CN112472866B - Preparation method of starch microsphere and human recombinant tissue factor lipidated compound hemostatic material - Google Patents
Preparation method of starch microsphere and human recombinant tissue factor lipidated compound hemostatic material Download PDFInfo
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- CN112472866B CN112472866B CN202011481864.1A CN202011481864A CN112472866B CN 112472866 B CN112472866 B CN 112472866B CN 202011481864 A CN202011481864 A CN 202011481864A CN 112472866 B CN112472866 B CN 112472866B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
- A61L24/08—Polysaccharides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/001—Use of materials characterised by their function or physical properties
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/001—Use of materials characterised by their function or physical properties
- A61L24/0042—Materials resorbable by the body
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/04—Materials for stopping bleeding
Abstract
A preparation method of a starch microsphere and human recombinant tissue factor lipidated compound hemostatic material comprises the steps of dissolving potato starch in deionized water, mixing the potato starch with emulsified soybean oil according to the mass ratio of 1:2.0-1:2.5 of water phase to oil phase solution, and forming starch microspheres with mutually condensed hydroxyl groups under the action of an initiator; further dispersing the obtained starch microspheres in anhydrous 1, 4-dioxane, modifying by N, N' -disuccinimidyl carbonate (DSC), adding human recombinant tissue factor lipidated substance, combining the tissue factor and the starch microspheres in a covalent bonding mode, centrifuging, cleaning and drying in vacuum to obtain the starch microsphere and human recombinant tissue factor lipidated substance composite hemostatic material, and realizing rapid blood coagulation effect by utilizing the starting blood coagulation function of the tissue factor.
Description
Technical Field
The field relates to a hemostatic compound, which is a rapid hemostatic dry powder material for external use.
Background
Accidental bleeding, as well as surgical bleeding, can cause shock and even death due to improper handling. Clinical cases caused by excessive blood loss account for a large proportion, hemostasis is a core link of treatment, and a good hemostasis technology is a successful guarantee of the link. The development and improvement of hemostatic materials have become the research focus in the field of modern medicine, and with the progress of medical technology, novel medical materials and related technologies have been developed well.
The effective hemostatic material adopts absorbable material with good biocompatibility to stop bleeding, and has high safety, no allergy and no irritation. At present, hemostatic materials in the market mainly include fibrin glue, collagen, oxidized cellulose hemostatic gauze, chitosan hemostatic sponge, starch polysaccharide or chitosan hemostatic powder, and the like. The materials have good effects in animal experiments and clinical application, but have some defects, the gelatin is absorbed slowly to increase the infection risk of wounds, the animal-derived heterologous protein has potential allergenicity, the cellulose materials have long degradation time and poor adhesion and can cause re-bleeding, the Arista absorbable starch hemostatic microspheres produced by Medador corporation in America have complex preparation process, high price and great popularization difficulty, the pure chitosan materials have limited hemostatic effect, and the pure chitosan materials have poor adhesion and slow hemostasis for wounds with large bleeding amount. Therefore, the development of a novel hemostatic material which has the characteristics of rapid hemostasis, no animal source, good biocompatibility, no toxicity and the like becomes a necessity for the development of a hemostatic material.
The starch has good biocompatibility, is degradable, renewable and low in price and can be widely used in the medical field. The starch metabolism mechanism is clear, the biologically absorbable starch microspheres are prepared, and the human recombinant tissue factor lipidosome is quantitatively added, so that the hemostatic dressing has the characteristics of good biocompatibility and short hemostatic time, and is worthy of being developed. The hemostatic material prepared by combining the starch microspheres as the basis with the human recombinant tissue factor lipidosome has the advantages of convenient development and use and obvious effect, and is an ideal hemostatic dressing with short blood coagulation time.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide the composite hemostatic material which is prepared by quantitatively adding the human recombinant tissue factor lipidosome on the basis of the starch microspheres and has good biocompatibility, short hemostasis time and good effect and the preparation method thereof.
The preparation method of the starch microsphere and human recombinant tissue factor lipidated composite hemostatic material comprises the following steps:
(1) preparing an aqueous phase: adding potato starch into a deionized water solution, controlling the mass ratio of the potato starch in the deionized water to be 15-20%, adjusting the pH value to about 8 by using a NaOH solution, heating to 60-80 ℃, and cooling to room temperature after the starch is dissolved;
(2) preparing an oil phase: adding emulsifier into soybean oil, heating to 60 deg.C for complete dissolution, and cooling to room temperature;
(3) emulsification: slowly adding the starch solution obtained in the step (1) into the oil phase solution obtained in the step (2) according to the mass ratio of the water phase solution to the oil phase solution of 1:2.0-1:2.5, and fully stirring to obtain an emulsified mixed solution;
(4) adding an initiator into the emulsified mixed solution obtained in the step (3), continuously and fully stirring, and mutually condensing the hydroxyl groups of the starch under the action of the initiator to perform starch crosslinking to finally form a mixed solution containing starch microspheres;
(5) separating the mixed solution obtained in the step (4) by a centrifugal machine to obtain precipitates, sequentially washing the precipitates by ethyl acetate and ethanol, and further drying to obtain starch microspheres;
(6) dispersing the starch microspheres obtained in the step (5) in a proper amount of anhydrous 1, 4-dioxane, adding N, N' -disuccinimidyl carbonate (DSC), and reacting the DSC with the starch microspheres for 10 hours under the conditions of nitrogen protection, 4 ℃ and magnetic stirring, wherein the hydroxyl groups on the surfaces of the starch microspheres are modified by the DSC;
(7) and (3) adding a pH 7.4 Burriptan-Robinson buffer solution into the reaction solution obtained in the step (6), adding a human recombinant tissue factor lipidosome with the volume fraction of 0.3-0.5% into the buffer solution, continuing stirring for 12 hours at the temperature of 4 ℃ under the protection of nitrogen, centrifuging by using a centrifugal machine, discarding the supernatant, collecting the precipitate, sequentially washing the precipitate with ethanol and deionized water, and performing vacuum drying to obtain the final product, namely the starch microsphere and the human recombinant tissue factor lipidosome composite hemostatic dry powder material.
The mass of the starch microspheres in the step (6) is 15-20% of that of the anhydrous 1, 4-dioxane; the DSC dosage is 0.5-1% of the mass of the starch microspheres. The proportion of the human recombinant tissue factor lipidated substance to the starch microspheres in the step (7) is that 0.02 ml of human recombinant tissue factor lipidated substance is added into each gram of starch microspheres. The vacuum drying condition is 0.17 atmosphere and 40 ℃ for 6 h. The human recombinant tissue factor lipidosome is purchased from Taiyuan Boott Biotechnology Limited.
The emulsifier is Span60 (Span 60) with the mass being 4-8% of the mass of the soybean oil; or Tween (Tween 60) 9-10% of soybean oil. The initiator is epichlorohydrin, and the added mass is 6-7% of the using amount of starch; or sodium trimetaphosphate is added, and the mass of the sodium trimetaphosphate is 5-7% of the amount of the starch.
The potato starch has strong water absorption capacity, the specific surface area is increased after the starch is micro-spheroidized, the water absorption performance is improved, and simultaneously, the blood platelet and the blood cell can be gathered to improve the blood coagulation. The starch hemostatic microspheres prepared by the method have good biocompatibility, the degradation capability is remarkably improved, the large specific surface area can also increase the contact area of alpha-amylase, and the starch hemostatic microspheres are favorably degraded.
According to the invention, potato starch is prepared into starch microspheres by a reverse microemulsion method, and the tissue factor is combined with the starch microspheres in a covalent bonding manner, so that the relative physical adsorption is firmer, the blood coagulation starting function of the tissue factor is utilized, the thrombin is rapidly activated, and the physical action of the starch microspheres for aggregating platelets and erythrocytes is combined to prepare the hemostatic material, so that rapid blood coagulation is realized. The human recombinant tissue factor lipidosome is quantitatively added on the basis of the starch microspheres, so that the method has the effects of good biocompatibility, short hemostasis time and good effect, is simple in preparation process, and is expected to be widely applied to hemostasis by accidental bleeding.
Drawings
FIG. 1 is a graph showing the results of 24h and 72h tests on rabbit skin irritation test using the hemostatic material of the present invention.
Detailed Description
In order to illustrate the invention more clearly, the invention is further elucidated below with reference to a preferred embodiment and the accompanying drawing. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention, but rather includes any combination of the specific embodiments, which is calculated by those skilled in the art from the foregoing description and any equivalent variations which are within the spirit and scope of the invention as defined by the appended claims.
Example one
(1) Preparing water phase, adding 1.5 g of potato starch into 8 mL of deionized water solution, adjusting the pH value to about 8.0 by using NaOH solution, heating to 80 ℃, and cooling to room temperature after the starch is dissolved.
(2) The oil phase was prepared by placing 1 g Span60 into 20 mL soybean oil, heating to 60 deg.C for complete dissolution, and cooling to room temperature.
(3) Emulsifying, slowly adding the starch solution obtained in the step (1) into the oil phase solution obtained in the step (2), and fully stirring.
(4) And adding 0.10 g of sodium trimetaphosphate to complete starch crosslinking and form starch microspheres.
(5) And (4) centrifugally separating the solution in the step (4) to obtain a precipitate, sequentially washing the precipitate with ethyl acetate and ethanol, and drying to obtain the starch microspheres.
(6) Dispersing 1.5 g of the starch microspheres obtained in the step (5) in 10 mL of anhydrous 1, 4-dioxane solution, adding 0.015 g of DSC, and adding into N2And (4) protecting, magnetically stirring at 4 ℃ for 10 hours, and modifying the surface of the starch microsphere by DSC.
(7) 10 mL of pH 7.4 Burriptan-Robinson buffer containing 0.3% by volume fraction of human recombinant tissue factor lipidate was added to the reaction solution of step (6). Continue at N2Protecting, reacting for 12h at 4 ℃ to obtain a mixed solution of the starch microspheres and the human recombinant tissue factor lipidated covalent bonding material, centrifugally separating and precipitating, sequentially washing and precipitating with ethanol and deionized water, and drying in vacuum to obtain the starch microsphere and human recombinant tissue factor lipidated composite hemostatic dry powder material.
Example two
(1) Adding 20 g of potato starch into 100 mL of deionized water solution, adjusting the pH value to about 8 by using NaOH solution, heating to 80 ℃ until the starch is dissolved, and cooling to room temperature.
(2) 15 g of Span60 was placed in 240 mL of soybean oil, heated to 60 ℃ to completely dissolve, and cooled to room temperature.
(3) Slowly adding the starch solution obtained in the step (1) into the oil phase solution obtained in the step (2), and fully stirring.
(4) Adding 1 g of sodium trimetaphosphate, stirring for 5 hours to complete starch crosslinking, and forming starch microspheres.
(5) And (4) centrifugally separating the solution in the step (4) to obtain a precipitate, sequentially washing the precipitate with ethyl acetate and ethanol, and drying to obtain the starch microspheres.
（6）Dispersing 20 g of starch microspheres obtained in the step (5) in 100 mL of anhydrous 1, 4-dioxane solution, adding 0.2 g of DSC, and reacting in N2And (4) protecting, magnetically stirring at 4 ℃ for 10 hours, and modifying the surface of the starch microsphere by DSC.
(7) 100 mL of pH 7.4 Beriten-Robinson buffer solution containing a volume fraction of 0.5% of human recombinant tissue factor lipidate was added to the reaction solution of step (6). Continue at N2Protecting, reacting for 12h at 4 ℃ to obtain a covalent bonding material solution of the starch microspheres and the human recombinant tissue factor lipidosome, centrifugally separating and precipitating, sequentially washing and precipitating by using ethanol and deionized water, and drying in vacuum to obtain the starch microsphere and human recombinant tissue factor lipidosome composite hemostatic dry powder material.
Test 1: the hemostatic materials prepared in the above examples were subjected to an in vitro whole blood clotting time test: respectively weighing the hemostatic material prepared in the first embodiment of the invention, the hemostatic material prepared in the second embodiment of the invention and the same amount of potato starch, comparing, and uniformly scattering in 1cm2On a non-woven fabric. 0.5 ml of fresh human plasma is respectively measured and dripped on the non-woven fabric sprinkled with the hemostatic material. The stopwatch records plasma clotting time. The blood coagulation time and the effect are shown in table one, the blood coagulation time of the hemostatic material prepared by the invention is obviously shorter than that of potato starch, and the hemostatic effect is good (see table 1).
TABLE 1 Table of the results of the in vitro whole blood coagulation time test of the hemostatic materials of the present invention
And (3) testing 2: the hemostatic material prepared in the first embodiment of the invention is used for rabbit skin irritation experiments to verify the biocompatibility of the composite hemostatic material.
Experimental materials: the composite hemostatic material prepared according to the first embodiment, sterile normal saline, syringe and rabbit.
The experimental method comprises the following steps: the composite hemostatic material is accurately weighed and prepared into 5 percent, 10 percent and 20 percent solution by using normal saline. Two healthy rabbits were selected and normally raised for 3 days before the start of the experiment to adapt the animals to the environment, and before the experiment, the hairs on both sides of the spinal column of the rabbit were shaved off and observed as an injection area. 4 points are arranged on the left side of the spinal column for intradermal injection, parallel control is carried out at the same position on the right side, each point is injected with 0.1ml of injection, a control group is injected with 0.1ml of sterile physiological saline, and the conditions of each injection part are observed and recorded at 24h, 48h and 72 h.
The experimental results are as follows: the hemostatic material of the invention has no stimulation to rabbit skin and has good biocompatibility (see figure 1 and table 2).
TABLE 2 Rabbit skin irritation test results of the hemostatic material prepared in the first embodiment of the present invention
| Group of | Addition amount (ml) | Phenomenon(s) |
| Control group (sterile physiological saline) | 0.1 | No red swelling and |
| Hemostatic material | ||
| 5% solution | 0.1 | No red swelling and |
| Hemostatic material | ||
| 10% solution | 0.1 | No red swelling and |
| Hemostatic material | ||
| 20% solution | 0.1 | No red swelling and erythema |
Claims (6)
1. A preparation method of a starch microsphere and human recombinant tissue factor lipidated composite hemostatic material is characterized by comprising the following steps:
(1) preparing an aqueous phase: adding potato starch into a deionized water solution, controlling the mass ratio of the potato starch in the deionized water to be 15-20%, adjusting the pH value to about 8 by using a NaOH solution, heating to 60-80 ℃, and cooling to room temperature after the starch is dissolved;
(2) preparing an oil phase: adding emulsifier into soybean oil, heating to 60 deg.C for complete dissolution, and cooling to room temperature;
(3) emulsification: slowly adding the starch solution obtained in the step (1) into the oil phase solution obtained in the step (2) according to the mass ratio of the water phase solution to the oil phase solution of 1:2.0-1:2.5, and fully stirring to obtain an emulsified mixed solution;
(4) adding an initiator into the emulsified mixed solution obtained in the step (3), continuously and fully stirring, and mutually condensing the hydroxyl groups of the starch under the action of the initiator to perform starch crosslinking to finally form a mixed solution containing starch microspheres;
(5) separating the mixed solution obtained in the step (4) by a centrifugal machine to obtain precipitates, sequentially washing the precipitates by ethyl acetate and ethanol, and further drying to obtain starch microspheres;
(6) dispersing the starch microspheres obtained in the step (5) in a proper amount of anhydrous 1, 4-dioxane, adding N, N' -disuccinimidyl carbonate DSC, and reacting the DSC with the starch microspheres for 10 hours under the conditions of nitrogen protection, 4 ℃ and magnetic stirring, wherein the hydroxyl groups on the surfaces of the starch microspheres are modified by the DSC;
(7) and (3) adding a pH 7.4 Burriptan-Robinson buffer solution into the reaction solution obtained in the step (6), adding a human recombinant tissue factor lipidosome with the volume fraction of 0.3-0.5% into the buffer solution, continuing stirring for 12 hours at the temperature of 4 ℃ under the protection of nitrogen, centrifuging by using a centrifugal machine, discarding the supernatant, collecting the precipitate, sequentially washing the precipitate with ethanol and deionized water, and performing vacuum drying to obtain the final product, namely the starch microsphere and the human recombinant tissue factor lipidosome composite hemostatic dry powder material.
2. The preparation method of the starch microsphere and human recombinant tissue factor lipidated composite hemostatic material according to claim 1, wherein the emulsifier is Span60, and the mass of the emulsifier is 4-8% of the mass of soybean oil; or Tween60 with the mass being 9-10% of the mass of the soybean oil.
3. The preparation method of the starch microsphere and human recombinant tissue factor lipidated composite hemostatic material according to claim 1 or 2, characterized in that the initiator is epichlorohydrin, and the added mass is 6-7% of the amount of starch; or sodium trimetaphosphate is adopted, and the adding amount is 5-7% of the using amount of starch.
4. The preparation method of the starch microsphere and human recombinant tissue factor lipidated composite hemostatic material according to claim 1 or 2, characterized in that the mass of the starch microsphere is 15-20% of the mass of anhydrous 1, 4-dioxane; the DSC dosage is 0.5-1% of the mass of the starch microspheres.
5. The preparation method of the starch microsphere and human recombinant tissue factor lipidated composite hemostatic material according to claim 1 or 2, characterized in that the ratio of the human recombinant tissue factor lipidated compound to the starch microsphere is that 0.02 ml of human recombinant tissue factor lipidated compound is added in each gram of the starch microsphere.
6. The method for preparing the starch microsphere and human recombinant tissue factor lipidated composite hemostatic material according to claim 1 or 2, wherein the vacuum drying condition is 0.17 atmosphere and 40 ℃ for 6 hours.
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