WO2018205996A1 - 一种可注射型自愈合止血材料及其制备方法和应用 - Google Patents

一种可注射型自愈合止血材料及其制备方法和应用 Download PDF

Info

Publication number
WO2018205996A1
WO2018205996A1 PCT/CN2018/086502 CN2018086502W WO2018205996A1 WO 2018205996 A1 WO2018205996 A1 WO 2018205996A1 CN 2018086502 W CN2018086502 W CN 2018086502W WO 2018205996 A1 WO2018205996 A1 WO 2018205996A1
Authority
WO
WIPO (PCT)
Prior art keywords
gelatin
aqueous solution
hemostatic
particles
microgel particles
Prior art date
Application number
PCT/CN2018/086502
Other languages
English (en)
French (fr)
Inventor
王华楠
Original Assignee
深训华诺生物科技有限公司
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 深训华诺生物科技有限公司 filed Critical 深训华诺生物科技有限公司
Publication of WO2018205996A1 publication Critical patent/WO2018205996A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/001—Use of materials characterised by their function or physical properties
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/001—Use of materials characterised by their function or physical properties
    • A61L24/0015—Medicaments; Biocides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/001—Use of materials characterised by their function or physical properties
    • A61L24/0031—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/046—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/08—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/10—Polypeptides; Proteins
    • A61L24/104—Gelatin
    • 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
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A61L2300/414—Growth factors
    • 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
    • A61L2300/602—Type of release, e.g. controlled, sustained, slow
    • A61L2300/604—Biodegradation
    • 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, relates to a hemostatic material and a preparation method thereof, and in particular to a human and other breastfeeding which can be directly applied to a surgical operation (including minimally invasive surgery) and trauma.
  • hemostatic materials are a very important type of biomedical materials, which have important research and development significance.
  • the hemostatic materials commonly used in clinical practice can be divided into three categories: the first type of hemostatic material accelerates blood clotting (such as hemostatic gauze, high molecular polysaccharides, inorganic zeolites, etc.) through physical or chemical action of materials.
  • the second type is to provide external coagulation components directly or indirectly, to accelerate the realization of coagulation by stimulating self-coagulation mechanism (such as fibrin hemostatic materials, hemostatic materials containing thrombin and coagulation factors);
  • the third type is to use materials for tissue Strong adhesion directly seals the wound to achieve hemostasis (such as alpha cyanoacrylates).
  • self-coagulation mechanism such as fibrin hemostatic materials, hemostatic materials containing thrombin and coagulation factors
  • the third type is to use materials for tissue Strong adhesion directly seals the wound to achieve hemostasis (such as alpha cyanoacrylates).
  • the disadvantage is that they are only effective for bleeding with a small flow rate, so the hemostatic forceps must be used in conjunction with the hemostatic material, which often requires the surgeon to use the above-mentioned hemostatic material. Predict the amount of bleeding in advance or use the hemostat repeatedly.
  • the thrombin-like protein molecule easily overflows into a normal blood vessel, and thus there is a risk of inducing normal blood flow to cause blood clot to produce a thrombus, and thus does not depend on the body's coagulation mechanism and can Hemostatic products that quickly seal wounds are more popular in surgery.
  • Hemostasis products that achieve hemostasis through physical action need to physically compress the bleeding wounds during the hemostasis process, while the material absorbs water in the blood to promote coagulation, and the hemostasis is slow and the effect is poor.
  • such materials are only effective for bleeding with a small flow rate, so the hemostatic forceps must be used in combination, which makes the above-mentioned hemostatic material often require the surgeon to predict the amount of bleeding in advance or repeatedly use the hemostat.
  • traditional hemostatic materials (such as cotton yarn, bandages) are not ideal for hemostasis of irregular wounds such as irregular shape, deep, narrow, and arterial rupture.
  • hemostasis requires long-term external force to achieve physical hemostasis, for fragile nerves Or brain tissue is not applicable.
  • Another type of inorganic hemostatic material including zeolite, kaolin, etc., which utilizes the characteristics of the porous structure and high specific surface area of natural aluminosilicate materials, and absorbs the moisture in the blood to concentrate the local coagulation component of the wound. Quickly stop bleeding.
  • the artificially prepared mesoporous silicon material also has the characteristics of porous structure, high specific surface area, etc., and has the advantages of composition, particle size, and pore size controllability compared with the natural aluminosilicate material, and can effectively achieve hemostasis.
  • such materials have little effect on bleeding with large blood flow, and have limited effect on large-area wounds; and although such materials are benign in the local tissue reaction, mesoporous silicon particles entering the vascular and tissue organs cause serious Systemic toxicity. Therefore, the development of new, rapid, safe and effective hemostatic materials for on-site and pre-hospital first aid has become an important topic in the fields of medicine and biomaterials science.
  • Injectable gel-type hemostatic products have gradually entered clinical hemostasis applications due to their clinical ease of operation and suitable for minimally invasive surgery.
  • the hemostatic gel products used in clinical practice in China mainly rely on imports, of which the American Johnson & Johnson Company Occupy the main market share.
  • the material consists of gelatin, which quickly forms a foamy gel after water dissolution, and the product has good injectability.
  • the mechanical strength of the gel is poor, and the elastic modulus measured by the rheometer is ⁇ 1 kPa. For hemorrhagic tissue with large bleeding volume and high blood vessel pressure, hemostasis cannot be achieved.
  • the present invention provides an injectable self-healing hemostatic material and a method of preparing the same.
  • the hemostatic material prepared by the method of the invention has good injectability, self-healing property and good mechanical strength, and can achieve rapid hemostasis for bleeding tissue with large bleeding volume and high blood pressure.
  • An injectable self-healing hemostatic material prepared by the following method:
  • gelatin as a raw material, heating and dissolving in deionized water, disposing a gelatin aqueous solution having a concentration of 1 to 10 w/v%, adjusting the pH to 1-6 or 8-14, and adding >2 times solution to the solution.
  • the gelatin microgel particles have a zeta potential of -30 to +30 mV, and the gelatin microgel particles have a diameter of 20 nm to 2 ⁇ m;
  • the gelatin microgel particles having a surface zeta potential >+10 mV prepared in the step (1) are dispersed in an acidic aqueous solution having a pH of ⁇ 5 or an alkaline aqueous solution having a pH of >9 to obtain a positively charged gelatin.
  • the dispersion of the microgel particles is further blended with the negatively charged polymer particle dispersion at a particle ratio of 1:10 to 10:1, or with a negatively charged hydrophilic polymer having an isoelectric point of ⁇ 6.
  • the aqueous solution is blended according to the mass ratio of the gelatin microgel particles to the hydrophilic polymer 1:10 to 10:1, adjusted to pH 7.0 with a pH adjuster, and freeze-dried to obtain gelatin microgel particles lyophilized powder I;
  • the gelatin microgel particles having a surface zeta potential of -10 to +10 mV prepared in the step (1) are dispersed in a neutral aqueous solution, and then another polymer having a surface zeta potential of -10 to +10 mV.
  • the particle dispersion is blended according to the particle ratio of 1:10 to 10:1, or the hydrophilic polymer aqueous solution having the isoelectric point of 6-8 is in accordance with the mass ratio of the gelatin microgel particles to the hydrophilic polymer 1:10. ⁇ 10:1 blending, freeze-drying, to obtain gelatin microgel particles lyophilized powder III;
  • gelatin microgel particles lyophilized powder I, gelatin microgel particles lyophilized powder II or gelatin microgel particles lyophilized powder III are respectively blended with an aqueous solution to obtain an injectable self-healing hemostatic material;
  • the surface charge of the positively charged polymer particles is +5 to +60 mV
  • the surface charge of the negatively charged polymer particles is -5 to -60 mV
  • the diameter of the polymer particles is 100 nm to 20 ⁇ m.
  • the percentage of the colloidal particles in the total volume of the injectable self-healing hemostatic material obtained in the step (5) is 50 vol% to 150 vol%; the pros in the steps (3), (4) and (5)
  • the molecular weight of the aqueous polymer is from 1 k to 500 kDa.
  • the gelatin microgel particles prepared in the step (1), the lyophilized powder of the gelatin microgel particles obtained by freeze-drying are blended with the aqueous solution,
  • One or more of the polymers of the isoelectric points 6-8 are one or more of collagen, albumin, gelatin, polyvinyl alcohol, and polyethylene glycol.
  • the polar organic solvent described in the step (1) is one or a combination of methanol, ethanol, isopropanol, butanol, acetone, acetonitrile, tetrahydrofuran;
  • the crosslinking agent is one or more of glutaraldehyde, glyceraldehyde, formaldehyde, carbodiimide, dihaloalkane, isocyanate, diisocyanate, glutamine transaminase, and genipin.
  • the molar ratio of the crosslinking agent to the amino group in the gelatin in the reaction system of the crosslinking reaction described in the step (1) is 0.1 to 10.
  • the molar ratio of the cross-linking agent to the amino group in the gelatin affects the degree of cross-linking of the formed gelatin microgel particles, the cross-linking degree is too high, the gelatin microfluid strength is higher, the surface charge is more negatively charged, and the cross-linking degree is excessive.
  • the low gelatin microspheres have low strength and the surface charge depends on the isoelectric point of the gelatin raw material.
  • the degree of crosslinking is relatively low.
  • the molar ratio of the group should be controlled at 0.5 to 5.
  • the ion concentration contained in the acidic aqueous solution and the alkaline aqueous solution described in the steps (2) and (3) is less than 200 mM.
  • the kind of the ion contained in the acidic aqueous solution and the alkaline aqueous solution is not particularly limited, and reagents for adjusting acidity or basicity such as hydrochloric acid, sulfuric acid, acetic acid, calcium hydroxide, and potassium hydroxide which are conventional in the art can be used. , ammonia, sodium carbonate, etc.
  • the pH adjusting agent described in the step (2) and the step (3) includes an acidic substance and a basic substance, the acidic substance being gluconolactone, HCl, HNO 3 , H 2 SO 4 in one or several of the basic substance is a combination of urease and urea, or sodium hydroxide, calcium hydroxide, potassium hydroxide, ammonia, one or several.
  • the aqueous solution described in the step (5) is an aqueous solution having an ion concentration of 120 to 200 mM, a pH of 6 to 8, an aqueous solution of a hydrophilic polymer, and a non-aqueous nanoparticle.
  • the dispersions One or a combination of the dispersions.
  • the aqueous solution contains a biologically active substance and/or an excipient.
  • the excipient is a solvent, a dispersion medium, a coating agent, a surfactant, an antioxidant, a preservative, an isotonic agent, a binder, a lubricant, a pigment, and combinations or the like thereof;
  • the active substance is at least one of a blood coagulation agent, an anti-infective drug, an anti-inflammatory drug, and a biologically active protein drug.
  • the blood clotting agent is selected from the group consisting of collagen, gelatin, oxidized cellulose, carboxymethyl cellulose, chitosan, hyaluronic acid, sodium alginate, kaolin, thrombin, fibrin, calcium, protamine, polypeptide, One or a combination of a peptide, an amino acid selected from the group consisting of an antibiotic, an antibacterial agent, an antiviral agent, an antifungal drug, an antiulcer drug, a traditional Chinese medicine preparation, or a combination thereof; the anti-inflammatory drug It is selected from a non-steroidal, steroidal anti-inflammatory drug, an antiulcer drug, a traditional Chinese medicine preparation or a combination thereof.
  • the invention also provides the injectable self-healing hemostatic material described above for preparing hemostatic wounds, anti-adhesion, anti-infection, promoting tissue healing and/or sealing wounds of blood wounds for surface tissue, body tissues and body tissues.
  • the hemostatic product described therein may be a hemostatic powder, a hemostatic granule, a hemostatic globule, a hemostatic aerosol, a hemostatic sponge, a hemostatic gel, a hemostatic membrane, a thrombus stop or a hemostatic patch.
  • hemostatic materials are a very important type of biomedical materials, which have important research and development significance.
  • the hemostatic materials commonly used in clinical practice can be divided into three categories: the first type of hemostatic material accelerates blood clotting (such as hemostatic gauze, high molecular polysaccharides, inorganic zeolites, etc.) through physical or chemical action of materials.
  • the second type is to provide external coagulation components directly or indirectly, to accelerate the realization of coagulation by stimulating self-coagulation mechanism (such as fibrin hemostatic materials, hemostatic materials containing thrombin and coagulation factors);
  • the third type is to use materials for tissue Strong adhesion directly seals the wound to achieve hemostasis (such as alpha cyanoacrylates).
  • self-coagulation mechanism such as fibrin hemostatic materials, hemostatic materials containing thrombin and coagulation factors
  • the third type is to use materials for tissue Strong adhesion directly seals the wound to achieve hemostasis (such as alpha cyanoacrylates).
  • hemostatic products that use hemostasis to achieve blood coagulation, the disadvantage is that they are only effective for bleeding with a small flow rate, so they must be used with a hemostatic clamp, which makes the hemostatic material often require the surgeon to advance in use. Predict blood loss or use hemostat repeatedly.
  • the thrombin-like protein molecule easily overflows into a normal blood vessel, and thus there is a risk of inducing normal blood flow to cause blood clot to produce a thrombus, and thus does not depend on the body's coagulation mechanism and can Hemostatic products that quickly seal wounds are more popular in surgery.
  • Hemostasis products that achieve hemostasis through physical action need to physically compress the bleeding wounds during the hemostasis process, while the material absorbs water in the blood to promote coagulation, and the hemostasis is slow and the effect is poor.
  • such materials are only effective for bleeding with a small flow rate, so the hemostatic forceps must be used in combination, which makes the above-mentioned hemostatic material often require the surgeon to predict the amount of bleeding in advance or repeatedly use the hemostat.
  • traditional hemostatic materials (such as cotton yarn, bandages) are not ideal for hemostasis of irregular wounds such as irregular shape, deep, narrow, and arterial rupture.
  • hemostasis requires long-term external force to achieve physical hemostasis, for fragile nerves Or brain tissue is not applicable.
  • Another type of inorganic hemostatic material including zeolite, kaolin, etc., which utilizes the characteristics of the porous structure and high specific surface area of natural aluminosilicate materials, and absorbs the moisture in the blood to concentrate the local coagulation component of the wound. Quickly stop bleeding.
  • the artificially prepared mesoporous silicon material also has the characteristics of porous structure, high specific surface area, etc., and has the advantages of composition, particle size, and pore size controllability compared with the natural aluminosilicate material, and can effectively achieve hemostasis.
  • such materials have little effect on bleeding with large blood flow, and have limited effect on large-area wounds; and although such materials are benign in the local tissue reaction, mesoporous silicon particles entering the vascular and tissue organs cause serious Systemic toxicity.
  • the injectable self-healing hemostatic material provided by the present invention is a colloidal gel material obtained by self-assembly of electrostatic interaction between particles by using surface-charged polymer microgel particles. Since the electrostatic interaction between the microgels is physically crosslinked and reversible, when the colloidal gel is destroyed by destructive shearing force, the electrostatic interaction between the microgel particles is destroyed by external force, and colloidal condensation The conversion of glue from a rigid solid material to a fluid material with fluidity is known as shear thinning behavior. When the external force is canceled, the interaction between the colloids is quickly restored, and the colloidal particles are assembled into a colloidal gel by re-forming physical cross-linking.
  • the hemostatic material of the present invention has injectability and self-healing properties, and the self-healing performance test shows that the self-healing rate after shear failure exceeds 85%. More importantly, the hemostatic material of the present invention does not need to introduce a chemical crosslinking reaction during the injection and curing process, and is different from the chemical polymer, and does not introduce a small molecule cross-linking agent, has good biocompatibility, can be degraded and absorbed, and has no toxic side effects. , good security, making it widely used in the field of biomedicine.
  • the hemostatic material provided by the invention has good mechanical strength, and the elastic modulus of the gel-like hemostatic material is greater than 10 kPa, and rapid hemostasis can be achieved for bleeding tissue with large bleeding volume and high blood pressure. Further, the high specific surface area of the colloidal particles of the hemostatic material of the present invention and the stable strength of the colloidal gel are advantageous for the closure of the wound.
  • Hemostatic products are one of the essential biomedical materials in surgery.
  • the combination of hemostatic materials and functional drugs is used to improve clinical hemostasis and promote wound healing.
  • These coagulation drugs, antibacterial and anti-inflammatory drugs, or bioactive drugs that promote tissue repair and regeneration include small molecule drugs and protein-based macromolecular drugs. These drugs are uncontrollable in drug release in combination with hemostatic products, and molecular drugs are easy to use. Inactivation problem. Therefore, the development of hemostatic materials that can load drugs and control release drugs has important medical application value.
  • Conventional hemostatic materials usually use pre-processed hemostatic products (such as hemostatic membrane, hemostatic powder, hemostatic sponge, etc.), and the materials are directly mixed into the drug solution, and the drug is loaded by physical adsorption of the drug on the surface of the stent. This approach usually results in the release of the drug in question when the hemostatic material is in use, and does not achieve long-term sustained release of protein drugs that promote wound healing.
  • pre-processed hemostatic products such as hemostatic membrane, hemostatic powder, hemostatic sponge, etc.
  • Microgel particles with micro-nano size are the basic unit, which has higher specific surface area than traditional porous scaffold materials, so the amount of protein adsorbed on the surface is higher; 2) loading of growth factors is microgel particles The lyophilized powder is directly blended with the aqueous growth factor solution. During the swelling process, the protein molecules enter the microgel particle network under osmotic pressure, so the release of the protein is mainly controlled by the degradation rate of the microgel; 3) growth The release rate of the factor is mainly controlled by the degradation rate of the gelatin microgel. Therefore, by controlling the degree of cross-linking of the microgel, the release rate of the loaded growth factor can be controlled, and different growth factors can be further loaded into different cross-linking degrees.
  • the colloidal particles can achieve orderly and controlled release of various growth factors.
  • Figure 1 is a scanning electron micrograph of a type A gelatin microgel particle prepared by the method of Example 1.
  • Figure 2 is a graph showing the rheological test results of the self-repairing behavior of a composite colloidal gel composed of oppositely charged colloidal particles of type A and type B gelatin prepared by the method of Example 1.
  • Figure 3 is a graph showing the rheological test results of the self-repairing behavior of a composite colloidal gel composed of oppositely charged calcium alginate and type A gelatin colloidal particles prepared by the method described in Example 2.
  • Figure 4 shows the hemostatic material prepared by the method described in Example 6 to achieve the ordered release of the bioactive protein drug.
  • Fig. 5 is a view showing the hemostatic effect of the hemostatic material of the present invention confirmed by animal experiments as described in Example 7.
  • the colloidal particles were freeze-dried to obtain lyophilized powders of the respective colloidal particles, wherein the freeze-drying conditions were: freeze-drying the colloidal particles at -60 ° C, ⁇ 300 Pa for 2-3 days.
  • type A gelatin as raw material, it is dissolved in deionized water by heating at 40 ° C, and a gelatin aqueous solution having a concentration of 5 w/v% is disposed, the pH value of the gelatin aqueous solution is adjusted to 3, and then 3 times solution volume of ethanol is added to the solution to generate a dispersion of gelatin microgel particles; respectively, adding 25 wt% aqueous solution of glutaraldehyde to the dispersion to crosslink the gelatin microgel particles, and the amount of glutaraldehyde added is 65 ⁇ L per gram of gelatin corresponding to 25 wt% glutaraldehyde.
  • the crosslinking reaction time was 12 hr, and centrifugal dispersion was carried out to obtain a dispersion of type A gelatin microgel particles having a positive zeta potential.
  • a dispersion of B-type gelatin microgel particles having a negative zeta potential on the surface was prepared using B-type gelatin as a raw material.
  • the microgel particle size and zeta potential data measured by a laser particle size analyzer are shown in Table 1.
  • Lyophilized powders (labeled as GelA) of type A gelatin particles and lyophilized powders of type B gelatin particles (labeled as GelB) were obtained by lyophilization.
  • the morphology of the gelatin particles was observed by a scanning electron microscope, and the results are shown in Fig. 1.
  • the prepared type A gelatin particles have a narrow particle size distribution and a size in the range of 150 to 300 nm.
  • the dispersion is thoroughly mixed and stirred to obtain a dispersion in which two different microgel particles are dispersed, wherein the ratio of the particles of type A gelatin to type B gelatin is 1:1; and 100 mM is added to the dispersion.
  • the hydrochloric acid was adjusted to a pH of 7.0, stirred and mixed, and lyophilized to obtain a lyophilized powder containing two different gelatin colloidal particles, labeled as GelA+B, which was used as a hemostatic powder.
  • the lyophilized powder of the above mixture was separately blended with an appropriate amount of 1 mM NaCl solution, and rapidly stirred and mixed to obtain an injectable self-healing colloidal gel, and a colloidal gel containing a volume fraction of colloidal particles of different microgels was obtained.
  • injectable hemostatic gel The resulting colloidal gel of the different components was prepared, and the viscoelastic properties of the resulting hemostatic gel material were evaluated by a rheometer. The results are shown in Table 2. As the colloidal volume fraction increases, the elastic modulus of the gel increases; at the same volume fraction, the gel elastic modulus of the oppositely charged colloidal particles is significantly stronger than that of the single component colloidal gel.
  • the gelatin gel modulus of the GelA+B component was about 38 kPa at a microgel colloidal particle volume fraction of 120 vol%.
  • the self-repairing behavior of the colloidal gel is characterized by a rheometer, and the specific test method is as follows. Continuous rheological testing of the colloidal gel: firstly, the oscillating time scan is performed, and an external force of 1 Hz and a strain of 0.5% is applied to the sample, and the storage modulus (or elastic modulus, G') and the loss mode of the test sample are tested. The amount (or viscous modulus, G"), at which point the gel exhibits a rigid behavior of the solid under low shear conditions, so the storage modulus G' is greater than the loss modulus G" and remains stable. The G' value at this stage is the initial elastic modulus of the sample. Then gradually increase the applied strain from 0.1% to 1000%.
  • the elastic modulus of the gel recovers instantaneously after shear failure, and the self-repairing elastic modulus is restored to more than 85% of the initial modulus within 5 minutes. And thus the self-repairing behavior can occur repeatedly: applying more to the sample During the cyclic shear failure process, each time the external force is removed, the elastic modulus of the gel recovers rapidly and returns to more than 80% of the initial elastic modulus.
  • the injectable self-healing hemostatic material of the present invention can also be obtained, but its performance is inferior to that of the injectable self-healing colloidal gel prepared by other methods of the present invention.
  • the preparation can be different.
  • the concentration of the aqueous gelatin solution is preferably from 1 to 10 w/v%, more preferably from 2.5 to 5 w/v%; and the amount of the polar organic solvent added is preferably >2 times, more preferably 3 to 4 parts of the gelatin aqueous solution. 6 times, the diameter of the gelatin microgel particles is preferably from 20 nm to 5 ⁇ m, more preferably from 100 nm to 2000 nm.
  • the dispersion of the gelatin microgel particles and the number of particles in each organic polymer particle dispersion affects the elastic modulus and the self-repairing efficiency of the prepared colloidal gel.
  • the ratio of the number of particles in the two dispersions in the present invention is preferably from 1:10 to 10:1, more preferably from 1:5 to 5:1.
  • an injectable self-healing hemostatic material with higher elastic modulus and self-repairing efficiency can be obtained. If the difference in diameter is too large, the obtained The colloidal gel has a reduced modulus of elasticity and a reduced self-healing efficiency.
  • the prepared gelatin microgel particle lyophilized powder I, gelatin microgel particle lyophilized powder II or gelatin microgel particle lyophilized powder III is directly applied as a hemostatic material
  • the injectable self-healing hemostatic material having a colloidal gel shape can be prepared by blending with an appropriate amount of the aqueous solution, and has an elastic modulus of 1 Pa to 100 kPa, preferably 20 kPa to 100 kPa.
  • the positively charged polymer particle dispersion is in chitosan, type A gelatin, polyacrylamide, poly(N-isopropylacrylamide), polyethyleneimine.
  • the one or more of the negatively charged polymer particle dispersions are prepared by using one or more of hyaluronic acid, alginic acid, type A gelatin, type B gelatin or polyacrylic acid. It is prepared that the polymer particle dispersion having a surface zeta potential of -10 to +10 mV is prepared by using one or more of collagen, albumin and gelatin as raw materials.
  • the positively charged polymer particle dispersion or the negatively charged polymer particle dispersion or the polymer particle dispersion having a surface zeta potential of -10 to +10 mV which can be prepared by a person skilled in the art according to the conventional preparation of the polymer particles. The techniques are prepared and will not be described in detail in this application.
  • the positively charged hydrophilic polymer having an isoelectric point of >8 is one of chitosan, type A gelatin, polyacrylamide, poly(N-isopropylacrylamide), and polyethyleneimine. Or several kinds, the negatively charged hydrophilic polymer having the isoelectric point ⁇ 6 is hyaluronic acid, alginic acid, type A gelatin, type B gelatin or polyacrylic acid
  • a type gelatin was used as a raw material, dissolved at 40 ° C, and a concentration of 5 w/v% of A-type gelatin aqueous solution was adjusted to adjust the pH to 11, and then 3.5 times of ethanol was added to the solution to form a type A gelatin micro.
  • a gel particle suspension adding 25 wt% aqueous solution of glutaraldehyde to the suspension to crosslink the gelatin microgel particles, and adding glutaraldehyde in an amount of 66 ⁇ L of 25 wt% glutaraldehyde per g gelatin, and the crosslinking reaction time is 12 hr.
  • glycine was added to neutralize the unreacted aldehyde group, and centrifugally washed to obtain type A gelatin microgel particles, and the particle size and surface zeta point parameters are shown in Table 4.
  • the alginic acid microgel particles are prepared by an emulsion method, and the preparation method is as follows: a 1 wt% aqueous solution of sodium alginate is added dropwise to a 50 mM aqueous solution of calcium chloride and stirring is continued at a high speed (stirring speed>5000 rpm) to obtain calcium alginate.
  • Microgel particles, particle size and surface zeta point parameters are shown in Table 4. Dispersing type A gelatin and calcium alginate microgel particles in 10 mM acidic acetic acid solution respectively, respectively, to obtain a dispersion of positively charged type A gelatin microgel particles, and a negatively charged alginate microgel particles.
  • Dispersion II the dispersion I and the dispersion II are thoroughly mixed and stirred to obtain a dispersion III, wherein the ratio of the particles of the type A gelatin and the calcium alginate microgel particles is 2:1; to the dispersion III
  • the pH was adjusted to 7.0 by the addition of 100 mM sodium hydroxide, stirred and mixed, and lyophilized to obtain a lyophilized powder in which two phases of different colloidal particles were mixed.
  • microgel particle lyophilized powder is blended with a volume of 1 mM NaCl salt solution, and rapidly stirred and mixed uniformly to obtain an injectable self-healing colloidal gel, that is, the hemostatic material of the present invention, wherein the microgel
  • the colloidal particle volume fraction is 50 vol% or 100 vol% of the colloidal gel volume.
  • the mechanical parameters of the colloidal gel were evaluated using a rheometer. The results are shown in Figure 3 and Table 5.
  • the elastic modulus of the colloidal gel obtained by blending oppositely charged calcium alginate and type A gelatin colloidal particles
  • the colloidal particle volume fraction increases with an increase in the volume fraction of 100 vol%, and the storage (elastic) modulus G' exceeds 12 kPa.
  • the self-healing efficiency also increased with the increase of the volume fraction of colloidal particles within 5 minutes after shear failure. When the volume fraction was 100 vol%, the self-healing efficiency of the elastic modulus G' exceeded 80%.
  • B-type gelatin was used as a raw material, dissolved at 40 ° C, and a B-type gelatin aqueous solution having a concentration of 5 w/v% was prepared to adjust the pH to 3, and then 3 volumes of ethanol were added to the solution to form a B-type gelatin micro.
  • a gel particle suspension adding 25 wt% aqueous solution of glutaraldehyde to the suspension to crosslink the gelatin microgel particles, and adding glutaraldehyde in an amount of 80 ⁇ L per gram of gelatin corresponding to 25 wt% glutaraldehyde, and the crosslinking reaction time is 12 hr.
  • the cells were repeatedly centrifuged and resuspended in deionized water to obtain B-type gelatin microgel particles.
  • the particle size and surface zeta point parameters are shown in Table 6.
  • the chitosan quaternary ammonium salt was dissolved in deionized water to a concentration of 2.5 w/v% chitosan quaternary ammonium salt aqueous solution.
  • the gelatin B microgel particles are dispersed in a 20 mM acidic acetic acid aqueous solution, and the chitosan quaternary ammonium salt aqueous solution is further thoroughly blended with the gelatin B microgel particle dispersion, and stirred to obtain a dispersion III, wherein the type B gelatin micro
  • the mass ratio of the gel particles to the polymer chitosan quaternary ammonium salt is 10:1, 100 mM sodium hydroxide is added to the dispersion III to adjust the pH to 7.0, and then freeze-dried to obtain B-type gelatin colloidal particles and shell aggregation.
  • a composite lyophilized powder of a sugar quaternary ammonium salt is blended with a volume of 10 mM NaCl salt solution, and rapidly stirred and mixed uniformly to obtain an injectable self-healing colloidal gel, that is, the hemostatic material of the present invention, wherein the gelatin microgel particle volume fraction
  • the volume fraction from the healing gel is 100 vol%.
  • the mechanical parameters of the colloidal gel were evaluated using a rheometer. The results are shown in Table 7.
  • the colloidal gel obtained by blending oppositely charged B-type gelatin particles and chitosan quaternary ammonium salt has a colloidal particle volume fraction of 100 vol.
  • the storage (elastic) modulus G' is about 48 kPa.
  • a type gelatin was used as a raw material, dissolved at 40 ° C, and a concentration of 10 w/v% of A-type gelatin aqueous solution was adjusted to adjust the pH to 11, and then 2 volumes of ethanol were added to the solution to form a type A gelatin.
  • a microgel particle suspension adding 25 wt% aqueous solution of glutaraldehyde to the suspension to crosslink the gelatin microgel particles, and adding glutaraldehyde in an amount of 264 ⁇ L per gram of gelatin corresponding to 25 wt% glutaraldehyde, crosslinking reaction time After 12 hr, repeated centrifugation and resuspension in deionized water to obtain type A gelatin microgel particles, the particle size and surface zeta point parameters are shown in Table 8.
  • Polyethylene glycol (PEG, molecular weight 2 kDa) was dissolved in deionized water to a concentration of 5 w/v% PEG aqueous solution.
  • the PEG aqueous solution obtained by dispersing the type A gelatin microgel particles prepared by the above method is further thoroughly blended with the A type gelatin microgel dispersion, and stirred to obtain a dispersion III, wherein the type A gelatin microgel The mass ratio of particles to PEG is 1:2.
  • the pH of the dispersion III was adjusted to 7.0, followed by freeze-drying to obtain a composite lyophilized powder of gelatin colloidal particles of type A and PEG.
  • the above lyophilized powder is blended with a volume of 10 mM NaCl salt solution, and rapidly stirred and mixed uniformly to obtain a self-healing colloidal gel, that is, the hemostatic material of the present invention, wherein the gelatin microgel particle volume fraction accounts for self-healing
  • the gel volume fraction was 100 vol%.
  • the mechanical parameters of the colloidal gel were evaluated using a rheometer.
  • the volume fraction of the type A gelatin colloidal particles in the colloidal gel was 100 vol%
  • the storage (elastic) modulus G' was about 19 kPa.
  • the self-healing efficiency within 5 minutes after shear failure, when the colloidal particle volume fraction is 100 vol%, the self-healing efficiency of the elastic modulus G' is about 83%.
  • a type gelatin was used as a raw material, dissolved at 40 ° C, and a type A gelatin aqueous solution having a concentration of 5 w/v% was prepared, and the pH was adjusted to 11, and then 3.5 times of ethanol was added to the solution to form a type A gelatin.
  • the microgel particle suspension then adding 25 wt% aqueous solution of glutaraldehyde to crosslink the gelatin microgel particles, the amount of glutaraldehyde added is 66 ⁇ L corresponding to 25 wt% glutaraldehyde per g gelatin, the crosslinking reaction time is 12 hr, and the mixture is centrifuged to obtain Type A gelatin microgel particles, particle size and surface zeta point parameters are shown in Table 10.
  • the alginic acid microgel particles are prepared by an emulsion method, and the preparation method is as follows: adding 1 wt% of an aqueous solution of sodium alginate to an aqueous solution of calcium chloride and continuing high-speed stirring (stirring speed>5000 rpm) to obtain calcium alginate particles, particles
  • the size and surface zeta point parameters are shown in Table 10.
  • Dispersion II, the dispersion I and the dispersion II are thoroughly mixed and stirred to obtain a dispersion III, wherein the ratio of the particles of the type A gelatin and the alginic acid microgel particles is 2:1; and the dispersion III is added.
  • the pH was adjusted to 7.0 with 100 mM sodium hydroxide, stirred and mixed, and lyophilized to obtain a composite lyophilized powder in which two phases of different colloidal particles were mixed.
  • a type of gelatin microgel particles were prepared by the anti-solvent method described in Example 1 using A-type gelatin as a raw material.
  • the cross-linking concentration of glutaraldehyde was 65 ⁇ L per 25 g of glutaraldehyde per g of gelatin, and the preparation was positive.
  • Charge type A gelatin microgel particles; Form B gelatin microgel particles were prepared by the anti-solvent method described in Example 1 using B type gelatin as a raw material, wherein the glutaraldehyde cross-linking concentration was 25 wt% per g gelatin.
  • the amount of glutaraldehyde was 264 ⁇ L, and a negatively charged type B gelatin microgel particle was prepared.
  • the preparation parameters and the obtained microgel particle size and zeta point data are shown in Table 12.
  • bFGF basic fibroblast growth factor
  • BMP-2 bone morphogenetic protein-2
  • a dispersion of the type A gelatin particles carrying bFGF and the type B gelatin microgel particles carrying BMP-2 was obtained.
  • the above two gelatin particle dispersions were thoroughly mixed according to a particle ratio of 1:1, and lyophilized to obtain two gelatin particle lyophilized powders carrying different growth factors.
  • microgel particle lyophilized powder is blended with a volume of 1 mM NaCl salt solution, and rapidly stirred and uniformly mixed to obtain an injectable self-healing colloidal gel carrying different growth factors, wherein the microgel colloid
  • the particle volume fraction is 100 vol% of the volume of the colloidal gel.
  • the release profile of in vitro release kinetics of two different growth factors from colloidal gel carrier materials is shown in Figure 4.
  • the type A gelatin loaded with bFGF has a faster degradation rate due to the lower degree of cross-linking, so the release rate of bFGF is faster.
  • B-type gelatin loaded with BMP-2 has a slow rate of degradation due to high degree of cross-linking, so the release rate of BMP-2 is relatively slow; the results show that the colloidal gel of the present invention can achieve the orderly release of different growth factor drugs.
  • the type A gelatin and the type B gelatin microgel particle dispersion were obtained by the preparation method in the first embodiment, and the two gelatin particles were respectively dispersed in a 20 mM aqueous hydrochloric acid solution to obtain a positively charged type A gelatin microgel. a dispersion of particles and negatively charged type B gelatin microgel particles, the two are thoroughly mixed and stirred to obtain a dispersion in which two different microgel particles are dispersed, wherein the type A gelatin and the type B gelatin are mixed.
  • the quantitative ratio was 1:1; 100 mM sodium hydroxide was added to the dispersion to adjust the pH to 7.0, stirred and mixed, and lyophilized to obtain a lyophilized powder containing two different gelatin colloidal particles.
  • the above mixture lyophilized powder is blended with phosphate buffer solution PBS, and rapidly stirred and mixed uniformly to obtain an injectable self-healing colloidal gel, that is, the hemostatic material of the present invention, wherein the volume fraction of the two colloidal particles accounts for the colloidal gel. 100 vol% of volume.
  • the New Zealand rabbit back spine bone defect bleeding wound was used as an animal model to make a 2.5-4 mm hemorrhage wound.
  • the gelatin-based hemostatic gel was directly injected into the bleeding wound by a common medical syringe. After 1 minute of light compression, the bleeding point was closed and the hemostasis time was stopped. It is 1 min.
  • the process of the animal experiment is shown in Figure 5.

Landscapes

  • Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Surgery (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Materials Engineering (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Dispersion Chemistry (AREA)
  • Materials For Medical Uses (AREA)
  • Medicinal Preparation (AREA)

Abstract

一种止血材料及其制备方法和应用。使用带表面电荷的高分子微凝胶颗粒，通过颗粒间或颗粒与高分子间的静电相互作用自组装得到注射型自愈合止血材料。静电作用是物理交联且具有可逆性，在注射和固化过程中没有引入小分子交联剂；得到的止血材料具有较好的力学强度，对于出血量大、血管压力高的出血组织也可以实现快速止血。

Description

一种可注射型自愈合止血材料及其制备方法和应用 技术领域
本发明属于生物医用材料技术领域，涉及一种止血材料及其制备方法，具体而言，本发明涉及一种可直接施用于外科手术(含微创手术)以及外伤等原因造成的人及其它哺乳动物的组织器官创面，用于止血、封闭伤口、减少组织渗出、促进组织修复以及保护创面组织等目的的明胶基止血粉剂和止血凝胶材料的制备方法。
背景技术
外科手术过程中需要对不同原因造成的伤口进行止血和封闭，因此止血材料是非常重要的一类生物医用材料，具有重要的研发和应用意义。目前临床常用的止血材料从作用机制上可分为三类：第一类止血材料是通过材料的物理或化学作用使伤口部位加速凝血(如止血纱布、高分子多糖类、无机类沸石等)；第二类是直接或间接提供外来的凝血成分、利用激发自身凝血机制加速实现凝血(如纤维蛋白类止血材料、含有凝血酶和凝血因子类止血材料)；第三类是利用材料对组织很强的粘着力直接封闭创面，从而实现止血(如α氰基丙烯酸酯类材料)。然而不同类型的止血材料都存在诸多瓶颈。
对于利用止血机制来实现血凝的之血制品而言，其缺点是它们仅对流速较小的出血有效，因此使用时必须配合止血钳夹，这就使得上述止血材料在使用时常常需要外科医生提前预测出血量或者反复多次使用止血钳。另外，载有凝血酶类凝血剂的止血制品在使用过程中，凝血酶类蛋白分子易外溢到正常血管中，因此存在诱发正常血流发生凝血产生血栓的风险，因此不依赖机体凝血机制并能迅速封闭创面的止血制品在外科手术中更受青睐。
而通过物理作用实现止血的止血制品，如止血纱布、海绵等，在止血过程需要通过对出血创面进行物理压迫，同时材料通过吸收血液中的水，促使凝血，止血速度慢，效果较差。并且，这类材料仅对流速较小的出血有效，因此使用时必须配合止血钳夹，这就使得上述止血材料在使用时常常需要外科医生提前预测出血量或者反复多次使用止血钳。同时，传统止血材料(如棉纱、绷带)对于不规则形状、深、窄、动脉破裂等现场常见创伤的止血效果很不理想。另 外，还存在其他的缺点：1)影响术后缝合，增加新的创面出血；2)不能自然降解，在体内形成残留；3)止血过程需要长时间的外力按压实现物理止血，对于脆弱的神经或脑组织不适用。另一类无机物止血材料，包括沸石、高岭土等，这类材料利用了天然硅铝酸盐材料多孔结构、高比表面积等特性，通过吸收血液中的水分，浓缩创面局部的凝血成分，从而实现快速止血。而人工制备的介孔硅材料同样具有多孔结构、高比表面积等特性，而且相比天然硅铝酸盐材料具有成分、粒径、孔径可控等优势，同样可实现有效止血。然而，这类材料对于血流大的出血无能为力，对于大面积创面效果也有限；并且这类材料虽然介局部组织反应是良性的，但是进入脉管和组织器官的介孔硅颗粒会引起严重的全身毒性。因此，开发针对现场和院前急救用的，快速、安全、有效的新型止血材料成为医学和生物材料科学领域中的重要课题。
可注射的凝胶类止血制品因其临床易操作性、适于微创手术近年来渐渐进入临床止血应用。目前国内临床上使用的止血凝胶类产品主要依靠进口，其中美国强生公司的
Figure PCTCN2018086502-appb-000001
占据了主要的市场份额。该材料由明胶组成，水溶后快速形成泡沫状凝胶物，产品具有很好的可注射性。但在止血过程中存在诸多问题。首先，凝胶力学强度差，流变仪测得弹性模量<1kPa，对于出血量大、血管压力高的出血组织无法实现止血；其次，该材料与组织间黏性差，对于临床出血量稍大的创面，难以实现快速止血。因此，开发具有良好可注射性、可塑性等临床操作性能，可用于血流量大、血压高的组织器官创面止血的新型凝胶材料具有重要的实用意义。
发明内容
鉴于上述所述的止血材料在现有技术中存在的问题，本发明提供一种可注射型自愈合止血材料及其制备方法。本发明的方法制备得到的止血材料具有良好的可注射性、自愈合性能和较好的力学强度，对于出血量大、血管压力高的出血组织也可以实现快速止血。
本发明的技术方案如下：
一种可注射型自愈合止血材料，由如下方法制备得到：
(1)以明胶为原料，使其在去离子水中加热溶解，配置浓度为1～10w/v％的明胶水溶液，调pH值为1-6或8-14，向溶液中加入>2倍溶液体积的极性有 机溶剂，生成明胶微凝胶颗粒分散液，加入交联剂交联反应1～12h，离心、清洗得到明胶微凝胶颗粒；
其中，所述明胶微凝胶颗粒的zeta电势为-30～+30mV，所述明胶微凝胶颗粒的直径为20nm～2μm；
(2)将步骤(1)制备得到的表面zeta电势>+10mV的明胶微凝胶颗粒，分散在pH<5的酸性水溶液或pH>9的碱性水溶液中，得分散有带正电荷的明胶微凝胶颗粒的分散液，再与带负电荷的高分子颗粒分散液按照颗粒数比1:10～10:1共混，或者与等电点<6的带负电荷的亲水性高分子水溶液按照明胶微凝胶颗粒与亲水性高分子的质量比1:10～10:1共混，用pH调节剂调pH至7.0，冷冻干燥，得明胶微凝胶颗粒冻干粉末I；
(3)将步骤(1)制备得到的表面zeta电势<-10mV的明胶微凝胶颗粒，分散在pH<5的酸性水溶液或pH>9的碱性水溶液中，得明胶微凝胶颗粒的分散液，再与带正电荷的高分子颗粒分散液按照颗粒数比1:10～10:1混合，或者与等电点>8的带正电荷的亲水性高分子水溶液按照明胶微凝胶颗粒与亲水性高分子的质量比1:10～10:1共混，用pH调节剂调pH至7.0，冷冻干燥，得明胶微凝胶颗粒冻干粉末II；
(4)将步骤(1)制备得到的表面zeta电势为-10～+10mV的明胶微凝胶颗粒分散在中性水溶液中，再与另一种表面zeta电势在-10～+10mV的高分子颗粒分散液按照颗粒数比1:10～10:1共混，或者与等电点6～8的亲水性高分子水溶液按照明胶微凝胶颗粒与亲水性高分子的质量比1:10～10:1共混，冷冻干燥，得明胶微凝胶颗粒冻干粉末Ⅲ；
(5)将明胶微凝胶颗粒冻干粉末I、明胶微凝胶颗粒冻干粉末II或明胶微凝胶颗粒冻干粉末Ⅲ分别与水性溶液共混，得可注射型自愈合止血材料；
其中，所述的带正电荷的高分子颗粒的表面电荷为+5～+60mV，带负电荷的高分子颗粒的表面电荷为-5～-60mV，所述高分子颗粒的直径为100nm～20μm，在步骤(5)中得到的可注射型自愈合止血材料中胶体颗粒占总体积的百分比为50vol％～150vol％；在步骤(3)、(4)和(5)中所述的亲水性高分子的分子量为1k～500kDa。
本发明上述可注射型自愈合止血材料的制备方法中，将步骤(1)制备得到的明胶微凝胶颗粒，冷冻干燥得到的明胶微凝胶颗粒冻干粉末与水性溶液共混， 中的一种或几种，所述的等电点6～8的高分子为胶原蛋白、白蛋白、明胶、聚乙烯醇、聚乙二醇中的一种或几种。
进一步地，在上述技术方案中，在步骤(1)中所述的极性有机溶剂为甲醇、乙醇、异丙醇、丁醇、丙酮、乙腈、四氢呋喃中的一种或几种的组合；所述的交联剂为戊二醛、甘油醛、甲醛、碳二亚胺、二卤代烷、异氰酸酯、二异氰酸酯、谷氨酰胺转胺酶、京尼平中的一种或几种。
进一步地，在上述技术方案中，在步骤(1)中所述的交联反应的反应体系中交联剂与明胶中氨基基团的摩尔比为0.1～10。交联剂与明胶中氨基基团的摩尔比影响所形成的明胶微凝胶颗粒的交联度，交联度过高明胶微流强度更高、表面电荷更倾向于带负电，交联度过低明胶微球强度低、表面电荷取决于明胶原料的等电点，本发明优选的技术方案中交联度较低时较为好，优选的，本发明中所述交联剂与明胶中氨基基团的摩尔比要控制在0.5～5。
进一步地，在上述技术方案中，在步骤(2)和步骤(3)中所述的酸性水溶液和碱性水溶液中所含有的离子浓度均小于200mM。所述的酸性水溶液和碱性水溶液中所含有的离子的种类不特别限定，可以采用本领域常规的用于调节酸性或碱性的试剂，如盐酸、硫酸、醋酸、氢氧化钙、氢氧化钾、氨水、碳酸钠等。
进一步地，在上述技术方案中，在步骤(2)和步骤(3)中所述的pH调节剂包括酸性物质和碱性物质，所述酸性物质为葡萄糖酸内酯、HCl、HNO 3、H 2SO 4中的一种或几种，所述碱性物质为尿素和脲酶的组合、或者氢氧化钠、氢氧化钙、氢氧化钾、氨水中的一种或几种。
进一步地，在上述技术方案中，在步骤(5)中所述的水性溶液是离子浓度120～200mM、pH值为6～8的任意水溶液、亲水性高分子的水溶液、不水溶的纳米颗粒分散液中的一种或几种的组合。
进一步地，在上述技术方案中，所述水性溶液中含有生物活性物质和/或赋形剂。其中所述赋形剂为溶剂、分散介质、包被剂、表面活性剂、抗氧化剂、防腐剂、等渗剂、粘合剂、润滑剂、颜料，以及它们的组合或类似物；所述生物活性物质为凝血剂、抗感染药物、消炎药、生物活性蛋白药物中的至少一种。所述凝血剂选自胶原蛋白、明胶、氧化纤维素、羧甲基纤维素、壳聚糖、透明质酸、海藻酸钠、高岭土、凝血酶、纤维蛋白、钙剂、鱼精蛋白、多肽、缩氨 酸、氨基酸中的一种或其组合；所述抗感染药物选自抗生素、抗菌制剂、抗病毒制剂、抗真菌药物、抗溃疡药物、中药制剂的一种或其组合；所述消炎药物选自非类固醇类、固醇类消炎药、抗溃疡药物、中药制剂的一种或其组合。
本发明还提供上述所述的可注射型自愈合止血材料在制备用于体表组织、体腔内组织器官的有血创面的止血、防黏连、防感染、促进组织愈合和/或封闭伤口的止血制品中的应用。其中所述的止血制品可采用止血粉剂、止血颗粒、止血球、止血气雾剂、止血海绵、止血凝胶、止血膜、止血栓或止血贴等方式。
本发明的有益效果：
1.和传统止血材料的区别和优势；
外科手术过程中需要对不同原因造成的伤口进行止血和封闭，因此止血材料是非常重要的一类生物医用材料，具有重要的研发和应用意义。目前临床常用的止血材料从作用机制上可分为三类：第一类止血材料是通过材料的物理或化学作用使伤口部位加速凝血(如止血纱布、高分子多糖类、无机类沸石等)；第二类是直接或间接提供外来的凝血成分、利用激发自身凝血机制加速实现凝血(如纤维蛋白类止血材料、含有凝血酶和凝血因子类止血材料)；第三类是利用材料对组织很强的粘着力直接封闭创面，从而实现止血(如α氰基丙烯酸酯类材料)。然而不同类型的止血材料都存在诸多瓶颈。
对于利用止血机制来实现血凝的止血制品而言，其缺点是它们仅对流速较小的出血有效，因此使用时必须配合止血钳夹，这就使得上述止血材料在使用时常常需要外科医生提前预测出血量或者反复多次使用止血钳。另外，载有凝血酶类凝血剂的止血制品在使用过程中，凝血酶类蛋白分子易外溢到正常血管中，因此存在诱发正常血流发生凝血产生血栓的风险，因此不依赖机体凝血机制并能迅速封闭创面的止血制品在外科手术中更受青睐。
而通过物理作用实现止血的止血制品，如止血纱布、海绵等，在止血过程需要通过对出血创面进行物理压迫，同时材料通过吸收血液中的水，促使凝血，止血速度慢，效果较差。并且，这类材料仅对流速较小的出血有效，因此使用时必须配合止血钳夹，这就使得上述止血材料在使用时常常需要外科医生提前预测出血量或者反复多次使用止血钳。同时，传统止血材料(如棉纱、绷带)对于不规则形状、深、窄、动脉破裂等现场常见创伤的止血效果很不理想。另外，还存在其他的缺点：1)影响术后缝合，增加新的创面出血；2)不能自然 降解，在体内形成残留；3)止血过程需要长时间的外力按压实现物理止血，对于脆弱的神经或脑组织不适用。另一类无机物止血材料，包括沸石、高岭土等，这类材料利用了天然硅铝酸盐材料多孔结构、高比表面积等特性，通过吸收血液中的水分，浓缩创面局部的凝血成分，从而实现快速止血。而人工制备的介孔硅材料同样具有多孔结构、高比表面积等特性，而且相比天然硅铝酸盐材料具有成分、粒径、孔径可控等优势，同样可实现有效止血。然而，这类材料对于血流大的出血无能为力，对于大面积创面效果也有限；并且这类材料虽然介局部组织反应是良性的，但是进入脉管和组织器官的介孔硅颗粒会引起严重的全身毒性。
而本发明提供的可注射型自愈合止血材料为使用带表面电荷的高分子微凝胶颗粒，通过颗粒间的静电相互作用自组装得到的胶体凝胶材料。由于微凝胶之间的静电作用是物理交联且具有可逆性，因此当这种胶体凝胶受到破坏性的剪切力破坏时，微凝胶颗粒间的静电相互作用被外力破坏，胶体凝胶从具有刚性的固体材料向具有流动性的流体材料转变，这一过程被称为剪切变稀行为。当外力取消时，胶体间的相互作用快速恢复，胶体颗粒通过重新形成物理交联组装成胶体凝胶。因此本发明所述的止血材料具有可注射性和自愈合性能，自愈合性能测试显示，剪切破坏后的自修复率超过85％以上。更重要的是，本发明的止血材料在注射和固化过程无需引入化学交联反应，不同于化学高分子，也没有引入小分子交联剂，生物相容性好，可降解吸收，无毒副作用，安全性好，使其在生物医学领域的广泛应用成为可能。
另外，本发明提供的止血材料有较好的力学强度，凝胶状止血材料的弹性模量大于10kPa，对于出血量大、血管压力高的出血组织也可以实现快速止血。还有本发明止血材料的胶体颗粒的高比表面积、胶体凝胶的稳定强度有利于创口的封闭。
2.在药物缓释方面的优势
止血制品是外科手术中必不可少的生物医用材料之一，为促进止血、控制手术创面感染等风险、加速催化创口愈合，将止血材料与功能性药物联合使用是提高临床止血、促进创面愈合的有效手段之一。这些凝血药物、抗菌消炎药物、或促进组织修复再生的生物活性药物包括小分子药物和蛋白类大分子药物，这些药物在与止血制品复合使用的过程中都存在药物释放不可控，分子类药物 易失活的问题。因此，开发可加载药物并可控释放药物的止血材料具有重要的医学应用价值。传统的止血材料通常利用预加工好的止血制品(如止血膜、止血粉、止血海绵等)，将材料直接混入药物溶液，通过药物在支架的表面物理吸附实现药物的加载。这种方式通常导致止血材料在使用时，所载药物的暴释，对于促进创伤修复的蛋白类药物不能实现长期持续的释放。
本发明的止血材料对比与传统载体材料具有更多优势。1)具有微纳米尺寸的微凝胶颗粒为基本单元，相比传统多孔支架材料具有更高比表面积，因此可表面吸附的蛋白量更高；2)生长因子的加载是将微凝胶颗粒的冻干粉直接与生长因子水溶液共混，在颗粒溶胀过程中蛋白分子在渗透压作用下进入微凝胶颗粒网络内部，因此蛋白的释放主要是由微凝胶的降解速率控制的；3)生长因子的释放速率主要由明胶微凝胶的降解速率调控，因此通过控制微凝胶的交联度可以实现对加载的生长因子的释放速率进行调控，进一步将不同的生长因子加载入不同交联度的胶体颗粒，可以实现多种生长因子的有序可控释放。
附图说明
图1为实施例1方法制备的A型明胶微凝胶颗粒的扫描电镜照片。
图2为实施例1方法制备的由带相反电荷的A型和B型明胶胶体颗粒组成的复合胶体凝胶的自修复行为的流变学测试结果。
图3为实施例2所述的方法制备的带相反电荷的海藻酸钙和A型明胶胶体颗粒组成的复合胶体凝胶的自修复行为的流变学测试结果。
图4表示实施例6中所述方法制备的止血材料实现生物活性蛋白药物的有序释放。
图5表示实施例7所述的通过动物实验证实本发明止血材料的止血功效。
具体实施方式
下述非限制性实施例可以使本领域的普通技术人员更全面地理解本发明，但不以任何方式限制本发明。下述实施例中，如无特殊说明，所使用的实验方法均为常规方法，所用材料、试剂等均可从生物或化学公司购买。
下面实施例中将胶体颗粒冷冻干燥后得各胶体颗粒的冻干粉末，其中所述冷冻干燥条件为：将胶体颗粒在-60℃、<300Pa下冷冻干燥2-3天。
实施例1
以A型明胶为原料，使其在去离子水中加热40℃溶解，配置浓度为5w/v％的明胶水溶液，调节明胶水溶液pH值为3，随后向溶液中加入3倍溶液体积的乙醇，生成明胶微凝胶颗粒的分散液；分别向分散液中加入25wt％戊二醛水溶液，使明胶微凝胶颗粒交联，加入戊二醛的量分别为每克明胶对应25wt％戊二醛66μL，交联反应时间12hr，离心清洗得到表面zeta电势位正的A型明胶微凝胶颗粒的分散液。同样方法，以B型明胶为原料制备得到表面zeta电势为负的B型明胶微凝胶颗粒的分散液。利用激光粒度仪测得制备微凝胶颗粒尺寸和zeta电势数据如表1所示。经冷冻干燥分别得到A型明胶颗粒的冻干粉末(标记为GelA)和B型明胶颗粒的冻干粉末(标记为GelB)。通过扫描电子显微镜对明胶颗粒的形貌进行观测，结果如图1所示。制备得到的A型明胶颗粒的粒径分布窄，尺寸在150-300nm范围内。
表1.不同类型明胶颗粒的性能参数
Figure PCTCN2018086502-appb-000002
将A型明胶和B型明胶微凝胶颗粒分别分散在20mM的NaOH碱性水溶液中，分别得到分散有带正电荷的A型明胶微凝胶颗粒和带负电荷的B型明胶微凝胶颗粒的分散液，将两者充分混合、搅拌，得分散有两种不同微凝胶颗粒的分散液，其中A型明胶和B型明胶混合的颗粒数量比为1:1；向分散液中加入100mM的盐酸调节pH值至7.0，搅拌混合，冷冻干燥，得到含有两种不同明胶胶体颗粒的冻干粉末，标记为GelA+B，可作为止血粉剂。将上述混合物冻干粉末分别与适当量的1mM的NaCl溶液共混，并快速搅拌混合均匀得到可注射型自愈合胶体凝胶，得到含不同微凝胶胶体颗粒体积分数的胶体凝胶，得到可注射型止血凝胶。制备所得不同组分的胶体凝胶，通过流变仪对所得止血凝胶材料的粘弹性能进行评价。结果如表2所示，胶体体积分数增加，凝胶的弹性模量增加；在体积分数相同时，带相反电荷胶体颗粒组成的凝胶弹性模量显著强于单一组分的胶体凝胶。在微凝胶胶体颗粒体积分数为120vol％时，GelA+B组分的胶体凝胶弹性模量约为38kPa。
胶体凝胶的自修复行为是通过流变仪进行表征，具体测试方法如下。对胶体凝胶进行连续的流变测试：首先进行震荡时间扫描，对样品施加频率为1Hz和应变为0.5％的外力，测试样品的储能模量(或弹性模量，G’)和损耗模量(或粘性模量，G”)，此时凝胶在低剪切力情况下表现出固体的刚性行为，因此储能模量G’大于损耗模量G”且保持稳定。这一阶段的G’值即为样品的初始弹性模量。随后逐渐增加施加的应变从0.1％至1000％，此过程中通过施加外力将样品破坏，弹性模量G’逐渐降低，最终低于G”，即胶体体系从刚性固体向粘性流体发生转变，结构被破坏。随后立即取消外力作用，考察样品弹性模量的恢复情况。将外力释放后，样品恢复的储能(弹性)模量与其初始储能弹性模的百分比(％)定量考察凝胶的自修复效率。凝胶的自修复效率如表3所示，由带相反电荷胶体颗粒组成的凝胶弹性模量显著强于单一组分的胶体凝胶。GelA+B胶体凝胶的自修复过程如图2所示，凝胶在剪切破坏后其弹性模量瞬间恢复，5分钟内自修复弹性模量恢复到初始模量超过85％。并且这样自修复行为可以反复发生：在对样品施加多个循环的剪切破坏过程中，每次取消外力后，凝胶的弹性模量都会快速恢复，并恢复到初始弹性模量的80％以上。
表2.实施例1中制备的不同胶体凝胶含有不同体积分数的微凝胶颗粒所得到凝胶材料的弹性模量G'
Figure PCTCN2018086502-appb-000003
表3.实施例1中制备的不同胶体凝胶不同体积分数的微凝胶颗粒所得的凝胶材料的自修复效率
Figure PCTCN2018086502-appb-000004
也可以得到本发明所述的可注射型自愈合止血材料，但其性能差于本发明中其他方法制备得到的可注射型自愈合胶体凝胶。
本发明上述可注射型自愈合止血材料的制备方法中，在步骤(1)中，根据调整明胶水溶液的浓度、极性有机溶剂的加入量、交联反应时间等，可以制备得到带有不同zeta电势和直径大小的明胶微凝胶颗粒。本发明上述技术方案中，明胶水溶液浓度优选为1～10w/v％，更优选为2.5～5w/v％；极性有机溶剂的加入量优选为明胶水溶液的>2倍，更优选为3～6倍，明胶微凝胶颗粒直径优选为20nm～5μm，更优选为100nm～2000nm。
本发明上述可注射型自愈合止血材料的制备方法中，步骤(2)、(3)和(4)中，明胶微凝胶颗粒的分散液和各有机高分子颗粒分散液中的颗粒数的比例影响所制备胶体凝胶的弹性模量和自修复效率，本发明中两种分散液中颗粒数的比例优选为1:10～10:1，更优选为1:5～5:1，共混的两种颗粒直径比为1:5～5:1时，可得到具有更高弹性模量和自修复效率的可注射型自愈合止血材料，若直径的差异过大，则所得到的胶体凝胶弹性模量下降，自修复效率降低。
在本发明的上述技术方案中，将制备得到的明胶微凝胶颗粒冻干粉末I、明胶微凝胶颗粒冻干粉末II或明胶微凝胶颗粒冻干粉末Ⅲ直接作为止血材料来应用，也可以与适量水性溶液共混，制备得到胶体凝胶状的可注射型自愈合止血材料，其弹性模量为1Pa～100kPa，优选20kPa～100kPa。
进一步地，在上述技术方案中，所述的带正电荷的高分子颗粒分散液以壳聚糖、A型明胶、聚丙烯酰胺、聚(N-异丙基丙烯酰胺)、聚乙烯亚胺中的一种或几种作为原料制备得到，所述的带负电荷的高分子颗粒分散液以透明质酸、海藻酸、A型明胶、B型明胶或聚丙烯酸中的一种或几种作为原料制备得到，所述的表面zeta电势在-10～+10mV的高分子颗粒分散液以胶原蛋白、白蛋白、明胶中的一种或几种作为原料制备得到。所述带正电荷的高分子颗粒分散液或带负电荷的高分子颗粒分散液或表面zeta电势在-10～+10mV的高分子颗粒分散液，本领域技术人员可以根据高分子颗粒的常规制备技术来制备得到，在本申请中不再详细陈述。
所述的等电点>8的带正电荷的亲水性高分子为壳聚糖、A型明胶、聚丙烯酰胺、聚(N-异丙基丙烯酰胺)、聚乙烯亚胺中的一种或几种，所述等电点<6的带负电荷的亲水性高分子为透明质酸、海藻酸、A型明胶、B型明胶或聚丙烯酸
*注：自修复效率是采用1000％的应变持续你剪切凝胶材料60s后，检测应力释放后5min内的弹性模量恢复的百分比(％)。
实施例2
以A型明胶为原料，在加热40℃下溶解，配置得到浓度为5w/v％的A型明胶水溶液，调节pH值为11，随后向溶液中加入3.5倍体积的乙醇，生成A型明胶微凝胶颗粒悬浮液；向悬浮液中加入25wt％戊二醛水溶液，使明胶微凝胶颗粒交联，加入戊二醛的量为每g明胶对应25wt％戊二醛66μL，交联反应时间12hr，随后加入甘氨酸中和未反应的醛基，离心清洗得到A型明胶微凝胶颗粒，颗粒尺寸和表面zeta点位参数如表4所示。
通过乳液法制备海藻酸微凝胶颗粒，具体制备方法如下：将1wt％的海藻酸钠的水溶液逐滴加入50mM氯化钙水溶液中并持续高速搅拌(搅拌速度>5000rpm)，即得到海藻酸钙微凝胶颗粒，颗粒尺寸和表面zeta点位参数如表4所示。将A型明胶和海藻酸钙微凝胶颗粒分别分散在10mM的醋酸酸性水溶液中，分别得到带正电荷的A型明胶微凝胶颗粒的分散液Ⅰ、带负电荷的海藻酸微凝胶颗粒的分散液Ⅱ，将分散液Ⅰ和分散液Ⅱ充分混合、搅拌，得分散液III，其中A型明胶和海藻酸钙微凝胶颗粒混合的颗粒数量比为2:1；向分散液III中加入100mM的氢氧化钠调节pH值至7.0，搅拌混合，冷冻干燥，得到混有两相不同胶体颗粒的冻干粉末。将上述微凝胶颗粒冻干粉末与一定体积的1mM的NaCl盐溶液共混，并快速搅拌混合均匀得到可注射型自愈合胶体凝胶，即本发明所述的止血材料，其中微凝胶胶体颗粒体积分数占胶体凝胶体积的50vol％或100vol％。使用流变仪对胶体凝胶的力学参数进行评价，结果如图3和表5所示，带相反电荷的海藻酸钙和A型明胶胶体颗粒共混得到的胶体凝胶的弹性模量随着胶体颗粒体积分数的增加而增加，体积分数100vol％时，储存(弹性)模量G’超过12kPa。剪切破坏后5分钟内自愈合效率同样随着胶体颗粒体积分数的增加而增加，体积分数100vol％时，弹性模量G’自愈合效率超过80％。
表4.实施例2中制备的A型明胶和海藻酸微凝胶的性能参数
Figure PCTCN2018086502-appb-000005
表5.实施例2中制备的自愈合胶体凝胶在不同胶体体积分数情况下的力学强度 (流变测试弹性模量G')和自愈合效率。
Figure PCTCN2018086502-appb-000006
*注：自修复效率是采用1000％的应变持续你剪切凝胶材料60s后，检测应力释放后5min内的弹性模量恢复的百分比(％)。
实施例3
以B型明胶为原料，在加热40℃下溶解，配置得到浓度为5w/v％的B型明胶水溶液，调节pH值为3，随后向溶液中加入3倍体积的乙醇，生成B型明胶微凝胶颗粒悬浮液；向悬浮液中加入25wt％戊二醛水溶液，使明胶微凝胶颗粒交联，加入戊二醛的量为每g明胶对应25wt％戊二醛80μL，交联反应时间12hr，反复离心并在去离子水中重悬，得到B型明胶微凝胶颗粒，颗粒尺寸和表面zeta点位参数如表6所示。
将壳聚糖季铵盐溶于去离子水配置浓度为2.5w/v％壳聚糖季铵盐水溶液。将明胶B微凝胶颗粒分散在20mM的醋酸酸性水溶液中，进一步将壳聚糖季铵盐水溶液与明胶B微凝胶颗粒分散液充分共混，搅拌，得分散液III，其中B型明胶微凝胶颗粒与高分子壳聚糖季铵盐的质量比为10:1，向分散液III中加入100mM的氢氧化钠调节pH值至7.0，然后冷冻干燥，得到B型明胶胶体颗粒和壳聚糖季铵盐的复合冻干粉末。将上述冻干粉末与一定体积的10mM的NaCl盐溶液共混，并快速搅拌混合均匀得到可注射型自愈合胶体凝胶，即本发明所述的止血材料，其中明胶微凝胶颗粒体积分数占自愈合凝胶体积分数为100vol％。使用流变仪对胶体凝胶的力学参数进行评价，结果如表7所示，带相反电荷的B型明胶颗粒和壳聚糖季铵盐共混得到的胶体凝胶在胶体颗粒体积分数为100vol％时，储存(弹性)模量G’约48kPa。剪切破坏后5分钟内自愈合效率在胶体颗粒体积分数为100vol％时，弹性模量G’自愈合效率超过89％。
表6.实施例3中制备的B型明胶微凝胶颗粒的性能参数
Figure PCTCN2018086502-appb-000007
表7.实施例3中制备的B型明胶微凝胶颗粒和壳聚糖季铵盐共混制备的自愈合凝胶的力学强度(流变测试弹性模量G')和自愈合效率。
Figure PCTCN2018086502-appb-000008
*注：自修复效率是采用1000％的应变持续你剪切凝胶材料60s后，检测应力释放后5min内的弹性模量恢复的百分比(％)。
实施例4
以A型明胶为原料，在加热40℃下溶解，配置得到浓度为10w/v％的A型明胶水溶液，调节pH值为11，随后向溶液中各加入2倍体积的乙醇，生成A型明胶微凝胶颗粒悬浮液；向悬浮液中加入25wt％戊二醛水溶液，使明胶微凝胶颗粒交联，加入戊二醛的量为每g明胶对应25wt％戊二醛264μL，交联反应时间12hr，反复离心并在去离子水中重悬，得到A型明胶微凝胶颗粒，颗粒尺寸和表面zeta点位参数如表8所示。
将聚乙二醇(PEG，分子量2kDa)溶于去离子水配置浓度为5w/v％PEG水溶液。将上述方法制备的A型明胶微凝胶颗粒分散得到的PEG水溶液中，进一步将PEG水溶液与A型明胶微凝胶分散液充分共混，搅拌，得分散液III，其中A型明胶微凝胶颗粒与PEG的质量比为1:2。随后将分散液III的pH值调节至7.0，然后冷冻干燥，得到A型明胶胶体颗粒和PEG的复合冻干粉末。将上述冻干粉末与一定体积的10mM的NaCl盐溶液共混，并快速搅拌混合均匀得到自愈合胶体凝胶，即本发明所述的止血材料，其中明胶微凝胶颗粒体积分数占自愈合凝胶体积分数为100vol％。使用流变仪对胶体凝胶的力学参数进行评价，结果如表9所示，当胶体凝胶中A型明胶胶体颗粒体积分数为100vol％时，储存(弹性)模量G’约19kPa。剪切破坏后5分钟内自愈合效率在胶体颗粒体积分数为100vol％时，弹性模量G’自愈合效率约为83％。
表8.实施例5中制备的A型明胶微凝胶颗粒的性能参数
Figure PCTCN2018086502-appb-000009
表9.实施例4中制备的A型明胶微凝胶颗粒和PEG共混制备的自愈合凝胶的力 学强度(流变测试弹性模量G')和自愈合效率。
Figure PCTCN2018086502-appb-000010
*注：自修复效率是采用1000％的应变持续你剪切凝胶材料60s后，检测应力释放后5min内的弹性模量恢复的百分比(％)。
实施例5
以A型明胶为原料，在加热40℃下溶解，配置得到浓度为5w/v％的A型明胶水溶液，调节pH值为11，随后向溶液中各加入3.5倍体积的乙醇，生成A型明胶微凝胶颗粒悬浮液；随后加入25wt％戊二醛水溶液交联明胶微凝胶颗粒，加入戊二醛的量为每g明胶对应25wt％戊二醛66μL，交联反应时间12hr，离心清洗得到A型明胶微凝胶颗粒，颗粒尺寸和表面zeta点位参数如表10所示。
通过乳液法制备海藻酸微凝胶颗粒，具体制备方法如下：将1wt％的海藻酸钠的水溶液加入氯化钙水溶液中并持续高速搅拌(搅拌速度>5000rpm)，即得到海藻酸钙颗粒，颗粒尺寸和表面zeta点位参数如表10所示。
将A型明胶和海藻酸钙微凝胶颗粒分别分散在10mM的醋酸酸性水溶液中，分别得到带正电荷的A型明胶微凝胶颗粒的分散液Ⅰ、带负电荷的海藻酸微凝胶颗粒的分散液Ⅱ，将分散液Ⅰ和分散液Ⅱ充分混合、搅拌，得分散液III，其中A型明胶和海藻酸微凝胶颗粒混合的颗粒数量比为2:1；向分散液III中加入100mM的氢氧化钠调节pH值至7.0，搅拌混合，冷冻干燥，得到混有两相不同胶体颗粒的复合冻干粉末。将聚乙二醇(PEG，分子量2kDa)溶于去离子水配置浓度为5w/v％PEG水溶液，与上述复合冻干粉末共混，并快速搅拌得到自愈合凝胶，即本发明所述的止血材料，其中两种胶体颗粒体积分数占胶体凝胶体积的100vol％。使用流变仪考察胶体凝胶的力学参数，结果如表11所示，当胶体颗粒体积分数100vol％时，储存(弹性)模量G’超过87kPa。剪切破坏后5分钟内自愈合效率同样随着胶体颗粒体积分数的增加而增加，体积分数100vol％时，弹性模量G’自愈合效率超过91％。
表10.实施例5中制备的A型明胶和海藻酸钙微凝胶的性能参数
Figure PCTCN2018086502-appb-000011
Figure PCTCN2018086502-appb-000012
表11.实施例5中制备的自愈合胶体凝胶在不同胶体体积分数情况下的力学强度(流变测试弹性模量G')和自愈合效率。
Figure PCTCN2018086502-appb-000013
*注：自修复效率是采用1000％的应变持续你剪切凝胶材料60s后，检测应力释放后5min内的弹性模量恢复的百分比(％)。
实施例6
以A型明胶为原料，通过实施例1所述反溶剂法制备A型明胶微凝胶颗粒，戊二醛交联浓度为每g明胶使用25wt％戊二醛的量为66μL，制备得到带正电荷的A型明胶微凝胶颗粒；以B型明胶为原料，通过实施例1所述的反溶剂法制备B型明胶微凝胶颗粒，其中戊二醛交联浓度为每g明胶使用25wt％戊二醛的量为264μL，制备得到带负电荷的B型明胶微凝胶颗粒，制备参数和所得微凝胶颗粒尺寸和zeta点位数据如表12。
表12.实施例6中制备的不同类型明胶微凝胶颗粒的性能参数
Figure PCTCN2018086502-appb-000014
将A型明胶颗粒分散在含100ng/ml浓度的碱性成纤维生长因子(bFGF)的水溶液中，将B型明胶颗粒分散在含100ng/ml浓度的骨形态发生蛋白-2(BMP-2)的水溶液中，分别得到载有bFGF的A型明胶颗粒和载有BMP-2的B型明胶微凝胶颗粒的分散液。将上述两种明胶颗粒分散液按照颗粒数比1:1充分混合，并冷冻干燥，得到载有不同生长因子的两种明胶颗粒冻干粉末。将上述微凝胶颗粒冻干粉末与一定体积的1mM的NaCl盐溶液共混，并快速搅拌混合均匀，得到载有不同生长因子的、可注射型自愈合胶体凝胶，其中微凝胶胶体颗粒体积分数占胶体凝胶体积的100vol％。两种不同的生长因子从胶体凝胶载体材料中体 外释放动力学的释放曲线如图4所示，载有bFGF的A型明胶由于交联度低降解速率较快，因此bFGF释放速率较快，载有BMP-2的B型明胶由于交联度高降解速率慢，因此BMP-2释放速率较为缓慢；结果表明本发明所述的胶体凝胶可实现不同生长因子药物的有序释放。
实施例7
通过实施1中的制备方法得到A型明胶和B型明胶微凝胶颗粒分散液，将两种明胶颗粒分别分散在20mM的盐酸水溶液中，分别得到分散有带正电荷的A型明胶微凝胶颗粒和带负电荷的B型明胶微凝胶颗粒的分散液，将两者充分混合、搅拌，得分散有两种不同微凝胶颗粒的分散液，其中A型明胶和B型明胶混合的颗粒数量比为1:1；向分散液中加入100mM的氢氧化钠调节pH值至7.0，搅拌混合，冷冻干燥，得到含有两种不同明胶胶体颗粒的冻干粉末。将上述混合物冻干粉末与磷酸缓冲溶液PBS共混，并快速搅拌混合均匀得到可注射型自愈合胶体凝胶，即本发明所述的止血材料，其中两种胶体颗粒体积分数占胶体凝胶体积的100vol％。以新西兰兔背部脊柱骨缺损出血创面为动物实验模型，制造2.5-4mm的出血创口，将明胶基止血凝胶通过普通医用注射器直接注射在出血创面，轻轻压迫1min后出血点被封闭，止血时间为1min。动物实验的过程如图5所示。

Claims (10)

  1. 一种可注射型自愈合止血材料，由如下方法制备得到：
    (1)以明胶为原料，使其在去离子水中加热溶解，配置浓度为1～10w/v％的明胶水溶液，调pH值为1-6或8-14，向溶液中加入>2倍溶液体积的极性有机溶剂，生成明胶微凝胶颗粒分散液，加入交联剂交联反应1～12h，离心、清洗得到明胶微凝胶颗粒；
    其中，所述明胶微凝胶颗粒的zeta电势为-30～+30mV，所述明胶微凝胶颗粒的直径为20nm～2μm；
    (2)将步骤(1)制备得到的表面zeta电势>+10mV的明胶微凝胶颗粒，分散在pH<5的酸性水溶液或pH>9的碱性水溶液中，得分散有带正电荷的明胶微凝胶颗粒的分散液，再与带负电荷的高分子颗粒分散液按照颗粒数比1:10～10:1共混，或者与等电点<6的带负电荷的亲水性高分子水溶液按照明胶微凝胶颗粒与亲水性高分子的质量比1:10～10:1共混，用pH调节剂调pH至7.0，冷冻干燥，得明胶微凝胶颗粒冻干粉末I；
    (3)将步骤(1)制备得到的表面zeta电势<-10mV的明胶微凝胶颗粒，分散在pH<5的酸性水溶液或pH>9的碱性水溶液中，得明胶微凝胶颗粒的分散液，再与带正电荷的高分子颗粒分散液按照颗粒数比1:10～10:1混合，或者与等电点>8的带正电荷的亲水性高分子水溶液按照明胶微凝胶颗粒与亲水性高分子的质量比1:10～10:1共混，用pH调节剂调pH至7.0，冷冻干燥，得明胶微凝胶颗粒冻干粉末II；
    (4)将步骤(1)制备得到的表面zeta电势为-10～+10mV的明胶微凝胶颗粒分散在中性水溶液中，再与另一种表面zeta电势在-10～+10mV的高分子颗粒分散液按照颗粒数比1:10～10:1共混，或者与等电点6～8的亲水性高分子水溶液按照明胶微凝胶颗粒与亲水性高分子的质量比1:10～10:1共混，冷冻干燥，得明胶微凝胶颗粒冻干粉末Ⅲ；
    (5)将明胶微凝胶颗粒冻干粉末I、明胶微凝胶颗粒冻干粉末II或明胶微凝胶颗粒冻干粉末Ⅲ分别与水性溶液共混，得可注射型自愈合止血材料；
    其中，所述的带正电荷的高分子颗粒的表面电荷为+5～+60mV，带负电荷的高分子颗粒的表面电荷为-5～-60mV，所述高分子颗粒的直径为100nm～20μm，在步骤(5)中得到的可注射型自愈合止血材料中胶体颗粒占总体积的百分比为50vol％～150vol％；在步骤(3)、(4)和(5)中所述的亲水性高分子的分子 量为1k～500kDa。
  2. 根据权利要求1所述的可注射型自愈合止血材料，其特征在于，所述的带正电荷的高分子颗粒分散液以壳聚糖、A型明胶、聚丙烯酰胺、聚(N-异丙基丙烯酰胺)、聚乙烯亚胺中的一种或几种作为原料制备得到，所述的带负电荷的高分子颗粒分散液以透明质酸、海藻酸、A型明胶、B型明胶或聚丙烯酸中的一种或几种作为原料制备得到，所述的表面zeta电势在-10～+10mV的高分子颗粒分散液以胶原蛋白、白蛋白、明胶、聚乙烯醇、聚乙二醇中的一种或几种作为原料制备得到；所述的等电点>8的带正电荷的亲水性高分子为壳聚糖、A型明胶、聚丙烯酰胺、聚(N-异丙基丙烯酰胺)、聚乙烯亚胺中的一种或几种，所述等电点<6的带负电荷的亲水性高分子为透明质酸、海藻酸、A型明胶、B型明胶或聚丙烯酸中的一种或几种，所述的等电点6～8的高分子为胶原蛋白、白蛋白、明胶、聚乙烯醇、聚乙二醇中的一种或几种。
  3. 根据权利要求1所述的可注射型自愈合止血材料，其特征在于，在步骤(1)中所述的极性有机溶剂为甲醇、乙醇、异丙醇、丁醇、丙酮、乙腈、四氢呋喃中的一种或几种的组合；所述的交联剂为戊二醛、甘油醛、甲醛、碳二亚胺、二卤代烷、异氰酸酯、二异氰酸酯、谷氨酰胺转胺酶、京尼平中的一种或几种。
  4. 根据权利要求1所述的可注射型自愈合止血材料，其特征在于，在步骤(1)中所述的交联反应的反应体系中交联剂与明胶中氨基基团的摩尔比0.1～10。
  5. 根据权利要求1所述的可注射型自愈合止血材料，其特征在于，在步骤(2)和步骤(3)中所述的酸性水溶液和碱性水溶液中所含有的离子浓度均小于200mM。
  6. 根据权利要求1所述的可注射型自愈合止血材料，其特征在于，在步骤(2)和步骤(3)中所述的pH调节剂包括酸性物质和碱性物质，所述酸性物质为葡萄糖酸内酯、HCl、HNO 3、H 2SO 4中的一种或几种，所述碱性物质为尿素和脲酶的组合、或者氢氧化钠、氢氧化钙、氢氧化钾、氨水中的一种或几种。
  7. 根据权利要求1所述的可注射型自愈合止血材料，其特征在于，在步骤(5)中所述的水性溶液是离子浓度120～200mM、pH值为6～8的任意水溶液、亲水性高分子的水溶液、非水溶性的纳米颗粒分散液中的一种或几种的组合。
  8. 根据权利要求1所述的可注射型自愈合止血材料，其特征在于，所述水 性溶液中含有生物活性物质。
  9. 权利要求1～8的任一项所述的可注射型自愈合止血材料在制备用于体表组织、体腔内组织器官的有血创面的止血、防黏连、防感染、促进组织愈合和/或封闭伤口的止血制品中的应用。
  10. 根据权利要求1所述的应用，其特征在于，所述的止血制品为止血粉剂、止血颗粒、止血球、止血气雾剂、止血海绵、止血凝胶、止血膜、止血栓或止血贴。
PCT/CN2018/086502 2017-05-12 2018-05-11 一种可注射型自愈合止血材料及其制备方法和应用 WO2018205996A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201710335184.0 2017-05-12
CN201710335184.0A CN106983905B (zh) 2017-05-12 2017-05-12 一种可注射型自愈合止血材料及其制备方法和应用

Publications (1)

Publication Number Publication Date
WO2018205996A1 true WO2018205996A1 (zh) 2018-11-15

Family

ID=59419456

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/086502 WO2018205996A1 (zh) 2017-05-12 2018-05-11 一种可注射型自愈合止血材料及其制备方法和应用

Country Status (2)

Country Link
CN (1) CN106983905B (zh)
WO (1) WO2018205996A1 (zh)

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106983905B (zh) * 2017-05-12 2019-10-11 深圳华诺生物科技有限公司 一种可注射型自愈合止血材料及其制备方法和应用
JP7245235B2 (ja) * 2017-10-02 2023-03-23 アレオ ビーエムイー，インコーポレイテッド 水系組織接着剤
CN108310448B (zh) * 2018-02-07 2021-03-02 广州迈普再生医学科技股份有限公司 一种流体明胶止血材料的制备方法
CN109438727B (zh) * 2018-10-17 2020-08-07 中国人民解放军总医院 一种荧光响应的自愈性水凝胶及其制备方法
CN111068111B (zh) * 2018-10-19 2022-03-22 胡尚秀 可注射型自组装微球凝胶、其用途及制备方法
CN110527115B (zh) * 2019-08-01 2020-07-28 华南理工大学 多功能自修复水凝胶及其制备方法与在生物胺检测中的应用
CN110760076B (zh) * 2019-11-06 2022-04-08 大连理工大学 一种基于胶体颗粒-iPRF双网络结构的可注射、高强度复合水凝胶及其制备方法和应用
CN110743038B (zh) * 2019-11-06 2021-09-10 大连理工大学 一种双网络结构复合水凝胶及其制备方法和应用
CN114848668B (zh) * 2021-01-20 2024-03-26 香港中文大学 具有促进伤口愈合和快速止血功能的组合物
CN113134113B (zh) * 2021-04-07 2022-07-01 赛克赛斯生物科技股份有限公司 可吸收止血流体明胶的制备方法及可吸收止血流体明胶
CN114028606B (zh) * 2021-10-26 2023-06-13 湘雅生物医药(湖州)有限公司 一种壳聚糖、鱼精蛋白抗菌止血微球及其制备方法
CN115845121A (zh) * 2022-12-07 2023-03-28 华南理工大学 止血、抗菌、促愈合的微凝胶组装体粉末及其制备方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102688525A (zh) * 2012-05-07 2012-09-26 东南大学 一种生物大分子水凝胶及其制备方法
CN104918606A (zh) * 2012-11-01 2015-09-16 乔治·D.·福卢什 对用于不可按压出血的组织封闭剂的改进
US20160121017A1 (en) * 2014-11-04 2016-05-05 Biomedica Management Corp SINGLE SOLUTION of Gel-LIKE FIBRIN HEMOSTAT
CN105796478A (zh) * 2016-03-22 2016-07-27 王华楠 由纳米胶体颗粒组装的、高强度、自修复、可注射复合胶体凝胶材料及其制备方法和应用
CN106983905A (zh) * 2017-05-12 2017-07-28 王华楠 一种可注射型自愈合止血材料及其制备方法和应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009533455A (ja) * 2006-04-12 2009-09-17 マサチューセッツ インスティテュート オブ テクノロジー 癒着を阻害するための組成物および方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102688525A (zh) * 2012-05-07 2012-09-26 东南大学 一种生物大分子水凝胶及其制备方法
CN104918606A (zh) * 2012-11-01 2015-09-16 乔治·D.·福卢什 对用于不可按压出血的组织封闭剂的改进
US20160121017A1 (en) * 2014-11-04 2016-05-05 Biomedica Management Corp SINGLE SOLUTION of Gel-LIKE FIBRIN HEMOSTAT
CN105796478A (zh) * 2016-03-22 2016-07-27 王华楠 由纳米胶体颗粒组装的、高强度、自修复、可注射复合胶体凝胶材料及其制备方法和应用
CN106983905A (zh) * 2017-05-12 2017-07-28 王华楠 一种可注射型自愈合止血材料及其制备方法和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SHARMA ANSHU ET AL.: "Self-healing Gelatin lonogels", INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, vol. 95, 28 November 2016 (2016-11-28), pages 603 - 307, XP055612176 *

Also Published As

Publication number Publication date
CN106983905A (zh) 2017-07-28
CN106983905B (zh) 2019-10-11

Similar Documents

Publication Publication Date Title
WO2018205996A1 (zh) 一种可注射型自愈合止血材料及其制备方法和应用
US11446409B2 (en) Agent for biological damage repair or hemostasis and the method thereof
CN107007881B (zh) 可用于药物加载和释放的可注射型自愈合凝胶及其制备方法和应用
WO2022160518A1 (zh) 一种两场耦合交联的、可注射、可塑形、可打印颗粒水凝胶材料及其制备方法和应用
Li et al. Emerging materials for hemostasis
US9694101B2 (en) Flowable collagen-based hemostat and methods of use
JP6235104B2 (ja) 止血組成物
Li et al. Preparation and the hemostatic property study of porous gelatin microspheres both in vitro and in vivo
AU2003231147B2 (en) Chemically activated carboxypolysaccharides and uses
CN114349990B (zh) 一种动态特性可调水凝胶及其制备方法与应用
CN110433327B (zh) 一种骨修复材料及其制备方法
EP2362789A2 (en) Tissue adhesive
JP2000186048A (ja) 止血材
CN112300418B (zh) 一种可粘附高效止血微球及其制备方法
CN114848668B (zh) 具有促进伤口愈合和快速止血功能的组合物
US20220054529A1 (en) Hemostatic agent and method of production thereof
JP6877360B2 (ja) 止血組成物
US20230041921A1 (en) Nano fibrous polyelectrolyte complex for rapid control of hemorrhage
Hu et al. Sodium alginate/carboxycellulose/polydopamine composite microspheres for rapid hemostasis of deep irregular wounds
WO2024131926A1 (zh) 蛋白质响应性自组装肽及其应用
WO2024140931A1 (zh) 多糖基高分子交联剂、多糖基生物材料及制备方法与应用
Pogorielov et al. Development of chitosan hemostatic sponges with different solvents and tranexamic acid
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
AU2022381171A1 (en) Synthetic surgical hemostat
CN117018268A (zh) 一种基于明胶颗粒的3d生物打印墨水的制备方法及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18797768

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18797768

Country of ref document: EP

Kind code of ref document: A1