CN109498832B - Crosslinked porous hemostatic microsphere and preparation method thereof - Google Patents
Crosslinked porous hemostatic microsphere and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a cross-linked porous hemostatic microsphere and a preparation method thereof. The starch particles prepared by the method have rough surfaces, porous structures, pores extending towards the interior of the microspheres, extremely large specific surface area and specific pore volume, high water absorption rate and high water absorption speed, and can play a role of a molecular sieve when being used as a hemostatic material to achieve the effect of immediate hemostasis.
Description
Technical Field
The invention belongs to the technical field of medical supplies, and relates to a starch-based cross-linked porous hemostatic microsphere and a preparation method thereof.
Background
Generally, blood wounds are formed in surgical operations and trauma, a large amount of blood is lost in the process, the operation time is prolonged due to excessive bleeding in the operation, serious complications such as hemorrhagic shock occur seriously, and even death of a patient is caused, so that a good hemostasis technology is a key for ensuring the success of the operation. The mechanical hemostasis is carried out on the wound surfaces of parenchymal organs with rich blood flow and large fragility, such as brain, liver, kidney, spleen and the like, so that the tissue cutting damage, the split bleeding or the needle eye bleeding are easily caused, and even the functions of important organs are influenced.
The hemostatic materials in the market mainly comprise collagen, oxidized regenerated cellulose, alginates, zeolite, chitosan and the like, and the hemostatic principles of various materials are different. At present, a blood factor concentration type hemostatic product for hemostasis by a molecular sieve water absorption mechanism is a hot point for research and development of hemostatic materials at home and abroad, and a part of products on the market play an extremely important role in clinic and military. QuikClot, manufactured by Z-Medica, USA, for example, is a powdered body composed of a porous zeolite composite. When this powder is applied directly to the bleeding site, a hemostatic scab forms rapidly on the wound surface, preventing the escape of internal blood. The porous structure of the physical hemostatic material gelatin sponge can absorb blood 45 times heavier than the gelatin sponge, activate platelets and promote the formation of clot, thereby achieving the purpose of rapid hemostasis. The absorbable starch hemostatic microspheres produced by Medador corporation in America have good safety and good hemostatic effect. However, the existing market products have some disadvantages, such as QuikClot produced by U.S. Z-medical company can emit a large amount of heat when in use, so that wound inflammation is caused, and wound tissues are damaged; the physical hemostatic material of the gelatin sponge may increase wound infection, and has poor adhesion and easy shedding; the absorbable starch hemostatic microspheres produced by Medador in the United states are expensive and have insufficient adhesion and liquid absorption as an internal hemostatic material, and particularly, an epichlorohydrin crosslinking agent can have potential toxicity.
In addition, Chinese patent application CN2011100567422 also discloses a starch hemostatic microsphere and a preparation method thereof, wherein natural starch is used as a raw material, the raw material of the starch is prepared into a starch mixed solution, then alkali liquor is activated, and a reverse suspension polymerization method is carried out to prepare the composite starch microsphere. The method comprises the following steps:
1) preparing starch alkaline solution: dissolving bean starch, potato starch and cereal starch in distilled water of which the weight is 5-20 times of that of the bean starch, the potato starch and the cereal starch according to the weight ratio of 1: 2-5, adjusting the pH value to 8.5-10.5 by using alkali liquor, and stirring to obtain an activated starch alkaline solution;
2) preparing a vegetable oil-starch mixed solution: mixing a surfactant and vegetable oil =1: 50-500 g/mL, stirring for 0.5-2 h in a water bath at 60-80 ℃, adding the mixture into the starch alkaline solution obtained in the step (1), and stirring and mixing for 0.5-4 h, wherein the volume ratio of the vegetable oil to the starch alkaline solution is 5-20: 1, so as to obtain a vegetable oil-starch mixed solution;
3) and (3) crosslinking treatment: dropwise adding a cross-linking agent into the solution prepared in the step (2), wherein the ratio of the amount of the cross-linking agent to starch is 1: 5-10 g/mL, the dropwise adding time is 1-4 h, a reaction system is formed, the reaction is carried out for 2-10 h, the temperature is 40-60 ℃, and the stirring speed is 300-1000 r/min;
4) refining microspheres: and (4) carrying out suction filtration on the reaction product obtained in the step (3) to obtain a solid, washing the solid for 2-5 times, drying, screening and sterilizing to obtain the starch hemostatic microspheres.
The starch microsphere prepared by the patent application CN2011100567422 has no porous structure on the surface, so the specific surface area is small, and the starch microsphere only has the characteristic of water absorption as a hemostatic material, and the hemostasis cannot be realized by utilizing the molecular sieve principle (molecular sieve hemostasis is that tangible components with the volume larger than the pore diameter of a pore channel in blood, such as platelet, erythrocyte, blood protein, thrombin, fibrin and the like, are gathered on the surface of particles to form a gelatinous mixture, so the immediate hemostasis effect is realized).
Disclosure of Invention
The invention aims to provide a cross-linked porous hemostatic microsphere which has the advantages of large specific surface area, good water absorption, molecular sieve hemostasis, good biocompatibility, low cost and easy obtainment, and provides a hemostatic method with quick hemostasis and convenient use for various wound and operation tissue wound bleeding areas as a blood factor concentrated hemostatic material.
Specifically, the invention adopts the following technical scheme:
the cross-linked porous hemostatic microsphere is characterized by being a cross-linked porous starch microsphere prepared from starch serving as a raw material, wherein the particle size of the starch microsphere is 20-150 mu m, and the specific surface area of the starch microsphere is 0.998-1.279 m2A pore diameter of 0.2 to 1.5 μm, and a water absorption of 850-.
Preferably, the starch raw material comprises one or a mixture of more than two of potato starch, tapioca starch, corn starch, wheat starch, rice starch, mung bean starch, pea starch or sorghum starch.
The invention also discloses a method for preparing the cross-linked porous hemostatic particles, which is characterized by comprising the following steps:
(1) starch solution gelatinization: weighing starch in a certain weight unit, adding 0.1-5% sodium hydroxide water solution in an amount which is 5-50 times of the weight of the starch, and gelatinizing;
(2) crosslinking of gelatinized starch: adding a cross-linking agent into the gelatinized starch, and stirring for reaction for 0.1-2h, wherein the mass ratio of the cross-linking agent to the starch raw material is 0.05-2: 1;
(3) reverse suspension emulsification: mixing an emulsifier, namely an organic phase dispersant =1:10-120g/mL, fully stirring for 5-30min in a constant-temperature water bath kettle at 30-70 ℃, slowly injecting the gelatinized cross-linked starch solution in the step (2), and continuously stirring for 2-8h, wherein the volume ratio of the organic phase dispersant to the gelatinized cross-linked starch is 1-5: 1;
(4) washing and drying to obtain a starch microsphere intermediate: standing and layering the reaction product obtained in the step (3), pouring out an upper oil phase, adding a detergent, repeatedly washing, finally performing suction filtration, drying, screening and sterilization to obtain a starch microsphere intermediate;
(5) preparation of enzyme solution: weighing amylase with a certain weight unit, dissolving in a disodium hydrogen phosphate-citric acid buffer solution with the pH =2-7 to prepare 0.1-10% of amylase liquid, wherein the mass ratio of endo-amylase to exo-amylase (the endo-amylase comprises alpha-amylase, and the exo-amylase comprises beta-amylase, gamma-amylase, glucose glucoamylase and the like) is 1: 1-5;
(6) enzymolysis of a starch microsphere intermediate: and (3) placing the starch microsphere intermediate obtained in the step (4) into a disodium hydrogen phosphate-citric acid buffer solution with the pH =2-7 to prepare a 5-50% microsphere suspension, preheating in a constant-temperature water bath kettle at the temperature of 30-55 ℃ for 5-45min, then adding the amylase liquid prepared in the step (5), reacting in the constant-temperature water bath kettle at the temperature of 30-55 ℃ for 1-24h, finally adding a sodium hydroxide solution with the concentration of 4% until the reaction liquid is neutral, and stopping the reaction. Wherein the volume ratio of the amylase liquid to the microsphere suspension is 0.01-0.2: 1;
(7) washing and drying to obtain the cross-linked porous hemostatic microspheres: and (4) centrifuging the reaction product obtained in the step (6), adding a detergent into the precipitate for repeated washing, and finally performing suction filtration, drying, screening and sterilization to obtain the cross-linked porous hemostatic microspheres.
Preferably, the starch raw material used in step (1) comprises one or more than two of potato starch, tapioca starch, corn starch, wheat starch, rice starch, mung bean starch, pea starch or sorghum starch.
In addition, the cross-linking agent used in the step (2) comprises one or more than two of epichlorohydrin, sodium trimetaphosphate, sodium hexametaphosphate, sodium tripolyphosphate, phosphorus oxychloride and glutaraldehyde.
Further, the organic phase dispersant used in step (3) comprises one or more of liquid paraffin, dichloromethane, cyclohexane, soybean oil, peanut oil, olive oil or sesame oil.
Further preferably, the emulsifier used in step (3) comprises one or more of span 60, span 80, tween 60 and tween 80.
In addition, the enzyme used in step (5) includes one or more of α -amylase, β -amylase, γ -amylase, pullulanase and glucose-saccharifying enzyme.
In another preferred embodiment, the detergent used in the method comprises one or more of methanol, ethanol, acetone, ethyl acetate and petroleum ether.
Further, the drying method used in the method includes spray drying, freeze drying, vacuum drying or atmospheric drying.
Has the advantages that: the cross-linked porous hemostatic microsphere prepared by the invention has a rough surface, a porous structure and pores extending to the interior of the microsphere, so that the cross-linked porous hemostatic microsphere has a very large specific surface area and specific pore volume and high water absorption rate, can quickly absorb water in blood, promotes blood concentration and realizes the purpose of quick hemostasis; the surface of the cross-linked porous hemostatic microsphere has a porous structure, and can play a role of a molecular sieve when used as a hemostatic material, and visible components such as platelets, erythrocytes, blood proteins, thrombin, fibrin and the like with the volume larger than the pore diameter of a pore passage in blood are gathered on the surface of particles to form a gelatinous mixture, so that the effect of immediate hemostasis is achieved; the raw materials selected by the cross-linked porous hemostatic microspheres are plant sources, so that the safety is good, the price is low, and the obtained cross-linked porous hemostatic microspheres are safe, effective and good in biocompatibility; the hemostatic material as a blood factor concentrated hemostatic material provides a hemostatic method with rapid hemostasis and convenient use for the hemorrhage areas of wound surfaces of various wounds and surgical tissues.
Drawings
FIG. 1: the starch microsphere intermediate has the grain diameter of 20-150 mu m, and the surface is uneven, so that adsorption sites are provided for enzymolysis;
FIG. 2: the local detail photo of the starch microsphere intermediate shows that the surface roughness of the microsphere is high, and an adsorption site is provided for enzymolysis;
FIG. 3: the cross-linked porous hemostatic microspheres have the particle size of 20-150 mu m, the surfaces of the microspheres have porous structures, and pores extend towards the interior of the microspheres;
FIG. 4: a photograph of a detail of a cross-linked porous hemostatic microsphere reveals the internal porosity of the microparticle.
Detailed Description
The invention relates to a starch-based hemostatic material, which is prepared by taking natural starch as a raw material, preparing a starch mixed solution from the starch raw material, carrying out alkali liquor gelatinization and crosslinking treatment, preparing a starch microsphere intermediate by a reverse suspension method, and carrying out enzymolysis treatment on the starch microsphere intermediate to obtain a crosslinked porous hemostatic microsphere.
The method comprises the following steps:
(1) starch solution gelatinization: weighing starch in a certain weight unit, adding 0.1-5% sodium hydroxide water solution in an amount which is 5-50 times of the weight of the starch, and gelatinizing;
(2) crosslinking of gelatinized starch: adding a cross-linking agent into the gelatinized starch, and stirring for reacting for 0.1-2h, wherein the mass ratio of the cross-linking agent to the starch raw material is 0.05-2: 1;
(3) reverse suspension emulsification: mixing an emulsifier, namely an organic phase dispersant =1:10-120g/mL, fully stirring for 5-30min in a constant-temperature water bath kettle at 30-70 ℃, slowly injecting the gelatinized cross-linked starch solution in the step (2), and continuously stirring for 2-8h, wherein the volume ratio of the organic phase dispersant to the gelatinized cross-linked starch is 1-5: 1;
(4) washing and drying to obtain a starch microsphere intermediate: standing and layering the reaction product obtained in the step (3), pouring out an upper oil phase, adding a detergent, repeatedly washing, finally performing suction filtration, drying, screening and sterilization to obtain a starch microsphere intermediate;
(5) preparing amylase liquid: weighing amylase with a certain weight unit, and dissolving in disodium hydrogen phosphate-citric acid buffer solution with pH =2-7 to obtain 0.1-10% amylase solution;
(6) enzymolysis of a starch microsphere intermediate: and (3) placing the starch microsphere intermediate obtained in the step (4) into a disodium hydrogen phosphate-citric acid buffer solution with the pH =2-7 to prepare a 5-50% microsphere suspension, preheating in a constant-temperature water bath kettle at the temperature of 30-55 ℃ for 5-45min, then adding the amylase liquid prepared in the step (5), reacting in the constant-temperature water bath kettle at the temperature of 30-55 ℃ for 1-24h, finally adding a sodium hydroxide solution with the concentration of 4% until the reaction liquid is neutral, and stopping the reaction. Wherein the volume ratio of the amylase liquid to the microsphere suspension is 0.01-0.2: 1;
(7) washing and drying to obtain the cross-linked porous hemostatic microspheres: and (4) centrifuging the reaction product obtained in the step (6), adding a detergent into the precipitate for repeated washing, and finally performing suction filtration, drying, screening and sterilization to obtain the cross-linked porous hemostatic microspheres.
The cross-linked porous hemostatic microsphere of the invention comprises the following raw materials of starch: potato starch, tapioca starch, corn starch, wheat starch, rice starch, mung bean starch, pea starch or sorghum starch.
The cross-linked porous hemostatic microsphere of the invention comprises the following cross-linking agents: one or more than two of epichlorohydrin, sodium trimetaphosphate, sodium hexametaphosphate, sodium tripolyphosphate, phosphorus oxychloride and glutaraldehyde.
The cross-linked porous hemostatic microsphere of the invention comprises the following organic phase dispersant: liquid paraffin, methylene chloride, cyclohexane, soybean oil, peanut oil, olive oil or sesame oil.
The cross-linked porous hemostatic microsphere of the invention comprises the following emulsifiers: span 60, span 80, tween 60 and tween 80.
The above-mentioned cross-linked porous hemostatic microsphere of the present invention, wherein the amylase comprises: one or more than two of alpha-amylase, beta-amylase, gamma-amylase, pullulanase and glucose saccharifying enzyme.
The above-mentioned cross-linked porous hemostatic microsphere of the present invention comprises: one or more of methanol, ethanol, acetone, ethyl acetate and petroleum ether.
The above-mentioned cross-linked porous hemostatic microsphere of the present invention comprises the following steps: spray drying, freeze drying, vacuum drying or drying under normal pressure.
The cross-linked porous hemostatic microspheres have the advantages that the selected raw materials are plant sources, the safety is good, the sources are wide, the price is low, and the obtained cross-linked porous hemostatic microspheres are safe, effective and good in biocompatibility. Can be used for hemostasis in surgical operation, and can be directly sprayed on blood wound of human, mammal, etc. for hemostasis.
The action mechanism of the invention is as follows: the cross-linked porous hemostatic microspheres are dry, sterile and white powder, the surfaces of the microspheres are rough and have porous structures, and pores extend into the microspheres, so the cross-linked porous hemostatic microspheres have extremely large specific surface area and specific pore volume, and high water absorption rate, can quickly absorb water in blood, promote blood concentration and realize the purpose of quick hemostasis; the surface of the cross-linked porous hemostatic microsphere has a porous structure, and can play a role of a molecular sieve when used as a hemostatic material, and visible components such as platelets, erythrocytes, blood proteins, thrombin, fibrin and the like with the volume larger than the pore diameter of a pore passage in blood are gathered on the surface of particles to form a gelatinous mixture, so that the effect of immediate hemostasis is achieved.
The present invention is further illustrated by the following specific examples.
EXAMPLE 1 preparation of crosslinked porous hemostatic microspheres
(1) Starch solution gelatinization: weighing 10.0g of corn starch, and adding 200mL of 2% sodium hydroxide aqueous solution for gelatinization;
(2) crosslinking of gelatinized starch: adding 3.0g of sodium trimetaphosphate into the gelatinized corn starch, and stirring for reaction for 10 min;
(3) reverse suspension emulsification: mixing 30.0g of span 80 with 600mL of soybean oil, fully stirring for 10min in a constant-temperature water bath kettle at 40 ℃, slowly injecting the gelatinized cross-linked starch solution in the step (2), and continuously stirring for 5 h;
(4) washing and drying to obtain a starch microsphere intermediate: standing and layering the reaction product obtained in the step (3), pouring out an upper oil phase, adding ethanol for repeated washing, and finally performing suction filtration, vacuum drying, screening and sterilization to obtain a starch microsphere intermediate;
(5) preparing amylase liquid: weighing 250mg of alpha-amylase and 500mg of glucose saccharifying enzyme, dissolving in a 10mL volumetric flask by using a disodium hydrogen phosphate-citric acid buffer solution with pH =5.0, fixing the volume, and shaking up;
(6) enzymolysis of a starch microsphere intermediate: weighing 15g of the starch microsphere intermediate product obtained in the step (4), placing the starch microsphere intermediate product into a disodium hydrogen phosphate-citric acid buffer solution with the pH =5.0 to prepare 150mL of 10% microsphere suspension, preheating the suspension in a 45 ℃ constant-temperature water bath for 10min, then adding 15mL of amylase solution prepared in the step (5), reacting for 5h in the 45 ℃ constant-temperature water bath, finally adding a 4% sodium hydroxide solution until the reaction solution is neutral, and stopping the reaction.
(7) Washing and drying to obtain the cross-linked porous hemostatic microspheres: and (4) centrifuging the reaction product obtained in the step (6), adding ethanol into the precipitate for repeated washing, and finally performing suction filtration, spray drying, screening and sterilization to obtain the cross-linked porous hemostatic microspheres.
Example 2: preparation method of crosslinked porous hemostatic microspheres
(1) Starch solution gelatinization: weighing 10.0g of potato starch, adding 200mL of 1% sodium hydroxide aqueous solution for gelatinization;
(2) crosslinking of gelatinized starch: adding 10.0g of sodium trimetaphosphate into the gelatinized potato starch, and stirring to react for 30 min;
(3) reverse suspension emulsification: mixing 30.0g of span 80 with 400mL of liquid paraffin, fully stirring for 30min in a constant-temperature water bath kettle at 55 ℃, slowly injecting the gelatinized cross-linked starch solution in the step (2), and continuously stirring for 5 h;
(4) washing and drying to obtain a starch microsphere intermediate: standing and layering the reaction product obtained in the step (3), pouring out an upper oil phase, sequentially adding methanol and petroleum ether, repeatedly washing, finally performing suction filtration, vacuum drying, screening and sterilizing to obtain a starch microsphere intermediate;
(5) preparing amylase liquid: weighing 250mg of alpha-amylase and 500mg of beta-amylase, dissolving the alpha-amylase and the beta-amylase in a 10mL volumetric flask by using a disodium hydrogen phosphate-citric acid buffer solution with pH =5.4, fixing the volume, and shaking up;
(6) enzymolysis of a starch microsphere intermediate: weighing 30g of the starch microsphere intermediate product obtained in the step (4), placing the product in a disodium hydrogen phosphate-citric acid buffer solution with the pH =5.4 to prepare 150mL of 20% microsphere suspension, preheating the suspension in a 50 ℃ constant-temperature water bath for 10min, then adding 15mL of amylase solution prepared in the step (5), reacting for 8h in the 50 ℃ constant-temperature water bath, finally adding a 4% sodium hydroxide solution until the reaction solution is neutral, and stopping the reaction.
(7) Washing and drying to obtain the cross-linked porous hemostatic microspheres: and (4) centrifuging the reaction product obtained in the step (6), sequentially adding methanol and petroleum ether into the precipitate for repeated washing, and finally performing suction filtration, spray drying, screening and sterilization to obtain the cross-linked porous hemostatic microspheres.
Example 3: preparation method of crosslinked porous hemostatic microspheres
(1) Starch solution gelatinization: weighing 10.0g of wheat starch, and adding 200mL of 1% sodium hydroxide aqueous solution for gelatinization;
(2) crosslinking of gelatinized starch: adding 16g of epoxy chloropropane into the gelatinized wheat starch, and stirring for reacting for 30 min;
(3) reverse suspension emulsification: mixing 20.0g of span 80 and 5g of Tween 60 with 200mL of cyclohexane, fully stirring for 30min in a constant-temperature water bath kettle at 50 ℃, slowly injecting the gelatinized cross-linked starch solution in the step (2), and continuously stirring for 8 h;
(4) washing and drying to obtain a starch microsphere intermediate: standing and layering the reaction product obtained in the step (3), pouring out an upper oil phase, sequentially adding petroleum ether and ethanol, repeatedly washing, finally performing suction filtration, drying under normal pressure, screening and sterilizing to obtain a starch microsphere intermediate;
(5) preparing amylase liquid: weighing 300mg of alpha-amylase and 450mg of gamma-amylase, dissolving the alpha-amylase and the gamma-amylase in a 10mL volumetric flask by using a disodium hydrogen phosphate-citric acid buffer solution with pH =4.6, fixing the volume, and shaking up;
(6) enzymolysis of a starch microsphere intermediate: weighing 30g of the starch microsphere intermediate product obtained in the step (4), placing the product in a disodium hydrogen phosphate-citric acid buffer solution with the pH =4.6 to prepare 150mL of 20% microsphere suspension, preheating the suspension in a constant-temperature water bath kettle at 40 ℃ for 10min, then adding 15mL of amylase solution prepared in the step (5), reacting in the constant-temperature water bath kettle at 40 ℃ for 8h, finally adding a sodium hydroxide solution with the concentration of 4% until the reaction solution is neutral, and stopping the reaction.
(7) Washing and drying to obtain the cross-linked porous hemostatic microspheres: and (4) centrifuging the reaction product obtained in the step (6), sequentially adding petroleum ether and ethanol into the precipitate, repeatedly washing, finally performing suction filtration, spray drying, screening and sterilization to obtain the cross-linked porous hemostatic microspheres.
Example 4: measurement of particle diameter and specific surface area
Measured using a Malvern Mastersizer2000, UK. Absolute ethyl alcohol is used as a dispersing agent, automatic analysis is carried out according to the principle of a laser diffraction method, and the results are shown in table 1 after automatic processing and analysis by computer software.
TABLE 1 particle size and specific surface area of crosslinked porous hemostatic microspheres
| Volume average particle diameter (μm) | d（0.5）（μm） | Specific surface area (m)2/g） | |
| Crosslinked porous hemostatic microspheres | 119.234 | 73.485 | 1.033 |
| Native starch | 14.748 | 8.524 | 0.403 |
The result shows that the particle size of the cross-linked porous hemostatic microsphere prepared by the invention is increased compared with that of the original starch, and the specific surface area is obviously improved.
Example 5: determination of pore size
Measured by scanning electron microscope using Hitachi SU8010 of Japan. The average of representative 50 pore sizes was taken and the results are shown in table 2.
TABLE 2 average pore diameter of crosslinked porous hemostatic microspheres
| Average pore diameter (μm) | |
| Crosslinked porous hemostatic microspheres | 0.859 |
The result shows that the average pore diameter of the cross-linked porous hemostatic microsphere prepared by the invention is 0.859 μm, which is far smaller than the average diameter (2-8 μm) of human platelets.
Example 6: water absorption test
Water absorption experiments were conducted on the products of examples 1-3, and the results are shown in Table 3.
0.1g of sample (m) is weighed1) About 10.0g of distilled water (m) was added2) Standing for swelling for 5min, filtering with 45 μm sieve, collecting the rest water, and weighing (m)3）。
Water absorption = (m)2-m3)/m1×100%
Table 3: water absorption test results of examples 1 to 3 (
±S，n=3）
The result shows that the water absorption of the cross-linked porous hemostatic microspheres prepared by the method is obviously improved compared with that of the original starch.
Example 7: animal experiments
The invention aims to evaluate the hemostatic effect of the crosslinked porous hemostatic microspheres on a mouse liver hemorrhage model.
Animal ICR mice (provided by Qinglong mountain animal breeding farm in Jianning district of Nanjing city) are 6 animals per group, 12 animals are in total, the weight is 18-22g, and the sex is not limited.
The tested medicine is the cross-linked porous hemostatic microsphere prepared by the invention.
The method comprises the following steps: mice were randomly grouped into 6 mice each, dosing and blank control groups. The mice were anesthetized by ether inhalation, mounted on a laboratory bench, abdomened layer by sterile procedure, the mouse liver was exposed, and a 1cm long wound was cut in the liver with a surgical blade. When the blood flows out, the mice in the administration group are immediately sprayed with the crosslinked porous hemostatic microspheres, the hemostatic condition is observed, and the hemostatic time is recorded. When blood flows out of the blank control group of mice, medical gauze is laid on the wound surface, the hemostasis condition is observed, and the hemostasis time is recorded. The results are shown in Table 4.
Compared with a blank control group, the wound hemostasis time of the mice in the administration group is obviously shortened, and the difference has statistical significance (P < 0.05).
Table 4: hemostasis test of crosslinked porous hemostatic microspheres on mouse liver hemorrhage model: (
±S，n=6）
| Group of | Hemostasis time(s) |
| Administration set | 4.0±1.55 |
| Blank control group | 29.3±0.82γγ |
And (4) conclusion: the cross-linked porous hemostatic microspheres prepared by the invention can obviously shorten the hemostatic time of the bleeding wound surface and have good hemostatic effect.
The cross-linked porous hemostatic microspheres prepared by the method have rough surfaces and porous structures, greatly increase the specific surface area of the microspheres, and have good promotion effect on the water absorption of the hemostatic microspheres. The surface of the cross-linked porous hemostatic microsphere prepared by the invention is distributed with micropores, and the cross-linked porous hemostatic microsphere can be used as a hemostatic material, and can effectively use a molecular sieve hemostatic mechanism to perform hemostasis, namely, the micropores on the surface of the cross-linked porous hemostatic microsphere can gather visible components with a volume larger than the pore diameter of a pore passage in blood, such as platelet, erythrocyte, blood protein, thrombin, fibrin and the like, on the surface of particles to form a gel mixture, so that the effect of immediate hemostasis is achieved. In addition, the cross-linked porous hemostatic microspheres prepared by the invention are prepared from plant-derived raw materials, and have the advantages of good safety, wide sources and low price, and the obtained cross-linked porous hemostatic microspheres are safe, effective and good in biocompatibility.
While the embodiments of the present invention have been described in detail with reference to the drawings and the specific examples, the present invention is not limited to the embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art.
Claims (9)
1. The cross-linked porous hemostatic microsphere is characterized by being a cross-linked porous starch microsphere prepared from starch serving as a raw material, wherein the particle size of the starch microsphere is 20-150 mu m, and the specific surface area of the starch microsphere is 0.998-1.279 m2A pore diameter of 0.2 to 1.5 μm, a water absorption of 850-,
wherein the cross-linked porous hemostatic microsphere is prepared by the following method:
(1) starch solution gelatinization: weighing starch in a certain weight unit, adding 0.1-5% sodium hydroxide water solution in an amount which is 5-50 times of the weight of the starch, and gelatinizing;
(2) crosslinking of gelatinized starch: adding a cross-linking agent into the gelatinized starch, and stirring for reaction for 0.1-2h, wherein the mass ratio of the cross-linking agent to the starch raw material is 0.05-2: 1;
(3) reverse suspension emulsification: mixing an emulsifier, namely an organic phase dispersant =1:10-120g/mL, fully stirring for 5-30min in a constant-temperature water bath kettle at 30-70 ℃, slowly injecting the gelatinized cross-linked starch solution in the step (2), and continuously stirring for 2-8h, wherein the volume ratio of the organic phase dispersant to the gelatinized cross-linked starch is 1-5: 1;
(4) washing and drying to obtain a starch microsphere intermediate: standing and layering the reaction product obtained in the step (3), pouring out an upper oil phase, adding a detergent, repeatedly washing, finally performing suction filtration, drying, screening and sterilization to obtain a starch microsphere intermediate;
(5) preparation of enzyme solution: weighing amylase in a certain weight unit, dissolving the amylase in a disodium hydrogen phosphate-citric acid buffer solution with the pH =2-7 to prepare 0.1-10% of amylase liquid, wherein the mass ratio of endo-amylase to exo-amylase is 1:1-5, the endo-amylase comprises alpha-amylase, and the exo-amylase comprises one or more than two of beta-amylase, gamma-amylase, pullulanase and glucose glucoamylase;
(6) enzymolysis of a starch microsphere intermediate: placing the starch microsphere intermediate obtained in the step (4) into a disodium hydrogen phosphate-citric acid buffer solution with the pH =2-7 to prepare a 5-50% microsphere suspension, preheating the microsphere suspension in a constant-temperature water bath kettle at the temperature of 30-55 ℃ for 5-45min, then adding the amylase liquid prepared in the step (5), reacting for 1-24h in the constant-temperature water bath kettle at the temperature of 30-55 ℃, finally adding a sodium hydroxide solution with the concentration of 4% until the reaction liquid is neutral, and stopping the reaction, wherein the volume ratio of the amylase liquid to the microsphere suspension is 0.01-0.2: 1;
(7) washing and drying to obtain the cross-linked porous hemostatic microspheres: and (4) centrifuging the reaction product obtained in the step (6), adding a detergent into the precipitate for repeated washing, and finally performing suction filtration, drying, screening and sterilization to obtain the cross-linked porous hemostatic microspheres.
2. The crosslinked porous hemostatic microsphere of claim 1, wherein the starch material comprises one or a mixture of two or more of potato starch, tapioca starch, corn starch, wheat starch, rice starch, mung bean starch, pea starch, or sorghum starch.
3. A method of making crosslinked porous hemostatic microparticles, comprising the steps of:
(1) starch solution gelatinization: weighing starch in a certain weight unit, adding 0.1-5% sodium hydroxide water solution in an amount which is 5-50 times of the weight of the starch, and gelatinizing;
(2) crosslinking of gelatinized starch: adding a cross-linking agent into the gelatinized starch, and stirring for reaction for 0.1-2h, wherein the mass ratio of the cross-linking agent to the starch raw material is 0.05-2: 1;
(3) reverse suspension emulsification: mixing an emulsifier, namely an organic phase dispersant =1:10-120g/mL, fully stirring for 5-30min in a constant-temperature water bath kettle at 30-70 ℃, slowly injecting the gelatinized cross-linked starch solution in the step (2), and continuously stirring for 2-8h, wherein the volume ratio of the organic phase dispersant to the gelatinized cross-linked starch is 1-5: 1;
(4) washing and drying to obtain a starch microsphere intermediate: standing and layering the reaction product obtained in the step (3), pouring out an upper oil phase, adding a detergent, repeatedly washing, finally performing suction filtration, drying, screening and sterilization to obtain a starch microsphere intermediate;
(5) preparation of enzyme solution: weighing amylase in a certain weight unit, dissolving the amylase in a disodium hydrogen phosphate-citric acid buffer solution with the pH =2-7 to prepare 0.1-10% of amylase liquid, wherein the mass ratio of endo-amylase to exo-amylase is 1:1-5, the endo-amylase comprises alpha-amylase, and the exo-amylase comprises one or more than two of beta-amylase, gamma-amylase, pullulanase and glucose glucoamylase;
(6) enzymolysis of a starch microsphere intermediate: placing the starch microsphere intermediate obtained in the step (4) into a disodium hydrogen phosphate-citric acid buffer solution with the pH =2-7 to prepare a 5-50% microsphere suspension, preheating the microsphere suspension in a constant-temperature water bath kettle at the temperature of 30-55 ℃ for 5-45min, then adding the amylase liquid prepared in the step (5), reacting for 1-24h in the constant-temperature water bath kettle at the temperature of 30-55 ℃, finally adding a sodium hydroxide solution with the concentration of 4% until the reaction liquid is neutral, and stopping the reaction, wherein the volume ratio of the amylase liquid to the microsphere suspension is 0.01-0.2: 1;
(7) washing and drying to obtain the cross-linked porous hemostatic microspheres: and (4) centrifuging the reaction product obtained in the step (6), adding a detergent into the precipitate for repeated washing, and finally performing suction filtration, drying, screening and sterilization to obtain the cross-linked porous hemostatic microspheres.
4. The method of preparing crosslinked porous hemostatic microparticles according to claim 3, wherein the starch material used in step (1) comprises one or more of potato starch, tapioca starch, corn starch, wheat starch, rice starch, mung bean starch, pea starch, or sorghum starch.
5. The method for preparing crosslinked porous hemostatic microparticles according to claim 3, wherein the crosslinking agent used in step (2) comprises one or more of epichlorohydrin, sodium trimetaphosphate, sodium hexametaphosphate, sodium tripolyphosphate, phosphorus oxychloride, and glutaraldehyde.
6. The method for preparing crosslinked porous hemostatic microparticles according to claim 3, wherein the organic phase dispersant used in step (3) comprises one or more of liquid paraffin, dichloromethane, cyclohexane, soybean oil, peanut oil, olive oil or sesame oil.
7. The method for preparing crosslinked porous hemostatic microparticles according to claim 3, wherein the emulsifier used in step (3) comprises one or more of span 60, span 80, tween 60 and tween 80.
8. The method of preparing crosslinked porous hemostatic microparticles according to claim 3, wherein the detergent used in the method comprises one or more of methanol, ethanol, acetone, ethyl acetate, and petroleum ether.
9. The method of making crosslinked porous hemostatic microparticles according to claim 3, wherein the drying method used in the method comprises spray drying, freeze drying, vacuum drying or atmospheric drying.
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