CN113952499A - Natural polysaccharide sponge and preparation method and application thereof - Google Patents

Natural polysaccharide sponge and preparation method and application thereof Download PDF

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Publication number
CN113952499A
CN113952499A CN202111135070.4A CN202111135070A CN113952499A CN 113952499 A CN113952499 A CN 113952499A CN 202111135070 A CN202111135070 A CN 202111135070A CN 113952499 A CN113952499 A CN 113952499A
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polysaccharide
solution
sponge
parts
natural polysaccharide
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徐婷
王荣
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Ningbo Institute of Material Technology and Engineering of CAS
Cixi Institute of Biomedical Engineering CNITECH of CAS
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Ningbo Institute of Material Technology and Engineering of CAS
Cixi Institute of Biomedical Engineering CNITECH of CAS
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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
    • 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
    • A61L24/0036—Porous materials, e.g. foams or sponges
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/02—Surgical adhesives or cements; Adhesives for colostomy devices containing inorganic materials
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
    • C08J9/28—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof by elimination of a liquid phase from a macromolecular composition or article, e.g. drying of coagulum
    • 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
    • A61L2300/10—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing inorganic materials
    • A61L2300/102—Metals or metal compounds, e.g. salts such as bicarbonates, carbonates, oxides, zeolites, silicates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/23—Carbohydrates
    • A61L2300/232—Monosaccharides, disaccharides, polysaccharides, lipopolysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/418—Agents promoting blood coagulation, blood-clotting agents, embolising agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/04—Materials for stopping bleeding
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2201/00—Foams characterised by the foaming process
    • C08J2201/04—Foams characterised by the foaming process characterised by the elimination of a liquid or solid component, e.g. precipitation, leaching out, evaporation
    • C08J2201/048—Elimination of a frozen liquid phase
    • C08J2201/0484—Elimination of a frozen liquid phase the liquid phase being aqueous
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
    • C08J2305/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2405/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2401/00 or C08J2403/00
    • C08J2405/02—Dextran; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2405/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2401/00 or C08J2403/00
    • C08J2405/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof

Abstract

The application discloses a natural polysaccharide sponge and a preparation method and application thereof, and belongs to the field of biomedical high polymer materials. The preparation method of the natural polysaccharide sponge comprises the following steps: (1) preparing a solution A containing polysaccharide I with amino and an inorganic compound; (2) preparing a solution B containing polysaccharide II with aldehyde groups; (3) mixing the solution A and the solution B, and carrying out crosslinking reaction to obtain hydrogel; (4) freezing the hydrogel at-80 to-196 ℃ for 12 to 72 hours, and then carrying out freeze drying treatment to obtain the natural polysaccharide sponge. The natural polysaccharide sponge provided by the application has a good hemostatic effect, is light in weight and strong in liquid absorption, and has a relatively low hemolytic rate, and the hemolytic rate of the natural polysaccharide sponge is less than 5% and meets the biological safety requirement on biological materials in the national standard.

Description

Natural polysaccharide sponge and preparation method and application thereof
Technical Field
The invention relates to the field of biomedical polymer materials, in particular to a natural polysaccharide sponge and a preparation method and application thereof.
Background
Uncontrolled bleeding poses a great threat to the life safety of people. The materials such as hemostatic gauze, hemostatic bandage and hemostatic fiber which are commonly used in clinic have the problems of non-absorption by tissues, low hemostatic speed, easy adhesion with wounds to cause secondary injury and the like, and are particularly difficult to play a role in the non-compressible parts. The development of a novel rapid and effective hemostatic material can relieve the pain of patients and save the lives of the patients, and is one of the key research directions in the field of biomedical materials.
In recent years, a hemostatic sponge with a porous structure draws more and more attention, and the sponge has good liquid absorption capacity, can rapidly absorb blood and exudates on the surface of a wound, effectively activates platelets and realizes rapid hemostasis; however, at present, there is still a lack of a sponge which is degradable, safe, nontoxic and excellent in hemostatic performance, and further research and development are needed.
Disclosure of Invention
Aiming at the defects of the prior art, the application provides a natural polysaccharide sponge and a preparation method and application thereof, the natural polysaccharide sponge has good hemostatic effect, light weight, strong liquid absorption and lower hemolytic rate, and the hemolytic rate of the natural polysaccharide sponge is less than 5% and meets the biological safety requirement of the national standard on biological materials.
According to one aspect of the present application, there is provided a natural polysaccharide sponge, the composition of which comprises a polysaccharide I having amino groups, a polysaccharide II having aldehyde groups, and an inorganic compound.
The Blood Coagulation Index (BCI) of the natural polysaccharide sponge is 6-15%; the hemolysis rate is 1.6-4.5%; the porosity is 52.9-76.3%, and the pore size is 20-80 μm.
Optionally, the natural polysaccharide sponge comprises 15-80 parts by weight of polysaccharide I with amino groups, 11-74 parts by weight of polysaccharide II with aldehyde groups and 3-53 parts by weight of inorganic compounds.
Optionally, the polysaccharide I with amino groups is carboxymethyl chitosan, and the polysaccharide II with aldehyde groups is aldehyde-group natural polysaccharide; the inorganic compound is kaolin.
Optionally, the aldehyde-based natural polysaccharide is selected from at least one of aldehyde-based hyaluronic acid, aldehyde-based chondroitin sulfate, and aldehyde-based dextran.
Specifically, the amount of carboxymethyl chitosan may be independently selected from 15 parts, 18 parts, 20 parts, 23 parts, 25 parts, 28 parts, 30 parts, 35 parts, 38 parts, 40 parts, 43 parts, 45 parts, 48 parts, 50 parts, 53 parts, 55 parts, 58 parts, 60 parts, 63 parts, 65 parts, 68 parts, 70 parts, 73 parts, 75 parts, 78 parts, 80 parts, or any number therebetween.
Specifically, the content of the aldehydic natural polysaccharide may be independently selected from 11 parts, 13 parts, 15 parts, 18 parts, 20 parts, 23 parts, 25 parts, 28 parts, 30 parts, 33 parts, 35 parts, 38 parts, 40 parts, 43 parts, 45 parts, 48 parts, 50 parts, 53 parts, 55 parts, 58 parts, 60 parts, 63 parts, 65 parts, 68 parts, 70 parts, 73 parts, 74 parts, or any value between the two.
Specifically, the amount of kaolin can be independently selected from 3 parts, 5 parts, 8 parts, 10 parts, 13 parts, 15 parts, 18 parts, 20 parts, 23 parts, 25 parts, 28 parts, 30 parts, 33 parts, 35 parts, 38 parts, 40 parts, 43 parts, 45 parts, 48 parts, 50 parts, 53 parts, or any number therebetween.
According to yet another aspect of the present application, there is provided a method of preparing a natural polysaccharide sponge, the method comprising the steps of:
(1) preparing a solution A containing polysaccharide I with amino and an inorganic compound;
(2) preparing a solution B containing polysaccharide II with aldehyde groups;
(3) mixing the solution A and the solution B, and carrying out crosslinking reaction to obtain hydrogel;
(4) freezing the hydrogel at the temperature of-80-196 ℃ for 12-72 hours, and then carrying out freeze drying treatment to obtain the natural polysaccharide sponge.
Optionally, the solution a and the solution B comprise a water-soluble solvent, and the water-soluble solvent is selected from one of phosphate buffered saline solution or physiological saline; the pH of the phosphate buffered saline solution is 7.2-7.4.
Optionally, the polysaccharide I with amino groups is carboxymethyl chitosan, and the polysaccharide II with aldehyde groups is aldehyde-group natural polysaccharide; the inorganic compound is kaolin.
The aldehyde natural polysaccharide is at least one selected from aldehyde hyaluronic acid, aldehyde chondroitin sulfate and aldehyde dextran.
The aldehyde-based hyaluronic acid, aldehyde-based chondroitin sulfate and aldehyde-based glucan can be prepared by the prior art.
Optionally, in solution a, the concentration of the polysaccharide i having amino groups is from 2 wt.% to 8 wt.%, and the concentration of the inorganic compound is from 0.5 wt.% to 4 wt.%; in solution B, the concentration of polysaccharide ii having aldehyde groups is 1.5 wt.% to 7 wt.%.
Optionally, the volume ratio of the solution A to the solution B is 1-9: 9-1.
Optionally, the time of the crosslinking reaction is 5 s-50 min, and the temperature of the crosslinking reaction is 20-25 ℃.
Specifically, the amount of carboxymethyl chitosan may be independently selected from 2 wt.%, 2.5 wt.%, 3 wt.%, 3.5 wt.%, 4 wt.%, 4.5 wt.%, 5 wt.%, 5.5 wt.%, 6 wt.%, 6.5 wt.%, 7 wt.%, 7.5 wt.%, 8 wt.%, or any number therebetween.
Specifically, the amount of the aldehydized natural polysaccharide may be independently selected from 1.5 wt.%, 2 wt.%, 2.5 wt.%, 3 wt.%, 3.5 wt.%, 4 wt.%, 4.5 wt.%, 5 wt.%, 5.5 wt.%, 6 wt.%, 6.5 wt.%, 7 wt.%, or any value therebetween.
Specifically, the amount of kaolin can be independently selected from 0.5 wt.%, 1 wt.%, 1.5 wt.%, 2 wt.%, 2.5 wt.%, 3 wt.%, 3.5 wt.%, 4 wt.%, or any value therebetween.
Specifically, the volume ratio of solution a to solution B may be independently selected from 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, or any ratio therebetween.
The crosslinking reaction process is also referred to as gelation of the hydrogel, and the crosslinking time is the gelation time of the hydrogel.
Specifically, the time of the crosslinking reaction is 5 s-5 min, 6 min-10 min, 11 min-15 min, 16 min-20 min, 21 min-25 min, 26 min-30 min, 31 min-35 min, 36 min-40 min, 41 min-45 min or 46 min-50 min.
Specifically, the time of the crosslinking reaction is 5s, 7s, 10s, 12s, 15s, 20s, 25s, 30s, 35s, 40s, 45s, 50s, 55s, 1min, 65s, 70s, 75s, 80s, 85s, 90s, 95s, 100s, 105s, 110s, 115s, 2min, 130s, 150s, 3min, 200s, 4min, 4.5min, 5min, 5.5min, 6min, 7.5min, 8min, 8.5min, 9min, 9.5min, 10min, 10.5min, 11min, 11.5min, 12min, 12.5min, 13min, 13.5min, 14min, 14.5min, 15min, 15.5min, 16min, 16.5min, 17min, 17.5min, 18min, 18.5min, 19min, 20min, 5min, 23.5min, 22min, 5.5min, 22min, 22.5min, 22min, 5min, 22.5min, 23min, 24min, 5min, 25.5min, 22min, 24min, 25.5min, 26min, 25.5min, 24min, 25.5min, 26min, 25.5min, 25min, 25.5min, 24min, 25.5min, 25min, 25.5min, 25min, 25.5min, 25min, 24min, 25min, 25.5min, 24min, 25.5min, 25min, 25.5min, 25min, 25.5min, 25min, 25.5min, 25min, 25.5, 31min, 31.5min, 32min, 32.5min, 33min, 33.5min, 34min, 34.5min, 35min, 35.5min, 36min, 36.5min, 37min, 37.5min, 38min, 38.5min, 39min, 40min, 41min, 42min, 43min, 44min, 45min, 46min, 47min, 48min, 49min, 50min, or any value therebetween.
Alternatively, the hydrogel has a double cross-linked network structure,
the first re-crosslinking structure forms a polysaccharide network through dynamic covalent crosslinking of polysaccharide I with amino and polysaccharide II with aldehyde groups;
the second cross-linked structure is formed by physically cross-linking the inorganic compound with the polysaccharide network.
According to another aspect of the application, the invention also provides the application of the natural polysaccharide sponge in the aspect of hemostatic materials, and the in vitro coagulation index of the natural polysaccharide sponge is evaluated.
Optionally, the natural polysaccharide sponge has a Blood Coagulation Index (BCI) of 6% to 15%; the hemolysis rate is 1.6-4.5%; the porosity is 52.9-76.3%, and the pore size is 20-80 μm.
Specifically, the coagulation index of the native polysaccharide is 6%, 6.79%, 7%, 8%, 9.09%, 9.83%, 10%, 10.82%, 11%, 11.87%, 12%, 12.85%, 13.69%, 14%, 15%, or any value between the two.
The natural polysaccharide has a hemolysis rate of 1.6%, 2.0%, 2.2%, 2.5%, 2.8%, 3.2%, 3.5%, 3.8%, 4.0%, 4.3%, 4.5%, or any value therebetween.
The natural polysaccharide has a porosity of 52.9%, 55.6%, 58.6%, 60%, 63.5%, 65%, 67.4%, 69.3%, 71.3%, 74%, 76.3%, or any value therebetween.
The natural polysaccharide has a pore size of 20-45 μm, 25-50 μm, 30-65 μm, 40-80 μm, 50-80 μm, 55-70 μm or 60-80 μm.
Compared with the prior art, the invention has the following beneficial effects:
(1) the carboxymethyl chitosan, the aldehyde-based natural polysaccharide and the kaolin adopted by the invention have the characteristics of safety, no toxicity, excellent biocompatibility, biodegradability and the like. The aldehyde-based natural polysaccharide is used as a natural cross-linking agent, forms a hydrogel network with amino on carboxymethyl chitosan through Schiff base dynamic covalent bonds, and is further subjected to freeze drying to prepare the sponge, so that the problem of high toxicity caused by a common glutaraldehyde cross-linking agent is solved, and the problem that the visualization of the sponge hemostasis process is influenced by material discoloration caused by a genipin cross-linking agent is also solved.
(2) The invention adopts the rapid cooling at the temperature of minus 80 ℃ to minus 196 ℃, solves the problem that the inorganic particles are settled to the bottom, and is beneficial to the uniform dispersion of the inorganic particles.
(3) The natural polysaccharide sponge provided by the invention has light weight, strong liquid absorption and lower hemolysis rate, and the hemolysis rate of the natural polysaccharide sponge is less than 5 percent and meets the biosafety requirement of national standards on biological materials. Meanwhile, the natural polysaccharide sponge provided by the invention has multiple hemostatic effects and a good hemostatic effect, and firstly, the porous structure of the natural polysaccharide sponge and the inorganic particle kaolin can quickly absorb water in blood, concentrate platelets and thrombin in the blood, and enable coagulation factors and the platelets to be gathered and deposited, so that the hemostatic purpose is achieved; chitosan has a certain amount of positive charges, and directly activates platelets on the wound surface to initiate blood coagulation reaction, so that blood coagulation is promoted, and the hemostatic effect is achieved; thirdly, kaolin is contacted with blood, which can directly excite coagulation factor XII, start intrinsic coagulation pathway, and then activate coagulation factor XI, so that fibrin forms fibrin monomer, and the fibrin monomer is combined into fibrin polymer to form water-insoluble blood clot.
In addition, in the absence of the blood coagulation factor, the kaolin can directly start the blood coagulation factor XI, thereby achieving the purpose of hemostasis. The natural polysaccharide sponge provides a carrier for the inorganic particles, and the problems that the inorganic particles are difficult to take out and cause secondary damage to wounds when used independently are avoided.
Drawings
FIG. 1(a) is a diagram showing a natural polysaccharide sponge according to example 1 of the present invention;
FIG. 1(b) is a scanning electron microscope image of the natural polysaccharide sponge of example 1 of the present invention at a magnification of 2000;
FIG. 2 is a chart showing the hemolysis ratio data of natural polysaccharide sponges in examples 1-8 of the present invention.
FIG. 3 is a data chart of blood coagulation indexes of the natural polysaccharide sponges and gauzes according to examples 1-8 of the present invention.
Detailed Description
The invention will now be further described with reference to the following examples, which are intended to illustrate the invention without limiting it in any way.
(1) Preparing aldehyde natural polysaccharide: weighing 1-5 g of chondroitin sulfate, sodium hyaluronate or dextran, pouring into deionized water, and stirring at room temperature until the chondroitin sulfate, the sodium hyaluronate or the dextran is completely dissolved to obtain a polysaccharide aqueous solution with the concentration of 1% (wt/v); then, adding sodium periodate according to the mass ratio of the natural polysaccharide to the sodium periodate of 1-10: 1, and reacting for 1-3 hours in a dark place; then, adding excessive ethylene glycol, stopping the reaction, and continuing stirring for 1-2 hours; and finally, filling the solution into a dialysis bag, dialyzing in ultrapure water for 3-6 days, and freeze-drying at-65 ℃ to obtain aldehyde-based natural polysaccharide solid powder, and refrigerating at-18 ℃ for later use.
(2) Testing the hemolysis rate: the rabbit arterial blood was taken, 20mL of PBS buffer (pH7.4) was added to 10mL of rabbit whole blood, and after gentle mixing, the mixture was centrifuged at 3000rpm for 6 minutes. Then, Red Blood Cells (RBC) were collected from the bottom of the centrifuged tube, and the same protocol as above was repeated 5 times at a volume ratio of 1:2 of RBC to PBS buffer (pH 7.4). Next, RBC suspensions were prepared by diluting red blood cells at a concentration of 5% v/v in fresh PBS buffer (pH 7.4). The native polysaccharide sponge of this application was dispersed at 400. mu.g/mL in PBS buffer (pH7.4) and the resulting suspension was then gently mixed with the RBC suspension at a ratio of 1:1v/v to achieve a final concentration of 200. mu.g/mL. After incubation at room temperature for 130 minutes, the samples were centrifuged at 3000rpm for 7 minutes. The supernatant (measured in 150. mu.L) was transferred to a 96-well plate, and the absorbance of the sample was recorded at 545nm using a microplate reader. The hemolysis rate of each sample was calculated using the following method:
hemolysis ratio (%) ((a)s-Ab)/(Ap-Ab))*100％
AsAs absorbance of the sample, AbThe absorbance of the negative control is Ap, and the absorbance of the positive control is Ap; the negative control is that no natural polysaccharide sponge group is added, and the PBS buffer solution and the RBC suspension are mixed according to the ratio of 1:1 v/v; the positive control is no natural polysaccharide sponge added, deionized water is mixed with the RBC suspension at a ratio of 1:1 v/v.
(3) Blood coagulation index test: pre-weighed 50mg of the natural polysaccharide sponges of the application and pre-weighed 50mg of gauze (control) were placed in centrifuge tubes, respectively, and preheated at 37 ℃ for 5 minutes. Subsequently, anticoagulated rabbit blood (100. mu.L) was gently added dropwise to the surface of the sample, followed by addition of 12.5. mu.L of CaCl2(0.2 mol/L). After incubation at 37 ℃ for 120 seconds, 10mL of deionized water was added very gently to the centrifuge tube and then centrifuged at 1000rpm for 5 minutes. Then, the microplate reader obtained the absorbance of the supernatant at 545 nm. The coagulation index was calculated using the following method:
BCI(％)＝(As/Ar)×100％
wherein A issIs the absorbance of the supernatant of a sample treated with a natural polysaccharide sponge or gauze, ArIs the absorbance of the supernatant in the blank; the blank group is prepared without adding sponge, gauze and CaCl2And (6) processing the groups.
In this experiment, lower BCI indicates better clotting ability.
(4) And (3) porosity testing: 15mg of sponge was immersed in absolute ethanol and kept fully saturated in ethanol. The percent porosity after immersion of the sample was calculated as follows:
porosity (%) ═ (W)s-Wd)/(Vs×ρ)
WsFor the weight of the sample immersed in absolute ethanol, WdWeight of the freeze-dried sample, VsThe volume of the sample after freeze-drying, ρ is the density of ethanol.
Example 1
Preparing natural polysaccharide sponge:
(1) dissolving carboxymethyl chitosan and kaolin in physiological saline to give solution a containing 2 wt.% CMCS and 0.5 wt.% KA;
(2) preparing aldehyde hyaluronic acid, wherein the mass ratio of sodium hyaluronate to sodium periodate is 1:1, the dark reaction time is 2 hours, the molecular weight cut-off of a dialysis bag is 3500, and dissolving the aldehyde hyaluronic acid in physiological saline to obtain a solution B containing 5 wt.% of aldehyde hyaluronic acid;
(3) mixing the solution A and the solution B in a volume ratio of 3:7, and gelatinizing for 12s at room temperature to obtain hydrogel;
(4) freezing the hydrogel at-80 deg.C for 48h, and freeze drying to obtain natural polysaccharide sponge.
The natural polysaccharide sponge comprises 14 parts of carboxymethyl chitosan, 3.5 parts of kaolin and 83 parts of aldehyde hyaluronic acid in parts by weight. The material diagram and the scanning electron microscope diagram of the obtained natural polysaccharide sponge are shown in figure 1, and the sponge can be seen from figure 1 to have a porous structure, and the pore diameter is in the range of 25-50 mu m.
The native polysaccharide sponge was then tested for hemolysis and coagulation index, the sponge having a hemolysis of 2.5% and a coagulation index of 11.87%, with gauze as a control and a coagulation index of 86.9%. The hemolysis rate below 5% is indicated by the national standard as a safe biomaterial, from which it can be seen that the sponge is safe, non-toxic and has excellent hemostatic capabilities, see in particular figures 2 and 3. Finally, the porosity of the sponge is tested, and the porosity of the sponge is calculated to be 63.5%.
Example 2
Preparing natural polysaccharide sponge:
(1) dissolving carboxymethyl chitosan and kaolin in physiological saline to give solution a containing 4 wt.% CMCS and 2 wt.% KA;
(2) preparing an aldehyde chondroitin sulfate, wherein the mass ratio of the chondroitin sulfate to sodium periodate is 3:1, the dark reaction time is 1h, the cut-off molecular weight of a dialysis bag is 8000-14000, and dissolving the aldehyde chondroitin sulfate in physiological saline to obtain a solution B containing 4 wt.% of the aldehyde chondroitin sulfate;
(3) mixing the solution A and the solution B in a volume ratio of 5:5, and gelatinizing at room temperature for 125s to obtain hydrogel;
(4) freezing the hydrogel at-80 deg.C for 72h, and freeze drying to obtain natural polysaccharide sponge.
The natural polysaccharide sponge comprises, by weight, 40 parts of carboxymethyl chitosan, 20 parts of kaolin and 40 parts of aldehyde chondroitin sulfate. The sponge has a hemolysis rate of 1.6% and a coagulation index of 9.09%, see in particular fig. 2 and 3; the porosity of the sponge is 52.9%, and the pore size is 20-45 μm.
Example 3
Preparing natural polysaccharide sponge:
(1) dissolving carboxymethyl chitosan and kaolin in a PBS solution at pH 7.2, resulting in solution a containing 8 wt.% CMCS and 4 wt.% KA;
(2) preparing aldehyde dextran, wherein the mass ratio of dextran to sodium periodate is 10:1, the dark reaction time is 3 hours, the cut-off molecular weight of a dialysis bag is 3500, and the aldehyde dextran is dissolved in a PBS solution with the pH value of 7.2 to obtain a solution B containing 6 wt.% of aldehyde dextran;
(3) mixing the solution A and the solution B in a volume ratio of 8:2, and gelatinizing at room temperature for 36min to obtain hydrogel;
(4) freezing the hydrogel at-80 deg.C for 12h, and freeze drying to obtain natural polysaccharide sponge.
The natural polysaccharide sponge comprises 59 parts of carboxymethyl chitosan, 29.5 parts of kaolin and 11.5 parts of aldehyde dextran in parts by weight. The sponge has a hemolysis rate of 3.2% and a coagulation index of 6.79%, see in particular fig. 2 and 3; the porosity of the sponge is 71.3%, and the pore size is 40-80 μm.
Example 4
Preparing natural polysaccharide sponge:
(1) dissolving carboxymethyl chitosan and kaolin in a PBS solution at pH 7.2, resulting in solution a containing 7 wt.% CMCS and 1 wt.% KA;
(2) preparing aldehyde hyaluronic acid, wherein the mass ratio of sodium hyaluronate to sodium periodate is 5:1, the dark reaction time is 3 hours, the cut-off molecular weight of a dialysis bag is 3500, and the aldehyde hyaluronic acid is dissolved in a PBS solution with the pH of 7.2 to obtain a solution B containing 3 wt.% of aldehyde hyaluronic acid;
(3) mixing the solution A and the solution B in a volume ratio of 1:9, and gelatinizing at room temperature for 150s to obtain hydrogel;
(4) freezing the hydrogel at-196 deg.C for 12h, and freeze drying to obtain natural polysaccharide sponge.
The natural polysaccharide sponge comprises, by weight, 38.5 parts of carboxymethyl chitosan, 12.5 parts of kaolin and 49 parts of aldehyde hyaluronic acid. The sponge had a hemolysis rate of 3.8% and a coagulation index of 13.69%, see in particular fig. 2 and 3; the porosity of the sponge is 58.6%, and the pore size is 30-65 μm.
Example 5
Preparing natural polysaccharide sponge:
(1) dissolving carboxymethyl chitosan and kaolin in a PBS solution at pH7.4, resulting in solution a containing 3 wt.% CMCS and 1.5 wt.% KA;
(2) preparing aldehyde dextran, wherein the mass ratio of dextran to sodium periodate is 10:1, the dark reaction time is 3 hours, the cut-off molecular weight of a dialysis bag is 3500, and the aldehyde dextran is dissolved in a PBS solution with the pH value of 7.4 to obtain a solution B containing 5 wt.% of aldehyde dextran;
(3) mixing the solution A and the solution B in a volume ratio of 2:8, and gelatinizing for 7s at room temperature to obtain hydrogel;
(4) freezing the hydrogel at-80 deg.C for 12h, and freeze drying to obtain natural polysaccharide sponge.
The natural polysaccharide sponge comprises 12 parts of carboxymethyl chitosan, 6 parts of kaolin and 82 parts of aldehyde dextran in parts by weight. The sponge has a hemolysis rate of 4.0% and a coagulation index of 15%, see in particular fig. 2 and 3; the porosity of the sponge is 76.3%, and the pore size is 60-80 μm.
Example 6
Preparing natural polysaccharide sponge:
(1) dissolving carboxymethyl chitosan and kaolin in physiological saline to give solution a containing 5 wt.% CMCS and 3 wt.% KA;
(2) preparing an aldehyde chondroitin sulfate, wherein the mass ratio of the chondroitin sulfate to sodium periodate is 8:1, the dark reaction time is 3 hours, the cut-off molecular weight of a dialysis bag is 8000-14000, and dissolving the aldehyde chondroitin sulfate in physiological saline to obtain a solution B containing 6 wt.% of the aldehyde chondroitin sulfate;
(3) mixing the solution A and the solution B in a volume ratio of 4:6, and gelatinizing for 130s at room temperature to obtain hydrogel;
(4) freezing the hydrogel at-80 deg.C for 12h, and freeze drying to obtain natural polysaccharide sponge.
The natural polysaccharide sponge comprises 29.5 parts of carboxymethyl chitosan, 17.5 parts of kaolin and 53 parts of aldehyde chondroitin sulfate in parts by weight. The sponge had a hemolysis rate of 4.3% and a coagulation index of 12.85%, see in particular fig. 2 and 3; the porosity of the sponge is 67.4%, and the pore size is 50-80 μm.
Example 7
Preparing natural polysaccharide sponge:
(1) dissolving carboxymethyl chitosan and kaolin in physiological saline to give solution a containing 6 wt.% CMCS and 2.5 wt.% KA;
(2) preparing aldehyde hyaluronic acid, wherein the mass ratio of sodium hyaluronate to sodium periodate is 3:1, the dark reaction time is 3 hours, the cut-off molecular weight of a dialysis bag is 3500, and the aldehyde hyaluronic acid is dissolved in a PBS solution with the pH of 7.2 to obtain a solution B containing 6 wt.% of aldehyde hyaluronic acid;
(3) mixing the solution A and the solution B in a volume ratio of 6:4, and gelatinizing at room temperature for 8.5min to obtain hydrogel;
(4) freezing the hydrogel at-196 deg.C for 24h, and freeze drying to obtain natural polysaccharide sponge.
The natural polysaccharide sponge comprises 48 parts of carboxymethyl chitosan, 20 parts of kaolin and 32 parts of aldehyde hyaluronic acid in parts by weight. The sponge had a hemolysis rate of 4.5% and a coagulation index of 9.83%, see in particular fig. 2 and 3; the porosity of the sponge is 55.6%, and the pore size is 25-45 μm.
Example 8
Preparing natural polysaccharide sponge:
(1) dissolving carboxymethyl chitosan and kaolin in a PBS solution at pH 7.2, resulting in solution a containing 2.5 wt.% CMCS and 3.5 wt.% KA;
(2) preparing aldehyde dextran, wherein the mass ratio of dextran to sodium periodate is 10:1, the dark reaction time is 3 hours, the cut-off molecular weight of a dialysis bag is 3500, and the aldehyde dextran is dissolved in a PBS solution with the pH value of 7.2 to obtain a solution B containing 6.5 wt.% of aldehyde dextran;
(3) mixing the solution A and the solution B in a volume ratio of 9:1, and gelatinizing at room temperature for 28min to obtain hydrogel;
(4) freezing the hydrogel in an ultralow temperature refrigerator at-80 ℃ for 48h, and then carrying out freeze drying treatment to obtain the natural polysaccharide sponge.
The natural polysaccharide sponge comprises, by weight, 37 parts of carboxymethyl chitosan, 52 parts of kaolin and 11 parts of aldehyde dextran. The sponge has a hemolysis rate of 2.2% and a coagulation index of 10.82%, see in particular fig. 2 and 3; the porosity of the sponge is 69.3%, and the pore size is 55-70 μm.
As can be seen from examples 1-8, the crosslinking reaction time is gradually shortened as the proportion of the aldehydized natural polysaccharide in the natural polysaccharide spongin component is increased.
As can be seen from FIG. 2, the hemolysis rates of the natural polysaccharide sponges prepared in examples 1-8 are all within 5% relative to the 100% hemolysis rate of the positive control group, and meet the biosafety requirements of national standards for biomaterials.
As can be seen from FIG. 3, the blood coagulation index of the natural polysaccharide sponges prepared in examples 1 to 8 is greatly reduced relative to the blood coagulation index of gauze of 86.9%, and the natural polysaccharide sponges exhibit good blood coagulation ability.
Comparative example 1
The procedure was as in example 1 except that KA was not added in step (1). The obtained natural polysaccharide sponge was tested, and the hemolysis rate was 4.8%, and the blood coagulation index was 35.8%. It can be seen that, although the sponge meets the requirements of national standard on biological safety of biological materials, the blood coagulation capacity of the sponge is obviously lower than that of the natural polysaccharide sponge.
Although the present application has been described with reference to a few embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the application as defined by the appended claims.

Claims (10)

1. A preparation method of natural polysaccharide sponge is characterized by comprising the following steps:
(1) preparing a solution A containing polysaccharide I with amino and an inorganic compound;
(2) preparing a solution B containing polysaccharide II with aldehyde groups;
(3) mixing the solution A and the solution B, and carrying out crosslinking reaction to obtain hydrogel;
(4) freezing the hydrogel at-80 to-196 ℃ for 12 to 72 hours, and then carrying out freeze drying treatment to obtain the natural polysaccharide sponge.
2. The preparation method according to claim 1, wherein the polysaccharide I having amino groups is carboxymethyl chitosan, the polysaccharide II having aldehyde groups is an aldehydized natural polysaccharide, and the inorganic compound is kaolin;
preferably, the aldehyde-based natural polysaccharide is at least one selected from aldehyde-based hyaluronic acid, aldehyde-based chondroitin sulfate and aldehyde-based dextran.
3. The method according to claim 1, wherein the solution A and the solution B comprise a water-soluble solvent selected from phosphate buffered saline or physiological saline; the pH value of the phosphate buffer salt solution is 7.2-7.4.
4. The method according to claim 1, wherein the concentration of the polysaccharide i having amino groups in the solution a is 2 to 8 wt.%, and the concentration of the inorganic compound is 0.5 to 4 wt.%;
in the solution B, the concentration of the polysaccharide II with aldehyde groups is 1.5-7 wt.%.
5. The method according to claim 1, wherein the volume ratio of the solution A to the solution B is 1-9: 9-1.
6. The preparation method according to claim 1, wherein the time of the crosslinking reaction is 5s to 50min, and the temperature of the crosslinking reaction is 20 to 25 ℃.
7. The method according to claim 1, wherein the hydrogel has a double cross-linked network structure,
the first re-crosslinking structure is formed by dynamically and covalently crosslinking polysaccharide I with amino and polysaccharide II with aldehyde groups to form a polysaccharide network;
the second cross-linked structure is formed by physically cross-linking an inorganic compound with a polysaccharide network.
8. The natural polysaccharide sponge prepared by the preparation method of any one of claims 1 to 7, wherein the natural polysaccharide sponge comprises the following components of polysaccharide I with amino groups, polysaccharide II with aldehyde groups and inorganic compounds;
preferably, the Blood Coagulation Index (BCI) of the natural polysaccharide sponge is 6-15%; the hemolysis rate is 1.6-4.5%; the porosity is 52.9-76.3%, and the pore size is 20-80 μm.
9. The natural polysaccharide sponge of claim 8, comprising in parts by weight:
15-80 parts of polysaccharide I with amino;
11-74 parts of polysaccharide II with aldehyde groups;
3-53 parts of an inorganic compound.
10. A natural polysaccharide sponge prepared by the preparation method of any one of claims 1 to 7 and application of the natural polysaccharide sponge of any one of claims 8 to 9 in a hemostatic material.
CN202111135070.4A 2021-09-27 2021-09-27 Natural polysaccharide sponge and preparation method and application thereof Pending CN113952499A (en)

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