are 0.45, 0.35, 0.35, 0.45 min, respectively, which shows that the trypsin chabazite hemostatic combination
  • the substance has a good coagulation effect on normal blood; it also has a good coagulation effect on blood lacking X, VIII, IX, XI coagulation factors, the coagulation time is greatly shortened, and it can promote coagulation in patients with hemophilia.
  • Example 4 When blood lacking X, VIII, IX, XI clotting factors is added to chabazite, the blood will not coagulate for a long time. From the comparison between Example 4 and Comparative Example 13, it can be obtained that the hemostatic effect of the trypsin chabazite hemostatic composition is far better than that of chabazite alone or trypsin alone, and the clotting time is greatly shortened.
  • beta zeolite Na type
  • the beta zeolite contains divalent magnesium ions and accounts for the content of metal cations outside the zeolite framework 95%, monovalent sodium ion accounts for 5% of the metal cations outside the zeolite framework.
  • beta zeolite Na type
  • the beta zeolite contains divalent magnesium ions and accounts for the content of metal cations outside the zeolite framework 95%, monovalent sodium ion accounts for 5% of the metal cations outside the zeolite framework.
  • the normal blood clotting time of the trypsin ⁇ zeolite hemostatic composition is 0.52 min, and the blood clotting times lacking X, VIII, IX, and XI clotting factors are 0.55, 0.55, 0.5, and 0.5 min, respectively, which shows that the trypsin ⁇ zeolite hemostatic combination
  • the substance has a good coagulation effect on normal blood; it also has a good coagulation effect on blood lacking X, VIII, IX, XI coagulation factors, the coagulation time is greatly shortened, and it can promote coagulation in patients with hemophilia.
  • the mordenite (Na type) with pores with a silica-alumina ratio of 10 as micropores is immersed in a 5M magnesium chloride solution at room temperature for 12 hours.
  • the resulting mordenite contains divalent magnesium ions and accounts for the content of metal cations outside the zeolite framework 95%, monovalent sodium ion accounts for 5% of the metal cations outside the zeolite framework.
  • the mordenite (Na type) with pores with a silica-alumina ratio of 10 as micropores is immersed in a 5M magnesium chloride solution at room temperature for 12 hours.
  • the resulting mordenite contains divalent magnesium ions and accounts for the content of metal cations outside the zeolite framework 95%, monovalent sodium ion accounts for 5% of the metal cations outside the zeolite framework.
  • the normal blood clotting time of the trypsin mordenite hemostatic composition is 0.45min, and the blood clotting times lacking X, VIII, IX, and XI clotting factors are 0.45, 0.45, 0.5, 0.5min, respectively, which shows that the trypsin mordenite hemostatic combination
  • the substance has a good coagulation effect on normal blood; it also has a good coagulation effect on blood lacking X, VIII, IX, XI coagulation factors, the coagulation time is greatly shortened, and it can promote coagulation in patients with hemophilia.
  • blood lacking X, VIII, IX, and XI clotting factors is added to mordenite, the blood does not coagulate for a long time. From the comparison between Example 6 and Comparative Example 15, it can be obtained that the hemostatic effect of the trypsin mordenite hemostatic composition is far better than that of mordenite or trypsin, and the coagulation time is greatly shortened.
  • 1X-type zeolite Na-type
  • pores of silicon-aluminum ratio of 1 being microporous
  • magnesium chloride solution 5M concentration of magnesium chloride solution at room temperature for 12h
  • monovalent sodium ions account for 5% of the metal cations outside the zeolite framework.
  • 1X-type zeolite Na-type
  • the obtained X-type zeolite contains divalent magnesium ions and it accounts for metal cations outside the zeolite framework
  • monovalent sodium ions account for 5% of the metal cations outside the zeolite framework.
  • the normal blood clotting time of the trypsin X-type zeolite hemostatic composition is 0.48 min, and the blood clotting times lacking X, VIII, IX, and XI clotting factors are 0.48, 0.45, 0.5, and 0.5 min, respectively.
  • the hemostatic composition has a good coagulation effect on normal blood; it also has a good coagulation effect on blood lacking X, VIII, IX, XI coagulation factors, the coagulation time is greatly shortened, and it can promote coagulation of hemophilia patients .
  • blood lacking X, VIII, IX, and XI clotting factors is added to type X zeolite, the blood will not coagulate for a long time.
  • Example 7 From the comparison between Example 7 and Comparative Example 16, it can be obtained that the coagulation effect of the trypsin X-type zeolite hemostatic composition is far better than that of X-type zeolite or trypsin, and the coagulation time is greatly shortened.
  • Micro-pore Y-type zeolite (Na type) with a silica-alumina ratio of 3 is impregnated with a 5M magnesium chloride solution at room temperature for 12 hours.
  • the obtained Y-type zeolite contains divalent magnesium ions and accounts for metal cations outside the zeolite framework At 95% of the content, monovalent sodium ions account for 5% of the metal cations outside the zeolite framework.
  • Micro-pore Y-type zeolite (Na type) with a silica-alumina ratio of 3 is impregnated with a 5M magnesium chloride solution at room temperature for 12 hours.
  • the obtained Y-type zeolite contains divalent magnesium ions and accounts for metal cations outside the zeolite framework At 95% of the content, monovalent sodium ions account for 5% of the metal cations outside the zeolite framework.
  • the normal blood clotting time of the trypsin Y-type zeolite hemostatic composition is 0.3 min, and the blood clotting times lacking X, VIII, IX, and XI clotting factors are 0.33, 0.4, 0.3, and 0.3 min, respectively.
  • the hemostatic composition has a good coagulation effect on normal blood; it also has a good coagulation effect on blood lacking X, VIII, IX, XI coagulation factors, the coagulation time is greatly shortened, and it can promote coagulation of hemophilia patients .
  • blood lacking X, VIII, IX, and XI clotting factors is added to Y-type zeolite, the blood will not coagulate for a long time.
  • Example 8 From the comparison between Example 8 and Comparative Example 17, it can be obtained that the hemostatic effect of the trypsin Y-type zeolite hemostatic composition is far better than that of Y-type zeolite or trypsin, and the coagulation time is greatly shortened.
  • 1ZSM-5 type zeolite (Na type) with pores of silicon-aluminum ratio of 20 as micropores is impregnated with 5M concentration of magnesium chloride solution at room temperature for 12 hours.
  • the obtained ZSM-5 type zeolite contains divalent magnesium ions and accounts for zeolite The content of metal cations outside the framework is 95%, and monovalent sodium ions account for 5% of the metal cations outside the framework of the zeolite.
  • 1ZSM-5 type zeolite (Na type) with pores of silicon-aluminum ratio of 20 as micropores is impregnated with 5M concentration of magnesium chloride solution at room temperature for 12 hours.
  • the obtained ZSM-5 type zeolite contains divalent magnesium ions and accounts for zeolite The content of metal cations outside the framework is 95%, and monovalent sodium ions account for 5% of the metal cations outside the framework of the zeolite.
  • the normal blood coagulation time of trypsin ZSM-5 type zeolite hemostatic composition is 0.4min, and the blood coagulation time lacking X, VIII, IX, XI clotting factors is 0.43, 0.42, 0.4, 0.4min, respectively, which shows that trypsin ZSM -Type 5 zeolite hemostatic composition has a good coagulation effect on normal blood; it also has a good coagulation effect on blood lacking X, VIII, IX, XI coagulation factors, the coagulation time is greatly shortened, and it can be very good for hemophilia patients Promotes coagulation.
  • Example 9 When blood lacking X, VIII, IX, and XI clotting factors is added to ZSM-5 zeolite, the blood will not coagulate for a long time. From the comparison between Example 9 and Comparative Example 18, it can be obtained that the hemostatic effect of the trypsin ZSM-5 zeolite hemostatic composition is far better than that of the ZSM-5 zeolite or trypsin, and the coagulation time is greatly shortened.
  • 1ZSM-5 type zeolite (Na type) with micro-pores of silica-alumina ratio of 20 and Y-type zeolite (Na type) with micro-pores of silica-alumina ratio of 4 are used at room temperature with 5M concentration of magnesium chloride solution at room temperature After being immersed for 12h, the resulting zeolite contains divalent magnesium ions and accounts for 95% of the content of metal cations outside the zeolite framework, and monovalent sodium ions account for 5% of the metal cations outside the zeolite framework.
  • the normal blood coagulation time of trypsin ZSM-5/Y type zeolite hemostatic composition is 0.34min, and the blood coagulation time lacking X, VIII, IX, XI clotting factors is 0.33, 0.32, 0.4, 0.34min, respectively.
  • the trypsin ZSM-5/Y type zeolite hemostatic composition has a good coagulation effect on normal blood; it also has a good coagulation effect on blood lacking X, VIII, IX, XI coagulation factors, and the coagulation time is greatly shortened, which is harmful to hemophilia Patients with disease can have a good role in promoting blood coagulation.
  • the hemostatic effect of the trypsin ZSM-5/Y zeolite hemostatic composition is far better than that of zeolite or trypsin, and the coagulation time is greatly shortened.
  • 1Mordenite zeolite (Na type) with a silica-alumina ratio of 20 and micropores and X-type zeolite (Na type) with a silica-alumina ratio of 1.2 and micropores are immersed in a 5M magnesium chloride solution at room temperature for 12 hours.
  • the resulting zeolite contains divalent magnesium ions and accounts for 95% of the content of metal cations outside the zeolite framework, and monovalent sodium ions account for 5% of the metal cations outside the zeolite framework.
  • the normal blood clotting time of the trypsin mordenite/X-type zeolite hemostatic composition is 0.4 min, and the blood clotting times lacking X, VIII, IX, and XI clotting factors are 0.43, 0.37, 0.4, and 0.36 min, respectively.
  • the mordenite/X type zeolite hemostatic composition has a good coagulation effect on normal blood; it also has a good coagulation effect on blood lacking X, VIII, IX, and XI coagulation factors, and the coagulation time is greatly shortened, which can have Very good to promote blood coagulation.
  • the coagulation effect of the trypsin mordenite/X-type zeolite hemostatic composition is far better than that of zeolite or trypsin, and the coagulation time is greatly shortened.
  • the mass ratio of the zeolite suspension of the zeolite suspension described in Examples 1-11 of the present invention and deionized water is 1:0.5 ⁇ 1:20; preferably, the zeolite suspension of the zeolite suspension and deionized water
  • the mass ratio of 1:0.8 ⁇ 1:10; preferably, the mass ratio of zeolite to deionized water of the zeolite suspension is 1:1 ⁇ 1:5; preferably, the suspension of the zeolite
  • the mass ratio of zeolite to deionized water in the turbid liquid is 1:1.5 ⁇ 1:2.5.
  • the suspension of the zeolite is subjected to ultrasonic treatment.
  • the ultrasonic treatment time described in Examples 1-11 of the present invention is 0.5-30 min, the ultrasonic frequency is 20-200 kHz, and the ultrasonic power is 200-5000 W.
  • the temperature at which the trypsin and the zeolite in Examples 1-11 of the present invention are contacted is 10 to 37°C; preferably, the temperature at which the trypsin and the zeolite are contacted is 15 to 30°C; preferably, the The temperature of the trypsin contacting with the zeolite is 20-25°C.
  • the method further includes the step of freeze-drying the zeolite suspension and trypsin.
  • the temperature of the freeze-drying is 0°C to -80 °C; preferably, the lyophilization temperature is -10 °C ⁇ -60 °C; preferably, the lyophilization temperature is -20 °C ⁇ -50 °C; preferably, the lyophilization temperature is -30 °C ⁇ -45°C.
  • a specific zeolite to trypsin mass ratio (1:200 to 4:10), a specific zeolite framework metal cation (divalent metal) Cations) and a specific zeolite pore size (micropores) are indispensable to form a hemostatic composition with excellent hemostatic properties according to the present invention.
  • the hemostatic properties of the hemostatic composition of trypsin and zeolite are equal to or even better than those of thrombin alone, which can greatly reduce the cost of hemostatic materials and have commercial prospects.
  • the invention overcomes the prejudice of the prior art and unexpectedly prepares for the first time an inorganic biological enzyme hemostatic material with rapid hemostasis, low cost and effective for hemophilia.
  • the coagulation effect of the hemostatic composition is far better than that of zeolite or trypsin alone, the coagulation time is greatly shortened, and the amount of blood loss is significantly reduced.
  • the zeolite (micropore channel structure, divalent metal cation, specific zeolite to trypsin mass ratio) in the hemostatic composition of the present invention positively regulates the spatial conformation and spatial orientation of trypsin, making the zeolite
  • the spatial conformation of the trypsin on the surface is more conducive to contact with prothrombin in the clotting channel (clotting cascade), thereby promoting the activity of prothrombin converted to thrombin during the clotting channel (clotting cascade), and improving the procoagulant activity.
  • the overall effect of the hemostatic composition of the present invention constituted by the synergistic effect of trypsin and zeolite is far better than that of trypsin alone or zeolite alone, and its procoagulant activity is much better than that of trypsin alone, and is also superior to The procoagulant activity of zeolite alone is even better than that of thrombin alone.
  • the hemostatic composition can effectively stop bleeding, reduce the risk of death from large artery bleeding, reduce the damage to important organs, and also have a good effect on promoting blood clotting in patients with hemophilia.

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Abstract

本发明提供一种具有止血组合物，包括胰蛋白酶和沸石，所述沸石的孔道为微孔，沸石包含二价金属阳离子，所述胰蛋白酶与沸石的质量比为1:200～4:10。本发明通过将胰蛋白酶与沸石特异性结合，使胰蛋白酶在沸石表面保持一定的构象，获得更高的促凝血活性，从而得到凝血效果极好的止血组合物。本发明止血组合物的制备方法简单，成本低、使用便利，可广泛用于创伤和手术止血，特别是用于血友病患者的紧急止血。

Description

一种止血组合物及其制备方法 技术领域
本发明属于生物医学材料技术领域，具体涉及一种止血组合物及其制备方法。
背景技术
在多种场合中人类(包括动物)可能受伤。在一些场合中，创伤和出血是轻微的，除施加简单急救外，仅要求常规的凝血功能就能正常止血。然而，不幸的是在很多意外场合中可能发生大量出血，例如，皮肤割破或者穿透伤(由刀割或者子弹引起)会导致主动脉损伤，一个正常人的大部分血会在几分钟中流失并死亡。因而，在日常生活中突发性事故的急救治疗、医院对病人的手术过程中的创伤止血、特别是战争中受伤战士的救护中，对患者有效的快速止血是非常重要。
目前，很多紧急止血材料被开发，例如脱水沸石/粘合剂(US4822349)、部分水合沸石(US7858123)、高岭土/纱布复合物(US7604819)以及沸石与高岭土的组合物(US8703634)。无论是正常功能的凝血还是利用止血材料来促进血液凝固，其凝血过程必须严格遵循凝血通道的每一步反应流程来达到血液凝固的目的，并且凝血通道的每一步需要消耗一定的时间，其详细过程如图1所示。所述凝血通道的每一步反应流程是指从一种凝血因子经反应形成活化凝血因子，例如图1中从凝血因子XII形成XIIa(XII的活化凝血因子)，由XIa激活凝血因子IX形成IXa(IX的活化凝血因子)。如果凝血通道的其中一步反应发生障碍，必然导致凝血通道不能正常进行，无法实现凝血功能。目前以沸石代表的无机止血材料，其促进血液凝固的过程需遵循内源性凝血通道(intrinsic pathway)，也就是必须严格按照以下反应流程进行：首先由沸石激活凝血因子XII形成XIIa，从而启动内源性凝血瀑布，然后由XIIa激活凝血因子XI形成XIa，再由XIa激活凝血因子IX形成IXa，再由IXa激活凝血因子X形成Xa；在凝血因子Xa处，活化凝血因子II形成凝血酶，然后凝血酶剪切纤维蛋白原，形成纤维蛋白单体，最后在凝血因子XIIIa作用下形成交联的纤维蛋白。由沸石激活凝血因子XII到最终形成交联的纤维蛋白的多步反应，总共需要花费2～6min。虽然沸石能促进凝血过程，但凝血时间过长，会导致患者失血过多，部分重要器官因失血会产生不可逆损伤，造成生命危险。
另外，血友病患者由于先天性缺乏凝血通道中的VIII、IX、XI凝血因子，无法通过凝血通道来实现凝血。对于血友病患者发生出血的情况，止血远比正常人困难，需要通过外加生物止血制剂来实现凝血，常用的生物止血制剂可以是凝血酶、纤维蛋白胶等。如图1所示，凝血酶起凝血作用的过程是：凝血酶直接剪切纤维蛋白原，形成纤维蛋白单体，最后在凝血因子XIIIa作用下形成交联的纤维蛋白，一般需要的凝血时间为0.5～1min。纤维蛋白胶能够提供凝血酶和纤维蛋白原，所以可以实现凝血功能，一般需要的凝血时间为0.5～1min。其中，凝血酶用于剪切纤维蛋白原，同时形成活化的凝血因子XIIIa；剪切后的纤维蛋白原形成纤维蛋白单体，其在XIIIa作用下彼此交联，形成稳定的交联纤维蛋白凝块，从而起到止血的作用。但是凝血酶或者纤维蛋白胶制取困难、纯度要求高、保存条件(-20℃)苛刻，成本非常高(凝血酶的市场价一般为2000元/mg；纤维蛋白胶的市场价一般为8000元/小包，一次用量)，因而导致其实用价值和商业价值低。
沸石是一种常用的无机材料，酶是一种促进生物反应的生物材料，无机与生物材料两者结合存在很大的挑战，其主要的原因是：沸石为“刚性”的无机材料，酶为“柔性”的生物大分子，当酶在接触到沸石表面时，刚性的沸石表面容易使得酶的“柔性”特性发生变化，即构象发生变化；并且与沸石结合后的酶，酶的其中一部分与沸石表面接触，导致与反应物接触的可能性降低，即反应速度变慢，酶与沸石的结合使得酶的活性降低。因此，在沸石表面引入酶，都会导致酶在沸石表面不同程度的失活。例如：沸石是一种无机止血材料，凝血酶是一 种高效的生物止血制剂；但是两者结合，大部分凝血酶(94％)在沸石表面失活，凝血酶的凝血效果从0.5min延长至2.2min(图4)，止血效果不佳，远远没有达到紧急止血的要求。目前，现有技术仍未发现止血快速、成本低、且对血友病有效的无机生物酶结合止血材料。
发明内容
针对现有技术的不足，本发明所要解决的技术问题是提供一种止血快速、成本低、且对血友病有效的无机生物酶止血组合物。在研究过程中，发明人通过选择特定种类的沸石、特定性能的酶和特定沸石与酶的质量比，以及对沸石的表面进行改性来控制沸石与酶之间的相互作用以及酶在沸石表面的构象，打破了传统学术上的思维定式，意外地首次制得了一种止血快速、成本低、且对血友病有效的无机生物酶止血材料。发明人通过控制孔道尺寸、沸石的二价金属阳离子的极化作用、胰蛋白酶与沸石按照质量比为1:200～4:10的配比，以上控制手段的综合作用来“规范”酶在沸石表面的构象，发明人意外发现胰蛋白酶不进入沸石的内部骨架，并且胰蛋白酶按照规律的构象和取向排布在沸石表面(图2)，特别是胰蛋白酶的活性部分不朝向沸石，有利于“捕捉”反应物。通过设计这种特殊的止血组合物，意外地实现了无机与生物材料的高效结合，克服了酶在沸石表面失活的技术难题。本发明止血组合物的胰蛋白酶活性不但没有降低，反而止血组合物的止血效果相对胰蛋白酶或沸石都大大提高，凝血时间大大降低(图3)。本发明的止血组合物不涉及凝血酶等昂贵生物制剂，仅需要廉价且易得的胰蛋白酶与具有微孔和二价阳离子的沸石特异性按特定比例组合，达到了止血快速、成本低、对血友病有效止血的目的。
本发明采用如下的技术方案：本发明用简单的方法得到了一种全新的材料，一种止血组合物，所述止血组合物至少包括沸石和胰蛋白酶，其中所述沸石的孔道为微孔，所述的沸石包含二价金属阳离子，所述胰蛋白酶与沸石的质量比为1:200～4:10(图2)。
进一步的，本发明所述的二价金属阳离子属于沸石骨架外金属阳离子；沸石骨架外金属阳离子是平衡沸石骨架负电荷的阳离子，位于沸石的孔道(channel)和笼(cage)之中。
本发明所述的微孔为沸石孔道尺寸在2nm以下。
进一步的，所述的二价金属阳离子选自钴离子、镍离子、钙离子、镁离子、锶离子的任意一种或者多种。
进一步的，所述胰蛋白酶与沸石的质量比为1:100～3:10；优选的，所述胰蛋白酶与沸石的质量为1:60～2.5:10；优选的，所述胰蛋白酶与沸石的质量比为1:50～2:10；优选的，所述胰蛋白酶与沸石的质量比为1:40～1.5:10；优选的，所述的胰蛋白酶与沸石的质量比为1:20～1:10。
进一步的，所述的二价金属阳离子占沸石骨架外金属阳离子的50％～95％；优选的，所述的二价金属阳离子占沸石骨架外金属阳离子的60％～90％；优选的，所述的二价金属阳离子占沸石骨架外金属阳离子的65％～85％；优选的，所述的二价金属阳离子占沸石骨架外金属阳离子的70％～80％；优选的，所述的二价金属阳离子占沸石骨架外金属阳离子的72％～78％。
进一步的，所述的沸石的硅铝比为1～20；优选的，所述的硅铝比为1.2～15；优选的，所述的硅铝比为1.5～4；优选的，所述的硅铝比为2～3。
进一步的，所述的沸石选自A型沸石、菱沸石、β沸石、丝光沸石、X型沸石、Y型沸石、ZSM-5型沸石的任意一种或者多种。
本发明的第二个目的是提供一种止血组合物的制备方法，包括如下步骤：
(1)制备一种沸石的悬浊液，所述沸石的孔道为微孔和沸石表面包含二价金属阳离子；
(2)将沸石的悬浊液与胰蛋白酶接触；
(3)使胰蛋白酶在沸石表面吸附，得到所述的止血组合物。
进一步的，所述沸石的悬浊液的沸石与去离子水的质量比为1:0.5～1:20；优选的，所 述沸石的悬浊液的沸石与去离子水的质量比为1:0.8～1:10；优选的，所述的沸石的悬浊液的沸石与去离子水的质量比为1:1～1:5；优选的，所述的沸石的悬浊液的沸石与去离子水的质量比1:1.5～1:2.5。
进一步的，对所述步骤(1)沸石的悬浊液进行超声处理。
进一步的，所述的超声处理的时间为0.5～30min，超声频率为20～200kHz，超声功率为200～5000W。
进一步的，所述的步骤(2)接触的温度为10～37℃；优选的，所述的步骤(2)接触的温度为15～30℃；优选的，所述的步骤(2)接触的温度为20～25℃。
进一步的，所述的步骤(3)胰蛋白酶在沸石表面吸附后还包括将沸石的悬浊液与胰蛋白酶冻干的步骤，优选的，所述冻干的温度为0℃～-80℃；优选的，所述冻干的温度为-10℃～-60℃；优选的，所述冻干的温度为-20℃～-50℃；优选的，所述冻干的温度为-30℃～-45℃。
进一步的，所述胰蛋白酶与沸石的质量比为1:200～4:10；所述胰蛋白酶与沸石的质量比为1:100～3:10；优选的，所述胰蛋白酶与沸石的质量为1:60～2.5:10；优选的，所述胰蛋白酶与沸石的质量比为1:50～2:10；优选的，所述胰蛋白酶与沸石的质量比为1:40～1.5:10；优选的，所述的胰蛋白酶与沸石的质量比为1:20～1:10。
本发明的第三个目的是提供一种止血复合材料，所述的止血复合材料包括如前所述任何一种形式的止血组合物和添加剂。
进一步的，所述添加剂选自载体、抗菌材料、抗静电材料、高分子多糖的任意一种或多种。
进一步的，所述的载体是用于使止血组合物接触于伤口的基体。
进一步的，所述的抗菌材料是自身具有杀灭或抑制微生物功能的材料。
进一步的，所述的载体选自棉、蚕丝、羊毛、塑料、纤维素、人造丝、聚酯、聚氨酯、聚乙烯泡沫、聚丙烯酸泡沫、低密度聚醚、聚乙烯醇、聚甲基丙烯酸甲酯的任意一种或者多种。
进一步的，所述的抗菌材料选自纳米银颗粒、香草醛、乙基香草醛类化合物的任意一种或者多种。
进一步的，所述的高分子多糖选自纤维素、木质素、淀粉、壳聚糖、琼脂糖的任意一种或多种。
本发明的第四个目的是提供如前所述任何一种形式的止血组合物或如前所述任何一种形式的止血复合材料在止血领域的用途。
本发明的有益效果是：
1.止血组合物的凝血效果远远好于单独的沸石或者单独的胰蛋白酶的凝血效果，凝血时间大大缩短；在遭遇突发意外大出血时，能非常高效止血，降低大动脉出血致死的风险，减少重要器官的损伤；对血友病患者也能在非常短的时间内止血。
2.目前，对于大动脉出血的紧急情况以及血友病患者出血的特殊情况，最为有效的止血材料是以凝血酶为代表的血液制品；但是以凝血酶为代表的血液制品存在着价格昂贵(凝血酶的价格为2000元/mg)，含量稀少(需要从人类或者动物血液中提取)，储存困难(需要低温保存，在体外容易发生失活)等缺点。胰蛋白酶(0.1元/mg)和沸石(300元/吨＝3×10 -7元/mg)都非常便宜，通过无机材料与生物大分子特异性结合，胰蛋白酶与沸石的止血组合物的凝血性能等同于、甚至优于凝血酶的性能，可以大大降低止血材料的成本，更具有商业化前景。尤其对于血友病患者，止血组合物可作为其随身携带的紧急止血材料，减少其出血不止的风险，降低血友病患者一生的止血用药费用。
附图说明
图1为凝血通道的示意图；
图2为本发明的止血组合物的结构示意图；
图3为自然凝血时间、胰蛋白酶、A型沸石、凝血酶以及本发明的胰蛋白酶A型沸石止血组合物的正常血液的凝血效果比较图；
图4为自然凝血时间、凝血酶、凝血酶沸石复合材料、胰蛋白酶以及本发明的胰蛋白酶A型沸石止血组合物的正常血液的凝血效果比较图；
图5为自然凝血时间、胰蛋白酶4A型沸石组合物、胰蛋白酶A型沸石(NH 4 +)组合物、本发明的胰蛋白酶A型沸石止血组合物的正常血液的凝血效果比较图。
具体实施方式
以下结合附图和实施例进一步说明本发明。
凝血效果的测定
①正常血液的凝血时间
取25mg样品加入到2mL的离心管,在37℃进行体外凝血实验，将正常血液预先在37℃水浴恒温半小时。然后加入20uL的0.2M CaCl 2，最后加入1mL恒温的正常血液，正常血液凝固时的时间记为该样品的凝血时间。
②缺乏X因子血液的凝血时间
取25mg样品加入到2mL的离心管,在37℃进行体外凝血实验，将缺乏X因子血液预先在37℃水浴恒温半小时。然后加入20uL的0.2M CaCl 2，最后加入1mL恒温的缺乏X因子血液，缺乏X因子血液凝固时的时间记为该样品的凝血时间。
③缺乏VIII因子血液的凝血时间
取25mg样品加入到2mL的离心管,在37℃进行体外凝血实验，将缺乏VIII因子血液预先在37℃水浴恒温半小时。然后加入20uL的0.2M CaCl 2，最后加入1mL恒温的缺乏VIII因子血液，缺乏VIII因子血液凝固时的时间记为该样品的凝血时间。
④缺乏IX因子血液的凝血时间
取25mg样品加入到2mL的离心管,在37℃进行体外凝血实验，将缺乏IX因子血液预先在37℃水浴恒温半小时。然后加入20uL的0.2M CaCl 2，最后加入1mL恒温的缺乏IX因子血液，缺乏IX因子血液凝固时的时间记为该样品的凝血时间。
⑤缺乏XI因子血液的凝血时间
取25mg样品加入到2mL的离心管,在37℃进行体外凝血实验，将缺乏XI因子血液预先在37℃水浴恒温半小时。然后加入20uL的0.2M CaCl 2，最后加入1mL恒温的缺乏XI因子血液，缺乏XI因子血液凝固时的时间记为该样品的凝血时间。
对比实施例1
自然凝血时间为正常人出血到凝血所需的时间。
自然凝血效果的测定：在37℃进行体外凝血实验，将正常血液预先在37℃水浴恒温半小时。体外实验时，取20μL的0.2M CaCl 2溶液加入到2mL的离心管中，然后加入1mL恒温的正常血液，正常血液凝固的时间记为自然凝血时间(9.6min)。
对比实施例2
A型沸石的凝血效果测定：
①将硅铝比为2的A型沸石(Na型，即一价钠离子占沸石骨架外金属阳离子的100％，微孔)，用5M浓度的氯化镁溶液室温下浸渍12h，得到的A型沸石包含二价镁离子且其占沸石骨架外金属阳离子含量的95％，一价钠离子占沸石骨架外金属阳离子的5％。
②将上述制备得到的25mgA型沸石加入到2mL的离心管,在37℃进行体外凝血实验，将正常血液预先在37℃水浴恒温半小时。然后加入20uL的0.2M CaCl 2，最后加入1mL恒温的正常血液，正常血液凝固时的时间记为A型沸石的凝血时间，A型沸石的凝血时间为2.8min(表1)，由此可见A型沸石的凝血效果一般。
取25mgA型沸石加入到2mL的离心管,在37℃进行体外凝血实验，将乏X、VIII、IX或XI因子血液预先在37℃水浴恒温半小时。然后加入20uL的0.2M CaCl 2，最后加入1mL恒温的乏X、VIII、IX或XI因子血液，乏X、VIII、IX或XI因子血液凝固时的时间记为A型沸石的凝血时间，但其过程中上述有缺陷的血液长时间没有发生凝固(表1)，由此可见A型沸石对血友病的患者并没有促凝血作用。
对比实施例3
胰蛋白酶的凝血效果测定：
①取25mg胰蛋白酶加入到2mL的离心管,在37℃进行体外凝血实验，将正常血液预先在37℃水浴恒温半小时。然后加入20uL的0.2M CaCl 2，最后加入1mL恒温的正常血液，正常血液凝固时的时间记为胰蛋白酶的凝血时间(4min)，由此可见胰蛋白酶的凝血效果一般。
②取25mg胰蛋白酶加入到2mL的离心管,在37℃进行体外凝血实验，将乏X、VIII、IX或XI因子血液预先在37℃水浴恒温半小时。然后加入20uL的0.2M CaCl 2，最后加入1mL恒温的乏X、VIII、IX或XI因子血液，乏X、VIII、IX或XI因子血液凝固时的时间分别为4、4.6、4、4min，由此可见胰蛋白酶的凝血效果一般。
对比实施例4
凝血酶凝血效果的测定：
①取凝血酶(Sigma T4648凝血酶，生产商Sigma-Aldrich公司)配制成1mg/mL溶液，取50uL加入到2mL的离心管,在37℃进行体外凝血实验，将正常血液预先在37℃水浴恒温半小时。然后加入20uL的0.2M CaCl 2，最后加入1mL恒温的正常血液，正常血液凝固时的时间记为凝血酶的凝血时间，凝血酶的凝血时间为0.5min，其凝血效果较好(见图3)。
②取凝血酶(Sigma T4648凝血酶，生产商Sigma-Aldrich公司)配制成1mg/mL溶液，取50uL加入到2mL的离心管,在37℃进行体外凝血实验，将乏X、VIII、IX或XI因子血液预先在37℃水浴恒温半小时。然后加入20uL的0.2M CaCl 2，最后加入1mL恒温的乏X、VIII、IX或XI因子血液，乏X、VIII、IX或XI因子血液凝固时的时间分别为0.5、0.52、0.51、0.54min，由此可见凝血酶的凝血效果较好(表1)。
对比实施例5
凝血酶沸石复合材料的制备：将5gA型沸石分散于30ml蒸馏水中；将凝血酶溶于pH为7.0的磷酸盐缓冲液，得到质量浓度为0.1％的凝血酶(Sigma T4648凝血酶，生产商Sigma-Aldrich公司)溶液10mL；然后将二者按体积比3:1混合，制得A型沸石与凝血酶的重量比例为10:1的混合溶液40mL。在搅拌的条件下，加入0.5g丙三醇和1g甘露醇，并逐滴加入质量浓度5％的氯化钙溶液50uL，混合均匀，再于搅拌状态下，逐滴加入体积浓度1％戊二醛溶液250uL进行交联，然后倾入适当形状的模具中，放置2小时，待形成凝胶后，-20℃冷冻24小时，再于-40℃真空冷冻干燥36h，倒模、磨碎、封装，得到凝血酶沸石复合材料。
凝血酶沸石复合材料凝血效果的测定：
①取25mg凝血酶沸石复合材料加入到2mL的离心管,在37℃进行体外凝血实验，将正常血液预先在37℃水浴恒温半小时。然后加入20uL的0.2M CaCl 2，最后加入1mL恒温的正常血液，正常血液凝固时的时间记为凝血酶沸石复合材料的凝血时间(2.2min)，固定凝血酶的凝血酶沸石复合材料的凝血效果降低。
②取25mg凝血酶沸石复合材料加入到2mL的离心管,在37℃进行体外凝血实验，将乏X、VIII、IX或XI因子血液预先在37℃水浴恒温半小时。然后加入20uL的0.2M CaCl 2，最后加入1mL恒温的乏X、VIII、IX或XI因子血液，乏X、VIII、IX或XI因子血液凝固时的时间分别为4、3.6、4、4min，固定凝血酶的凝血酶沸石复合材料的凝血效果降低(图4)。
凝血酶A型沸石复合材料2的制备：将硅铝比为2的A型沸石(Na型，即一价钠离子占沸石骨架外金属阳离子的100％，微孔)，用5M浓度的氯化镁溶液室温下浸渍12h。将上述制备得到的A型沸石在水溶液中分散均匀，形成A型沸石的悬浊液；使A型沸石与凝血酶充分接触(按凝血酶与A型沸石的质量比为1:10加入)，25℃混合30min，使凝血酶在A型沸石表面吸附，并将上述溶液在-20℃真空干燥5h，凝血酶分散于沸石表面上，得到凝血酶A型沸石复合材料2。
同凝血酶沸石复合材料的凝血测试，对凝血酶A型沸石复合材料2进行正常血液、乏X、VIII、IX和XI因子血液的凝血效果测试，凝血时间分别为：2，5，5，5.5，5.2min。
由对比实施例4与对比实施例5的比较可得：凝血酶与沸石结合形成的复合物凝血时间长于单独的凝血酶的凝血时间，即对应的凝血效果低于单独的凝血酶，并没有实现两种止血材料复合性能优于单独的止血材料(参见表1)。
实施例1
胰蛋白酶A型沸石止血组合物的制备：
①将硅铝比为2的A型沸石(Na型，即一价钠离子占沸石骨架外金属阳离子的100％，微孔)，用5M浓度的氯化镁溶液室温下浸渍12h，得到的A型沸石包含二价镁离子且其占沸石骨架外金属阳离子含量的95％，一价钠离子占沸石骨架外金属阳离子的5％。
②将上述制备得到的25mg的A型沸石在1mL水溶液中分散均匀，形成A型沸石的悬浊液；使A型沸石与胰蛋白酶充分接触(按胰蛋白酶与A型沸石的质量比为1:10加入)，25℃混合30min，使胰蛋白酶在A型沸石表面吸附，并将上述溶液在-20℃真空干燥5h，胰蛋白酶分散于沸石表面上，得到胰蛋白酶A型沸石止血组合物。
胰蛋白酶A型沸石止血组合物的正常血液凝血时间为0.4min，缺乏X、VIII、IX、XI凝血因子血液的凝血时间分别为0.5、0.4、0.4、0.45min，由此可见胰蛋白酶A型沸石止血组合物对正常血液的凝血效果很好；对缺乏X、VIII、IX、XI凝血因子的血液的凝血效果也很好，凝血时间大大缩短，对血友病患者能有很好的促进凝血作用，缺乏X、VIII、IX、XI凝血因子的血液加入A型沸石时，血液长时间不凝固(表1)。
由实施例1与对比实施例2、3的比较可得：胰蛋白酶A型沸石止血组合物的凝血效果远远好于A型沸石或者胰蛋白酶的凝血效果(图3)，凝血时间大大缩短。本发明所述的止血组合物中沸石(孔道结构及金属阳离子)对胰蛋白酶的空间取向进行正向的调控，使得在沸石表面的胰蛋白酶促进凝血通道(clotting cascade)过程中凝血酶原转化成凝血酶的活性提高，其促凝血活性优于单独胰蛋白酶的活性；更优于单独沸石的促凝血活性。在遭遇突发意外大出血时，能非常高效止血，降低大动脉出血致死的风险，减少重要器官的损伤。止血组合物的止血效果与凝血酶所起到的作用相当，可以大大降低止血材料的成本，更具有商业化前景。
表1胰蛋白酶A型沸石止血组合物、胰蛋白酶及A型沸石的凝血时间
Figure PCTCN2020074061-appb-000001
实施例2
不同二价阳离子的胰蛋白酶A型沸石止血组合物的制备：
①将硅铝比为2的孔道为微孔的A型沸石(Na型)，分别用5M浓度的氯化钴、氯化镍、氯化钙、氯化镁或氯化锶溶液一种或多种室温下浸渍12h，得到的A型沸石分别包含不同二价阳离子。
②将上述制备得到的25mg含不同二价阳离子的A型沸石在1mL水溶液中分散均匀，形成A型沸石的悬浊液；使A型沸石与胰蛋白酶充分接触(按胰蛋白酶与A型沸石的质量比为1:10加入)，25℃混合30min，使胰蛋白酶在沸石表面吸附，并将上述溶液在-20℃真空干燥5h，胰蛋白酶分散于沸石表面上，得到不同二价阳离子的胰蛋白酶A型沸石止血组合物。
不同的二价阳离子(钴，镍，钙，镁，锶)的胰蛋白酶A型沸石止血组合物(表2)与对比实施例1-3的凝血效果，二价阳离子的胰蛋白酶A型沸石止血组合物的凝血效果大大优于自然凝血、胰蛋白酶、A型沸石的凝血效果。对缺乏X、VIII、IX、XI凝血因子的血液的凝血效果也很好，凝血时间大大缩短。
表2不同二价阳离子的胰蛋白酶A型沸石止血组合物的凝血时间
Figure PCTCN2020074061-appb-000002
备注：1)胰蛋白酶Co-A型沸石止血组合物表示包含钴离子的A型沸石与胰蛋白酶组合的止血组合物。2)胰蛋白酶Co-A型/Ni-A型沸石止血组合物表示包含钴离子和镍离子的A型沸石与胰蛋白酶组合的止血组合物。3)表格中的一价阳离子为钠离子。
进一步的，所述实施例2所述的沸石一价阳离子也可选自钠离子、钾离子、锂离子、铵离子、氢离子的任意一种或者多种。当实施例2所述的一价阳离子替换成钾离子、锂离子、铵离子、氢离子的任意一种或者多种，对应的包含二价金属阳离子的胰蛋白酶沸石止血组合物也能实现本发明所述的凝血效果。
实施例3
不同胰蛋白酶与沸石质量比例的胰蛋白酶A型沸石止血组合物的制备：
①将硅铝比为2的孔道为微孔的A型沸石，用5M浓度的氯化镁溶液室温下浸渍12h，得到的A型沸石分别包含二价镁离子且其占沸石骨架外金属阳离子含量的95％，一价钠离子占沸石骨架外金属阳离子的5％。
②将上述制备得到的25mg的A型沸石在1mL水溶液中分散均匀，形成A型沸石的悬浊液；使A型沸石与胰蛋白酶充分接触(按胰蛋白酶与A型沸石的质量比为1:200，1:100，1:60，1:50，1:40，1:20，1:10，1.5:10，2:10，2.5:10，3:10，4:10加入)，25℃混合30min，使胰蛋白酶在沸石表面吸附，并将上述溶液在-20℃真空干燥5h，胰蛋白酶分散于沸石表面上，得到不同比例的胰蛋白酶A型沸石止血组合物(表3)。
表3不同比例的胰蛋白酶A型沸石止血组合物的凝血时间
Figure PCTCN2020074061-appb-000003
不同比例的胰蛋白酶A型沸石止血组合物(分别按1:200，1:100，1:60，1:50，1:40，1:20，1:10，1.5:10，2:10，2.5:10，3:10，4:10的质量比制备)与对比实施例1-3的凝血效果，在胰蛋白酶与沸石的质量比为1:200～4:10的范围内，不同比例的胰蛋白酶A型沸石止血组合物的凝血效果(表3)大大优于自然凝血、胰蛋白酶、沸石的凝血效果。对缺乏X、VIII、IX、XI凝血因子的血液的凝血效果也很好，凝血时间大大缩短。
对比实施例6
①将硅铝比为2的孔道为微孔的A型沸石(Na型)，用5M浓度的氯化钠溶液室温下浸渍24h，得到的沸石骨架外金属阳离子为Na的A型沸石(即4A型沸石)，一价钠离子占沸石骨架外金属阳离子的100％。
②将上述制备得到的25mg的4A型沸石在1mL水溶液中分散均匀，形成4A型沸石的悬浊液；使4A型沸石与胰蛋白酶充分接触(按胰蛋白酶与4A型沸石的质量比为1:10加入)，25℃混合30min，使胰蛋白酶在沸石表面吸附，并将上述溶液在-20℃真空干燥5h，胰蛋白酶分散于沸石表面上，得到胰蛋白酶4A型沸石止血组合物。
胰蛋白酶4A型沸石组合物的正常血液凝血时间为5min，缺乏X、VIII、IX、XI凝血 因子的血液凝血时间分别为6.5、6、6、6.5min，由此可见胰蛋白酶4A型沸石组合物对正常血液和缺乏X、VIII、IX、XI凝血因子的血液的凝血效果也不理想。
由实施例1与对比实施例6的比较可得：胰蛋白酶4A型沸石组合物的凝血效果远不如的胰蛋白酶A型沸石止血组合物(图5)，胰蛋白酶4A型沸石组合物甚至比4A型沸石或者胰蛋白酶的凝血效果差，表明胰蛋白酶在4A型沸石(无二价阳离子)活性减小，甚至全部失活；也导致沸石促进凝血的效果降低。
其中，当沸石骨架外金属阳离子为一价阳离子时(含量为100％)，沸石与胰蛋白酶所形成的组合物的促凝血活性低，不属于本发明的止血组合物。
所述的一价阳离子选自钠离子、钾离子、锂离子、铵离子、氢离子的任意一种或者多种。
对比实施例7
①将硅铝比为2的孔道为微孔的A型沸石(Na型)，用5M浓度的氯化铵溶液室温下浸渍24h，得到的A型沸石包含一价铵离子且其占沸石骨架外金属阳离子含量的60％，一价钠离子占沸石骨架外金属阳离子的40％。
②将上述制备得到的A型沸石(NH 4 +)在1mL水溶液中分散均匀，形成A型沸石(NH 4 +)的悬浊液；使A型沸石(NH 4 +)与胰蛋白酶充分接触(按胰蛋白酶与A型沸石(NH 4 +)的质量比为1:10加入)，25℃混合30min，使胰蛋白酶在沸石表面吸附，并将上述溶液在-20℃真空干燥5h，胰蛋白酶分散于沸石表面上，得到胰蛋白酶A型沸石(NH 4 +)组合物。
胰蛋白酶A型沸石(NH 4 +)组合物的正常血液凝血时间为5.2min，缺乏X、VIII、IX、XI凝血因子的血液凝血时间分别为6.1、6.4、6.4、6.5min，由此可见胰蛋白酶A型沸石(NH 4 +)组合物对正常血液和缺乏X、VIII、IX、XI凝血因子的血液的凝血效果也不理想。
由实施例1与对比实施例7的比较可得：胰蛋白酶A型沸石(NH 4 +)组合物的凝血效果远不如的胰蛋白酶A型沸石止血组合物(图5)，胰蛋白酶A型沸石(NH 4 +)组合物甚至比A型沸石(NH 4 +，2.8min)或者胰蛋白酶的凝血效果差，表明胰蛋白酶在A型沸石(NH 4 +，无二价阳离子)活性减小，甚至全部失活，也导致沸石促进凝血的效果降低。
对比实施例8
①将硅铝比为15的孔道为微孔的β沸石(Na型)，用0.1M浓度的氨水溶液室温下浸渍24h，得到的β沸石包含一价铵离子且其占沸石骨架外金属阳离子含量的40％，一价钠离子占沸石骨架外金属阳离子的60％。
②将上述制备得到的β沸石(NH 4 +)在1mL水溶液中分散均匀，形成β沸石(NH 4 +)的悬浊液；使β沸石(NH 4 +)与胰蛋白酶充分接触(按胰蛋白酶与β沸石(NH 4 +)的质量比为1:10加入)，25℃混合30min，使胰蛋白酶在沸石表面吸附，并将上述溶液在-20℃真空干燥5h，胰蛋白酶分散于沸石表面上，得到胰蛋白酶β沸石(NH 4 +)组合物。
胰蛋白酶β沸石(NH 4 +)组合物的正常血液凝血时间为5.6min，缺乏X、VIII、IX、XI凝血因子的血液凝血时间分别为6.1、6.4、6.3、6.5min，由此可见胰蛋白酶β沸石(NH 4 +)组合物对正常血液和缺乏X、VIII、IX、XI凝血因子的血液的凝血效果也不理想。胰蛋白酶β沸石(NH 4 +)组合物的凝血效果远不如的胰蛋白酶β沸石止血组合物，胰蛋白酶β沸石(NH 4 +，2.8min)组合物甚至比β沸石(NH 4 +)或者胰蛋白酶的凝血效果差，表明胰蛋白酶在β沸石(NH 4 +，无二价阳离子)活性减小，甚至全部失活，也导致沸石促进凝血的效果降低。
对比实施例9
①将硅铝比为2的孔道为微孔的A型沸石(Na型)，用5M浓度的氯化镁溶液室温下浸渍12h，得到的A型沸石包含二价镁离子且其占沸石骨架外金属阳离子含量的95％，一价钠离子占沸石骨架外金属阳离子的5％。
②将上述制备得到的25mgA型沸石在1mL水溶液中分散均匀，形成A型沸石的悬浊液；使A型沸石与0.83mg胰蛋白酶充分分散均匀(按胰蛋白酶与A型沸石的质量比为1:300)，25℃混合30min，使胰蛋白酶在沸石表面吸附，并将上述溶液在-20℃真空干燥5h，胰蛋白酶分散于沸石表面上，得到胰蛋白酶A型沸石组合物(1:300)。
胰蛋白酶A型沸石组合物(1:300)的正常血液凝血时间为2.8min，缺乏X、VIII、IX、XI凝血因子的血液在加入胰蛋白酶A型沸石组合物(1:300)后，血液长时间不凝固。由此可见，A型沸石与胰蛋白酶的比例需要特定，才能发挥较好的促凝血作用；当胰蛋白酶与沸石比例低于1:200时，促凝血效果差。
对比实施例10
①将硅铝比为2的孔道为微孔的A型沸石(Na型)，用5M浓度的氯化镁溶液室温下浸渍12h，得到的A型沸石包含二价镁离子且其占沸石骨架外金属阳离子含量的95％，一价钠离子占沸石骨架外金属阳离子的5％。
②将上述制备得到的A型沸石在1mL水溶液中分散均匀，形成A型沸石的悬浊液；使A型沸石与胰蛋白酶充分接触(按胰蛋白酶与A型沸石的质量比为5:10加入)，25℃混合30min，使胰蛋白酶在沸石表面吸附，并将上述溶液在-20℃真空干燥5h，胰蛋白酶分散于沸石表面上，得到胰蛋白酶A型沸石组合物(5:10)。
正常血液和缺乏X、VIII、IX、XI凝血因子的血液在加入胰蛋白酶A型沸石组合物(5:10)后，血液长时间不凝固。由此可见，A型沸石与胰蛋白酶的比例需要特定，才能发挥较好的促凝血作用；当胰蛋白酶与沸石比例高于4:10时，反而导致血液产生抗凝现象，加剧血液的大量流失。
由实施例1与对比实施例3、9、10的比较可得：本发明所述的组合物中的沸石与胰蛋白酶的质量比需要满足特定的质量比范围(1:200～4:10)，当两者的质量比在该特定的质量比范围之外(对比实施例9、对比实施例10)时，形成的沸石与胰蛋白酶复合物对血液的凝血时间长于单独胰蛋白酶的凝血时间，即促凝血活性低于单独胰蛋白酶的凝血活性，无法达到本发明高效的止血性能。
对比实施例11
将介孔二氧化硅MCM-41(孔道为2.8-4.5nm)在1mL水溶液中分散均匀，形成介孔二氧化硅MCM-41的悬浊液；使介孔二氧化硅MCM-41与胰蛋白酶充分接触(按胰蛋白酶与介孔二氧化硅MCM-41的质量比为1:10加入)，25℃混合30min，并将上述溶液在-20℃真空干燥5h，得到胰蛋白酶MCM-41组合物。
胰蛋白酶MCM-41组合物的正常血液凝血时间为5min，缺乏X、VIII、IX、XI凝血因子的血液凝血时间分别为5.5、6.3、6.2、6.1min，由此可见胰蛋白酶MCM-41组合物对正常血液和缺乏X、VIII、IX、XI凝血因子的血液的凝血效果也不理想。胰蛋白酶MCM-41组合物甚至比MCM-41(3min)或者胰蛋白酶的凝血效果差。
对比实施例12
将硅铝比为2的介孔A型沸石(Na型，孔道为5nm)，用5M浓度的氯化镁溶液室温下浸渍12h，得到的A型沸石包含二价镁离子且其占沸石骨架外金属阳离子含量的95％，一价钠离子占沸石骨架外金属阳离子的5％。
将上述制备得到的介孔A型沸石在1mL水溶液中分散均匀，形成介孔A型沸石的悬浊液；使介孔A型沸石与胰蛋白酶充分接触(按胰蛋白酶与介孔A型沸石的质量比为1:10加入)，25℃混合30min，并将上述溶液在-20℃真空干燥5h，得到胰蛋白酶介孔A型沸石组合物。
胰蛋白酶介孔A型沸石组合物的正常血液凝血时间为5.5min，缺乏X、VIII、IX、XI 凝血因子的血液凝血时间分别为5.6、5.3、5.2、5.1min，由此可见胰蛋白酶介孔A型沸石组合物对正常血液和缺乏X、VIII、IX、XI凝血因子的血液的凝血效果也不理想。胰蛋白酶介孔A型沸石组合物甚至比介孔A型沸石或者胰蛋白酶的凝血效果差。
由实施例1与对比实施例11、12的比较可得：胰蛋白酶介孔沸石组合物的促凝血效果低于本发明所述的胰蛋白酶沸石止血组合物，胰蛋白酶介孔沸石组合物比单独的介孔沸石或者单独的胰蛋白酶的凝血效果差。在相同的沸石与胰蛋白酶质量比、相同的二价金属阳离子的条件下，沸石孔径的大小对沸石与胰蛋白酶组成的止血组合物的止血性能起着关键的作用。这是由于胰蛋白酶会进入介孔沸石的介孔中，而生物大分子无法进入沸石的介孔孔道内；在接触血液时，介孔孔道内的胰蛋白酶无法接触血液中的凝血通道的凝血因子，无法快速激活凝血通道反应，使得促进凝血的效果降低。
本发明所述的介孔为沸石孔道尺寸为50nm以下，且大于微孔的尺寸。
对比实施例13
①将硅铝比为1.5的孔道为微孔的菱沸石(Na型)，用5M浓度的氯化镁溶液室温下浸渍12h，得到的菱沸石包含二价镁离子且其占沸石骨架外金属阳离子含量的95％，一价钠离子占沸石骨架外金属阳离子的5％。
②上述制备得到的菱沸石的凝血效果测定：取25mg菱沸石加入到2mL的离心管,在37℃进行体外凝血实验，将正常血液预先在37℃水浴恒温半小时。然后加入20uL的0.2MCaCl 2，最后加入1mL恒温的正常血液，正常血液凝固时的时间记为菱沸石的凝血时间，菱沸石的凝血时间为2min，由此可见菱沸石的凝血效果一般。
取上述制备得到的25mg菱沸石加入到2mL的离心管,在37℃进行体外凝血实验，将乏X、VIII、IX或XI因子血液预先在37℃水浴恒温半小时。然后加入20uL的0.2M CaCl 2，最后加入1mL恒温的乏X、VIII、IX或XI因子血液，乏X、VIII、IX或XI因子血液凝固时的时间记为菱沸石的凝血时间，但其过程中上述有缺陷的血液长时间没有发生凝固，由此可见菱沸石对血友病的患者并没有促凝血作用。
实施例4
胰蛋白酶菱沸石止血组合物的制备：
①将硅铝比为1.5的孔道为微孔的菱沸石(Na型)，用5M浓度的氯化镁溶液室温下浸渍12h，得到的菱沸石包含二价镁离子且其占沸石骨架外金属阳离子含量的95％，一价钠离子占沸石骨架外金属阳离子的5％。
②将上述制备得到的25mg的菱沸石在1mL水溶液中分散均匀，形成菱沸石的悬浊液；使菱沸石与胰蛋白酶充分接触(按胰蛋白酶与菱沸石的质量比为1:10加入)，25℃混合30min，使胰蛋白酶在沸石表面吸附，并将上述溶液在-20℃真空干燥5h，胰蛋白酶分散于沸石表面上，得到胰蛋白酶菱沸石止血组合物。
胰蛋白酶菱沸石止血组合物的正常血液凝血时间为0.35min，缺乏X、VIII、IX、XI凝血因子的血液凝血时间分别为0.45、0.35、0.35、0.45min，由此可见胰蛋白酶菱沸石止血组合物对正常血液的凝血效果很好；对缺乏X、VIII、IX、XI凝血因子的血液的凝血效果也很好，凝血时间大大缩短，对血友病患者能有很好的促进凝血作用。缺乏X、VIII、IX、XI凝血因子的血液加入菱沸石时，血液长时间不凝固。由实施例4与对比实施例13的比较可得：胰蛋白酶菱沸石止血组合物的凝血效果远远好于单独的菱沸石或者单独的胰蛋白酶的凝血效果，凝血时间大大缩短。
对比实施例14
①将硅铝比为15的孔道为微孔的β沸石(Na型)，用5M浓度的氯化镁溶液室温下浸渍12h，得到的β沸石包含二价镁离子且其占沸石骨架外金属阳离子含量的95％，一价钠离 子占沸石骨架外金属阳离子的5％。
②上述得到制备得到的β沸石的凝血效果测定：取25mgβ沸石加入到2mL的离心管,在37℃进行体外凝血实验，将正常血液预先在37℃水浴恒温半小时。然后加入20uL的0.2MCaCl 2，最后加入1mL恒温的正常血液，正常血液凝固时的时间记为β沸石的凝血时间，β沸石的凝血时间为3min，由此可见β沸石的凝血效果一般。
取25mgβ沸石加入到2mL的离心管,在37℃进行体外凝血实验，将乏X、VIII、IX或XI因子血液预先在37℃水浴恒温半小时。然后加入20uL的0.2M CaCl 2，最后加入1mL恒温的乏X、VIII、IX或XI因子血液，乏X、VIII、IX或XI因子血液凝固时的时间记为β沸石的凝血时间，但其过程中上述有缺陷的血液长时间没有发生凝固，由此可见β沸石对血友病的患者并没有促凝血作用。
实施例5
胰蛋白酶β沸石止血组合物的制备：
①将硅铝比为15的孔道为微孔的β沸石(Na型)，用5M浓度的氯化镁溶液室温下浸渍12h，得到的β沸石包含二价镁离子且其占沸石骨架外金属阳离子含量的95％，一价钠离子占沸石骨架外金属阳离子的5％。
②将上述制备得到的25mg的β沸石在1mL水溶液中分散均匀，形成β沸石的悬浊液；使β沸石与胰蛋白酶充分接触(按胰蛋白酶与β沸石的质量比为1:10加入)，25℃混合30min，使胰蛋白酶在沸石表面吸附，并将上述溶液在-20℃真空干燥5h，胰蛋白酶分散于沸石表面上，得到胰蛋白酶β沸石止血组合物。
胰蛋白酶β沸石止血组合物的正常血液凝血时间为0.52min，缺乏X、VIII、IX、XI凝血因子的血液凝血时间分别为0.55、0.55、0.5、0.5min，由此可见胰蛋白酶β沸石止血组合物对正常血液的凝血效果很好；对缺乏X、VIII、IX、XI凝血因子的血液的凝血效果也很好，凝血时间大大缩短，对血友病患者能有很好的促进凝血作用。缺乏X、VIII、IX、XI凝血因子的血液加入β沸石时，血液长时间不凝固。由实施例5与对比实施例14的比较可得：胰蛋白酶β沸石止血组合物的凝血效果远远好于β沸石或者胰蛋白酶的凝血效果，凝血时间大大缩短。
对比实施例15
①将硅铝比为10的孔道为微孔的丝光沸石(Na型)，用5M浓度的氯化镁溶液室温下浸渍12h，得到的丝光沸石包含二价镁离子且其占沸石骨架外金属阳离子含量的95％，一价钠离子占沸石骨架外金属阳离子的5％。
②取上述制备得到的丝光沸石的凝血效果测定：取25mg丝光沸石加入到2mL的离心管,在37℃进行体外凝血实验，将正常血液预先在37℃水浴恒温半小时。然后加入20uL的0.2M CaCl 2，最后加入1mL恒温的正常血液，正常血液凝固时的时间记为丝光沸石的凝血时间，丝光沸石的凝血时间为2.5min，由此可见丝光沸石的凝血效果一般。
取25mg丝光沸石加入到2mL的离心管,在37℃进行体外凝血实验，将乏X、VIII、IX或XI因子血液预先在37℃水浴恒温半小时。然后加入20uL的0.2M CaCl 2，最后加入1mL恒温的乏X、VIII、IX或XI因子血液，乏X、VIII、IX或XI因子血液凝固时的时间记为丝光沸石的凝血时间，但其过程中上述有缺陷的血液长时间没有发生凝固，由此可见丝光沸石对血友病的患者并没有促凝血作用。
实施例6
胰蛋白酶丝光沸石止血组合物的制备：
①将硅铝比为10的孔道为微孔的丝光沸石(Na型)，用5M浓度的氯化镁溶液室温下浸渍12h，得到的丝光沸石包含二价镁离子且其占沸石骨架外金属阳离子含量的95％，一价 钠离子占沸石骨架外金属阳离子的5％。
②将上述制备得到的25mg的丝光沸石在1mL水溶液中分散均匀，形成丝光沸石的悬浊液；使丝光沸石与胰蛋白酶充分接触(按胰蛋白酶与丝光沸石的质量比为1:10加入)，25℃混合30min，使胰蛋白酶在沸石表面吸附，并将上述溶液在-20℃真空干燥5h，胰蛋白酶分散于沸石表面上，得到胰蛋白酶丝光沸石止血组合物。
胰蛋白酶丝光沸石止血组合物的正常血液凝血时间为0.45min，缺乏X、VIII、IX、XI凝血因子的血液凝血时间分别为0.45、0.45、0.5、0.5min，由此可见胰蛋白酶丝光沸石止血组合物对正常血液的凝血效果很好；对缺乏X、VIII、IX、XI凝血因子的血液的凝血效果也很好，凝血时间大大缩短，对血友病患者能有很好的促进凝血作用。缺乏X、VIII、IX、XI凝血因子的血液加入丝光沸石时，血液长时间不凝固。由实施例6与对比实施例15的比较可得：胰蛋白酶丝光沸石止血组合物的凝血效果远远好于丝光沸石或者胰蛋白酶的凝血效果，凝血时间大大缩短。
对比实施例16
①将硅铝比为1的孔道为微孔的X型沸石(Na型)，用5M浓度的氯化镁溶液室温下浸渍12h，得到的X型沸石包含二价镁离子且其占沸石骨架外金属阳离子含量的95％，一价钠离子占沸石骨架外金属阳离子的5％。
②取上述制备得到的X型沸石的凝血效果测定：取25mgX型沸石加入到2mL的离心管,在37℃进行体外凝血实验，将正常血液预先在37℃水浴恒温半小时。然后加入20uL的0.2M CaCl 2，最后加入1mL恒温的正常血液，正常血液凝固时的时间记为X型沸石的凝血时间，X型沸石的凝血时间为2.1min，由此可见X型沸石的凝血效果一般。
取25mgX型沸石加入到2mL的离心管,在37℃进行体外凝血实验，将乏X、VIII、IX或XI因子血液预先在37℃水浴恒温半小时。然后加入20uL的0.2M CaCl 2，最后加入1mL恒温的乏X、VIII、IX或XI因子血液，乏X、VIII、IX或XI因子血液凝固时的时间记为X型沸石的凝血时间，但其过程中上述有缺陷的血液长时间没有发生凝固，由此可见X型沸石对血友病的患者并没有促凝血作用。
实施例7
胰蛋白酶X型沸石止血组合物的制备：
①将硅铝比为1的孔道为微孔的X型沸石(Na型)，用5M浓度的氯化镁溶液室温下浸渍12h，得到的X型沸石包含二价镁离子且其占沸石骨架外金属阳离子含量的95％，一价钠离子占沸石骨架外金属阳离子的5％。
②将上述制备得到的25mg的X型沸石在1mL水溶液中分散均匀，形成X型沸石的悬浊液；使X型沸石与胰蛋白酶充分接触(按胰蛋白酶与X型沸石的质量比为1:10加入)，25℃混合30min，使胰蛋白酶在沸石表面吸附，并将上述溶液在-20℃真空干燥5h，胰蛋白酶分散于沸石表面上，得到胰蛋白酶X型沸石止血组合物。
胰蛋白酶X型沸石止血组合物的正常血液凝血时间为0.48min，缺乏X、VIII、IX、XI凝血因子的血液凝血时间分别为0.48、0.45、0.5、0.5min，由此可见胰蛋白酶X型沸石止血组合物对正常血液的凝血效果很好；对缺乏X、VIII、IX、XI凝血因子的血液的凝血效果也很好，凝血时间大大缩短，对血友病患者能有很好的促进凝血作用。缺乏X、VIII、IX、XI凝血因子的血液加入X型沸石时，血液长时间不凝固。由实施例7与对比实施例16的比较可得：胰蛋白酶X型沸石止血组合物的凝血效果远远好于X型沸石或者胰蛋白酶的凝血效果，凝血时间大大缩短。
对比实施例17
①将硅铝比为3的孔道为微孔的Y型沸石(Na型)，用5M浓度的氯化镁溶液室温下浸渍12h，得到的Y型沸石包含二价镁离子且其占沸石骨架外金属阳离子含量的95％，一价钠离子占沸石骨架外金属阳离子的5％。
②取上述制备得到的Y型沸石的凝血效果测定：取25mgY型沸石加入到2mL的离心管,在37℃进行体外凝血实验，将正常血液预先在37℃水浴恒温半小时。然后加入20uL的0.2M CaCl 2，最后加入1mL恒温的正常血液，正常血液凝固时的时间记为Y型沸石的凝血时间，Y型沸石的凝血时间为2min，由此可见Y型沸石的凝血效果一般。
取25mgY型沸石加入到2mL的离心管,在37℃进行体外凝血实验，将乏X、VIII、IX或XI因子血液预先在37℃水浴恒温半小时。然后加入20uL的0.2M CaCl 2，最后加入1mL恒温的乏X、VIII、IX或XI因子血液，乏X、VIII、IX或XI因子血液凝固时的时间记为Y型沸石的凝血时间，但其过程中上述有缺陷的血液长时间没有发生凝固，由此可见Y型沸石对血友病的患者并没有促凝血作用。
实施例8
胰蛋白酶Y型沸石止血组合物的制备：
①将硅铝比为3的孔道为微孔的Y型沸石(Na型)，用5M浓度的氯化镁溶液室温下浸渍12h，得到的Y型沸石包含二价镁离子且其占沸石骨架外金属阳离子含量的95％，一价钠离子占沸石骨架外金属阳离子的5％。
②将上述制备得到的25mg的Y型沸石在1mL水溶液中分散均匀，形成Y型沸石的悬浊液；使Y型沸石与胰蛋白酶充分接触(按胰蛋白酶与Y型沸石的质量比为1:10加入)，25℃混合30min，使胰蛋白酶在沸石表面吸附，并将上述溶液在-20℃真空干燥5h，胰蛋白酶分散于沸石表面上，得到胰蛋白酶Y型沸石止血组合物。
胰蛋白酶Y型沸石止血组合物的正常血液凝血时间为0.3min，缺乏X、VIII、IX、XI凝血因子的血液凝血时间分别为0.33、0.4、0.3、0.3min，由此可见胰蛋白酶Y型沸石止血组合物对正常血液的凝血效果很好；对缺乏X、VIII、IX、XI凝血因子的血液的凝血效果也很好，凝血时间大大缩短，对血友病患者能有很好的促进凝血作用。缺乏X、VIII、IX、XI凝血因子的血液加入Y型沸石时，血液长时间不凝固。由实施例8与对比实施例17的比较可得：胰蛋白酶Y型沸石止血组合物的凝血效果远远好于Y型沸石或者胰蛋白酶的凝血效果，凝血时间大大缩短。
对比实施例18
①将硅铝比为20的孔道为微孔的ZSM-5型沸石(Na型)，用5M浓度的氯化镁溶液室温下浸渍12h，得到的ZSM-5型沸石包含二价镁离子且其占沸石骨架外金属阳离子含量的95％，一价钠离子占沸石骨架外金属阳离子的5％。
②取上述制备得到的ZSM-5型沸石的凝血效果测定：取25mgZSM-5型沸石加入到2mL的离心管,在37℃进行体外凝血实验，将正常血液预先在37℃水浴恒温半小时。然后加入20uL的0.2M CaCl 2，最后加入1mL恒温的正常血液，正常血液凝固时的时间记为ZSM-5型沸石的凝血时间，ZSM-5型沸石的凝血时间为2min，由此可见ZSM-5型沸石的凝血效果一般。
取25mgZSM-5型沸石加入到2mL的离心管,在37℃进行体外凝血实验，将乏X、VIII、IX或XI因子血液预先在37℃水浴恒温半小时。然后加入20uL的0.2M CaCl 2，最后加入1mL恒温的乏X、VIII、IX或XI因子血液，乏X、VIII、IX或XI因子血液凝固时的时间记为ZSM-5型沸石的凝血时间，但其过程中上述有缺陷的血液长时间没有发生凝固，由此可见ZSM-5型沸石对血友病的患者并没有促凝血作用。
实施例9
胰蛋白酶ZSM-5型沸石止血组合物的制备：
①将硅铝比为20的孔道为微孔的ZSM-5型沸石(Na型)，用5M浓度的氯化镁溶液室温下浸渍12h，得到的ZSM-5型沸石包含二价镁离子且其占沸石骨架外金属阳离子含量的95％，一价钠离子占沸石骨架外金属阳离子的5％。
②将上述制备得到的25mg的ZSM-5型沸石在1mL水溶液中分散均匀，形成ZSM-5型沸石的悬浊液；使ZSM-5型沸石与胰蛋白酶充分接触(按胰蛋白酶与ZSM-5型沸石的质量比为1:10加入)，25℃混合30min，使胰蛋白酶在沸石表面吸附，并将上述溶液在-20℃真空干燥5h，胰蛋白酶分散于沸石表面上，得到胰蛋白酶ZSM-5型沸石止血组合物。
胰蛋白酶ZSM-5型沸石止血组合物的正常血液凝血时间为0.4min，缺乏X、VIII、IX、XI凝血因子的血液凝血时间分别为0.43、0.42、0.4、0.4min，由此可见胰蛋白酶ZSM-5型沸石止血组合物对正常血液的凝血效果很好；对缺乏X、VIII、IX、XI凝血因子的血液的凝血效果也很好，凝血时间大大缩短，对血友病患者能有很好的促进凝血作用。缺乏X、VIII、IX、XI凝血因子的血液加入ZSM-5型沸石时，血液长时间不凝固。由实施例9与对比实施例18的比较可得：胰蛋白酶ZSM-5型沸石止血组合物的凝血效果远远好于ZSM-5型沸石或者胰蛋白酶的凝血效果，凝血时间大大缩短。
实施例10
胰蛋白酶ZSM-5型/Y型沸石止血组合物的制备：
①将硅铝比为20的孔道为微孔的ZSM-5型沸石(Na型)和硅铝比为4的孔道为微孔的Y型沸石(Na型)，用5M浓度的氯化镁溶液室温下浸渍12h，得到的沸石包含二价镁离子且其占沸石骨架外金属阳离子含量的95％，一价钠离子占沸石骨架外金属阳离子的5％。
②将上述制备得到的25mg的ZSM-5型沸石和Y型沸石(1:1)在1mL水溶液中分散均匀，形成ZSM-5型沸石和Y型沸石的悬浊液；使ZSM-5型沸石和Y型沸石与胰蛋白酶充分接触(按胰蛋白酶与ZSM-5型沸石和Y型沸石的质量比为1:10加入)，25℃混合30min，使胰蛋白酶在沸石表面吸附，并将上述溶液在-20℃真空干燥5h，胰蛋白酶分散于沸石表面上，得到胰蛋白酶ZSM-5型/Y型沸石止血组合物。
胰蛋白酶ZSM-5型/Y型沸石止血组合物的正常血液凝血时间为0.34min，缺乏X、VIII、IX、XI凝血因子的血液凝血时间分别为0.33、0.32、0.4、0.34min，由此可见胰蛋白酶ZSM-5型/Y型沸石止血组合物对正常血液的凝血效果很好；对缺乏X、VIII、IX、XI凝血因子的血液的凝血效果也很好，凝血时间大大缩短，对血友病患者能有很好的促进凝血作用。胰蛋白酶ZSM-5型/Y型沸石止血组合物的凝血效果远远好于沸石或者胰蛋白酶的凝血效果，凝血时间大大缩短。
实施例11
胰蛋白酶丝光沸石/X型沸石止血组合物的制备：
①将硅铝比为20的孔道为微孔的丝光沸石(Na型)和硅铝比为1.2的孔道为微孔的X型沸石(Na型)，用5M浓度的氯化镁溶液室温下浸渍12h，得到的沸石包含二价镁离子且其占沸石骨架外金属阳离子含量的95％，一价钠离子占沸石骨架外金属阳离子的5％。
②将上述制备得到的25mg的丝光沸石和X型沸石(1:1)在1mL水溶液中分散均匀，形成丝光沸石和X型沸石的悬浊液；使丝光沸石和X型沸石与胰蛋白酶充分接触(按胰蛋白酶与丝光沸石和X型沸石的质量比为1:10加入)，25℃混合30min，使胰蛋白酶在沸石表面吸附，并将上述溶液在-20℃真空干燥5h，胰蛋白酶分散于沸石表面上，得到胰蛋白酶丝光沸石/X型沸石止血组合物。
胰蛋白酶丝光沸石/X型沸石止血组合物的正常血液凝血时间为0.4min，缺乏X、VIII、IX、XI凝血因子的血液凝血时间分别为0.43、0.37、0.4、0.36min，由此可见胰蛋白酶丝光沸石/X型沸石止血组合物对正常血液的凝血效果很好；对缺乏X、VIII、IX、XI凝血因子 的血液的凝血效果也很好，凝血时间大大缩短，对血友病患者能有很好的促进凝血作用。胰蛋白酶丝光沸石/X型沸石止血组合物的凝血效果远远好于沸石或者胰蛋白酶的凝血效果，凝血时间大大缩短。
其中，本发明实施例1-11所述沸石的悬浊液的沸石与去离子水的质量比为1:0.5～1:20；优选的，所述沸石的悬浊液的沸石与去离子水的质量比为1:0.8～1:10；优选的，所述的沸石的悬浊液的沸石与去离子水的质量比为1:1～1:5；优选的，所述的沸石的悬浊液的沸石与去离子水的质量比1:1.5～1:2.5。
进一步的，本发明实施例1-11对所述沸石的悬浊液进行超声处理。
进一步的，本发明实施例1-11所述的超声处理的时间为0.5～30min，超声频率为20～200kHz，超声功率为200～5000W。
进一步的，本发明实施例1-11所述的胰蛋白酶与沸石接触的温度为10～37℃；优选的，所述的胰蛋白酶与沸石接触的温度为15～30℃；优选的，所述的胰蛋白酶与沸石接触的温度为20～25℃。
进一步的，本发明实施例1-11所述胰蛋白酶在沸石表面吸附后还包括将沸石的悬浊液与胰蛋白酶冻干的步骤，优选的，所述冻干的温度为0℃～-80℃；优选的，所述冻干的温度为-10℃～-60℃；优选的，所述冻干的温度为-20℃～-50℃；优选的，所述冻干的温度为-30℃～-45℃。
综上所述，为了达到本发明所述止血复合物的促凝血效果，必须由特定的沸石与胰蛋白酶质量比(1:200～4:10)、特定的沸石骨架外金属阳离子(二价金属阳离子)、特定的沸石孔径大小(微孔)的共同协同作用，三个条件缺一不可，才能形成本发明所述的止血性能优异的止血组合物。通过无机材料与生物大分子特异性结合，胰蛋白酶与沸石的止血组合物的凝血性能等同于、甚至优于单独的凝血酶的凝血性能，可以大大降低止血材料的成本，更具有商业化前景。
本发明克服了现有技术的偏见，意外地首次制得了一种止血快速、成本低、且对血友病有效的无机生物酶止血材料。所述的止血组合物的凝血效果远好于单独使用沸石或者胰蛋白酶的凝血性能，凝血时间大大缩短，失血量明显降低。本发明所述的止血组合物中的沸石(微孔的孔道结构、二价金属阳离子、特定的沸石与胰蛋白酶的质量比)对胰蛋白酶的空间构象、空间取向进行正向的调控，使得沸石表面的胰蛋白酶的空间构象更利于与凝血通道(clotting cascade)中的凝血酶原接触，从而促进凝血通道(clotting cascade)过程中凝血酶原转化成凝血酶的活性提高，促凝血活性提高。胰蛋白酶与沸石的协同作用构成的本发明所述止血组合物的整体效果远好于单独胰蛋白酶或者单独沸石的效果，其促凝血活性远优于单独使用胰蛋白酶的促凝血活性，也优于单独使用沸石的促凝血活性，甚至优于单独的凝血酶的凝血活性。在遭遇突发意外大出血时，止血组合物能高效止血，降低大动脉出血致死的风险，减少重要器官的损伤，对血友病患者也有很好的促进凝血作用。
以上实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (19)

  1. 一种止血组合物，其特征在于，所述止血组合物至少包含沸石和胰蛋白酶，其中所述沸石的孔道为微孔，所述的沸石包含二价金属阳离子，所述胰蛋白酶与沸石的质量比为1:200～4:10。
  2. 根据权利要求1所述的止血组合物，其特征在于，所述的二价金属阳离子选自钴离子、镍离子、钙离子、镁离子、锶离子的任意一种或者多种。
  3. 根据权利要求1所述的止血组合物，其特征在于，所述胰蛋白酶与沸石的质量比为1:100～3:10；优选的，所述胰蛋白酶与沸石的质量为1:60～2.5:10；优选的，所述胰蛋白酶与沸石的质量比为1:50～2:10；优选的，所述胰蛋白酶与沸石的质量比为1:40～1.5:10；优选的，所述的胰蛋白酶与沸石的质量比为1:20～1:10。
  4. 根据权利要求1所述的止血组合物，其特征在于，所述的二价金属阳离子占沸石骨架外金属阳离子的50％～95％；优选的，所述的二价金属阳离子占沸石骨架外金属阳离子的60％～90％；优选的，所述的二价金属阳离子占沸石骨架外金属阳离子的65％～85％；优选的，所述的二价金属阳离子占沸石骨架外金属阳离子的70％～80％；优选的，所述的二价金属阳离子占沸石骨架外金属阳离子的72％～78％。
  5. 根据权利要求1所述的止血组合物，其特征在于，所述的沸石的硅铝比为1～20；优选的，所述的硅铝比为1.2～15；优选的，所述的硅铝比为1.5～4；优选的，所述的硅铝比为2～3。
  6. 根据权利要求1所述的止血组合物，其特征在于，所述的沸石选自A型沸石、菱沸石、β沸石、丝光沸石、X型沸石、Y型沸石、ZSM-5型沸石的任意一种或者多种。
  7. 一种止血组合物的制备方法，其特征在于，包括如下步骤：
    (1)制备一种沸石的悬浊液，所述沸石的孔道为微孔和沸石表面包含二价金属阳离子；
    (2)将沸石的悬浊液与胰蛋白酶接触；
    (3)使胰蛋白酶在沸石表面吸附，得到所述的止血组合物。
  8. 根据权利要求7所述的制备方法，其特征在于，所述沸石的悬浊液的沸石与去离子水的质量比为1:0.5～1:20；优选的，所述沸石的悬浊液的沸石与去离子水的质量比为1:0.8～1:10；优选的，所述的沸石的悬浊液的沸石与去离子水的质量比为1:1～1:5；优选的，所述的沸石的悬浊液的沸石与去离子水的质量比1:1.5～1:2.5。
  9. 根据权利要求7所述的制备方法，其特征在于，对所述步骤(1)沸石的悬浊液进行超声处理。
  10. 根据权利要求9所述的制备方法，其特征在于，所述的超声处理的时间为0.5～30min，超声频率为20～200kHz，超声功率为200～5000W。
  11. 根据权利要求7所述的制备方法，其特征在于，所述的步骤(2)接触的温度为10～37℃；优选的，所述的步骤(2)接触的温度为15～30℃；优选的，所述的步骤(2)接触的温度为20～25℃。
  12. 根据权利要求7所述的制备方法，其特征在于，所述的步骤(3)胰蛋白酶在沸石表面吸附后还包括将沸石的悬浊液与胰蛋白酶冻干的步骤，优选的，所述冻干的温度为0℃～-80℃；优选的，所述冻干的温度为-10℃～-60℃；优选的，所述冻干的温度为-20℃～-50℃；优选的，所述冻干的温度为-30℃～-45℃。
  13. 根据权利要求7所述的制备方法，其特征在于，所述胰蛋白酶与沸石的质量比为1:200～4:10；优选的，所述胰蛋白酶与沸石的质量比为1:100～3:10；优选的，所述胰蛋白酶与沸石的质量为1:60～2.5:10；优选的，所述胰蛋白酶与沸石的质量比为1:50～2:10；优选的，所述胰蛋白酶与沸石的质量比为1:40～1.5:10；优选的，所述的胰蛋白酶与沸石的质量比为1:20～1:10。
  14. 一种止血复合材料，其特征在于，所述的止血复合材料包含权利要求1-6任一项所 述的止血组合物和添加剂。
  15. 根据权利要求14所述的止血复合材料，其特征在于，所述添加剂选自载体、抗菌材料、抗静电材料、高分子多糖的任意一种或多种。
  16. 根据权利要求14所述的止血复合材料，其特征在于，所述的载体选自棉、蚕丝、羊毛、塑料、纤维素、人造丝、聚酯、聚氨酯、聚乙烯泡沫、聚丙烯酸泡沫、低密度聚醚、聚乙烯醇、聚甲基丙烯酸甲酯的任意一种或者多种。
  17. 根据权利要求14所述的止血复合材料，其特征在于，所述的抗菌材料选自纳米银颗粒、香草醛、乙基香草醛类化合物的任意一种或者多种。
  18. 根据权利要求14所述的止血复合材料，其特征在于，所述的高分子多糖选自纤维素、木质素、淀粉、壳聚糖、琼脂糖的任意一种或多种。
  19. 权利要求1-6任一项所述的止血组合物或权利要求14-18任一项的止血复合材料在止血领域的用途。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4822349A (en) 1984-04-25 1989-04-18 Hursey Francis X Method of treating wounds
US7604819B2 (en) 2006-05-26 2009-10-20 Z-Medica Corporation Clay-based hemostatic agents and devices for the delivery thereof
US7858123B2 (en) 2005-04-04 2010-12-28 The Regents Of The University Of California Inorganic materials for hemostatic modulation and therapeutic wound healing
US8703634B2 (en) 2007-02-21 2014-04-22 The Regents Of The University Of California Hemostatic compositions and methods of use

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6500799B2 (en) * 1999-04-09 2002-12-31 Vladimir N. Filatov Wound dressing
UA68506A (en) * 2003-05-29 2004-08-16 Valentyna Vasylivna Trach Method for manufacturing dressing material
WO2008076407A2 (en) * 2006-12-15 2008-06-26 Lifebond Ltd. Gelatin-transglutaminase hemostatic dressings and sealants
CN101104080B (zh) * 2007-04-24 2011-06-22 深圳市鸿华投资有限公司 沸石止血敷料及其制备方法和用途
US20110086014A1 (en) * 2008-06-18 2011-04-14 Ishay Attar Method for enzymatic cross-linking of a protein
EP2307071B1 (en) * 2008-06-20 2015-08-12 Cook Biotech Incorporated Composite extracellular matrix materials and medical products formed therefrom
CN102049239A (zh) * 2010-12-07 2011-05-11 浙江大学 天然沸石离子交换改性制备的氮氧化物吸附剂
WO2014145271A1 (en) * 2013-03-15 2014-09-18 Stb, Ltd. Compositions having cylindrical volume, methods, and applicators for sealing injuries
CN107233614A (zh) * 2017-06-28 2017-10-10 常州武城服饰有限公司 一种促愈合型急救止血粉及其制备方法
CN108159476A (zh) * 2017-12-11 2018-06-15 江苏金秋弹性织物有限公司 一种医用抗菌绷带的制备方法
CN108785735B (zh) * 2018-09-10 2020-09-08 中山大学 止血材料

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4822349A (en) 1984-04-25 1989-04-18 Hursey Francis X Method of treating wounds
US7858123B2 (en) 2005-04-04 2010-12-28 The Regents Of The University Of California Inorganic materials for hemostatic modulation and therapeutic wound healing
US7604819B2 (en) 2006-05-26 2009-10-20 Z-Medica Corporation Clay-based hemostatic agents and devices for the delivery thereof
US8703634B2 (en) 2007-02-21 2014-04-22 The Regents Of The University Of California Hemostatic compositions and methods of use

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3991760A4

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