CN113304120B - Hemostatic micromotor and preparation method thereof - Google Patents

Hemostatic micromotor and preparation method thereof Download PDF

Info

Publication number
CN113304120B
CN113304120B CN202110656950.XA CN202110656950A CN113304120B CN 113304120 B CN113304120 B CN 113304120B CN 202110656950 A CN202110656950 A CN 202110656950A CN 113304120 B CN113304120 B CN 113304120B
Authority
CN
China
Prior art keywords
calcium carbonate
hemostatic
micromotor
magnetic calcium
magnetic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202110656950.XA
Other languages
Chinese (zh)
Other versions
CN113304120A (en
Inventor
蓝广芊
卢必涛
胡恩岭
陆飞
谢瑞琪
余堃
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Southwest University
Original Assignee
Southwest University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Southwest University filed Critical Southwest University
Priority to CN202110656950.XA priority Critical patent/CN113304120B/en
Publication of CN113304120A publication Critical patent/CN113304120A/en
Application granted granted Critical
Publication of CN113304120B publication Critical patent/CN113304120B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/501—Inorganic compounds
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • 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
    • A61K47/56—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 organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—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 organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/145—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic compounds
    • 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

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Dermatology (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Inorganic Chemistry (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Diabetes (AREA)
  • Hematology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicinal Preparation (AREA)

Abstract

The invention provides a preparation method of a hemostatic micromotor, which comprises the steps of preparing magnetic calcium carbonate microcapsules by using a Pickering emulsion method, mixing the prepared magnetic calcium carbonate microcapsules with thrombin, freezing and drying to obtain thrombin-loaded magnetic calcium carbonate microcapsules, and mixing the thrombin-loaded magnetic calcium carbonate microcapsules with protonated tranexamic acid to obtain the hemostatic micromotor. The invention also provides a hemostatic micromotor prepared by the method. Based on the torpedo attack principle, the medicine can be delivered to the deep part of the wound, the medicine diffusion is promoted inside the wound, and then the blood coagulation at the bleeding point is promoted.

Description

Hemostatic micromotor and preparation method thereof
Technical Field
The invention is applied to the technical field of hemostatic materials, and particularly relates to a hemostatic micromotor and a preparation method thereof.
Background
Acute traumatic hemorrhage is the leading cause of death in battlefields, traffic accidents and various accidental disasters. According to statistics, over 55% of soldiers in the war lose excessive blood and lose lives because of not being rescued in time; of the various accidents, over 150 million deaths per year are associated with massive blood loss. If the hemostatic device can provide timely hemostatic measures for the wounded in 30min, the survival rate of the negatively wounded can be improved by at least 40%. The existing first-aid hemostatic materials mainly comprise triangular towels, hemostatic gauze, hemostatic bandages and zeolite powder, and wound bleeding is controlled by matching with methods such as pressing, suturing and the like. Such methods are only applicable to superficial wounds, but are ineffective for irregular complex wounds that are deep, narrow, penetrating, and incompressible. Therefore, the development of an effective hemostatic strategy is crucial to save lives of victims in various accidents.
Among the many complex wounds caused by violence (e.g., gunshot, explosion), massive wound bleeding from gunshot or blast wounds is the most difficult to control. The reason is that after a bullet, a shrapnel or other fragments with impact force moving at high speed hit a human body, the bullet, the shrapnel or other fragments with impact force rotate in the wound under the resistance of human tissue, so that the wide-range injury of arterial blood vessels and venous blood vessels is caused, and a complex shape with a narrow entrance and expanded inside is formed. The bleeding source of such wounds is often hidden deep and the narrow entrance and high velocity of blood flow in the cavity severely prevents traditional hemostatic materials from entering the interior of the wound, resulting in the hemostatic material accelerating the clotting of surface blood only. Even if the wound surface can be temporarily occluded, the accumulation of blood in the blood chamber to form a relatively high pressure can break through the unstable thrombus on the superficial layer at any time, resulting in a failure to stop bleeding. In addition, the blood cavity is simply filled with flexible hemostatic materials such as hemostatic sponge and the like, so that hidden bleeding points cannot be effectively blocked, secondary damage can be caused to tissues, and huge pain is brought to the wounded.
Currently, some researchers prepare powder-type hemostatic microspheres with self-propelling capability through chemical modification, and can resist high-speed blood flow to deliver drugs to the deep part of a wound. The principle is that the microspheres quickly release bubbles after contacting blood, and the blood coagulation medicament is delivered to a deep wound by utilizing the reverse thrust generated in the moment of bubble detachment, so that the 'upstream' of the medicament is realized, the contact area of the blood and the medicament is increased, and the blood coagulation process is accelerated. However, the power provided by the bubbles in the method is not sustainable, and the bubbles floating upwards due to the buoyancy force can also prevent the medicine from continuously moving downwards; and the hemostatic material can not reach the bleeding point of the complex wound only by the power of the air bubbles, so as to stop bleeding from the bleeding source. Therefore, stable, continuous and controllable propulsive force is the key of the powder type micro drug delivery system in the hemostasis of complex wounds.
In order to solve the limitation existing in the current research, the invention designs a novel hemostasis micromotor delivery platform based on the torpedo attack principle, promotes the diffusion of the medicine in the wound cavity, increases the contact between the medicine and the bleeding site, and performs hemostasis from the source.
Disclosure of Invention
Aiming at the problems of insufficient stable, continuous and controllable propelling force of the hemostatic drug and the like in the prior art, the invention provides the hemostatic micromotor based on the torpedo attack principle, which can realize the delivery of the drug to the deep part of a wound, promote the diffusion of the drug in the wound and further promote the blood coagulation of a bleeding point.
The technical problem to be solved by the invention is realized by adopting the following technical scheme:
a preparation method of a hemostatic micromotor is characterized in that a Pickering emulsion method is utilized to prepare magnetic calcium carbonate microcapsules, the prepared magnetic calcium carbonate microcapsules are mixed with thrombin and then are frozen and dried to obtain thrombin-loaded magnetic calcium carbonate microcapsules, and the thrombin-loaded magnetic calcium carbonate microcapsules are mixed with protonated tranexamic acid to obtain the hemostatic micromotor.
Further, the magnetic calcium carbonate microcapsule and thrombin are mixed in 10-50 ml of PBS solution, and the precipitate is frozen and dried after being shaken for 1-3 hours to obtain the thrombin-loaded magnetic calcium carbonate microcapsule.
Furthermore, the ratio of the magnetic calcium carbonate microcapsule to the thrombin is 1g: 5U-1 g: 20U.
Furthermore, the controllable disintegration of the magnetic calcium carbonate microcapsule loaded with thrombin and the delayed release of the medicament can be realized by adjusting the doping amount of the protonated tranexamic acid. In order to realize delayed release of the micromotor in the wound, the mass ratio of the thrombin-loaded magnetic calcium carbonate microcapsule to the protonated tranexamic acid is preferably 1: 1-1: 3.
Further, the preparation method of the magnetic calcium carbonate microcapsule comprises the following steps: adding cation modified magnetic calcium carbonate into the anionic carbon nanodot solution, adding soybean oil after ultrasonic dispersion, stirring at a high speed to obtain uniform emulsion, separating and cleaning the suspension, adding n-butyl alcohol, and freeze-drying to obtain the magnetic calcium carbonate microcapsule.
Furthermore, the concentration of the cation modified magnetic calcium carbonate in the solution is 0.025-0.15 g/ml, and the concentration of the anionic carbon nanodots is 1-2 mg/ml. Based on the principle of an emulsion template method, cationic magnetic calcium carbonate and anionic carbon nanodots are assembled on the surface of a template oil drop to form a microcapsule, wherein the electrostatic interaction between cations and anions is the key of the stable structure of the microcapsule, and the stability of the final microcapsule can be controlled by the mixing ratio of the cationic modified magnetic calcium carbonate and the anionic carbon nanodots.
Further, the soybean oil is added in an amount of 50-300 mu l, and the mixed solution is stirred at a high speed by a high-speed emulsifying machine for 1-3 min.
Further, the uniformly stirred emulsion is placed for 6-12 hours before separation and cleaning.
Further, the separation and cleaning process of the suspension comprises the following steps: attracting the suspension to the bottom under a magnetic field, and removing the upper liquid; then adding tetrahydrofuran, uniformly mixing, attracting the suspension to the bottom under a magnetic field, removing the upper layer liquid, and repeating the operation for 3-5 times; and then centrifugally washing the suspension for 2-5 times by using a PBS (phosphate buffer solution).
Further, the preparation process of the cation modified magnetic calcium carbonate comprises the following steps: adding chitosan into sodium chloride solution, adding glacial acetic acid solution, and stirring at 60 deg.C for 1h to obtain chitosan solution; and (3) placing the chitosan solution to room temperature, adding magnetic calcium carbonate, placing the mixture into an oscillation incubator for oscillation for 30min after ultrasonic dispersion, and obtaining the cation modified magnetic calcium carbonate after centrifugal separation and drying.
Further, the ratio of the chitosan to the sodium chloride solution to the glacial acetic acid solution is 10-40 mg: 20 ml: 0.1ml, wherein the concentration of the sodium chloride solution is 0.5 mM.
Further, the preparation method of the magnetic calcium carbonate comprises the following steps: adding nano iron oxide into deionized water, performing ultrasonic dispersion for 30min, adding sodium carbonate, and continuing performing ultrasonic dispersion for 10min to obtain a sodium carbonate/iron oxide mixed solution; adding the calcium chloride solution into the sodium carbonate/ferric oxide mixed solution while stirring, centrifugally separating, and washing with alcohol to obtain the magnetic calcium carbonate.
Furthermore, the concentration of the nano iron oxide in the magnetic calcium carbonate is in direct proportion to the magnetic driving performance as a power source of the micro motor, but the excessive nano iron oxide can hinder the crystallization of the calcium carbonate, and the content of the nano iron oxide in the solution is preferably 1-10 mg/ml.
Further, the preparation method of the anionic carbon nanodots comprises the following steps: adding citric acid into a reaction kettle containing deionized water, adding ethylenediamine according to the molar ratio of the citric acid to the ethylenediamine of 1: 0.5-1: 1.2, reacting for 4 hours at 200 ℃, cooling, dialyzing for 3 days, and freeze-drying to obtain the anionic carbon nanodot. The surface charge property of the carbon nanodots depends on the reaction ratio between citric acid and ethylenediamine, and the molar ratio is selected to ensure that the surfaces of the carbon nanodots have negative charges.
The invention also provides a hemostatic micromotor prepared by the method.
The invention is based on a Pickering emulsion template method, magnetic calcium carbonate with cation modification and anionic carbon nanodots with negative charges are assembled on the surface of a micro-oil drop template, the template is removed to obtain the magnetic calcium carbonate microcapsule with a hollow structure, and thrombin is loaded in the magnetic calcium carbonate microcapsule by utilizing a physical adsorption method. And finally, mixing the protonated tranexamic acid and the magnetic calcium carbonate microcapsule loaded with thrombin according to a certain mass ratio in a physical doping mode to obtain the hemostatic micromotor. The specific working principle can be divided into two parts: imitating the torpedo launching and cruising stages: the external magnetic field provides continuous power, guides the hemostatic micro motor to resist the blood flow to move to the deep part of the wound, and changes the motion track of the micro motor by changing the magnetic field; simulating a torpedo explosion attack target process: the gradual dissolution of the protonated tranexamic acid forms a weakly acidic environment, stimulates calcium carbonate to generate carbon dioxide gas, and generates an effect similar to 'explosion' to promote the diffusion of the medicine and the micromotor in the wound. Because the dissolution and diffusion of tranexamic acid takes a certain time, the 'explosion' has a time delay effect, which ensures that the medicine can be released in deep wounds to exert the hemostatic effect to the maximum extent.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a powdery hemostasis micromotor based on a torpedo attack mode, which is a novel hemostasis strategy for actively delivering a medicament to enter a deep part of a wound and blocking bleeding from a bleeding source. The cationic modified magnetic calcium carbonate and the anionic carbon nanodots with negative charges are assembled on the surface of the micro-oil drop template, are hollow inside and have high loading efficiency on thrombin. The hemostatic micromotor is obtained by compounding the magnetic calcium carbonate microcapsule loaded with thrombin with protonated tranexamic acid. Wherein, the nanometer ferric oxide is used as a magnetic material to endow the micro motor with sustainable magnetic driving capability; the reaction between calcium carbonate and protonated tranexamic acid to generate carbon dioxide gas promotes the release of thrombin from the microcapsules and diffusion of the gas drives the diffusion of the drug across the internal wound. The beneficial combination of magnetic field navigation and gas propulsion accelerates the hemostatic micromotor into the wound and contacts the drug to the internal bleeding site by delaying the gas produced. The novel hemostasis micromotor based on the torpedo attack mode can be suitable for various complicated wounds.
The hemostatic micromotor is hemostatic powder consisting of micron-level small balls, the large specific surface area of the hemostatic micromotor is favorable for adsorbing thrombin and other medicaments, and meanwhile, the rough structure on the surface of the microspheres is favorable for activating platelets and adhering erythrocytes; in addition, the powder material has wide application range, is not limited by the shape of the wound, and can be suitable for various types of wounds.
The invention can accurately drive the micro motor by using an external magnetic field, so that the micro motor can move to the inside of the wound in high-speed blood flow, and the moving process of the micro motor not only promotes the contact area of the micro motor and the blood coagulation component, but also delivers the medicine to the deep wound.
The hemostatic micromotor is made of a material compatible with human body, is nontoxic and harmless to human tissue, can be completely degraded in the body, and avoids secondary wound caused by later debridement.
Drawings
Fig. 1 is a preparation process diagram of a hemostatic micromotor and a preparation method thereof.
Fig. 2 is a confocal microscope and a scanning electron microscope of a hemostatic micromotor and a method for manufacturing the same according to the present invention.
Fig. 3 is a diagram of the movement trace of the hemostatic micromotor in the simulated blood according to the hemostatic micromotor and the method for preparing the same of the present invention.
Fig. 4 is an image of the release of drugs by a hemostatic micromotor in a hemostatic micromotor and method of making the same of the present invention.
FIG. 5 is a comparison of hemostasis time and fluorescence staining of the present invention and commercially available products in a hemostatic micromotor and method of making the same.
Fig. 6 is a schematic view of the hemostasis of a hemostatic micromotor and a method for making the same according to the present invention.
Detailed Description
The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the techniques realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
Example 1:
a preparation method of a hemostatic micromotor comprises the following steps:
s1: preparation of magnetic calcium carbonate:
adding 50mg of ferric oxide into 50ml of deionized water, carrying out ultrasonic dispersion for 30min, adding 1.749g of sodium carbonate into the solution, and continuing ultrasonic dispersion for 10min to obtain a sodium carbonate/ferric oxide mixed solution. The solution was stirred at a constant speed using a mechanical stirrer, set at 250 rpm. The prepared calcium chloride solution (1.831g/50ml) was rapidly added to the continuously stirred sodium carbonate/iron oxide mixed solution, with the stirring time controlled at 0.5 min. The precipitate was separated by centrifugation (1500rpm), washed 3 times with alcohol and dried at 60 ℃ to obtain magnetic calcium carbonate.
S2: preparation of cation-modified magnetic calcium carbonate:
adding 50mg of chitosan into 100ml of sodium chloride solution (0.5mM), adding 0.5ml of glacial acetic acid solution, stirring at 60 deg.C for 1h to obtain chitosan solution, adjusting pH to 5.5 after it returns to room temperature, adding 1g of the magnetic calcium carbonate obtained in step S1 thereto, ultrasonically dispersing for 5min, and then placing into a shaking incubator, and shaking for 30 min. The magnetic calcium carbonate is separated by centrifugation (1500rpm), and the cation modified magnetic calcium carbonate is obtained after drying at 60 ℃.
S3: preparing the anionic carbon nanodots:
3.508g of citric acid is added into a reaction kettle containing 30ml of deionized water, ethylenediamine is added according to the molar ratio of the citric acid to the ethylenediamine of 1:0.8, and the reaction kettle is placed into an oven at 200 ℃ for reaction for 4 hours. After natural cooling, the mixture is put into a dialysis bag (3500Mn) and dialyzed for 3 days, and water is replaced every 4 hours. And (3) carrying out freeze drying for 24h at the temperature of-50 ℃ to obtain the anionic carbon nanodots.
S4: preparation of magnetic calcium carbonate microcapsules:
and (3) adding 0.1g of the cation modified magnetic calcium carbonate obtained in the step S2 into 2ml of the anionic carbon nanodot powder solution (2mg/ml) obtained in the step S3, performing ultrasonic dispersion for 60S, quickly adding 50-300 mu l of soybean oil, and stirring for 1-3 min (10000rpm) by using a high-speed emulsifying machine to obtain a uniform emulsion. Standing at room temperature for 12 h. A magnet was used to attract the suspension drop to the bottom and the upper liquid was carefully removed. Then 2ml tetrahydrofuran was added, mixed gently, left to stand for 15min and after a drop of the suspension was drawn to the bottom using a magnet, the upper liquid was carefully removed and the procedure was repeated 4 times. Then centrifugally washing (500rpm) for 3 times by using a PBS solution, finally adding 1ml of n-butyl alcohol, pre-freezing overnight at-80 ℃, and freeze-drying to obtain powdery magnetic calcium carbonate microcapsules.
S5: preparing the hemostatic micromotor:
and (3) mixing thrombin and the magnetic calcium carbonate microcapsules obtained in the step (S4) in a ratio of 5U:1g in 10ml of PBS solution, oscillating for 1h at 4 ℃, freeze-drying the precipitate for 24h at-50 ℃ to obtain thrombin-loaded magnetic calcium carbonate microcapsules, and mixing the thrombin-loaded magnetic calcium carbonate microcapsules and protonated tranexamic acid according to a mass ratio of 1:2 to obtain the hemostatic micromotor.
Example 2:
a preparation method of a hemostatic micromotor comprises the following steps:
s1: preparation of magnetic calcium carbonate:
adding 25mg of ferric oxide into 50ml of deionized water, carrying out ultrasonic dispersion for 30min, adding 1.749g of sodium carbonate into the solution, and continuing ultrasonic dispersion for 10min to obtain a sodium carbonate/ferric oxide mixed solution. The solution was stirred at a constant speed using a mechanical stirrer, set at 200 rpm. The prepared calcium chloride solution (1.831g/50ml) was quickly added to the continuously stirred sodium carbonate/iron oxide mixed solution, with the stirring time controlled at 1 min. The precipitate was separated by centrifugation (1500rpm), washed 3 times with alcohol and dried at 60 ℃ to obtain magnetic calcium carbonate.
S2: preparation of cation-modified magnetic calcium carbonate:
adding 100mg of chitosan into 100ml of sodium chloride solution (0.5mM), adding 0.5ml of glacial acetic acid solution, stirring at 60 ℃ for 1h to obtain chitosan solution, adjusting the pH to 5.5 after the chitosan solution is returned to room temperature, adding 1g of the magnetic calcium carbonate obtained in the step S1, performing ultrasonic dispersion for 5min, and then placing in a shaking incubator for 30min by shaking. The magnetic calcium carbonate is separated by centrifugation (1500rpm) and dried at 60 ℃ to obtain the cation modified magnetic calcium carbonate.
S3: preparing the anionic carbon nanodots:
3.508g of citric acid is added into a reaction kettle containing 30ml of deionized water, ethylenediamine is added according to the molar ratio of the citric acid to the ethylenediamine of 1:1.2, and the reaction kettle is placed into an oven at 200 ℃ for reaction for 4 hours. After natural cooling, the mixture is put into a dialysis bag (3500Mn) and dialyzed for 3 days, and water is replaced every 4 hours. And (3) freeze-drying for 24 hours at the temperature of 50 ℃ below zero to obtain the anionic carbon nanodots.
S4: preparation of magnetic calcium carbonate microcapsules:
adding 0.3g of the cation-modified magnetic calcium carbonate obtained in the step S2 into 2ml of the anionic carbon nanodot powder solution obtained in the step S3 (1mg/ml), ultrasonically dispersing for 60S, rapidly adding 50-300 μ l of soybean oil, and stirring for 1-3 min (10000rpm) by using a high-speed emulsifying machine to obtain a uniform emulsion. Standing at room temperature for 6 h. A magnet was used to attract the suspension drop to the bottom and the upper layer of liquid was carefully removed. Then 2ml tetrahydrofuran was added, mixed gently, left to stand for 15min and after a drop of the suspension was drawn to the bottom using a magnet, the upper liquid was carefully removed and the procedure was repeated 4 times. Then centrifugally washing (500rpm) for 3 times by using a PBS solution, finally adding 2ml of n-butyl alcohol, pre-freezing overnight at-80 ℃, and freeze-drying to obtain powdery magnetic calcium carbonate microcapsules.
S5: preparation of the hemostatic micromotor:
and (3) mixing thrombin and the magnetic calcium carbonate microcapsules obtained in the step (S4) in a ratio of 10U to 1g in 30ml of PBS solution, oscillating at 4 ℃ for 2h, freeze-drying the precipitate at-50 ℃ for 24h to obtain thrombin-loaded magnetic calcium carbonate microcapsules, and mixing the thrombin-loaded magnetic calcium carbonate microcapsules and protonated tranexamic acid according to a mass ratio of 1:2 to obtain the hemostatic micromotor.
Experimental example 1:
confocal microscopy and scanning electron microscopy were performed on hemostatic micromotors prepared in the examples of the invention. As a result, as shown in FIG. 2, it can be seen that the hemostatic micromotor has a rough surface structure and a hollow structure inside, which is advantageous for loading thrombin. In addition, the element analysis structure corresponding to the scanning electron microscope shows that the nano ferric oxide is distributed in the hemostatic micromotor, which shows that the material has good magnetic driving performance.
Experimental example 2:
preparing a glass of water, and placing a magnet at the bottom of the glass to enable the interior of the glass to have a magnetic field environment; the hemostatic micromotor prepared in the embodiment of the invention is put into water and observed by a high-speed camera. Referring to fig. 3, it can be seen that the micro motor rapidly moves towards the bottom under the action of the magnetic field, and starts to generate a large amount of bubbles when moving to the position close to the bottom, and the bubbles diffuse to the two sides, and finally fill the whole glass bottom, which is similar to the attack process of a torpedo. Therefore, the hemostatic micromotor enters the deep part of the wound under the action of an external magnetic field in the hemostatic process, starts to generate a large amount of bubbles after reaching the bottom of the wound, promotes the release of thrombin in the microcapsules under the driving of the bubbles, diffuses thrombin around the periphery, fills the wound, avoids the problem that hemostatic powder cannot reach the bleeding point in the deep part of the wound due to large blood flow, and is favorable for rapid hemostasis.
Experimental example 3:
the hemostatic micromotor is prepared according to the method, dextrorotatory polysaccharide (4KDa) modified by fluorescein isothiocyanate is loaded into the hemostatic micromotor as a mode drug, the obtained hemostatic micromotor is mixed with water, and the mixture is observed through a fluorescence microscope.
Referring to fig. 4, upon contact with water, the hemostatic micromotor was substantially completely disintegrated within 16 seconds and completely disintegrated within 30 seconds, as observed by a fluorescence microscope, with the concomitant generation of gas. Along with the collapse and disintegration of the hemostatic micro motor, the fluorescent area is gradually enlarged to the periphery. Therefore, due to the existence of the protonated tranexamic acid, the protonated tranexamic acid rapidly reacts with calcium carbonate in the hemostatic micromotor after contacting water to generate carbon dioxide gas, the hemostatic micromotor is rapidly disintegrated and is driven by the gas to diffuse around, so that the observed fluorescence range is gradually expanded, the rapid release process of the thrombin can be seen, and the complete release of the thrombin can be realized within 30 seconds of mixing with blood.
Experimental example 4:
the fluorescence-labeled hemostatic micromotor and the commercially available hemostatic material celex @ are respectively taken, the liver of a New Zealand white rabbit is taken to form two simulated gunshot wounds with the same shape and size, an internal expansibility complicated wound is formed, a certain amount of fluorescence-labeled hemostatic micromotor and the commercially available hemostatic material celex @ of the embodiment of the invention are respectively taken and directly scattered on the wound, hemostasis is performed by pressing, and the hemostatic effect of the hemostatic micromotor is verified by establishing an animal hemorrhage model. The hemostasis process of the hemostasis micromotor is carried out under the action of a magnetic field, and the hemostasis material celex @ on the market is used for hemostasis without the action of the magnetic field according to the product specification.
Referring to the animal experiment chart of figure 5, animal experiments prove that the hemostatic micromotor of the invention can complete hemostasis within 90s, and the commercially available hemostatic material celex @ needs 150s to complete hemostasis, so that the hemostatic efficiency of the hemostatic micromotor of the invention is far superior to that of the commercially available hemostatic material celex @. In addition, referring to the fluorescence staining graph in fig. 5, the fluorescence staining result after hemostasis by using the hemostatic micromotor of the invention shows that most of the hemostatic micromotors marked by fluorescence are distributed at the edge inside the wound, which fully indicates that the drugs can continuously diffuse around after entering the wound under the magnetic field guiding effect, so that the contact between the hemostatic drugs and the internal hidden bleeding point is increased, and hemostasis from the bleeding source of the wound is realized. The commercially available hemostatic material celox @ is basically dispersed on the surface of the wound, so that the commercially available hemostatic material celox @ can only perform hemostasis on the surface of the wound, cannot penetrate into the wound under the impact of blood flow to perform hemostasis from a bleeding source, and is slow in hemostasis speed.
Combining the above experimental results, the hemostasis principle of the micro motor is shown in fig. 6, which resists blood flow into the wound under the action of a magnetic field, and along with the dissolution of the protonated tranexamic acid, the micro motor rapidly releases gas, further accelerates the diffusion of the drug and the micro motor in the wound, and thus promotes the blood coagulation at the bleeding source. The invention directly delivers high-efficiency blood coagulation medicines (such as thrombin and batroxobin) to the deep part of a wound through a powder type micromotor, promotes the conversion of fibrinogen into fibrin at the bleeding source, and realizes the blockage of blood flow from the bleeding source. Compared with the shape memory sponge and the hemostatic materials such as the underwater adhesive hydrogel, the powder type hemostatic micromotor has huge specific surface area, is not limited by the shape of the wound, is easier to enter the deep wound, and thus has unique advantages in hemostasis of complex wounds. Compared with the existing powder type hemostatic material adopting bubbles as self-propelling power, the powder type hemostatic material has stable, continuous and controllable driving force, can directly reach bleeding points of various wounds and perform hemostasis from bleeding sources, has a greater hemostatic advantage especially for bent and complex wounds, and can realize rapid hemostasis.
The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above-described embodiments. It will be understood by those skilled in the art that various changes, substitutions of equivalents, and alterations can be made without departing from the spirit and scope of the invention.

Claims (10)

1. A preparation method of a hemostatic micromotor is characterized by comprising the following steps: the preparation method comprises the steps of preparing magnetic calcium carbonate microcapsules by using a Pickering emulsion method, obtaining the magnetic calcium carbonate microcapsules with hollow structures, mixing the prepared magnetic calcium carbonate microcapsules with hollow structures with thrombin, freezing and drying to obtain the magnetic calcium carbonate microcapsules loaded with the thrombin, and mixing the magnetic calcium carbonate microcapsules loaded with the thrombin with protonated tranexamic acid to obtain the hemostatic micromotor.
2. The method of claim 1, wherein the micro hemostatic motor comprises: and mixing the magnetic calcium carbonate microcapsule with thrombin in 10-50 ml of PBS solution, oscillating for 1-3 h, and freeze-drying the precipitate to obtain the thrombin-loaded magnetic calcium carbonate microcapsule.
3. The method of claim 1, wherein the micro hemostatic motor comprises: the ratio of the magnetic calcium carbonate microcapsule to the thrombin is 1g: 5U-1 g: 20U.
4. The method for preparing a hemostatic micromotor according to claim 1, wherein the magnetic calcium carbonate microcapsules are prepared by the following steps: adding cation modified magnetic calcium carbonate into the anionic carbon nanodot solution, adding soybean oil after ultrasonic dispersion, stirring at a high speed to obtain uniform emulsion, separating and cleaning the suspension, adding n-butyl alcohol, and freeze-drying to obtain the magnetic calcium carbonate microcapsule.
5. The method of claim 4, wherein the micro hemostatic motor comprises: the concentration of the cation-modified magnetic calcium carbonate in the solution is 0.025-0.15 g/ml, and the concentration of the anionic carbon nanodots is 1-2 mg/ml.
6. The method for preparing a hemostatic micromotor according to claim 4, wherein the separation and washing process of the suspension comprises: attracting the suspension to the bottom under a magnetic field, and removing the upper liquid; then adding tetrahydrofuran, uniformly mixing, attracting the suspension to the bottom under a magnetic field, removing the upper layer liquid, and repeating the operation for 3-5 times; and then centrifugally washing the suspension for 2-5 times by using a PBS (phosphate buffer solution).
7. The method of claim 5, wherein the cationic modified magnetic calcium carbonate is prepared by the following steps: adding chitosan into sodium chloride solution, adding glacial acetic acid solution, and stirring at 60 deg.C for 1h to obtain chitosan solution; and (3) placing the chitosan solution to room temperature, adding magnetic calcium carbonate, placing the mixture into an oscillation incubator for oscillation for 30min after ultrasonic dispersion, and obtaining the cation modified magnetic calcium carbonate after centrifugal separation and drying.
8. The method for preparing a hemostatic micromotor according to claim 7, wherein the method for preparing the magnetic calcium carbonate comprises: adding nano iron oxide into deionized water, performing ultrasonic dispersion for 30min, adding sodium carbonate, and continuing performing ultrasonic dispersion for 10min to obtain a sodium carbonate/iron oxide mixed solution; adding the calcium chloride solution into the sodium carbonate/ferric oxide mixed solution while stirring, centrifugally separating, and washing with alcohol to obtain the magnetic calcium carbonate.
9. The method for preparing a hemostatic micromotor according to claim 4, wherein the method for preparing the anionic carbon nanodots comprises: adding citric acid into a reaction kettle containing deionized water, adding ethylenediamine according to the molar ratio of the citric acid to the ethylenediamine of 1: 0.5-1: 1.2, reacting for 4 hours at 200 ℃, cooling, dialyzing for 3 days, and freeze-drying to obtain the anionic carbon nanodot.
10. A hemostatic micromotor prepared by the method of any one of claims 1 to 9.
CN202110656950.XA 2021-06-11 2021-06-11 Hemostatic micromotor and preparation method thereof Active CN113304120B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110656950.XA CN113304120B (en) 2021-06-11 2021-06-11 Hemostatic micromotor and preparation method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110656950.XA CN113304120B (en) 2021-06-11 2021-06-11 Hemostatic micromotor and preparation method thereof

Publications (2)

Publication Number Publication Date
CN113304120A CN113304120A (en) 2021-08-27
CN113304120B true CN113304120B (en) 2022-08-19

Family

ID=77378747

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110656950.XA Active CN113304120B (en) 2021-06-11 2021-06-11 Hemostatic micromotor and preparation method thereof

Country Status (1)

Country Link
CN (1) CN113304120B (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113797227A (en) * 2021-10-28 2021-12-17 浙江大学 Application of chemical self-driven micro-robot in biological sterilization
CN114748676B (en) * 2022-04-08 2022-11-18 四川大学 Chitosan-based efficient hemostatic with Janus structure and preparation method and application thereof
CN115212344B (en) * 2022-08-01 2023-08-22 西南大学 Self-expansion hemostatic aerogel and preparation method thereof

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100368071C (en) * 2004-04-29 2008-02-13 西安科技大学 Preparation of composite hollow nano-structure
CN103691376B (en) * 2013-12-24 2015-11-25 哈尔滨工业大学 Utilize laminated assembling technology to prepare the method for microcapsules using oil-in-water microemulsion as template
CN105642202B (en) * 2016-01-04 2017-11-28 哈尔滨工程大学 A kind of preparation method and application of functionalization graphene and calcium carbonate composite hollow microballoon
CN111135339B (en) * 2020-01-16 2021-03-16 西南大学 Preparation method of hemostatic agent with directional propelling function based on janus structure
CN112891612B (en) * 2021-01-28 2022-03-01 西南大学 Preparation method of janus structure hemostatic with directional driving function

Also Published As

Publication number Publication date
CN113304120A (en) 2021-08-27

Similar Documents

Publication Publication Date Title
CN113304120B (en) Hemostatic micromotor and preparation method thereof
JP5539959B2 (en) Drugs and bandages with electrospun dextran fibers
US11707550B2 (en) Polyphosphate-functionalized inorganic nanoparticles as hemostatic compositions and methods of use
US11266596B2 (en) Self-fueled particles for propulsion through flowing aqueous fluids
CN111135339B (en) Preparation method of hemostatic agent with directional propelling function based on janus structure
EP0102391A1 (en) Dual microcapsules
Lu et al. Microcluster colloidosomes for hemostat delivery into complex wounds: A platform inspired by the attack action of torpedoes
CN102408575A (en) Preparation method of injectable polypeptide hydrogel
CN112891612B (en) Preparation method of janus structure hemostatic with directional driving function
CN112300418A (en) Adhesive high-efficiency hemostatic microsphere and preparation method thereof
WO2023165052A1 (en) Directional driving hemostatic microsphere with puncture function and preparation method thereof
US20200352990A1 (en) Antimicrobial platelet-like particles
CN111035798B (en) Preparation method of diffusion mixed function hemostatic
US11311643B2 (en) Fibrin and/or dialdehyde starch hydrolysate materials, and preparation and use thereof
JP2010064956A (en) Particle and method for producing the same, and gel
RU2326655C1 (en) Method of incapsulation of protein bearing materials in microspheres from copolymer polylactide-polyglycolide
CN115212344B (en) Self-expansion hemostatic aerogel and preparation method thereof
US20240157022A1 (en) Polyphosphate-Functionalized Inorganic Nanoparticles as Hemostatic Compositions and Methods of Use
Chen et al. Swift Covalent Gelation Coupled with Robust Wet Adhesive Powder: A Novel Approach for Acute Massive Hemorrhage Control in Dynamic and High‐Pressure Wound Environments
Qiu et al. Research Article Dual-Driven Hemostats Featured with Puncturing Erythrocytes for Severe Bleeding in Complex Wounds
Cohen et al. Novel liposome-based formulations for prolonged delivery of proteins and vaccines
CN112891613A (en) Directional driving targeted hemostasis micromotor and preparation method thereof
EP4213865A1 (en) A system to improve haemostatic control
JPH041124A (en) Suspension liquid for ophthalmic therapy

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant