WO2023165052A1 - 一种具有穿刺功能的定向驱动止血微球及其制备方法 - Google Patents

一种具有穿刺功能的定向驱动止血微球及其制备方法 Download PDF

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WO2023165052A1
WO2023165052A1 PCT/CN2022/100351 CN2022100351W WO2023165052A1 WO 2023165052 A1 WO2023165052 A1 WO 2023165052A1 CN 2022100351 W CN2022100351 W CN 2022100351W WO 2023165052 A1 WO2023165052 A1 WO 2023165052A1
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pollen
microspheres
hollow
hemostatic
particles
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French (fr)
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蓝广芊
邱浩宇
谢瑞琪
胡恩岭
陆飞
余堃
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西南大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/4833—Thrombin (3.4.21.5)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/52—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an inorganic compound, e.g. an inorganic ion that is complexed with the active ingredient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
    • A61K9/0009—Galenical forms characterised by the drug release technique; Application systems commanded by energy involving or responsive to electricity, magnetism or acoustic waves; Galenical aspects of sonophoresis, iontophoresis, electroporation or electroosmosis
    • 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
    • A61L24/02—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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21005—Thrombin (3.4.21.5)
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/62—Encapsulated active agents, e.g. emulsified droplets
    • A61L2300/622—Microcapsules
    • 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 in particular relates to a directional driven hemostatic microsphere with a puncture function and a preparation method thereof.
  • Uncontrolled hemorrhage caused by trauma during natural disasters, accidents, wars, and surgical procedures is a major global problem that can result in massive deaths in military or civilian populations.
  • uncontrolled bleeding from large vessels and extensive bleeding from traumatized tissue is responsible for high mortality.
  • Timely and effective prehospital/emergency hemostasis techniques and hemostatic agents can buy valuable time for follow-up treatment, thereby reducing disability and mortality.
  • therapeutic drugs such as gauze, sponge, hydrogel, etc. can be quickly covered on the wound surface to stop bleeding.
  • the hemostatic materials on the domestic market such as: hemostatic gauze, tourniquet, elastic bandage, etc.
  • they have a certain hemostatic effect on heavy bleeding in emergencies, they are not suitable for irregular and deep wounds and aortic bleeding.
  • hemostatic materials such as: chitosan-based Celox TM dressings, zeolite-based gauze, and oxidized regenerated cellulose based Hemostatic agents can be used to control massive bleeding in wounds, but in deep and concealed perforated wounds, they still cannot quickly intercept the bleeding, and cannot touch deep and hidden bleeding sites, and can only form coagulation on superficial wounds. At the same time, these materials also have the disadvantages of being expensive and difficult to obtain.
  • the hemostatic agent For deep or perforated wounds with hidden bleeding sites, in order to effectively and quickly stop bleeding, the hemostatic agent should first fully and extensively contact the bleeding site, especially in the irregular and hollow bleeding cavity of the bleeding site. Second, it should have a sustained drive that allows it to fight blood flow into deep bleeding sites. Finally, the endogenous hemostatic pathway is rapidly activated. In order to fully and widely contact the bleeding site, the reported shape-adaptive hemostatic foam has the ability of rapid volume expansion and rapid liquid absorption when in contact with blood. When its shape is adapted to the wound cavity, effective hemostasis occurs rapidly in the blood cavity . Therefore, in recent years, researchers have carried out further research on the control of irregular and deep wound bleeding, and developed many new hemostatic materials, including: hemostatic foam for filling bleeding wounds and hemostatic microspheres with targeted wounds.
  • Porous materials have shown great potential in preventing massive bleeding due to their favorable properties, such as high surface area and porosity, ability to quickly absorb liquid, and good compatibility.
  • the self-expanding porous material stent or matrix material has good blood absorption capacity, sealing and filling wound function, and can quickly fill and seal the wound. It has a large and rich pore structure, has a very high specific surface area, and when in contact with blood, it can quickly absorb the water in the blood and gradually expand inside the wound to achieve the purpose of filling the wound surface; and because of its very high specific surface area , can enrich the coagulation factors in the blood, and then achieve the purpose of rapid hemostasis.
  • XSTAT a hemostatic dressing composed of chitosan-based cellulose sponge
  • the device consists of a stuffer filled with a highly compressed sponge that can expand axially several times its original volume in a short period of time after absorbing blood.
  • XSTAT has been experimentally proven to be effective in achieving hemostasis in various animal models of severe bleeding (groin, arteries, liver, etc.).
  • XSTAT is composed of microscopic cellulose sponges with volume expansion and limited mechanical strength
  • a large number of sponges are required to fill the wound cavity, and removal of the sponges after rehabilitation may be harmful. If the patient causes secondary injury, the time required for hemostasis and the time and difficulty of removing all sponges after the operation will inevitably increase accordingly.
  • Hemostatic microspheres capable of targeting wounds against blood flow movement have been reported in recent years.
  • Christian J. Kastrup et al. proposed a hemostatic material using calcium carbonate as a drug carrier.
  • the hemostatic material is composed of calcium carbonate and thrombin.
  • the hemostatic material is mixed with protonated tranexamic acid. When the mixture comes into contact with blood, CO2 is rapidly released to drive the movement of the particles.
  • microporous starch is used as a carrier, loaded with magnetic nano-Fe 3 O 4 particles, and has a targeting effect in a magnetic field, but due to the coverage of nano-Fe 3 O 4 particles, the water absorption capacity of micro-porous starch decreases, and the loaded coagulation
  • the number of enzymes is limited, and it is difficult to achieve an ideal level of hemostasis for rapid hemorrhage in a large area.
  • the hemostatic agent should first fully and widely contact the bleeding site, especially in the irregular and hollow bleeding cavity of the bleeding site. Second, it should have a sustained driving force, enabling it to resist blood flow into deep bleeding sites, precisely target bleeding points, as well as better drug delivery function and good biocompatibility.
  • the present invention provides a directional driven hemostatic microsphere with puncture function and its preparation method, which promotes the release of coagulation substances and adsorption of platelets, red blood cells and coagulation factors by puncturing red blood cells to promote fibrin network formation, so as to achieve rapid hemostasis on the wound.
  • the first object of the present invention is to provide a directional driven hemostatic microsphere with a puncture function, which is characterized in that it includes hollow pollen and a power system assembled on the surface of the hollow pollen, and the surface of the hollow pollen is needle-like or spike-like .
  • the hollow pollen is made from sunflower pollen.
  • the power system of the hemostatic microsphere is jointly composed of nanometer Fe 3 O 4 particles and CaCO 3 .
  • the directional-driven hemostatic microspheres further include thrombin, and the thrombin is loaded inside the hemostatic microspheres.
  • hemostatic microspheres are mixed with protonated tranexamic acid or an aqueous solution of protonated tranexamic acid with a pH less than 7.0 before application.
  • the second object of the present invention is to provide a preparation method of directional-driven hemostatic microspheres with puncture function, which is characterized in that the needle-like or spike-like natural pollen on the surface is degreased, and alkali hydrolysis-acidolysis-alkaline hydrolysis is used
  • the hollow pollen microspheres were obtained after removing the internal cytoplasm in a manner; a power system composed of nano-Fe 3 O 4 particles and CaCO 3 was grown on the surface of the hollow pollen microspheres, and then thrombin was loaded into the hollow pollen microspheres, The directional driven hemostatic microspheres with puncture function were obtained.
  • the degreasing process of natural pollen is as follows: mix natural pollen with acetone solution and reflux to separate large particles or insoluble impurities; Freeze-dry in a dish for 24-48 hours to obtain defatted pollen.
  • the process of removing the inner cytoplasm is as follows: freeze-dry the degreased pollen, mix and stir with the alkaline solution, and centrifuge after alkaline cracking to complete the first alkaline hydrolysis; stir the pollen after alkaline hydrolysis in the acid solution The acid hydrolysis is completed by centrifugation; the acid hydrolyzed pollen is mixed with the alkali solution, and the process of the first alkali hydrolysis is repeated to complete the second alkali hydrolysis to obtain hollow pollen microspheres.
  • 3-12% KOH solution is selected for the first alkaline hydrolysis and the second alkaline hydrolysis, and 70-95% w/v orthophosphoric acid is selected for acid hydrolysis.
  • the concentration of the added alkaline solution and the alkaline hydrolysis time have a great influence on the removal effect.
  • the concentration of the alkaline solution is low and the treatment time is short, the cytoplasm inside the pollen cannot be removed well; when the concentration of the alkaline solution is high and the treatment time is long, although the cytoplasm inside the pollen can be removed, the Under the alkaline solution, the pollen’s morphological structure is destroyed, the original hollow structure is lost, and the needle-like or spike-like structure is also greatly damaged, which is not conducive to the later drug loading and release and the puncture of red blood cells.
  • the concentration of KOH alkali solution is selected to be 6%-12%, the stirring temperature is 85° C., and the stirring time is 6-10 hours.
  • the concentration of the KOH alkali solution in the present invention is 6%, and the mixing and stirring time is 6 hours.
  • solutions used for centrifugation when removing the inner cytoplasm were hot deionized water, hot acetone, hot alcohol, 1mol/L hydrochloric acid, and 6% NaOH.
  • the process of growing a dynamic system composed of nano-Fe 3 O 4 particles and CaCO 3 on the surface of the hollow pollen microspheres is: assembling nano-Fe 3 O 4 onto the surface of the hollow pollen microspheres by in-situ growth , to obtain hollow pollen/nano-Fe 3 O 4 particles; CaCO 3 particles were clustered on the surface of hollow pollen/nano-Fe 3 O 4 particles by the soft template method, and then the unattached CaCO 3 particles were separated by a magnet to obtain hollow pollen /Nano Fe 3 O 4 /CaCO 3 particles.
  • the preparation process of the hollow pollen/nano-Fe 3 O 4 particles is as follows: After removing the inner cytoplasm of the hollow pollen microsphere suspension, nitrogen gas is introduced to exhaust the air, and FeCl 2 ⁇ 6H 2 O and FeCl 3 ⁇ 4H 2 are added O and stirred for 30 min to obtain hollow pollen/nano-Fe 3 O 4 particles.
  • the mass of FeCl 2 ⁇ 6H 2 O added is 1/5 of the mass of the hollow pollen microspheres, and the mass of FeCl 3 ⁇ 4H 2 O added is 1/10 of the mass of the hollow pollen microspheres.
  • the free Fe 2+ and Fe 3+ in the reaction solution will increase, and the free Fe 3 O 4 will increase, which increases the difficulty of removing free Fe 3 O 4 , and
  • the amount of Fe 3 O 4 on the surface of the hollow pollen microspheres is large, covering the nanoscale pores on the surface of the hollow pollen microspheres, which is not conducive to the loading and release of drugs in the later stage; the amount of Fe 3 O 4 adsorbed on the surface is too much, and the hollow pollen microspheres
  • the needle-like or spike-like structures on the surface are filled to affect the acupuncture effect during exercise.
  • the stirring time is 30 min, and the temperature is 50°C.
  • the hollow pollen microspheres adsorb Fe 2+ and Fe 3+ at a lower temperature, the adsorption amount of Fe 2+ and Fe 3+ will be reduced, resulting in a decrease in the loading of nano-Fe 3 O 4 on the hollow pollen microspheres.
  • the response of the pollen microspheres/Fe 3 O 4 to the magnetic field is weakened; if the temperature is too high, the physical structure of the hollow pollen microspheres will be destroyed, thereby affecting the loading of the hollow pollen microspheres on coagulation drugs, and the speed of hemostasis.
  • the adsorption time When the adsorption time is short, the adsorption amount of Fe 2+ and Fe 3+ is low, which also leads to a decrease in the loading of nano-Fe 3 O 4 on the hollow pollen microspheres, and the response of the hollow pollen microspheres/Fe 3 O 4 to the magnetic field is weakened ; If the adsorption time is long, the physical structure of the hollow pollen microspheres will be destroyed due to the influence of temperature.
  • the mass ratio of FeCl 2 ⁇ 6H 2 O to FeCl 3 ⁇ 4H 2 O is 2:1.
  • the hollow pollen/nano-Fe 3 O 4 particles were prepared, they were dispersed into 75% alcohol and washed with sufficient stirring, and then left to stand for 10 minutes to remove excess chemical reagents and free Fe 3 O 4 , and were washed 3 times in total .
  • the preparation process of hollow pollen/nano Fe 3 O 4 /CaCO 3 particles is as follows: dissolve CTAB and CaCl 2 in a mixture of ethylene glycol and water, then add the prepared hollow pollen/nano Fe 3 O 4 particles, add Na 2 CO 3 was reacted for 2 hours, and the unattached CaCO 3 particles were separated with a magnet, washed and transferred to a petri dish for 48 hours to freeze-dry to obtain dried hollow pollen/nano-Fe 3 O 4 /CaCO 3 particles.
  • the volume ratio of ethylene glycol to water is 1:1
  • the concentration of CTAB is 2 mg/ml
  • the concentration of Na 2 CO 3 and CaCl 2 is 0.22M
  • the volume ratio of CTAB to CaCl 2 1:1 is 0.22M
  • the process of loading thrombin is as follows: mix thrombin with hollow pollen/nano-Fe 3 O 4 /CaCO 3 particles in PBS at a ratio of 0.1U:1g-20U:1g, and load under vacuum at 4°C for 10 minutes, The process was repeated three times, and the precipitate was freeze-dried at -50°C for 48 hours to obtain directional-driven hemostatic microspheres with a puncture function.
  • the present invention aims at the defects of conventional hemostatic agents in the treatment of deep or perforated and concealed wounds, and provides directional driven hemostatic microspheres with puncture function, using hollow pollen as the carrier for delivering hemostatic drugs, and combining Fe 3 O 4 /CaCO 3 A new hemostatic material that composes a dynamic system and loads the hemostatic drug thrombin into the interior of pollen/nano Fe 3 O 4 /CaCO 3 microspheres.
  • the hemostatic microspheres of the present invention use the sunflower pollen microsphere material as a carrier for drug loading and delivery, and also as a substrate for the growth of Fe 3 O 4 and CaCO 3 .
  • Porous natural microspheres with high specific surface area and good biocompatibility.
  • the hollow pollen microsphere has a larger cavity, a larger specific surface area and a rougher surface, which can better load and release drugs, and can absorb and enrich More coagulation factors in the blood, coupled with the puncture effect of the acupuncture structure on red blood cells, can achieve the purpose of rapid hemostasis, which is faster than microporous starch as a carrier.
  • the hemostatic microspheres of the present invention are used after being mixed with protonated tranexamic acid. Due to the stimulation of protonated tranexamic acid, the hemostatic microspheres can quickly disperse in the blood and move deep into the wound under the action of a magnetic field. Targeting the bleeding point to deliver drugs, and because of its unique acupuncture structure and large specific surface area, it can enhance the collision with red blood cells during exercise, enhance the puncture effect on red blood cells, promote the release of coagulation substances and have a positive effect on platelets, Adsorption of red blood cells and coagulation factors promotes the formation of fibrin network, thereby realizing rapid hemostasis of wounds.
  • Fig. 1 is a flow chart for the preparation of a directional driven hemostatic microsphere with a puncture function in the present invention
  • Fig. 2 is a physical structure diagram of hollow pollen/nano- Fe3O4 for the preparation of a directional-driven hemostatic microsphere with puncture function in the present invention
  • Fig. 3 is a hemostasis time diagram of hollow pollen/nano Fe 3 O 4 loaded with different Fe 3 O 4 loads of a directional driven hemostatic microsphere with puncture function of the present invention
  • Fig. 4 is an electron micrograph and an element distribution diagram of a directional-driven hemostatic microsphere with a puncture function according to the present invention
  • Fig. 5 is a microscopic motion image diagram in water of a directionally driven hemostatic microsphere with a puncture function according to the present invention
  • Fig. 6 is a macro motion image diagram in water of a directionally driven hemostatic microsphere with a puncture function according to the present invention
  • Fig. 7 is a schematic diagram of the application of a directional driven hemostatic microsphere with a puncture function according to the present invention.
  • Fig. 8 is a scanning electron micrograph of a directional-driven hemostatic microsphere with a puncture function of the present invention at an alkali solution concentration of 15%.
  • Fig. 9 is a comparison chart of protein removal rates of pollen microspheres in each comparative example in Example 3 of a directional-driven hemostatic microsphere with a puncture function according to the present invention.
  • the invention relates to a directional driven hemostatic microsphere with a puncture function, which comprises hollow pollen and a power system assembled on the surface of the hollow pollen, and the surface of the hollow pollen is needle-like or spike-like.
  • the puncture effect on red blood cells can be realized through needle-like or spike-like hollow pollen, which can promote the release of coagulation substances and the adsorption of platelets, red blood cells and coagulation factors, and promote the formation of fibrin network; accelerate the hemostatic microspheres in the wound through the power system The fast movement can move to the inside of the wound as needed to achieve rapid hemostasis.
  • hollow pollen is made from sunflower pollen whose surface is needle-like or spike-like.
  • the power system of the hemostatic microsphere is composed of nanometer Fe 3 O 4 particles and CaCO 3 .
  • CaCO 3 grows on one side and cooperates with magnetic nano-Fe 3 O 4 particles to achieve directional and rapid movement in the wound.
  • the directional drive hemostatic microspheres In order to promote blood coagulation, accelerate hemostasis, and shorten the hemostatic time, the directional drive hemostatic microspheres also include thrombin, which is loaded inside the hemostatic microspheres. During the movement of the hemostatic microspheres, thrombin is released along with the gas to activate the fibrinogen in the blood to achieve the purpose of rapid coagulation.
  • the hemostatic microspheres are mixed with protonated tranexamic acid or a protonated tranexamic acid aqueous solution with pH less than 7.0 before application, and the free H + ions released by protonated tranexamic acid in the blood are combined with the CaCO 3 grown on one side A reaction occurs to generate CO 2 gas to realize one-way driving.
  • the present invention also provides a preparation method of directional-driven hemostatic microspheres with puncture function, which includes degreasing the needle-like or spike-like natural pollen on the surface, and removing the inner cytoplasm by alkaline hydrolysis-acidolysis-alkaline hydrolysis to obtain hollow microspheres.
  • Pollen microspheres a power system composed of nano-Fe 3 O 4 particles and CaCO 3 is grown on the surface of the hollow pollen microspheres, and then thrombin is loaded into the hollow pollen microspheres to obtain directional drive hemostasis with puncture function Microspheres.
  • Acupuncture-like or spike-like natural pollen is degreased and the internal cytoplasm is removed to form hollow pollen microspheres.
  • the hollow pollen microspheres have a larger cavity and a larger specific surface area, which is better for drug loading and release, and adsorption Enrich more coagulation factors in the blood.
  • magnetic nanometer Fe 3 O 4 particles and oriented growth of CaCO 3 particles were grown sequentially, and finally thrombin was loaded to form directional driven hemostatic microspheres with puncture function.
  • the prepared directional-driven hemostatic microspheres with puncture function can achieve directional drive and rapid hemostatic microspheres under the power of the needle-like or spike-like structure on the surface of the hollow pollen microspheres and the power system composed of nano-Fe 3 O 4 particles and CaCO 3 . stop bleeding.
  • a method for preparing a directional driven hemostatic microsphere with a puncture function of the present invention comprises the following steps:
  • Hollow pollen microspheres were prepared by a two-step method of degreasing and removing inner cytoplasm.
  • step (2) Remove internal cytoplasm: After 3 g of defatted pollen obtained in step (1) is freeze-dried, transfer it to a round-bottomed flask with a condenser, mix the defatted pollen with 100 ml of 6% KOH (w/v) aqueous solution, and stir magnetically (85°C, 500rpm) for 6h, and a suspension was obtained after alkaline lysis. Then transfer the pollen suspension into a 50ml centrifuge tube, wash with hot deionized water, hot acetone, hot alcohol, 1mol/L hydrochloric acid, 6% NaOH, and hot deionized water in a high-speed centrifuge (2min, 6500rpm) Fragments complete the first alkaline hydrolysis.
  • the first alkaline hydrolysis suspension was treated with 100 ml of orthophosphoric acid (85% w/v) at 85° C. under magnetic stirring at 500 rpm for 6 h. Then transfer the pollen suspension into a 50ml centrifuge tube, wash with hot deionized water, hot acetone, hot alcohol, 1mol/L hydrochloric acid, 6% NaOH, and hot deionized water in a high-speed centrifuge (2min, 6500rpm) . Finally, repeat the first alkaline lysis-washing step to complete the second alkaline hydrolysis to obtain hollow pollen microsphere suspension.
  • Magnetic nano-Fe 3 O 4 particles were assembled on the surface of hollow pollen microspheres by in situ growth technique.
  • CTAB Dissolve CTAB (2 mg/ml) and CaCl 2 (0.22M) in 50 ml of a mixture of ethylene glycol and water at a volume ratio of 1:1, and then mix the hollow pollen/nano-Fe 3 O 4 suspension obtained in step 2 with CTAB/CaCl 2 solutions were mixed according to the volume ratio of 1:1, then 50ml, 0.22M Na 2 CO 3 solution was added, and reacted at 35°C for 2 hours, and then the unattached CaCO 3 particles were separated with a magnet, washed three times, and the suspension was transferred to Freeze-dried in a petri dish for 48 hours to obtain dried hollow pollen/nano-Fe 3 O 4 /CaCO 3 particles.
  • Example 1 the element distribution on the surface of the hemostatic microspheres in Example 1 is analyzed. It can be seen that the O element is enriched on the entire surface of the hemostatic microspheres, the Fe element is mainly gathered on the surface of the hollow pollen, and the CaCO3 grown at one end The particle surface distribution was less; Ca element was mainly gathered on the surface of CaCO 3 particles growing at one end, and less distributed on the hollow pollen surface.
  • Example 1 The difference from Example 1 is that the amount, temperature, and adsorption time of Fe 2+ and Fe 3+ added in Step 2 were adjusted, and the adjustment of specific conditions is shown in Table 1.
  • hollow pollen/nanometer Fe 3 O 4 -1, hollow pollen/nano Fe 3 O 4 -2, hollow pollen/nano Fe 3 O 4 -3 samples and natural pollen step 1 of embodiment 1 obtained
  • the hollow pollen was observed by scanning electron microscope. It can be seen that compared with natural pollen and hollow pollen, the surfaces of the three samples have successfully assembled nanoparticles on the surface of pollen microspheres.
  • Hollow pollen/nano-Fe 3 O 4 -1, hollow pollen/ The Fe 3 O 4 particles on the surface of nano-Fe 3 O 4 -2 and hollow pollen/nano-Fe 3 O 4 -3 increased sequentially.
  • the hollow pollen/nano-Fe 3 O 4 particles Although the hollow pollen/nano-Fe 3 O 4 -1 well retained the nano-sized pores on the surface of the hollow pollen microspheres, the Fe 2+ , Fe 3+
  • the adsorption capacity of Fe 2+ and Fe 3+ on the hollow pollen microspheres is low, and the amount of Fe 3 O 4 on the surface of the hollow pollen microspheres is small, and the magnetic force in the magnetic field is small, which is not conducive to the target into the wound.
  • the hollow pollen microspheres When the hollow pollen microspheres adsorb Fe 2+ and Fe 3+ at a lower temperature, the adsorption amount of Fe 2+ and Fe 3+ will be reduced, resulting in a decrease in the loading of nano-Fe 3 O 4 on the hollow pollen microspheres.
  • the response of the pollen microspheres/Fe 3 O 4 to the magnetic field is weakened; if the temperature is too high, the physical structure of the hollow pollen microspheres will be destroyed, thereby affecting the loading of the hollow pollen microspheres on coagulation drugs, and the speed of hemostasis.
  • the adsorption time When the adsorption time is short, the adsorption amount of Fe 2+ and Fe 3+ is low, which also leads to a decrease in the loading of nano-Fe 3 O 4 on the hollow pollen microspheres, and the response of the hollow pollen microspheres/Fe 3 O 4 to the magnetic field is weakened ; If the adsorption time is long, the physical structure of the hollow pollen microspheres will be destroyed due to the influence of temperature.
  • the mass of FeCl 2 ⁇ 6H 2 O added is 1/10-2/5 of the mass of hollow pollen microspheres
  • the mass of FeCl 3 ⁇ 4H 2 O added is 1/20-1/5 of the mass of hollow pollen microspheres
  • FeCl When the mass ratio of 2 ⁇ 6H 2 O and FeCl 3 ⁇ 4H 2 O is 2:1, the stirring time is 30 min, and the temperature is 50°C, a good hemostatic effect can be obtained. The hemostatic effect is best when the mass of FeCl 2 ⁇ 6H 2 O is 1/5 of the mass of the hollow pollen microspheres, and the mass of FeCl 3 ⁇ 4H 2 O is 1/10 of the mass of the hollow pollen microspheres.
  • Hollow pollen/nano-Fe 3 O 4 -2 had the shortest hemostasis time, only 185s, and hollow pollen/nano-Fe 3 O 4 -1 and hollow pollen/nano-Fe 3 O 4 -3 needed 230s and 250s, respectively.
  • Example 2 The difference from Example 1 is that the conditions of the process of removing the internal cytoplasm in step 1 were adjusted, and the adjustment of specific conditions is shown in Table 2.
  • the cytoplasm removal effects of the hollow microspheres-2, the hollow microspheres-3, and the hollow microspheres-6 are all good, and the removal effect reaches more than 90%. It can be seen that when the concentration of the alkali solution is low (hollow microsphere-1, hollow microsphere-4), the cytoplasm inside the pollen cannot be removed well, and the removal rate is lower than 50%; when the alkali solution treatment time is shorter, the The removal of the cytoplasm inside the pollen also has a greater impact, see hollow microsphere-5 and hollow microsphere-6, when the treatment time is reduced to 5h, the removal rate drops significantly, only about 70%.
  • the concentration of the KOH alkali solution is selected to be 3% to 12%, the stirring temperature is 85° C., and the stirring time is 6 to 10 hours.
  • the concentration of KOH is 6%-12%, the stirring temperature is 85° C., and the stirring time is 6-10 h.
  • the present invention has greater influence on the needle - like or spike -like structure on the surface of the hemostatic microsphere through the adjustment of the concentration of the alkali solution and the treatment time;
  • the adsorption amount has a greater influence.
  • the hemostatic process of the directional driven hemostatic microspheres with puncture function of the present invention is as follows: in application, the hemostatic microspheres are mixed with protonated tranexamic acid or a protonated tranexamic acid aqueous solution with pH less than 7.0, when When the mixture comes into contact with blood, CO2 gas will be released rapidly to drive the movement of the particles, so that the hemostatic microspheres will disperse in the blood, and then under the action of an external magnetic field and CaCO3 to form a dynamic system, the hemostatic microspheres will generate power to move to the target position During exercise, the needle-like or spike-like structures on the surface of hemostatic microspheres form a puncture effect on red blood cells, promote the release of coagulation substances, and promote the formation of fibrin networks.
  • the larger specific surface area of hemostatic microspheres is enhanced during exercise.
  • the collision of red blood cells accelerates coagulation.
  • the movement direction of the microspheres can be changed to adapt to different types of wounds.
  • the needle-like or spike-like structure on the surface of the hemostatic microsphere of the present invention cooperates with the puncture effect on red blood cells and the dynamic system composed of Fe 3 O 4 /CaCO 3 . Time, adapt to various types of bleeding wounds.
  • the present invention assembles nano-Fe 3 O 4 onto the surface of hollow pollen microspheres by means of in-situ growth, the surface of hollow pollen/nano-Fe 3 O 4 particles can grow in blood and react with H + ions Particles of compounds that form gases.
  • CaCO 3 particles that can grow in clusters on one side NaHCO 3 particles and KHCO 3 particles that can grow on the surface of hollow pollen/nano-Fe 3 O 4 particles, etc., are all compound particles grown in this application with protonated ammonia
  • the free H + ions released by cyclic acid or its solution in the blood react to generate CO 2 gas, which is derived from the principle that the hemostatic microspheres are driven by the gas, and any kind of particles made on this basis All changes are within the protection scope of the present invention.

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Abstract

本发明提供一种具有穿刺功能的定向驱动止血微球，包括空心花粉、组装在空心花粉表面的动力系统，所述止血微球表面为针刺状或刺突状。本发明还提供了一种具有穿刺功能的定向驱动止血微球的制备方法，包括空心花粉微球的制备、动力系统在花粉微球上的生长、止血药物凝血酶的加载三步。本发明通过穿刺红细胞促进凝血物质的释放以及对血小板、红细胞和凝血因子吸附，促进纤维蛋白网络的形成，从而实现对伤口的快速止血。

Description

一种具有穿刺功能的定向驱动止血微球及其制备方法 技术领域
本发明属于生物医药材料技术领域，具体涉及一种具有穿刺功能的定向驱动止血微球及其制备方法。
背景技术
在自然灾害、意外事故、战争和外科手术过程中由创伤引起的无法控制的出血是一个重大的全球性问题，它可能导致军人或平民人口大量死亡。对于突发和意外大出血的患者，无法控制的大血管出血和外伤组织广泛出血是死亡率高的原因。及时有效的院前/急救止血技术和止血剂可以为后续治疗赢得宝贵的时间，从而降低残疾和死亡率。对于普通的伤口出血，例如表皮的组织损伤引起的出血，可以将治疗药物如纱布、海绵、水凝胶等快速覆盖在伤口表面进行止血。但对于深部或穿孔、出血部位隐蔽的伤口，普通的止血剂在止血过程中由于受到伤口血流的冲击，容易被血流冲出伤口，难以通过不规则而深的伤口通道对出血点/受损血管进行治疗。因此，如何针对不规则而深的伤口设计出一种能够在战场或医院手术过程中能够逆血流运动来控制不规则伤口出血的安全有效的止血剂至关重要。
目前，在止血材料领域，国内市场上的止血材料，如：止血纱布、止血带、弹性绷带等，虽然对于紧急情况的大出血有一定的止血效果，但是对于不规则而深的伤口以及大动脉出血等，由于它们作用在伤口表面，无法进入深部出血部位以促进止血，因此凝血作用只发生在伤口表面，难以对不规则而深的伤口进行及时、理想的治疗。对于动脉破裂的大出血，虽然商业止血材料，如：基于壳聚糖的Celox TM敷料，沸石基的
Figure PCTCN2022100351-appb-000001
纱布，和氧化再生纤维素基的
Figure PCTCN2022100351-appb-000002
止血剂可用于伤口的大出血控制，但是在出血部位较深、隐蔽的穿孔创面，仍不能迅速截流，无法触及深层、隐蔽的出血部位，只能在浅表创面形成凝血。同时这些材料还存在价格昂贵、难以获取的缺点。
对于深部或穿孔、出血部位隐蔽的伤口，为了有效、快速止血，止血剂首先应充分、广泛接触出血部位，尤其是在出血部位不规则、空洞的出血腔内。其次，它应该具有持续的驱动力，使其能够对抗血流进入深层出血部位。最后，快速激活内源性止血通路。为了充分和广泛地接触出血部位，已有报道的形状适应性止血泡沫与血液接触时具有快 速体积扩张能力和快速吸液能力，当其形状与创面腔相适应时，血腔内迅速发生有效止血。因此，近年来科研人员针对控制不规则而深的伤口出血进行了进一步研究，开发出许多新型止血材料，包括：填充出血创面的止血泡沫以及具有靶向伤口的止血微球等。
多孔材料由于其良好的特性，如高表面积和孔隙率、快速吸收液体的能力以及良好相容性，在阻止大出血方面显示出巨大的潜力。例如：自膨胀多孔材料支架或基质材料具有良好的血液吸收能力、密封和填塞伤口作用，能够快速填充、封闭伤口。其具有大量且丰富的孔道结构，具有极高的比表面积，在与血液接触时，能够快速吸收血液中的水分，逐渐在伤口内部膨胀以达到填充创面的目的；并且由于其极高的比表面积，能够富集血液中的凝血因子，进而达到快速止血的目的。随着自膨胀多孔材料逐渐作为支架或基质材料类止血材料或技术的发展，美国俄勒冈州威尔逊维尔的RevMedX公司生产的一种用于军事领域快速止血的膨胀多孔材料已获得FDA批准(XSTAT)，XSTAT是一种基于壳聚糖的纤维素海绵组成止血敷料，用于治疗四肢出血，在控制不可压缩的交界处出血方面也显示出前景。该装置由一个填充器组成，该填充器内部填充有高度压缩的海绵，吸收血液后，海绵可以在很短的时间内轴向膨胀数倍于其原始体积。XSTAT已被实验证明可有效地在多种严重出血动物模型(腹股沟、动脉和肝脏等中实现止血)。然而，由于XSTAT是由体积膨胀和机械强度有限的微型纤维素海绵组成，当应用于出血部位时，需要大量海绵(超过90个)来填充创腔，并且在康复后对海绵的去除可能会对患者造成二次伤害，其所需的止血时间为以及术后去除所有海绵的时间和难度必然会相应增加。
近年来，如何快速、精准地向深部或穿孔、出血部位隐蔽的伤口的出血点递送止血药物已成为当前止血领域的研究热点。能够逆血流运动靶向伤口的止血微球近年来已有所报导。如：Christian J.Kastrup等人提出了一种使用碳酸钙作为药物载体的止血材料。该止血材料由碳酸钙与凝血酶组成，在应用过程中通过将止血材料与质子化氨甲环酸混合，当混合物与血液接触时，会迅速释放CO 2推动颗粒的运动。然而，由于气体产生的方向的不确定性，因此对颗粒的推进并不全是向伤口内部运动，导致大部分颗粒只是停留在伤口表面，难以到达出血点。专利CN112891613A中采用微孔淀粉为载体，负载磁性纳米Fe 3O 4粒子，在磁场中产生靶向作用，但是由于纳米Fe 3O 4粒子的覆盖，微孔淀粉的吸水能力下降，且负载的凝血酶数量有限，对于大面积的快速出血止血速度难以达到理想的水平。
对于深部或穿孔、出血部位隐蔽的伤口，由于血液的阻力，大部分止血材料难以逆 血流运动对出血点进行靶向止血，导致止血时间长、失血多、难度大。而现阶段对于靶向或填充伤口内部的止血材料存在动力方向不一致、易造成二次伤害等问题。因此，为了有效、快速止血，止血剂首先应充分、广泛接触出血部位，尤其是在出血部位不规则、空洞的出血腔内。其次，它应该具有持续的驱动力，使其能够对抗血流进入深层出血部位，精确靶向出血点，以及较好的药物递送功能和良好的生物相容性。
发明内容
为了解决现有技术中的问题，本发明提供一种具有穿刺功能的定向驱动止血微球及其制备方法，通过穿刺红细胞促进凝血物质的释放以及对血小板、红细胞和凝血因子吸附，促进纤维蛋白网络的形成，从而实现对伤口的快速止血。
本发明第一个目的在于提供一种具有穿刺功能的定向驱动止血微球，其特征在于，包括空心花粉、组装在空心花粉表面的动力系统，所述空心花粉表面为针刺状或刺突状。
进一步的，所述空心花粉由向日葵花粉制得。
进一步的，所述止血微球的动力系统由纳米Fe 3O 4粒子与CaCO 3共同组成。
进一步的，所述定向驱动止血微球还包括凝血酶，所述凝血酶加载在止血微球的内部。
进一步的，所述止血微球在应用前与质子化氨甲环酸或PH小于7.0的质子化氨甲环酸水溶液混合。
本发明第二个目的在于提供一种具有穿刺功能的定向驱动止血微球的制备方法，其特征在于，将表面针刺状或刺突状的天然花粉脱脂、采用碱解-酸解-碱解的方式去除内部细胞质后得到空心花粉微球；在所述空心花粉微球表面生长由纳米Fe 3O 4粒子与CaCO 3共同组成的动力系统，然后将凝血酶加载到空心花粉微球的内部，得到具有穿刺功能的定向驱动止血微球。
进一步的，天然花粉脱脂的过程为：将天然花粉与丙酮溶液混合搅拌回流，分离出大颗粒或不溶性杂质；再用丙酮搅拌回流后，向过滤后的花粉中加入去离子水搅拌，转移到培养皿中冻干24～48h得到脱脂花粉。
进一步的，天然花粉脱脂时两次混合搅拌回流的时间为3～12h。
进一步的，所述去除内部细胞质的过程为：将脱脂后的花粉冷冻干燥后，与碱溶液混合搅拌，碱性裂解后离心完成第一次碱解；将碱解后的花粉在酸液中搅拌离心完成酸解；将酸解后的花粉与碱溶液混合搅拌，重复第一次碱解的过程完成第二次碱解，得到空心花粉微球。
进一步的，所述第一次碱解与第二次碱解时选用3～12％的KOH溶液，酸解时选用70～95％w/v的正磷酸。
去除内部细胞质时，加入碱溶液的浓度、碱解时间对去除效果影响较大。当碱溶液的浓度较低、处理时间较短时，花粉内部的细胞质不能很好的去除；当碱溶液的浓度较高、处理时间较长时，虽然能够去除花粉内部的细胞质，但是在高浓度碱溶液下，花粉的形态结构遭到破坏，丧失原有的空心结构，同时也对针刺状或刺突状结构有较大的破坏，不利于后期的药物加载释放以及对于红细胞的穿刺。进一步的，碱解时选用KOH碱溶液的浓度为6％～12％，搅拌温度85℃，搅拌时间6～10h。优选的，本发明中KOH碱溶液的浓度为6％，混合搅拌的时间为6h。
进一步的，去除内部细胞质时离心采用的溶液分别为热去离子水、热丙酮、热醇、1mol/L盐酸、6％NaOH。
进一步的，在所述空心花粉微球表面生长由纳米Fe 3O 4粒子与CaCO 3共同组成的动力系统的过程为：通过原位生长的方式将纳米Fe 3O 4组装到空心花粉微球表面，得到空心花粉/纳米Fe 3O 4颗粒；通过软模板法使空心花粉/纳米Fe 3O 4颗粒表面一侧团簇生长CaCO 3颗粒，再用磁铁分离未附着的CaCO 3颗粒，得到空心花粉/纳米Fe 3O 4/CaCO 3颗粒。
进一步的，空心花粉/纳米Fe 3O 4颗粒的制备过程为：经过去除内部细胞质后的空心花粉微球悬浮液，通入氮气以排出空气，加入FeCl 2·6H 2O和FeCl 3·4H 2O搅拌30min，得到空心花粉/纳米Fe 3O 4颗粒。
加入FeCl 2·6H 2O和FeCl 3·4H 2O搅拌使微孔淀粉充分吸附Fe 2+、Fe 3+，再逐滴加入NaOH水溶液，直到溶液变黑，得到空心花粉/纳米Fe 3O 4颗粒。通过原位生长的方式，调节加入Fe 2+、Fe 3+的含量及制备条件，增加止血微球的磁力，并利于后期药物的加载及释放。优选的，加入FeCl 2·6H 2O的质量为空心花粉微球质量的1/5，加入FeCl 3·4H 2O的质量为空心花粉微球质量的1/10。在空心花粉/纳米Fe 3O 4颗粒的组装过程中，Fe 2+、Fe 3+的投入量较低时，空心花粉微球对Fe 2+、Fe 3+的吸附量较低，空心花粉微球表面的Fe 3O 4量少，在磁场中受到的磁力较小，不利于靶向伤口内部。Fe 2+、Fe 3+的投入量较高时，反应液中的游离Fe 2+、Fe 3+增加，游离的Fe 3O 4会增多，这增加了去除游离Fe 3O 4的难度，并且空心花粉微球表面的Fe 3O 4量多，覆盖了空心花粉微球表面的纳米级小孔，不利于后期药物的加载以及释放；表面吸附的Fe 3O 4量过多，空心花粉微球表面的针刺状或刺突状结构被填充，影响运动过程中的针刺效果。
优选的，加入FeCl 2·6H 2O和FeCl 3·4H 2O后搅拌时间为30min，温度为50℃。当空 心花粉微球吸附Fe 2+、Fe 3+的温度较低时，会减少Fe 2+、Fe 3+的吸附量，导致纳米Fe 3O 4在空心花粉微球上的负载量减少，空心花粉微球/Fe 3O 4对磁场的响应减弱；若温度过高，则会破坏空心花粉微球的物理结构，进而影响空心花粉微球对凝血药物的负载，进而以及止血中的速度。当吸附时间短，则Fe 2+、Fe 3+的吸附量低，同样导致纳米Fe 3O 4在空心花粉微球上的负载量减少，空心花粉微球/Fe 3O 4对磁场的响应减弱；若吸附时间长，由于温度的影响，会破坏空心花粉微球的物理结构。
进一步的，所述FeCl 2·6H 2O和FeCl 3·4H 2O的质量比为2:1。
进一步的，所述空心花粉/纳米Fe 3O 4颗粒制成后分散到75％的酒精中充分搅拌洗涤，随后静置10min，去除过量的化学试剂以及游离的Fe 3O 4，共洗涤3次。
进一步的，空心花粉/纳米Fe 3O 4/CaCO 3颗粒的制备过程为：在乙二醇和水的混合物中溶解CTAB和CaCl 2，然后加入制得的空心花粉/纳米Fe 3O 4颗粒，加入Na 2CO 3反应2h，将未附着的CaCO 3颗粒用磁铁分离，洗涤后转移到培养皿中冻干48小时，得到干燥的空心花粉/纳米Fe 3O 4/CaCO 3颗粒。
进一步的，所述乙二醇与水的体积比为1:1，所述CTAB的浓度为2mg/ml，Na 2CO 3与CaCl 2的浓度为0.22M，所述CTAB与CaCl 2的体积比为1:1。
进一步的，加载凝血酶的过程为：将凝血酶与空心花粉/纳米Fe 3O 4/CaCO 3颗粒按照0.1U:1g-20U:1g的比例在PBS中混合，在4℃下真空加载10min，重复三次，将沉淀物在-50℃条件下冷冻干燥48h，得到具有穿刺功能的定向驱动止血微球。
本发明的有益技术效果为：
本发明针对常规止血剂对于深部或穿孔、出血部位隐蔽的伤口治疗的缺陷，提供具有穿刺功能的定向驱动止血微球，以空心花粉作为递送止血药物的载体，将Fe 3O 4/CaCO 3共同组成动力体系，再将止血药物凝血酶加载到花粉/纳米Fe 3O 4/CaCO 3微球的内部的新型止血材料。由于其独特的针刺结构以及较大的比表面积，在运动的过程中能够增强与红细胞的碰撞，加强对红细胞的穿刺效果，促进凝血物质的释放以及对血小板、红细胞和凝血因子吸附，促进纤维蛋白网络的形成，从而实现对伤口的快速止血。
本发明止血微球的使用向日葵花粉微球材料作为药物加载与递送的载体，同时也作为Fe 3O 4和CaCO 3生长的基体，其是一种具有纳米级孔以及针刺状或刺突状结构的多孔天然微球，具有高的比表面积以及良好的生物相容性。与现有技术中的微孔淀粉相比，空心花粉微球具有较大的空腔、较大的比表面积以及更为粗糙的表面，能够更好地进行药物的加载与释放，能够吸附富集更多血液中的凝血因子，再加上针刺结构对红细胞的 穿刺效果，从而达到快速止血的目的，较微孔淀粉作为载体止血速度更快。
本发明止血微球在使用时与质子化氨甲环酸混合后使用，由于质子化氨甲环酸的刺激，使止血微球能够迅速在血液中分散，在磁场作用下向伤口深处运动，靶向出血点递送药物，并且由于其独特的针刺结构以及较大的比表面积，在运动的过程中能够增强与红细胞的碰撞，加强对红细胞的穿刺效果，促进凝血物质的释放以及对血小板、红细胞和凝血因子吸附，促进纤维蛋白网络的形成，从而实现对伤口的快速止血。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，而可依照说明书的内容予以实施，并且为了让本发明的上述和其它目的、特征和优点能够更明显易懂，以下特举本发明的具体实施方式。
附图说明
图1为本发明一种具有穿刺功能的定向驱动止血微球的制备流程图；
图2为本发明一种具有穿刺功能的定向驱动止血微球的制备中空心花粉/纳米Fe 3O 4的物理结构图；
图3为本发明一种具有穿刺功能的定向驱动止血微球不同Fe 3O 4负载量空心花粉/纳米Fe 3O 4的止血时间图；
图4为本发明一种具有穿刺功能的定向驱动止血微球的电镜图和元素分布图；
图5为本发明一种具有穿刺功能的定向驱动止血微球的在水中的微观运动图象图；
图6为本发明一种具有穿刺功能的定向驱动止血微球的在水中的宏观运动图象图；
图7为本发明一种具有穿刺功能的定向驱动止血微球的应用示意图。
图8为本发明一种具有穿刺功能的定向驱动止血微球在碱溶液浓度15％时的扫描电镜图。
图9为本发明一种具有穿刺功能的定向驱动止血微球实施例3中各对比例的花粉微球蛋白质去除率对比图。
具体实施方式
以下结合附图和具体实施例对本发明技术方案作进一步详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。需要说明的是，本发明原料及试剂均为市售产品。
本发明一种具有穿刺功能的定向驱动止血微球，包括空心花粉、组装在空心花粉表面的动力系统，所述空心花粉表面为针刺状或刺突状。通过针刺状或刺突状的空心花粉 实现对红细胞的穿刺效果，促进凝血物质的释放以及对血小板、红细胞和凝血因子吸附，促进纤维蛋白网络的形成；通过动力系统加速止血微球在伤口内的快速移动，可以根据需要运动到伤口的内部，实现快速止血。
在一优选实施例中，空心花粉由表面为针刺状或刺突状的向日葵花粉制得。
在一优选实施例中，止血微球的动力系统由纳米Fe 3O 4粒子与CaCO 3共同组成。CaCO 3单侧生长，与磁性纳米Fe 3O 4粒子相互配合，实现在伤口内的定向快速移动。
为了促进凝血，加快止血，缩短止血时间，定向驱动止血微球还包括凝血酶，凝血酶加载在止血微球的内部。在止血微球运动过程中，凝血酶伴随着气体释放，激活血液中的纤维蛋白原，达到快速凝血的目的。
止血微球在应用前与质子化氨甲环酸或PH小于7.0的质子化氨甲环酸水溶液混合，质子化氨甲环酸在血液中释放出的游离H +离子与单侧生长的CaCO 3发生反应生成CO 2气体，实现单向驱动。
本发明还提供一种具有穿刺功能的定向驱动止血微球的制备方法，将表面针刺状或刺突状的天然花粉脱脂、采用碱解-酸解-碱解的方式去除内部细胞质后得到空心花粉微球；在所述空心花粉微球表面生长由纳米Fe 3O 4粒子与CaCO 3共同组成的动力系统，然后将凝血酶加载到空心花粉微球的内部，得到具有穿刺功能的定向驱动止血微球。
针刺状或刺突状的天然花粉脱脂、去除内部细胞质后形成空心花粉微球，空心花粉微球具有较大的空腔及较大的比表面积，更好地进行药物的加载与释放，吸附富集更多血液中的凝血因子。在空心花粉微球的表面依次生长磁性纳米Fe 3O 4粒子与定向生长CaCO 3颗粒，最后加载凝血酶，形成具有穿刺功能的定向驱动止血微球。制备得到的具有穿刺功能的定向驱动止血微球在空心花粉微球表面的针刺状或刺突状结构及纳米Fe 3O 4粒子与CaCO 3共同组成的动力系统的动力下实现定向驱动及快速止血。
实施例1
本发明一种具有穿刺功能的定向驱动止血微球的制备方法，如图1所示，包括如下步骤：
1、空心花粉微球的制备：
采用脱脂和去除内部细胞质两步法制备空心花粉微球。
(1)脱脂:取天然葵花花粉40g在砂磨中研碎后，用100ml丙酮进行磁性搅拌回流4h(50℃，500rpm)。然后，静置倾倒上清液，用60目尼龙网分离大颗粒或不溶性杂质。再用100ml丙酮进行磁性搅拌回流4h(50℃，500rpm)。静置倾倒上清液后，向花粉中加 入去离子水150ml，搅拌1h(50℃，400rpm)。将向日葵花粉转移到培养皿中冻干48小时，得到脱脂花粉。
(2)去除内部细胞质:将步骤(1)得到的脱脂花粉3g冷冻干燥后，转移到带冷凝器的圆底烧瓶中，将脱脂花粉与6％KOH(w/v)水溶液100ml混合，磁力搅拌(85℃，500rpm)6h，碱性裂解后得到悬浮液。然后将花粉悬浮液转入50ml的离心管中，分别用热去离子水、热丙酮、热醇、1mol/L盐酸、6％NaOH、热去离子水经高速离心机(2min,6500rpm)离心洗涤碎块完成第一次碱解。然后将第一次碱解悬浮液用100ml正磷酸(85％w/v)在85℃、500rpm的磁力搅拌下处理6h。再将花粉悬浮液转入50ml的离心管中，分别用热去离子水、热丙酮、热醇、1mol/L盐酸、6％NaOH、热去离子水经高速离心机(2min,6500rpm)离心洗涤。最后，重复第一次碱性裂解-洗涤步骤完成第二次碱解，得到空心花粉微球悬浮液。
2、纳米Fe 3O 4在空心花粉微球上的组装：
采用原位生长技术在空心花粉微球表面组装磁性纳米Fe 3O 4颗粒。在步骤1得到的100ml空心花粉微球悬液中，在40-70℃下通入氮气10-30min以排出空气，再加入0.4g FeCl 2·6H 2O和0.2g FeCl 3·4H 2O，磁力搅拌(50℃，500rpm)30分钟后，再通过微量注射器逐滴滴加1mol/L NaOH，直到溶液变黑停止滴加，连续搅拌30min，得到空心花粉/纳米Fe 3O 4颗粒，然后将空心微球/纳米Fe 3O 4颗粒分散到75％的酒精中充分搅拌洗涤，随后静置10min，去除过量的化学试剂以及游离的Fe 3O 4，共洗涤3次，得到空心花粉/纳米Fe 3O 4悬浮液。
3、空心花粉/纳米Fe 3O 4/CaCO 3颗粒的制备：
在50ml体积比1:1的乙二醇和水的混合物中溶解CTAB(2mg/ml)和CaCl 2(0.22M)，然后将步骤2得到的空心花粉/纳米Fe 3O 4悬浮液与CTAB/CaCl 2溶液按照体积比1:1混合，然后加入50ml、0.22M的Na 2CO 3溶液，在35℃下反应2h，再将未附着的CaCO 3颗粒用磁铁分离，洗涤三次，将悬浮液转移到培养皿中冻干48小时，得到干燥的空心花粉/纳米Fe 3O 4/CaCO 3颗粒。
4、凝血酶的加载：
将凝血酶与步骤3得到的空心花粉/纳米Fe 3O 4/CaCO 3颗粒按照0.1U:1g-20U:1g的比例在PBS中混合，在4℃下真空加载10min，重复三次，将沉淀物在-50℃条件下冷冻干燥48h，即得具有穿刺功能的定向驱动止血微球。
参见附图4，对实施例1的止血微球表面的元素分布进行分析，可以看出，O元素在 整个止血微球表面富集，Fe元素主要在空心花粉表面聚集，在一端生长的CaCO 3颗粒表面分布较少；Ca元素主要聚集在一端生长的CaCO 3颗粒表面，在空心花粉表面分布较少。
参见附图5、6，为了测试磁场对止血微球的作用，分别在宏观和微观上就止血微球对磁场的响应进行了表征。可见，在磁场的刺激下，能够控制止血微球的运动方向，图5中止血微球在3s内沿指定方向移动600μm，图6中分散的止血微球悬浊液在24s内运动聚集在磁铁处，外界磁场靶向作用好。
实施例2
与实施例1所不同的是，对步骤2中加入Fe 2+、Fe 3+的量、温度以及吸附时间进行调整，具体条件的调整见表1。
表1
Figure PCTCN2022100351-appb-000003
参见附图2，对空心花粉/纳米Fe 3O 4-1，空心花粉/纳米Fe 3O 4-2，空心花粉/纳米Fe 3O 4-3的样品与天然花粉、实施例1步骤1得到的空心花粉进行扫描电镜观察，可见与天然花粉、空心花粉相比，三个样品表面都已成功自主组装纳米粒子在花粉微球的表面，空心花粉/纳米Fe 3O 4-1，空心花粉/纳米Fe 3O 4-2，空心花粉/纳米Fe 3O 4-3表面的Fe 3O 4颗粒依次增多。在空心花粉/纳米Fe 3O 4颗粒的组装过程中，空心花粉/纳米Fe 3O 4-1虽然很好的保留了空心花粉微球表面的纳米级小孔，但是Fe 2+、Fe 3+的投入量较低时，空心花粉微球 对Fe 2+、Fe 3+的吸附量较低，空心花粉微球表面的Fe 3O 4量少，在磁场中受到的磁力较小，不利于靶向伤口内部。相反空心花粉/纳米Fe 3O 4-3中Fe 2+、Fe 3+的投入量较高时，反应液中的游离Fe 2+、Fe 3+增加，游离的Fe 3O 4会增多，这增加了去除游离Fe 3O 4的难度，并且空心花粉微球表面的Fe 3O 4量多，覆盖了空心花粉微球表面的纳米级小孔，不利于后期药物的加载以及释放。
当空心花粉微球吸附Fe 2+、Fe 3+的温度较低时，会减少Fe 2+、Fe 3+的吸附量，导致纳米Fe 3O 4在空心花粉微球上的负载量减少，空心花粉微球/Fe 3O 4对磁场的响应减弱；若温度过高，则会破坏空心花粉微球的物理结构，进而影响空心花粉微球对凝血药物的负载，进而以及止血中的速度。当吸附时间短，则Fe 2+、Fe 3+的吸附量低，同样导致纳米Fe 3O 4在空心花粉微球上的负载量减少，空心花粉微球/Fe 3O 4对磁场的响应减弱；若吸附时间长，由于温度的影响，会破坏空心花粉微球的物理结构。当加入FeCl 2·6H 2O的质量为空心花粉微球质量的1/10～2/5，加入FeCl 3·4H 2O的质量为空心花粉微球质量的1/20～1/5，FeCl 2·6H 2O和FeCl 3·4H 2O的质量比为2:1，搅拌时间为30min，温度为50℃时，均能获得较好的止血效果。加入FeCl 2·6H 2O的质量为空心花粉微球质量的1/5，加入FeCl 3·4H 2O的质量为空心花粉微球质量的1/10时止血效果最佳。
参见附图3，分别取兔股动脉切出1.5cm长的“J”型弯曲伤口作为出血模型，分别选用空心花粉/纳米Fe 3O 4-1，空心花粉/纳米Fe 3O 4-2，空心花粉/纳米Fe 3O 4-3的样品与实施例1步骤1得到的空心花粉的止血时间进行检测，可以看出空心花粉由于表面纳米小孔的存在，对血液具有较好的吸附作用，大大降低了止血时间，但仍需要280s。空心花粉/纳米Fe 3O 4-2止血时间最短，仅需要185s，空心花粉/纳米Fe 3O 4-1与空心花粉/纳米Fe 3O 4-3分别需要230s与250s。
实施例3
与实施例1所不同的是，对步骤1中去除内部细胞质过程条件进行调整，具体条件的调整见表2。
表2
  KOH浓度 搅拌温度 搅拌时间
空心微球-1 3％ 85℃ 6h
空心微球-2 6％ 85℃ 6h
空心微球-3 12％ 85℃ 6h
空心微球-4 3％ 85℃ 10h
空心微球-5 6％ 85℃ 5h
空心微球-6 6％ 85℃ 10h
参见附图9，空心微球-2、空心微球-3、空心微球-6细胞质的去除效果都较好，去除效果达到90％以上。可见当碱溶液的浓度较低时(空心微球-1、空心微球-4)，花粉内部的细胞质不能很好的去除，去除率低于50％；当碱溶液处理时间较短时，对花粉内部细胞质的去除也有较大的影响，参见空心微球-5及空心微球-6，当处理时间减小到5h时去除率有较明显的下降，只有70％左右。在实验中，当碱溶液的浓度较高、处理时间较长时，虽然能够去除花粉内部的细胞质，但是在高浓度碱溶液下，花粉的形态结构遭到破坏，丧失原有的空心结构，同时也对针刺状或刺突状结构有较大的破坏，不利于后期的药物加载释放以及对于红细胞的穿刺。参见附图8，为KOH的浓度为15％时花粉微球的扫描电镜图，可见针刺状或刺突状结构开始出现了较明显的破坏，部分结构开始脱落。本发明碱解时选用KOH碱溶液的浓度为3％～12％，搅拌温度85℃，搅拌时间6～10h。优选的，KOH的浓度为6％～12％，搅拌温度85℃，搅拌时间6～10h。
本发明通过对碱溶液浓度及处理时间的调整对止血微球表面的针刺状或刺突状结构具有较大的影响；且纳米Fe 3O 4颗粒的负载量、负载条件对止血微球的吸附量具有较大的影响。
参见附图7，本发明具有穿刺功能的定向驱动止血微球的止血过程为：在应用时将止血微球与质子化氨甲环酸或PH小于7.0的质子化氨甲环酸水溶液混合，当混合物与血液接触时，会迅速释放CO 2气体推动颗粒的运动，使止血微球在血液中分散，然后在外界磁场与CaCO 3共同组成动力体系的作用下，止血微球产生动力向目标位置运动，在运动过程中止血微球表面的针刺状或刺突状结构对红细胞形成穿刺效果，促进凝血物质释放，促进纤维蛋白网络的形成，止血微球较大的比表面积在运动过程中增强与红细胞的碰撞，加速凝血。当改变磁场的方向时，能够改变微球的运动方向，从而适应不同类型的伤口。本发明止血微球表面针刺状或刺突状结构对红细胞的穿刺效果与Fe 3O 4/CaCO 3共同组成动力体系相配合，与现有技术相比具有更加的止血效果，大大缩短了止血时间，适应各种类型的出血伤口。
需要说明的是，本发明在通过原位生长的方式将纳米Fe 3O 4组装到空心花粉微球表面后，在空心花粉/纳米Fe 3O 4颗粒表面可以生长在血液中与H +离子反应生成气体的化合 物颗粒。如可以一侧团簇生长的CaCO 3颗粒，可以在空心花粉/纳米Fe 3O 4颗粒表面生长的NaHCO 3颗粒、KHCO 3颗粒等，都是在本申请中生长的化合物颗粒与质子化氨甲环酸或其溶液在血液中释放出的游离H +离子发生反应生成CO 2气体，从而在气体的驱动下带动止血微球运动的原理下得出的，任何在此基础上做出的颗粒种类的变化都在本发明的保护范围之内。
上述本发明实施例序号仅仅为了描述，不代表实施例的优劣。
上面结合附图对本发明的实施例进行了描述，但是本发明并不局限于上述的具体实施方式，上述的具体实施方式仅仅是示意性的，而不是限制性的，本领域的普通技术人员在本发明的启示下，在不脱离本发明宗旨和权利要求所保护的范围情况下，还可做出很多形式，这些均属于本发明的保护之内。

Claims (10)

  1. 一种具有穿刺功能的定向驱动止血微球，其特征在于：包括空心花粉、组装在空心花粉表面的动力系统，所述空心花粉表面为针刺状或刺突状。
  2. 如权利要求1所述一种具有穿刺功能的定向驱动止血微球，其特征在于：所述动力系统由纳米Fe 3O 4粒子与CaCO 3共同组成。
  3. 如权利要求1所述一种具有穿刺功能的定向驱动止血微球，其特征在于：所述止血微球在应用前与质子化氨甲环酸或PH小于7.0的质子化氨甲环酸水溶液混合。
  4. 根据权利要求1-3中任一项所述一种具有穿刺功能的定向驱动止血微球的制备方法，其特征在于：将表面针刺状或刺突状的天然花粉脱脂、采用碱解-酸解-碱解的方式去除内部细胞质后得到空心花粉微球；在所述空心花粉微球表面生长由纳米Fe 3O 4粒子与CaCO 3共同组成的动力系统，然后将凝血酶加载到空心花粉微球的内部，得到具有穿刺功能的定向驱动止血微球。
  5. 如权利要求4所述一种具有穿刺功能的定向驱动止血微球的制备方法，其特征在于，天然花粉脱脂的过程为：将天然花粉与丙酮溶液混合搅拌回流，分离出大颗粒或不溶性杂质；再用丙酮搅拌回流后，向过滤后的花粉中加入去离子水搅拌，转移到培养皿中冻干24～48h得到脱脂花粉。
  6. 如权利要求4所述一种具有穿刺功能的定向驱动止血微球的制备方法，其特征在于，所述去除内部细胞质的过程为：将脱脂后的花粉冷冻干燥后，与碱溶液混合搅拌，碱性裂解后离心完成第一次碱解；将碱解后的花粉在酸液中搅拌离心完成酸解；将酸解后的花粉与碱溶液混合搅拌，重复第一次碱解的过程完成第二次碱解，得到空心花粉微球。
  7. 如权利要求6所述一种具有穿刺功能的定向驱动止血微球的制备方法，其特征在于：所述第一次碱解与第二次碱解时选用3～12％的KOH溶液，酸解时选用70～95％w/v的正磷酸。
  8. 如权利要求6所述一种具有穿刺功能的定向驱动止血微球的制备方法，其特征在于：去除内部细胞质时离心采用的溶液分别为热去离子水、热丙酮、热醇、1mol/L盐酸、 6％NaOH。
  9. 如权利要求4所述一种具有穿刺功能的定向驱动止血微球的制备方法，其特征在于，通过原位生长的方式将纳米Fe 3O 4组装到空心花粉微球表面，得到空心花粉/纳米Fe 3O 4颗粒；通过软模板法使空心花粉/纳米Fe 3O 4颗粒表面一侧团簇生长CaCO 3颗粒，再用磁铁分离未附着的CaCO 3颗粒，得到空心花粉/纳米Fe 3O 4/CaCO 3颗粒。
  10. 如权利要求4所述一种具有穿刺功能的定向驱动止血微球的制备方法，其特征在于，空心花粉/纳米Fe 3O 4颗粒的制备过程为：经过去除内部细胞质后的空心花粉微球悬浮液，通入氮气以排出空气，加入FeCl 2·6H 2O和FeCl 3·4H 2O搅拌30min，得到空心花粉/纳米Fe 3O 4颗粒。
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105688266A (zh) * 2016-01-22 2016-06-22 广西信业生物技术有限公司 一种复合医用生物纤维止血粉材料及其制备方法
US20160317434A1 (en) * 2013-12-16 2016-11-03 The University Of British Columbia Self-fueled particles for propulsion through flowing aqueous fluids
CN106943445A (zh) * 2017-05-15 2017-07-14 沈阳建筑大学 一种止血药物组合物及其应用
CN111135339A (zh) * 2020-01-16 2020-05-12 西南大学 一种基于janus结构的具有定向推进功能快速止血剂的制备方法
CN112891612A (zh) * 2021-01-28 2021-06-04 西南大学 一种具有定向驱动功能janus结构止血剂的制备方法
CN114470309A (zh) * 2022-03-04 2022-05-13 西南大学 一种具有穿刺功能的定向驱动止血微球及其制备方法

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2034223A1 (en) * 1969-02-26 1970-12-11 Saint Gobain Techn Nouvelles Shears for irradiated fuel elements
SI1031347T1 (en) * 1999-01-27 2002-10-31 Idea Ag Transnasal transport/immunisation with highly adaptable carriers
US6595215B2 (en) * 2000-03-13 2003-07-22 Innomed Technologies, Inc. Ventilation interface for sleep apnea therapy
CN1468859A (zh) * 2002-07-19 2004-01-21 复旦大学 蒲黄总黄酮提取物及其制备工艺和用途
US7455682B2 (en) * 2004-10-18 2008-11-25 Tyco Healthcare Group Lp Structure containing wound treatment material
GB0427520D0 (en) * 2004-12-16 2005-01-19 Univ Hull Magnetic resonance contrast media
WO2006097940A1 (en) * 2005-03-14 2006-09-21 Johnson & Johnson Limited A bandage with enhanced hemostatic effect
JP4637052B2 (ja) * 2005-06-06 2011-02-23 株式会社山田養蜂場本社 花粉荷を有効成分とする骨量増進組成物
CN104758728A (zh) * 2015-04-23 2015-07-08 龚灿锋 一种治疗小儿紫癜的中药
CN105816603A (zh) * 2016-05-25 2016-08-03 孙红梅 一种治疗产后出血的中药组合物
WO2019094700A1 (en) * 2017-11-10 2019-05-16 Cocoon Biotech Inc. Silk-based products and methods of use

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160317434A1 (en) * 2013-12-16 2016-11-03 The University Of British Columbia Self-fueled particles for propulsion through flowing aqueous fluids
CN105688266A (zh) * 2016-01-22 2016-06-22 广西信业生物技术有限公司 一种复合医用生物纤维止血粉材料及其制备方法
CN106943445A (zh) * 2017-05-15 2017-07-14 沈阳建筑大学 一种止血药物组合物及其应用
CN111135339A (zh) * 2020-01-16 2020-05-12 西南大学 一种基于janus结构的具有定向推进功能快速止血剂的制备方法
CN112891612A (zh) * 2021-01-28 2021-06-04 西南大学 一种具有定向驱动功能janus结构止血剂的制备方法
CN114470309A (zh) * 2022-03-04 2022-05-13 西南大学 一种具有穿刺功能的定向驱动止血微球及其制备方法

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